benzofurans and salvianolic-acid-B

It is estimated that over 44 million people across the globe have dementia, and half of these cases are believed to be Alzheimer's disease (AD). As the proportion of the global population which is over the age 60 increases so will the number of individuals living with AD. This will result in ever-increasing demands on healthcare systems and the economy. AD can be either sporadic or familial, but both present with similar pathobiology and symptoms. Three prominent theories about the cause of AD are the amyloid, tau and mitochondrial hypotheses. The mitochondrial hypothesis focuses on mitochondrial dysfunction in AD, however little attention has been given to the potential dysfunction of the mitochondrial ATP synthase in AD. ATP synthase is a proton pump which harnesses the chemical potential energy of the proton gradient across the inner mitochondrial membrane (IMM), generated by the electron transport chain (ETC), in order to produce the cellular energy currency ATP. This review presents the evidence accumulated so far that demonstrates dysfunction of ATP synthase in AD, before highlighting two potential pharmacological interventions which may modulate ATP synthase.

Topics: Alzheimer Disease; Animals; Benzofurans; Brain; Curcumin; Energy Metabolism; Humans; Mitochondria; Mitochondrial Proton-Translocating ATPases; Neuroprotective Agents

Salvia miltiorrhiza (Danshen), as an important traditional Chinese medicinal plant, has been used in China for the treatment of cardiovascular diseases for hundreds of years. Salvianolic acids (salvianolic acid A and salvianolic acid B) as the most abundant water-soluble component extracted from Salvia miltiorrhiza have attracted more and more attention from cardiovascular scientists due to its comprehensive cardiovascular actions.

Topics: Animals; Benzofurans; Caffeic Acids; Cardiovascular Diseases; Humans; Lactates; Medicine, Chinese Traditional; Mitogen-Activated Protein Kinases; Oxidative Stress; Reactive Oxygen Species; Signal Transduction

Nature has generously offered life-saving therapies to mankind by providing evolutionarily optimized drug-like entities in the form of natural products. These splendid gifts of nature have served as most suitable candidates for anti-cancer drug discovery due to their pleiotropic activity on target molecules. This review aims to provide an update on the natural sources and bioactivities of such gifts from nature, salvianolic acid A & B, which are major bioactive constituents of a traditional Chinses medicinal herb,

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzofurans; Caffeic Acids; Cell Cycle; Drugs, Chinese Herbal; Humans; Lactates; Polyphenols; Signal Transduction

Topics: Benzofurans; Drugs, Chinese Herbal; Humans; Neurodegenerative Diseases

In this review, the researches on Chinese herb components with anti-hepatic fibrosis activity in China in the recent 20 years were generalized. Almost thirty active herb components attracted author's attention, especially, salvianolic acid B and oxymatrine which were investigated comprehensively. Moreover, the author considered that, in view of the complex pathogenesis and the multi-pathway and multi-target superiority of Chinese medicine formula, the effective components formula investigations deserve more attention and probably prompt a potential researching direction.

Topics: Alkaloids; Animals; Attention; Benzofurans; Drugs, Chinese Herbal; Humans; Liver Cirrhosis; Quinolizines

As one of the main water-soluble composites of Radix Salviae, salvianolic acid B is a phenolic acid ingredient of the Chinese drug, which is rich content in the herb and has strong pharmaceutical activity. It is used to treat cardiocerebral vascular diseases, antagonize hepatic/renal fibrosis, prevent cancer, and promote stem cell proliferation and differentiation. In the researches of its acting mechanisms, rather deepened studies have been carried out for its application on cardiocerebral vascular diseases, but that for others are rather fewer.

Topics: Benzofurans; Biomedical Research; Disease; Humans; Medicine, Chinese Traditional; Stem Cells; Ventricular Remodeling

Salvianolic acids are the most abundant water-soluble compounds extracted from Radix Salvia miltiorrhiza (Danshen). In China, Danshen has been wildly used to treat cardiovascular diseases for hundreds of years. Salvianolic acids, especially salvianolic acid A (Sal A) and salvianolic acid B (Sal B), have been found to have potent anti-oxidative capabilities due to their polyphenolic structure. Recently, intracellular signaling pathways regulated by salvianolic acids in vascular endothelial cells, aortic smooth muscle cells, as well as cardiomyocytes, have been investigated both in vitro and in vivo upon various cardiovascular insults. It is discovered that the cardiovascular protection of salvianolic acids is not only because salvianolic acids act as reactive oxygen species scavengers, but also due to the reduction of leukocyte-endothelial adherence, inhibition of inflammation and metalloproteinases expression from aortic smooth muscle cells, and indirect regulation of immune function. Competitive binding of salvianolic acids to target proteins to interrupt protein-protein interactions has also been found to be a mechanism of cardiovascular protection by salvianolic acids. In this article, we review a variety of studies focusing on the above mentioned mechanisms. Besides, the target proteins of salvianolic acids are also described. These results of recent advances have shed new light to the development of novel therapeutic strategies for salvianolic acids to treat cardiovascular diseases.

Topics: Animals; Aorta; Benzofurans; Caffeic Acids; Drugs, Chinese Herbal; Endothelial Cells; Humans; Lactates; Myocytes, Cardiac; Myocytes, Smooth Muscle; Phenanthrolines; Reactive Oxygen Species; Salvia miltiorrhiza

Topics: Animals; Benzofurans; Bone Marrow Cells; Cell Differentiation; Drugs, Chinese Herbal; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Myocardial Infarction; Myocytes, Cardiac; Phytotherapy; Salvia miltiorrhiza

This review summarizes the recent advances in the chemical analysis of Danshen and its finished products, including the introduction of the identified bioactive components, analytical methods for quantitative determination of target analytes and fingerprinting authentication, quality criteria of Danshen crude herb and its preparations, as well as the pharmacokinetic and pharmacodynamic studies on the active components of Danshen and its finished products. Danshen contains mainly two types of constituents, the hydrophilic depsides and lipophilic diterpenoidal quinones and both of them are responsible for the pharmacological activities of Danshen. In order to monitor simultaneously both types of components which have different physicochemical properties, numerous analytical methods have been reported using various chromatographic and spectrophotometric technologies. In this review, 110 papers on analysis of Danshen are discussed, various analytical methods and their chromatographic conditions are briefly described and their advantages/disadvantages are compared. For obtaining a quick, accurate and applicable analytical approach for quality evaluation and establishing a harmonized criteria of Danshen and its finished products, the authors' suggestion and opinions are given, including the reasonable selection of marker compounds with high concentration and commercial availability, a simple sample preparation procedure with high recoveries of both the hydrophilic phenols and lipophilic tanshinones, and an optimized chromatographic condition with ideal resolutions of all the target components. The chemical degradation and transformation of the predominant constituent salvianolic acid B in Danshen during processing and manufacturing are also emphasized in order to assure the quality consistency of Danshen containing products.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Depsides; Diterpenes; Drugs, Chinese Herbal; Hydrophobic and Hydrophilic Interactions; Mass Spectrometry; Quinones; Salvia miltiorrhiza

The article reviewed the research on the pharmacological effects of Danshen on hemorheology. We discussed the action mechanism of Danshen and its preparations from the points of hemorheology, blood and vessel endothelium cell. Danshen and its preparations can obviously improve the hemorheological characteristic by various ways.

Topics: Abietanes; Animals; Benzofurans; Drugs, Chinese Herbal; Hemorheology; Hemostasis; Lactates; Phenanthrenes; Plant Roots; Plants, Medicinal; Platelet Aggregation; Salvia miltiorrhiza

Danshen, the dried root of Salvia miltiorrhiza, has been widely used in China and, to a lesser extent, in Japan, the United States, and other European countries for the treatment of cardiovascular and cerebrovascular diseases. In China, the specific clinical use is angina pectoris, hyperlipidemia, and acute ischemic stroke. The current review covers its traditional uses, chemical constituents, pharmacological activities, pharmacokinetics, clinical applications, and potential herb-drug interactions based on information obtained in both the English and Chinese literature. Although numerous clinical trials have demonstrated that certain Danshen products in China are effective and safe for the treatment of cardiovascular diseases, most of these lack sufficient quality. Therefore, large randomized clinical trials and further scientific research to determine its mechanism of actions will be necessary to ensure the safety, effectiveness, and better understanding of its action.

Topics: Abietanes; Angina Pectoris; Animals; Benzofurans; Drugs, Chinese Herbal; Fibrinolytic Agents; Herb-Drug Interactions; Humans; Hyperlipidemias; Lactates; Phenanthrenes; Phenanthrolines; Plant Extracts; Randomized Controlled Trials as Topic; Salvia miltiorrhiza; Stroke

Topics: Benzofurans; Caffeic Acids; Collagen; Humans; Lactates; Lipid Peroxidation; Liver Circulation; Liver Cirrhosis; Salvia miltiorrhiza; Tumor Necrosis Factor-alpha

The aim of this study was to investigate the effectiveness of triamcinolone acetonide (TA) and salvianolic acid B (SA-B) intralesional combined injection in the treatment of oral submucous fibrosis (OSF).. A randomized clinical trial was performed. TA, SA-B, and TA combined with SA-B were consecutively applied intralesionally weekly for 20 weeks. Mouth opening and burning sensation improvement as determined by a 100-mm visual analog scale were evaluated at weeks 10, 20, and 44.. Forty-two subjects fulfilled the study without obvious adverse reactions. The net gain in mouth opening was 2.00 ± 1.21 mm in the TA group, 3.48 ± 2.23 mm in the SA-B group, and 5.50 ± 1.80 mm in the TA + SA-B group at week 44. The burning sensation improved by 3.05 ± 0.76 in the TA group, 4.96 ± 0.97 in the SA-B group, and 6.11 ± 0.93 in the TA + SA-B group by the end of the study.. TA + SA-B intralesional injections improved mouth open and burning sensation in these OSF patients.

Topics: Adult; Anti-Inflammatory Agents; Benzofurans; Drug Combinations; Drugs, Chinese Herbal; Female; Follow-Up Studies; Glucocorticoids; Humans; Injections, Intralesional; Male; Mouth Mucosa; Oral Submucous Fibrosis; Pain Measurement; Paresthesia; Salvia miltiorrhiza; Treatment Outcome; Triamcinolone Acetonide

To evaluate the clinical efficacy of salvianolic acid B (SA-B) on liver fibrosis in chronic hepatitis B.. Sixty patients with definite diagnosis of liver fibrosis with hepatitis B were included in the trial. Interferon-gamma (IFN-gamma) was used as control drug. The patients took orally SA-B tablets or received muscular injection of IFN-gamma in the double blind randomized test. The complete course lasted 6 months. The histological changes of liver biopsy specimen before and after the treatment were the main evidence in evaluation, in combination with the results of contents of serum HA, LN, IV-C, P-III-P, liver ultrasound imaging, and symptoms and signs.. Reverse rate of fibrotic stage was 36.67 % in SA-B group and 30.0 % in IFN-gamma group. Inflammatory alleviating rate was 40.0 % in SA-B group and 36.67 % in IFN-gamma group. The average content of HA and IV-C was significantly lower than that before treatment. The abnormal rate also decreased remarkably. Overall analysis of 4 serological fibrotic markers showed significant improvement in SA-B group as compared with the IFN-gamma group. Score of liver ultrasound imaging was lower in SA-B group than in IFN-gamma group (HA 36.7 % vs 80 %, IV-C 3.3 % vs 23.2 %). Before the treatment, ALT AST activity and total bilirubin content of patients who had regression of fibrosis after oral administration of SA-B, were significantly lower than those of patients who had aggravation of fibrosis after oral administration of SA-B. IFN-gamma showed certain side effects (fever and transient decrease of leukocytes, occurrence rates were 50 % and 3.23 %), but SA-B showed no side effects.. SA-B could effectively reverse liver fibrosis in chronic hepatitis B. SA-B was better than IFN-gamma in reduction of serum HA content, overall decrease of 4 serum fibrotic markers, and decrease of ultrasound imaging score. Liver fibrosis in chronic hepatitis B with slight liver injury was more suitable to SA-B in anti-fibrotic treatment. SA-B showed no obvious side effects.

Topics: Adult; Benzofurans; Double-Blind Method; Drugs, Chinese Herbal; Female; Hepatitis B, Chronic; Humans; Interferon-gamma; Liver Cirrhosis; Male; Phytotherapy; Recombinant Proteins

Salvianolic acid B (SAB) can alleviate renal fibrosis and improve the renal function.. To investigate the effect of SAB on renal tubulointerstitial fibrosis and explore its underlying mechanisms.. Male C57 mice were subjected to unilateral ureteric obstruction (UUO) and aristolochic acid nephropathy (AAN) for renal fibrosis indication. Vehicle or SAB (10 mg/kg/d, i.p.) were given consecutively for 2 weeks in UUO mice while 4 weeks in AAN mice. The serum creatinine (Scr) and blood urine nitrogen (BUN) were measured. Masson's trichrome staining and the fibrotic markers (FN and α-SMA) were used to evaluate renal fibrosis. NRK-49F cells exposed to 2.5 ng/mL TGF-β were treated with SAB in the presence or absence of 20 μM 3-DZNep, an inhibitor of EZH2. The protein expression of EZH2, H3k27me3 and PTEN/Akt signaling pathway in renal tissue and NRK-49F cells were measured by Western blots.. SAB significantly improved the levels of Scr by 24.3% and BUN by 35.7% in AAN mice. SAB reduced renal interstitial collagen deposition by 34.7% in UUO mice and 72.8% in AAN mice. Both. SAB might have therapeutic potential on renal fibrosis of CKD through inhibiting EZH2, which encourages further clinical trials.

Topics: Animals; Benzofurans; Depsides; Enhancer of Zeste Homolog 2 Protein; Fibrosis; Histones; Kidney; Kidney Diseases; Male; Mice; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Transforming Growth Factor beta1; Ureteral Obstruction

Hypertrophic scars (HTSs) are a fibroproliferative disorder that occur following skin injuries. Salvianolic acid B (Sal-B) is an extractant from Salvia miltiorrhiza that has been reported to ameliorate fibrosis in multiple organs. However, the antifibrotic effect on HTSs remains unclear. This study aimed to determine the antifibrotic effect of Sal-B in vitro and in vivo.. In vitro, hypertrophic scar-derived fibroblasts (HSFs) were isolated from human HTSs and cultured. HSFs were treated with (0, 10, 50, 100 μmol/L) Sal-B. Cell proliferation and migration were evaluated by EdU, wound healing, and transwell assays. The protein and mRNA levels of TGFβI, Smad2, Smad3, α-SMA, COL1, and COL3 were detected by Western blots and real-time PCR. In vivo, tension stretching devices were fixed on incisions for HTS formation. The induced scars were treated with 100 μL of Sal-B/PBS per day according to the concentration of the group and followed up for 7 or 14 days. The scar condition, collagen deposition, and α-SMA expression were analyzed by gross visual examination, H&E, Masson, picrosirius red staining, and immunofluorescence.. In vitro, Sal-B inhibited HSF proliferation, migration, and downregulated the expression of TGFβI, Smad2, Smad3, α-SMA, COL1, and COL3 in HSFs. In vivo, 50 and 100 μmol/L Sal-B significantly reduced scar size in gross and cross-sectional observations, with decreased α-SMA expression and collagen deposition in the tension-induced HTS model.. Our study demonstrated that Sal-B inhibits HSFs proliferation, migration, fibrotic marker expression and attenuates HTS formation in a tension-induced HTS model in vivo.. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Topics: Animals; Benzofurans; Cicatrix, Hypertrophic; Cross-Sectional Studies; Fibroblasts; Fibrosis; Humans

Salvianolic acid B(Sal B) is the main water-soluble component of Salvia miltiorrhiza Bunge. Studies have found that Sal B has a good protective effect on blood vessels. Sal B can protect endothelial cells by anti-oxidative stress, inducing autophagy, inhibiting endoplasmic reticulum stress(ERS), inhibiting endothelial inflammation and adhesion molecule expression, inhibiting endothelial cell permeability, anti-thrombosis, and other ways. In addition, Sal B can alleviate endothelial cell damage caused by high glucose(HG). For vascular smooth muscle cell(VSMC), Sal B can reduce the synthesis and secretion of inflammatory factors by inhibiting cyclooxygenase. It can also play a vasodilatory role by inhibiting Ca~(2+) influx. In addition, Sal B can inhibit VSMC proliferation and migration, thereby alleviating vascular stenosis. Sal B also inhibits lipid deposition in the subendothelium, inhibits macrophage conversion to foam cells, and reduces macrophage apoptosis, thereby reducing the volume of subendothelial lipid plaques. For some atherosclerosis(AS) complications, such as peripheral artery disease(PAD), Sal B can promote angiogenesis, thereby improving ischemia. It should be pointed out that the conclusions obtained from different experiments are not completely consistent, which needs further research. In addition, previous pharmacokinetics showed that Sal B was poorly absorbed by oral administration, and it was unstable in the stomach, with a large first-pass effect in the liver. Sal B had fast distribution and metabolism in vivo and short drug action time. These affect the bioavailability and biological effects of Sal B, and the development of clinically valuable Sal B non-injectable delivery systems remains a great challenge.

Topics: Benzofurans; Endothelial Cells; Lipids; Oxidative Stress

Supramolecular natural product gels (NPGs) have emerged as promising biomaterials for scalable and adjustable drug delivery systems. These gels possess biocompatibility, biodegradability, and the ability to mimic the extracellular matrix. Salvianolic acid B (

Topics: Antioxidants; Benzofurans; Hydrogels; Phosphopeptides; Wound Healing

Induced vascular growth in the myocardium has been widely acknowledged as a promising intervention strategy for patients with ischemic coronary artery disease. Yet despite long-term efforts on gene, protein or cell-based pro-angiogenic therapies, the clinical translation remains challenging. Noticeably, multiple medicinal herbs have long-term documented effects in promoting blood circulation. Salvia miltiorrhiza and Ligusticum stratum are two representative traditional Chinese medicine herbs with suggested roles in enhancing organ blood supply, and Guanxinning Tablet (GXNT), a botanical drug which is formulated with these two herbs, exhibited significant efficacy against angina pectoris in clinical practices.. This study aimed to examine the pro-angiogenic activity of GXNT and its major components, as well as to explore their pharmacological mechanism in promoting angiogenesis.. In vitro, the pro-angiogenic effects of GXNT and its major components were examined on human umbilical vein endothelial cells by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch assay, and endothelial cell tube formation assay. In vivo, the pro-angiogenic effects were examined on the ponatinib-induced angiogenesis defective zebrafish model. The active compounds were identified through phenotype-based screening in zebrafish, and their pharmacological mechanism was explored in both in vitro and in vivo models by immunofluorescent staining, cell cycle analysis, quantitative PCR and whole embryo in-situ hybridization.. We demonstrated strong pro-angiogenic effects of GXNT in both human umbilical vein endothelial cells and zebrafish model. Moreover, through phenotype-based screening in zebrafish for active compounds, pro-angiogenic effects was discovered for salvianolic acid B (Sal B), a major component of Salvia miltiorrhiza, and its activity was further enhanced when co-administered with ferulic acid (FA), which is contained in Ligusticum stratum. On the cellular level, Sal B and FA cotreatment increased endothelial cell proliferation of sprouting arterial intersomitic vessels in zebrafish, as well as largely restored G1-S cell cycle progression and cyclin D1 expression in angiogenic defective HUVECs. Through quantitative transcriptional analysis, increased expression of vegfr2 (kdr, kdrl) and vegfr1 was detected after GXNT or SalB/FA treatment, together with upregulated transcription of their ligands including vegf-a, vegf-b, and pgfb. Bevacizumab, an anti-human VEGF-A monoclonal antibody, was able to significantly, but not completely, block the pro-angiogenic effects of GXNT or SalB/FA, suggesting their multi-targeting properties.. In conclusion, from a traditional Chinese medicine with effects in enhancing blood circulation, we demonstrated the synergistic pro-angiogenic effects of Sal B and FA via both in vitro and in vivo models, which function at least partially through regulating the expression of VEGF receptors and ligands. Future studies are warranted to further elaborate the molecular interaction between these two compounds and the key regulators in the process of neovascularization.

Topics: Animals; Animals, Genetically Modified; Benzofurans; Cell Cycle; Cell Proliferation; Cell Survival; Coumaric Acids; Drug Synergism; Embryo, Nonmammalian; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Neovascularization, Physiologic; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Vascular Endothelial Growth Factor A; Zebrafish

Salvianolic acid B (Sal B), the Q-marker in Salvia miltiorrhiza, was proved to present an obvious anti-diabetes effect when treated as a food intake. Until now, the metabolism feature, tissue distribution and anti-diabetes mechanism of Sal B have not been fully elucidated.. The metabolites of Sal B in rats were profiled using ultrahigh-performance liquid chromatography coupled with time-of-flight mass spectrometry. The potential anti-diabetes mechanism of Sal B was predicted by network pharmacology.. A total of 31 metabolites were characterized in rats after ingestion of Sal B at a dosage of 40 mg/kg, including 1 in plasma, 19 in urine, 31 in feces, 0 in heart, 0 in liver, 0 in spleen, 1 in lung, 1 in kidney and 0 in brain. Among them, 18 metabolites were reported for the first time. Phase I reactions of hydrolysis, hydrogenation, dehydroxylation, hydroxylation, decarboxylation and isomerization, and phase II reactions of methylation were found in Sal B. Notably, decarboxylation and dehydroxylation were revealed in Sal B for the first time. The pharmacology network results showed that Sal B and its metabolites could regulate ALB, PLG, ACE, CASP3, MMP9, MMP2, MTOR, etc. The above targets were involved in insulin signaling pathway, PI3K-Akt signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, etc. CONCLUSIONS: The metabolism feature of Sal B in vivo was systematically revealed, and its anti-diabetes mechanism for further pharmacological validations was predicted based on metabolite profiling and network pharmacology for the first time.

Topics: Animals; Benzofurans; Caspases; Chromatography, High Pressure Liquid; Diabetes Mellitus; Drugs, Chinese Herbal; Feces; Humans; Hypoglycemic Agents; Isomerism; Kidney; Liver; Lung; Male; Mass Spectrometry; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Network Pharmacology; Rats; Salvia miltiorrhiza

Salvianolic acid B (SAB) is one of the main active ingredients of Salvia Miltiorrhiza. It has significant skin anti-aging, whitening, and sun protection properties.. The study aimed at studying the mechanism underlying the effect of salvianolic acid Bon collagen synthesis, which has good anti-aging efficacy and modulates microcirculation.. This study employed available public databases, bioinformatics methodologies, and the inverse docking approach to explore the effectiveness of SAB in the regulating collagen synthesis, and then used an human dermal fibroblast (HDF)- Human dermal microvascular endothelial cell (HDMEC) in vitro model to validate the predicted mechanism of SAB in influencing collagen synthesis.. The results showed that NO production in SAB-treated HDMEC-conditioned medium was increased compared to that in control media, and the same tendency was also observed for growth factor production. SAB also upregulated HDMEC cellular eNOS and VEGF. When SAB-treated HDMEC conditioned medium was transferred to HDFs, the expression of collagen I, collagen III, and elastin in HDFs was upregulated and MMP-1 was downregulated.. The results show that SAB regulates collagen through the HDMEC-HDF pathway. Furthermore, the mechanisms might be closely related to the microcirculation factors NO and VEGF.

Topics: Benzofurans; Cells, Cultured; Collagen; Culture Media, Conditioned; Endothelial Cells; Fibroblasts; Humans; Skin; Vascular Endothelial Growth Factor A

Topics: Animals; Benzofurans; Catechol O-Methyltransferase; Olive Oil; Phospholipids; Rats

Astrocytic glycogen serves as an important glucose reserve, and its degradation provides extra support for neighboring neurons during energy deficiency. Salvianolic acid B (SAB) exerts a neuroprotective effect on reperfusion insult after cerebrovascular occlusion, but the effect of SAB on astrocytic glycogen and its relationship with neuroprotection are not completely understood. Here, we knocked down astrocyte-specific glycogen phosphorylase (GP, the rate-limiting enzyme in glycogenolysis) in vitro and in vivo and investigated the changes in key enzymes in glycogen metabolism by performing immunoblotting in vitro and immunofluorescence in vivo. Neurobehavioral and morphological assessments were conducted to uncover the outcomes during brain reperfusion. SAB accelerated astrocytic glycogenolysis by upregulating GP activity but not GP expression after reperfusion. Suppression of astrocytic glycogenolysis weakened SAB-mediated neuroprotection against the reperfusion insult. In addition, activation of glycogenolysis by SAB contributed to the survival of astrocytes and surrounding neurons by increasing antioxidant levels in astrocytes. Our data reveal that astrocytic GP represents an important metabolic target in SAB-induced protection against brain damage after cerebrovascular recanalization.

Topics: Animals; Antioxidants; Astrocytes; Behavior, Animal; Benzofurans; Cell Survival; Female; Glycogen; Glycogen Phosphorylase; Glycogenolysis; Ischemic Stroke; Male; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Reperfusion Injury

Topics: Adolescent; Adult; Animals; Benzofurans; Disease Models, Animal; Drugs, Chinese Herbal; Humans; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spermatic Cord Torsion; Testis; Young Adult

Salvianolic acid B (SAB) is a widely used cardioprotective agent, while its clinical application was limited by poor intestinal absorption and low oral bioavailability. In this study, SAB phospholipid (SP) complex was first prepared to improve the lipophilicity of SAB and then combined with d-glucose to further enhance intestinal absorption. Compared with free SAB, SP or the mixture of SAB and d-glucose, combination of SP and d-glucose showed higher intestinal absorption evidenced by increased effective permeation coefficient (

Topics: Administration, Oral; Animals; Benzofurans; Biological Availability; Drug Implants; Glucose; Intestinal Absorption; Phospholipids; Rats; Rats, Sprague-Dawley

To determine whether salvianolic acid B (Sal B) exerts protective effects on diabetic peripheral neuropathy by attenuating apoptosis and pyroptosis.. RSC96 cells were primarily cultured with DMEM (5.6 mmol/L glucose), hyperglycemia (HG, 125 mmol/L glucose) and Sal B (0.1, 1, and 10 µ mol/L). Cells proliferation was measured by 3-(4, 5-cimethylthiazol-2-yl)-2, 5-dilphenyltetrazolium bromide assay. Reactive oxygen species (ROS) generation and apoptosis rate were detected by flow cytometry analysis. Western blot was performed to analyze the expressions of poly ADP-ribose polymerase (PARP), cleaved-caspase 3, cleaved-caspase 9, Bcl-2, Bax, NLRP3, ASC, and interleukin (IL)-1β.. Treatment with HG at a concentration of 125 mmol/L attenuated cellular proliferation, while Sal B alleviated this injury (P<0.05). In addition, Sal B inhibited HG-induced ROS production and apoptosis rate (P<0.05). Furthermore, treatment with Sal B down-regulated HG-induced PARP, cleaved-caspase 3, cleaved-caspase 9, Bax, NLRP3, ASC, and IL-1β expression, but mitigated HG-mediated down-regulation of Bcl-2 expression (P<0.05).. Sal B may protect RSC96 cells against HG-induced cellular injury via the inhibition of apoptosis and pyroptosis activated by ROS.

Topics: Apoptosis; Benzofurans; Oxidative Stress; Pyroptosis; Reactive Oxygen Species

Salvianolic acid B (SalB) is effective for treating cardiovascular diseases. However, the molecular mechanisms underlying its therapeutic effects remain unclear. Mechanosensitive Piezo1 channels play important roles in vascular biology, although their pharmacological properties are poorly defined. Here, we aimed to identify novel Piezo1 inhibitors and gain insights into their mechanisms of action.. Intracellular Ca. Salvianolic acid B inhibited Yoda1-induced Ca. Our study provides novel mechanistic insights into the inhibitory role of salvianolic acid B against Piezo1 channels and improves our understanding of salvianolic acid B in preventing atherosclerotic lesions.

Topics: Animals; Atherosclerosis; Benzofurans; Endothelial Cells; Ion Channels; Mice; RAW 264.7 Cells

Topics: AMP-Activated Protein Kinases; Benzofurans; Cell Cycle Proteins; Endothelial Progenitor Cells; Forkhead Transcription Factors; Humans; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; rac1 GTP-Binding Protein; Signal Transduction; Syndecan-4

Topics: A549 Cells; Benzofurans; Carcinoma, Non-Small-Cell Lung; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Signal Transduction; Smad2 Protein

Pain comorbid with depression occurred frequently in clinical settings. This study aims to explore the molecular mechanism underlying antidepressant and analgetic effect of salvianolic acid B (SalB) in comorbid pain in depression induced by chronic restraint stress (CRS), which associates with GABAergic neuron activation in the amygdala and the ERK-CREB-BDNF signaling pathway. The differentially expressed genes related to comorbid pain in CRS-induced depression were screened through bioinformatics analysis. After CRS treatment for 3 weeks, depression-like behaviors were developed in GAD2-tdT mice. The retrograde tracer cholera toxin B subunit combined with retrograde tracer CTB-488 was injected into the parafascicular nucleus of thalamus to project GABAergic neurons to observe the labeling of neurons in the whole brain. After treatment with SalB and ERK-CREB-BDNF signaling pathway inhibitor, CRS mice showed a variety of depression-like behaviors, accompanied by enhanced activity of GABAergic neurons in the amygdala projecting to parafascicular nucleus of thalamus. BDNF underexpression occurred in the CRS mice. Overexpressed BDNF activated ERK-CREB-BDNF signaling pathway to alleviate comorbid pain in CRS-induced depression. After intraperitoneal injection of SalB, the depression-like behaviors and pain threshold in CRS mice were alleviated, the effects of which could be eliminated by ERK-CREB-BDNF signaling pathway antagonist. Collectively, SalB inhibits the excitation of GABAergic neurons in the amygdala and activates the ERK-CREB-BDNF signaling pathway through the parafascicular nucleus of thalamus, whereby alleviating comorbid pain in CRS-induced depression in mice.

Topics: Animals; Benzofurans; Brain-Derived Neurotrophic Factor; CREB-Binding Protein; Depression; Extracellular Signal-Regulated MAP Kinases; GABAergic Neurons; Hippocampus; Humans; Mice; Pain; Stress, Psychological

Topics: Animals; Antagomirs; Autophagy; Benzofurans; Glomerulonephritis, Membranous; Inflammation; Kidney; MicroRNAs; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction

As the key stromal cells that mediate the desmoplastic reaction, tumor-associated fibroblasts (TAFs) play a critical role in the limited nanoparticle penetration and suppressive immune tumor microenvironment. Herein, we found that salvianolic acid B-loaded PEGylated liposomes (PEG-SAB-Lip) can interfere with the activation of TAFs by inhibiting the secretion of TGF-β1. After inhibiting the activation of TAFs, collagen deposition in tumors was reduced, and the penetration of nanoparticles in tumors was enhanced. The results of RT-qPCR and immunofluorescence staining showed the high expression of Th1 cytokines and chemokines (CXCL9 and CXCL10) and the recruitment of CD4

Topics: Benzofurans; Cancer-Associated Fibroblasts; CD8-Positive T-Lymphocytes; Fibroblasts; Humans; Immunologic Factors; Immunotherapy; Liposomes; Neoplasms; Smad Proteins; Transforming Growth Factor beta1; Tumor Microenvironment

Tumor vasculature is characterized by aberrant structure and function, resulting in immune suppressive profiles of tumor microenvironment (TME) through limiting immune cell infiltration into tumors. The defective vascular perfusion in tumors also impairs the delivery and efficacy of chemotherapeutic agents. Targeting abnormal tumor blood vessels has emerged as an effective therapeutic strategy to improve the outcome of chemotherapy and immunotherapy. In this study, we demonstrated that Salvianolic acid B (SalB), one of the major ingredients of Salvia miltiorriza elicited vascular normalization in the mouse models of breast cancer, contributing to improved delivery and response of chemotherapeutic agent cisplatin as well as attenuated metastasis. Moreover, SalB in combination with anti-PD-L1 blockade retarded tumor growth, which was mainly due to elevated infiltration of immune effector cells and boosted delivery of anti-PD-L1 into tumors. Mechanistically, tumor cell enhancer of zeste homolog 2 (Ezh2)-driven cytokines disrupted the endothelial junctions with diminished VE-cadherin expression, which could be rescued in the presence of SalB. The restored vascular integrity by SalB via modulating the interactions between tumor cells and endothelial cells (ECs) offered a principal route for achieving vascular normalization. Taken together, our data elucidated that SalB enhanced sensitivity of tumor cells to chemotherapy and immunotherapy through triggering tumor vascular normalization, providing a potential therapeutic strategy of combining SalB and chemotherapy or immunotherapy for patients with breast cancer.

Topics: Animals; Antineoplastic Agents; Benzofurans; Cisplatin; Endothelial Cells; Enhancer of Zeste Homolog 2 Protein; Immunologic Factors; Immunotherapy; Mice; Neoplasms; T-Lymphocytes; Tumor Microenvironment

Topics: Animals; Benzofurans; Inflammation; Mice; Myeloid Differentiation Factor 88; NLR Family, Pyrin Domain-Containing 3 Protein; NLR Proteins; Oxidative Stress; Particulate Matter; Signal Transduction; Toll-Like Receptor 4

Salvianolic acid B (SalB) has been extensively investigated in our laboratory for myocardial ischemia (MI) disease. This study mainly aimed to illustrate the relationship between SIRT1 and the therapeutic effect of SalB on MI in rats and hypoxia damage in H9c2 cells. Furthermore, whether the antagonism of NLRP3 by SalB in the injuries mentioned above is related to SIRT1-AMPK-PGC-1α pathway-mediated mitochondrial biogenesis was further investigated. In vivo, 24 h after MI surgery, we found that SalB effectively reduced ST-segment elevation, myocardial infarct size enlargement, cardiac injury markers, myocardial structural abnormalities, and myocardial apoptotic cells in MI injury rats. In vitro, after 4 h of hypoxia exposure, SalB alleviated cell injury, inhibited the production of ROS and IL-1β, and prevented the loss of mitochondrial membrane potential (MMP). Besides, SalB downregulated the critical components of the NLRP3 inflammasome and upregulated the SIRT1-AMPK-PGC-1α signaling pathway-related molecules in myocardial tissues and H9c2 cells. However, all the above protective effects of SalB on MI could be offset by EX527. Taken together, our findings indicated that SalB could attenuate MI injury by targeting NLRP3, which is at least partially dependent on the SIRT1/AMPK/PGC-1α signaling pathway.

Topics: AMP-Activated Protein Kinases; Animals; Benzofurans; Cardiomegaly; Hypoxia; Inflammasomes; Myocardial Ischemia; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirtuin 1

Atherosclerosis (AS) is the greatest contributor to pathogenesis of atherosclerotic cardiovascular disease (ASCVD), which is associated with increased mortality and reduced quality of life. Early intervention to mitigate AS is key to prevention of ASCVD. Salvianolic acid B (Sal B) is mainly extracted from root and rhizome of Salvia Miltiorrhiza Bunge, and exerts anti-atherosclerotic effect. The purpose of this study was to screen for anti-AS targets of Sal B and to characterize immune cell infiltration in AS.. We identified targets of Sal B using SEA ( http://sea.bkslab.org/ ) and SIB ( https://www.sib.swiss/ ) databases. GSE28829 and GSE43292 datasets were obtained from Gene Expression Omnibus database. We identified differentially expressed genes (DEGs) and performed enrichment analysis. Weighted gene co-expression network analysis (WGCNA) was used to determine the most relevant module associated with atherosclerotic plaque stability. Intersecting candidate genes were evaluated by generating receiver operating characteristic (ROC) curves and molecular docking. Then, immune cell types were identified using CIBERSOFT and single-sample gene set enrichment analysis (ssGSEA), the relationship between candidate genes and immune cell infiltration was evaluated. Finally, a network-based approach to explore the candidate genes relationship with microRNAs (miRNAs) and Transcription factors (TFs).. MMP9 and MMP12 were been selected as candidate genes from 64 Sal B-related genes, 81 DEGs and turquoise module with 220 genes. ROC curve results showed that MMP9 (AUC = 0.815, P<0.001) and MMP12 (AUC = 0.763, P<0.001) were positively associated with advanced atherosclerotic plaques. The results of immune infiltration showed that B cells naive, B cells memory, Plasma cells, T cells CD8, T cells CD4 memory resting, T cells CD4 memory activated, T cells regulatory (Tregs), T cells gamma delta, NK cells activated, Monocytes, and Macrophages M0 may be involved in development of AS, and the candidate genes MMP9 and MMP12 were associated with these immune cells to different degrees. What' s more, miR-34a-5p and FOXC1, JUN maybe the most important miRNA and TFs.. The anti-AS effects of Sal B may be related to MMP9 and MMP12 and associated with immune cell infiltration, which is expected to be used in the early intervention of AS.

Topics: Atherosclerosis; Benzofurans; Computational Biology; Gene Expression Profiling; Gene Regulatory Networks; Humans; Matrix Metalloproteinase 12; Matrix Metalloproteinase 9; MicroRNAs; Molecular Docking Simulation; Plaque, Atherosclerotic; Protein Interaction Maps; Quality of Life

Non-alcoholic fatty liver disease (NAFLD) has high occurrence in the global world, which poses serious threats to human health. Salvianolic acid B (SalB), an extract of the root of Salvia miltiorrhiza, has the protective effect on metabolic homeostasis. However, the mechanism is still unknown. In this study, we used ob/ob mice, a model of NAFLD, to explore the hepatoprotective effects of SalB. The results showed that SalB significantly reduced the body weights and liver weights, and ameliorated plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), hepatic free fatty acid (FFA), total cholesterol (TC) levels, and hepatic TG and TC levels in ob/ob mice. SalB reduced the number of lipid droplets and inhibited hepatic lipogenesis by regulating peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), stearoyl-Co A desaturase 1 (SCD1), and cluster of differentiation 36 (CD36). Compared to ob/ob mice, the lower expressions of the pro-inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and F4/80, were observed after SalB treatment. Importantly, SalB treatment inhibited the activation of NLRP3 inflammasome and reduced the severity of liver inflammation. Our findings suggested that SalB improved NAFLD pathology in ob/ob mice by reducing hepatic lipid accumulation and NLRP3 inflammasome activation, which might be the potential hepatoprotective mechanism of SalB.

Topics: Animals; Benzofurans; Depsides; Humans; Inflammasomes; Liver; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Non-alcoholic Fatty Liver Disease; Triglycerides

Salvianolic acid B (SalB) can attenuate myocardial ischemia/reperfusion (I/R) injury, but the mechanisms are not entirely known.. Our study investigates if SalB protects cardiomyocytes against I/R injury by regulating Tripartite motif (TRIM) protein.. AC16 cardiomyocytes were treated with I/R, and then with SalB (10, 25 and 50 μM) for 24 h, while control cells were cultured under normal conditions. Female Sprague-Dawley rats were subjected to I/R injury, and then intravenously injected with 20, 40, or 60 mg/kg SalB or saline, as a control, rats received sham operation and saline injection.

Topics: Animals; Apoptosis; Benzofurans; Carrier Proteins; Cell Line; Depsides; Female; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Humans; Myocardial Reperfusion Injury; Nerve Tissue Proteins; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

M1 macrophages secrete a large number of proinflammatory factors and promote the expansion of atherosclerotic plaques and processes. Salvianolic acid B (Sal B) exerts anti-inflammatory, antitumor and other effects, but no study has addressed whether Sal B can regulate the polarization of macrophages to exert these anti-atherosclerotic effects. Therefore, we investigated the inhibition of Sal B in M1 macrophage polarization and the underlying mechanism. The effects of different treatments on cell viability, gene expression and secretion of related proteins, phenotypic markers and cytokines were detected by MTT and western blot assays, RT‒qPCR and ELISAs. Cell viability was not significantly changed when the concentration of Sal B was less than 200 μM, and Lipopolysaccharide (LPS) (100 ng/mL) + interferon-γ (IFN-γ) (2.5 ng/mL) successfully induced M1 polarization. RT‒qPCR and ELISAs indicated that Sal B can downregulate M1 marker (Inducible Nitric Oxide Synthase (iNOS), Tumor Necrosis Factor-α (TNF-α), and Interleukin-6 (IL-6)) and upregulate M2 marker (Arginase-1 (Arg-1) and Interleukin-10 (IL-10)) expression. Western blotting was performed to measure the expression of Nuclear Factor-κB (NF-κB), p-Akt, p-mTOR, LC3-II, Beclin-1, and p62, and the results suggested that Sal B inhibits the M1 polarization of RAW264.7 macrophages by promoting autophagy via the NF-κB signalling pathway. The study indicated that Sal B inhibits M1 macrophage polarization by inhibiting NF-κB signalling pathway activation and downregulating Akt/mTOR activation to promote autophagy.

Topics: Animals; Benzofurans; Lipopolysaccharides; Mice; NF-kappa B; Phenotype; Proto-Oncogene Proteins c-akt; RAW 264.7 Cells; TOR Serine-Threonine Kinases

Ischemic stroke is the second leading cause of death and the third leading cause of disability worldwide. Salvianolic acid B (SAB), a water-soluble phenolic acid derived from the traditional Chinese medicine Salvia miltiorrhiza, exerted protective effects on cerebral ischemia-reperfusion injury. However, the efficacy of SAB is seriously hindered by poor blood brain barrier (BBB) permeability and short biological half-life in plasma. Brain targeted biomimetic nanoparticle delivery systems offer much promise in overcoming these limitations.. A brain targeted biomimetic nanomedicine (RR@SABNPs) was developed, which comprised of SAB loaded bovine serum albumin nanoparticles and functionalized red blood cell membrane (RBCM) with Arg-Gly-Asp (RGD). The characterization parameters, including particle size, zeta potential, morphology, Encapsulation Efficiency (EE), Drug Loading (DL), release behavior, stability, and biocompatibility, were investigated. Moreover, the middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model was used to assess the therapeutic efficacy of RR@SABNPs on ischemic stroke. Finally, the reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) were detected by DHE and JC‑1 staining in oxygen-glucose deprivation/reperfusion (OGD/R) and H. RR@SABNPs exhibited spheric morphology with core-shell structures and good stability and biocompatibility. Meanwhile, RR@SABNPs can significantly prolong SAB circulation time by overcoming the reticuloendothelial system (RES) and actively targeting ischemic BBB. Moreover, RR@SABNPs had comprehensive protective effects on MCAO/R model mice, manifested as a reduced infarct volume and improved neurological and sensorimotor functions, and significantly scavenged excess ROS and maintained MMP.. The designed brain targeted biomimetic nanomedicine RR@SABNPs can significantly prolong the half-time of SAB, deliver SAB into the ischemic brain and exhibit good therapeutic effects on MCAO/R model mice.

Topics: Animals; Benzofurans; Brain Ischemia; Erythrocyte Membrane; Hydrogen Peroxide; Infarction, Middle Cerebral Artery; Ischemic Stroke; Mice; Nanoparticles; Rats; Reactive Oxygen Species; Reperfusion Injury

Vascular endothelial cells and oxidation reduction system play an important role in the pathogenesis of atherosclerosis (AS). If these conditions are disordered, it will inevitably lead to plaque formation and even rupture. Astragaloside IV (AsIV) and salvianolic acid B (Sal B) are the main active ingredients of Astragalus membranaceus and Salvia miltiorrhiza, respectively, and found to ameliorate vascular endothelial dysfunction and protect against oxidative stress in recent studies. However, it is still unknown if the combination of AsIV and Sal B (AsIV + Sal B) can inhibit the development of plaque through amplifying the protective effect of vascular endothelial cells and anti-oxidative stress effect. To clarify the role of AsIV + Sal B in AS, we observed the efficacy of each group (Control, Model, AsIV, Sal B, and AsIV + Sal B) by biomolecular assays, such as observing the pathological morphology of the aorta by oil red O staining, evaluating the level of oxidative stress and endothelial cells in the serum by the Elisa test, and analyzing the changes of all small molecule metabolites in liver tissue by UPLC-QTOF-MS. Results showed that AsIV, Sal B and AsIV + Sal B decreased the deposition of lipid in the arterial wall, so as to exert the effect of anti-oxidant stress and vascular endothelial protection, where the inhibitory effect of AsIV + Sal B was the most obvious. Metabonomics analysis showed that Sal B regulated the metabolic pathways of arginine and proline. AsIV regulated glycerol metabolism and saturated fatty acid biosynthesis metabolism. AsIV + Sal B is mainly related to the regulation of the citrate cycle (TCA cycle), alanine, aspartic acid, and glutamate metabolism, cysteine, and methionine metabolism. Succinic acid and methionine are synergistic metabolites that exert an enhancing effect when AsIV and Sal B were used in combination. In conclusion, we demonstrated that AsIV acompanied with Sal B can be successfully used for anti-oxidative stress and vascular endothelial protection of AS, and succinic acid and methionine are the synergistic metabolites.

Topics: Antioxidants; Atherosclerosis; Benzofurans; Endothelial Cells; Humans; Methionine; Saponins; Succinic Acid; Triterpenes

Renal interstitial fibrosis (RIF) is the main pathological feature of end-stage renal disease (ESRD) caused by various chronic kidney diseases (CKD), and is closely related to renal dysfunction and patient prognosis. Salvianolic acid A (Sal A) and salvianolic acid B (Sal B), isolated from traditional Chinese medicine Salviae miltiorrhizae, have been confirmed to have anti-fibrotic effects on liver, cardiac and kidney. However, the precise molecular mechanism underlying the nephroprotective effects of Sal A and Sal B, and whether there is a difference between the two in RIF are still unclear.. This study investigated the pharmacological effects of Sal A and Sal B in RIF and explore the underlying mechanisms by in vivo and in vitro experiments.. The nephroprotective effects of Sal A, Sal B and Sal A+B were evaluated by assessing the parameters related to kidney function such as renal histology, renal function, urinary protein NAG, urinary β2 microglobulin. In addition, RIF-related markers such as CTCF and Par3 were also detected. Thereafter, the related protein or gene levels of PDGF-C/PDGFR-α signaling pathways, apoptosis and endoplasmic reticulum stress (ERS) were determined by western blot, real-time PCR, flow cytometry or immunofluorescence staining.. In vivo, the results showed that Sal A, Sal B and Sal A+B partially improved kidney dysfunction, increased the expression of Par-3 and reduced the expression of CTGF, PDGF-C and PDGFR-α. In vitro, the results also showed that Sal A, Sal B and Sal A+B reversed apoptosis and ERS in HSA-induced HK-2 cells via regulating PDGF-C/PDGFR-α signaling pathway.. This article revealed a novel mechanism linking PDGF-C/PDGFR-α signaling pathway to RIF and suggested that Sal A, Sal B and Sal A+B were considered as potential therapeutic agents for the amelioration of RIF.

Topics: Benzofurans; Caffeic Acids; Depsides; Fibrosis; Humans; Kidney Diseases; Lactates; Lymphokines; Platelet-Derived Growth Factor; Signal Transduction

Long non-coding RNA (LncRNAs) have been reported to play an important role in liver fibrosis and are closely associated with hepatic stellate cell (HSC) activation. We previously found that salvianolic acid B (Sal B) improves liver fibrosis by regulating the NF-κB signaling pathway. However, whether the LncRNA, regulator of reprogramming (LncRNA-ROR) plays a role in Sal B-mediated anti-fibrosis effects via the NF-κB signaling pathway remain unclear.. This study aimed to evaluate the effects of Sal B on HSC activation and liver fibrosis and investigate its mechanism from the perspective of LncRNA-ROR-mediated NF-κB signaling pathways.. In this study, a lower level of LncRNA-ROR was found during Sal B attenuating HSC activation in HSCs. Mechanistically, Sal B impeded the NF-κB signaling pathway to inhibit HSC proliferation and activation by downregulating LncRNA-ROR. Additionally, Sal B upregulated miR-6499-3p to target LncRNA-ROR for degradation. Functionally, Sal B treatment ameliorated CCl. Sal B suppresses HSC activation and liver fibrosis via regulation of miR-6499-3p/LncRNA-ROR-mediated NF-κB signaling pathway. These results reveal a new molecular mechanism of Sal B on liver fibrosis from the insight of LncRNAs.

Topics: Animals; Benzofurans; Depsides; Hepatic Stellate Cells; Liver Cirrhosis; Male; Mice; MicroRNAs; NF-kappa B; RNA, Long Noncoding; Signal Transduction

Salvianolic acid B(Sal B), tanshinone Ⅱ_A(TSN Ⅱ_A), and glycyrrhetinic acid(GA) lipid emulsion(GTS-LE) was prepared by the high-speed dispersion method combined with ultrasonic emulsification.The preparation process of the emulsion was optimized by single-factor method and D-optimal method with appearance, centrifugal stability, and particle size of the emulsion as evalua-tion indexes, followed by verification.In vitro release of Sal B, TSN Ⅱ_A, and GA in GTS-LE was performed by reverse dialysis.In vivo pharmacokinetic evaluation was carried out in mice.The acute liver injury model was induced by acetaminophen.The effect of oral GTS-LE on the acute liver injury was investigated by serum liver function indexes and pathological changes in liver tissues of mice.The results showed that under the optimal preparation process, the average particle size of GTS-LE was(145.4±9.25) nm and the Zeta potential was(-33.6±1.45) mV.The drug-loading efficiencies of Sal B, TSN Ⅱ_A, and GA in GTS-LE were above 95%, and the drug release in vitro conformed to the Higuchi equation.The pharmacokinetic results showed that the C_(max) of Sal B, TSN Ⅱ_A, and GA in GTS-LE was 3.128, 2.7, and 2.85 times that of the GTS-S group, and AUC_(0-t) of Sal B, TSN Ⅱ_A, and GA in GTS-LE was 3.09, 2.23, and 1.9 times that of the GTS-S group.After intragastric administration of GTS-LE, the activities of alanine aminotransferase and aspartate aminotransferase were significantly inhibited, the content of malondialdehyde was reduced, and the structure of hepatocytes recovered to normal.In conclusion, GTS-LE can delay the release of Sal B and promote the release of TSN Ⅱ_A and GA.The encapsulation of three drug components in the emulsion can improve the oral bioavailability to varying degrees and can effectively prevent the acute liver injury caused by acetaminophen.

Topics: Abietanes; Acetaminophen; Alanine Transaminase; Animals; Antipyretics; Aspartate Aminotransferases; Benzofurans; Chemical and Drug Induced Liver Injury; Depsides; Emulsions; Glycyrrhetinic Acid; Liver; Malondialdehyde; Mice

The SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor play vital roles in plant growth and development. Although 15 SPL family genes have been recognized in the model medical plant

Topics: Benzofurans; Gene Expression Regulation, Plant; Plant Roots; Salvia miltiorrhiza; Transcription Factors

Progressive macrophage dysfunction and apoptosis are some of the major events that occur during atherogenesis. To further investigate the intrinsic association between atherosclerosis (AS) and macrophage apoptosis and autophagy, cholesterol crystals (CHCs) were used to stimulate RAW264.7 macrophages to establish a macrophage model of advanced AS. Cells in the CHC group were treated with salvianolic acid B (Sal B) to evaluate its protective effects and reveal its underlying molecular mechanism. The results demonstrated that treatments with Sal B significantly improved autophagy dysfunction and reduced the apoptotic rate of CHC‑induced macrophages. Furthermore, Sal B significantly attenuated CHC‑induced release of proinflammatory factors (TNF‑α and IL‑6) by macrophages. Treatment of macrophages with a specific inhibitor of autophagy (3‑methyladenine) significantly reversed Sal B‑mediated effects on autophagy, suggesting that Sal B‑induced autophagy may display a protective effect in CHC‑induced macrophages. Furthermore, pretreatment of CHC‑induced macrophages with insulin significantly decreased Sal B‑induced autophagy, indicating that the Akt/mTOR signaling pathway may serve as a critical mediator in regulating Sal B‑mediated cell death. Taken together, the present study demonstrated that Sal B improved autophagic dysfunction and reduced the apoptosis of CHC‑induced macrophages via inhibiting the Akt/mTOR signaling pathway.

Topics: Animals; Apoptosis; Atherosclerosis; Autophagy; Benzofurans; Drugs, Chinese Herbal; Macrophages; Mice; Protective Agents; Proto-Oncogene Proteins c-akt; RAW 264.7 Cells; Salvia miltiorrhiza; TOR Serine-Threonine Kinases

The dried root of

Topics: Benzofurans; Cyclopentanes; Gene Expression Regulation, Plant; Indoleacetic Acids; Oxylipins; Plant Roots; Salvia miltiorrhiza; Transcription Factors

Topics: Benzofurans; Cell Differentiation; Cell Proliferation; Cells, Cultured; Humans; Mesenchymal Stem Cells; Osteogenesis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt

Our previous study proved that Salvia miltiorrhiza could enhance fat graft survival by promoting adipogenesis. However, the effect of salvianolic acid B (Sal-B), the most abundant and bioactive water-soluble compound in Salvia miltiorrhiza, on fat graft survival has not yet been investigated.. This study aims to investigate whether salvianolic acid B could improve fat graft survival and promote preadipocyte differentiation. The underlying mechanism has also been studied.. In vivo, 0.2 ml of Coleman fat was transplanted into nude mice with salvianolic acid B. The grafts were evaluated by HE and IF at 2 and 4 weeks posttransplantation and by micro-CT at 4 weeks posttransplantation. In vitro, the adipogenesis and proliferative activities of salvianolic acid B were analyzed in cultured human adipose-derived stem cells (h-ADSCs) and 3T3-L1 cells to detect the mechanism by which salvianolic acid B affects graft survival.. In vivo, the weights and volumes of the fat grafts in the Sal-B-treated groups were significantly higher than those of the fat grafts in the control group. In addition, higher fat integrity and more viable adipocytes were observed in the Sal-B-treated groups. In vitro, salvianolic acid B showed the ability to promote 3T3-L1 and h-ADSC proliferation and adipogenesis.. Our in vitro experiments demonstrated that salvianolic acid B can promote the proliferation of adipose stem cells and enhance the differentiation of adipose stem cells. Simultaneously, in vivo experiments showed that salvianolic acid B can improve the survival rate of fat transplantation. Therefore, our research shed light on the potential therapeutic usage of salvianolic acid B in improving the survival rate of fat transplantation.

Topics: Adipogenesis; Adipose Tissue; Animals; Benzofurans; Cell Proliferation; Graft Survival; Mice; Mice, Nude

This study investigated the roles of bone morphogenetic protein-4 (BMP4) and ROS in diabetic endothelial dysfunction and explored whether Salvianolic acid B (Sal B) improved endothelial function by affecting BMP4-ROS in diabetic mice.. db/db mice were orally administrated with Sal B (10 mg/kg/day) for one week while db/m + mice were injected with adenoviral vectors delivering BMP4 (3 × 10. We first revealed the existence of a BMP4-ROS cycle in db/db mice, which stimulated p38 MAPK/JNK/caspase 3 and thus participated in endothelial dysfunction. One week-treatment or 24 h-incubation with Sal B disrupted the cycle, suppressed p38 MAPK/JNK/caspase 3 cascade, and improved endothelium-dependent relaxations (EDRs) in db/db mouse aortas. Importantly, in vivo Sal B treatment also improved flow-mediated dilatation in db/db mouse second order mesenteric arteries. Furthermore, in vivo BMP4 overexpression induced oxidative stress, stimulated p38 MAPK/JNK/caspase 3, and impaired EDRs in db/m + mouse aortas, which were all reversed by Sal B.. The present study demonstrates that Sal B ameliorates endothelial dysfunction through breaking the BMP4-ROS cycle and subsequently inhibiting p38 MAPK/JNK/caspase 3 in diabetic mice and provides evidence for the additional new mechanism underlying the benefit of Sal B against diabetic vasculopathy.

Topics: Animals; Aorta; Benzofurans; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Caspase 3; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Disease Models, Animal; Endothelial Cells; Endothelium, Vascular; Male; MAP Kinase Signaling System; Mesenteric Arteries; Mice; Mice, Inbred C57BL; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Vascular Diseases; Vasodilation

Mesoporous Bioactive Glass (MBG) has been widely studied because of its excellent histocompatibility and degradability. However, due to the lack of good osteoinductive activity, the pure MBG scaffold is not effective in repairing large-scale bone defects.. To observe the repair effect of MBG scaffolds delivering Salvianolic acid B (SB) on critical bone defects in rats.. In this study, MBG scaffolds were used as delivery vehicle. SB, a small molecular active drug with good osteogenic differentiation ability, was loaded into the MBG scaffolds at low, medium and high doses. The effect of SB released from the MBG scaffolds on osteogenic differentiation of rat Bone Marrow Mesenchymal Stem Cells (rBMSCs) was investigated using alkaline phosphatase staining, alizarin red staining and real-time quantitative polymerase chain reaction. Moreover, 8 weeks after implantation of the scaffolds, the bone regeneration was evaluated by micro- CT, sequential fluorescence labeling and histological staining analysis.. The in vitro results showed that different doses of SB had similar release rate from scaffolds and could be released from scaffolds continuously. The middle dose (MBG/MSB) and high dose (MBG/HSB) groups significantly promoted the osteogenic differentiation of rBMSCs when compared with a low dose (MBG/LSB) group. Moreover, SB produced significant increases in newly formed bone of calvarial bone defects in rats.. It is concluded that the use of MBG scffolds delivering SB is an effective strategy for the treatment of bone defects.

Topics: Animals; Benzofurans; Bone Regeneration; Glass; Osteogenesis; Porosity; Rats; Tissue Scaffolds

The combined use of two or more different drugs can better promote nerve recovery and its prognosis for treatment of stroke. The salvianolate lyophilized injection (SLI) and Xueshuantong Injection (XST) are two standardized Chinese medicine injections which have been widely used in the treatment of cerebrovascular diseases. Salvianolic acid B (Sal B) and Notoginsenoside R1 (NR1) is respectively one of the active constituents of SLI and XST, which have certain effects on stroke. In this study, we established a co-culture of endothelial cells and pericytes for oxygen-glucose deprivation/reperfusion (OGD/R) injury model to study the effects of SLI and Sal B or XST and NR1 alone, or with their combinations (1S1X) in regulation of BBB function. The results showed that compared with the OGD/R group, treatment with SLI, XST and SalB and NR1 can significantly increase the TEER, reduce the permeability of Na-Flu, enhance the expression of tight junctions (TJs) between cells, and stabilize the basement membrane (BM) composition. In addition, the combination of 1S1X is superior to the XST or SLI alone in enhancing the TJs between cells and stabilizing the BM. And the active components SalB and NR1 can play a strong role in these two aspects, even with the whole effects. Furthermore, the study showed that XST, Sal B and NR1 increases in Ang-1and Tie2, while decrease in Ang-2 and VEGF protein expressions. Overall, these findings suggest that SLI combined with XST (1X1S) has protective effects on co-culture of endothelial cells and pericytes after OGD/R. Moreover, its protective effect might be associated with increase of TJs and BMs through activation of Ang/Tie-2 system signaling pathway.

Topics: Animals; Astrocytes; Benzofurans; Blood-Brain Barrier; Cell Culture Techniques; China; Coculture Techniques; Drugs, Chinese Herbal; Endothelial Cells; Ginsenosides; Glucose; Mice; Models, Biological; Oxygen; Pericytes; Plant Extracts; Reperfusion Injury; Signal Transduction; Tight Junctions

Topics: Animals; Apoptosis; Benzofurans; Cell Movement; Cell Proliferation; Gene Expression Regulation; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mutation; Phosphorylation; Random Allocation; Smad3 Protein; Up-Regulation

To determine the effects of Salvianolic acid B (Sal B) on new bone formation in the orthopedically expanded premaxillary sutures in rats.. The sample consisting of Sprague Dawley rats (male, n = 14) was split in half by random selection: the experiment group (Sal B) and the control group. The premaxillary suture of each rat was expanded by bonding an open-loop spring to two maxillary incisors, each end to one tooth. A 5-day expansion period followed by a 12-day retention period was conducted. The 17-day intraperitoneal administration of Sal B was performed daily for the experiment group at a dose of 40 mg/kilo. The trial was completed after sacrificing the rats and dissection of the premaxillae for histological analysis. The amount of new bone, quantity of capillaries and intensity of inflammatory cells were histomorphometrically determined while the quantities of osteoblasts and osteoclasts were determined immunohistochemically.. The Sal B group was significantly different from the control group and had greater quantities of new bone, capillaries, inflammatory cells, osteoblasts, and osteoclasts.. Salvianolic acid B displays a positive effect during premaxillary expansion with a greater number of capillaries potentially in association with higher bone formation and improved angiogenesis in rats.

Topics: Animals; Benzofurans; Male; Osteogenesis; Palatal Expansion Technique; Rats; Rats, Sprague-Dawley; Rats, Wistar; Sutures

In this work, a smartphone-based device was constructed for thin-layer chromatography (TLC) detection and semi-quantitative analysis of the components of Salvia miltiorrhiza. The key construction and shooting parameters were investigated by the relative peak area and signal-to-noise ratio. The best conditions were as follows: shooting height, 17 cm; angle between the UV lamp and TLC plate, 58°; exposure compensation, 0~0.2 EV; and shutter speed under daylight and UV 365 nm, 1/50 s and 1/5 s, respectively. These ideal conditions could be replicated by smartphones from different brands with different versions of software. With good precision, repeatability and stability, the developed device was used for the semi-quantitative analysis of salvianolic acid B, rosmarinic acid, cryptotanshinone, tanshinone I, tanshinone IIA, and miltirone in the TLC analysis of 10 batches of S. miltiorrhiza. The results were compared with those obtained by a TLC densitometric scanner and two common types of image processing software, i.e., Gelanalyzer and ImageJ. Except for salvianolic acid B in the TLC densitometric scanner, all results were not significantly different among these methods, which suggested that smartphones might be a useful tool for the quality control of traditional Chinese medicines.

Topics: Abietanes; Benzofurans; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cinnamates; Depsides; Drugs, Chinese Herbal; Phenanthrenes; Quality Control; Rosmarinic Acid; Salvia miltiorrhiza; Smartphone

SIRT2 belongs to a class III of Histone Deacetylase (HDAC) and has crucial roles in neurodegeneration and malignancy.. The objective of this study is to discover structurally novel natural-product-derived SIRT2 inhibitors.. Structure-based pharmacophore modeling integrated with validated QSAR analysis was implemented to discover structurally novel SIRT2 inhibitors from the natural products database. The targeted QSAR model combined molecular descriptors with structure-based pharmacophore capable of explaining bioactivity variation of structurally diverse SIRT2 inhibitors. Manually built pharmacophore model, validated with receiver operating characteristic curve, and selected using the statistically optimum QSAR equation, was applied as a 3Dsearch query to mine AnalytiCon Discovery database of natural products.. Experimental in vitro testing of highest-ranked hits identified asperphenamate and salvianolic acid B as active SIRT2 inhibitors with IC. New chemical scaffolds of SIRT2 inhibitors have been identified that could serve as a starting point for lead-structure optimization.

Topics: Benzofurans; Biological Products; Dose-Response Relationship, Drug; Drug Discovery; Humans; Molecular Structure; Phenylalanine; Protein Kinase Inhibitors; Quantitative Structure-Activity Relationship; Sirtuin 2

Autophagy plays an important role in cellular homeostasis. Oxidative stress stimulated endothelial excessive autophagy has been proposed as a major risk factor for cardiovascular diseases (CVD). Danhong injection (DHI), the most prescribed traditional Chinese medicine for the treatment of CVD, has been shown to elicit vascular protective effects. However, its underlying mechanisms remain poorly defined. This study aimed to uncover the protective effects of DHI and its main bioactive components on autophagy injury of human umbilical vein endothelial cells (HUVECs) induced by H. HUVECs were treated with different concentrations of DHI or its components, after exposed to H. DHI and its components all had anti-autophagy effects. And Sal B (0.5 μg/ml) inhibited HUVECs autophagy via miR-19a/SIRT1 pathway.

Topics: Abietanes; Apoptosis; Autophagy; Beclin-1; Benzofurans; Drugs, Chinese Herbal; Human Umbilical Vein Endothelial Cells; Humans; Hydrogen Peroxide; MicroRNAs; Oxidative Stress; Sirtuin 1

Since December 2019, the new coronavirus (also known as severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2, 2019-nCoV])-induced disease, COVID-19, has spread rapidly worldwide. Studies have reported that the traditional Chinese medicine Salvia miltiorrhiza possesses remarkable antiviral properties; however, the anti-coronaviral activity of its main components, salvianolic acid A (SAA), salvianolic acid B (SAB), and salvianolic acid C (SAC) is still debated. In this study, we used Cell Counting Kit-8 staining and flow cytometry to evaluate the toxicity of SAA, SAB, and SAC on ACE2 (angiotensin-converting enzyme 2) high-expressing HEK293T cells (ACE2

Topics: Alkenes; Angiotensin-Converting Enzyme 2; Benzofurans; Caffeic Acids; Cell Survival; COVID-19 Drug Treatment; HEK293 Cells; Humans; Lactates; Molecular Structure; Polyphenols; Protein Binding; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Virus Internalization

Salvia bowleyana is a traditional Chinese medicinal plant that is a source of nutritional supplements rich in salvianolic acid B and a potential experimental system for the exploration of salvianolic acid B biosynthesis in the Labiatae. Here, we report a high-quality chromosome-scale genome assembly of S. bowleyana covering 462.44 Mb, with a scaffold N50 value of 57.96 Mb and 44,044 annotated protein-coding genes. Evolutionary analysis revealed an estimated divergence time between S. bowleyana and its close relative S. miltiorrhiza of ~3.94 million years. We also observed evidence of a whole-genome duplication in the S. bowleyana genome. Transcriptome analysis showed that SbPAL1 (PHENYLALANINE AMMONIA-LYASE1) is highly expressed in roots relative to stem and leaves, paralleling the location of salvianolic acid B accumulation. The laccase gene family in S. bowleyana outnumbered their counterparts in both S. miltiorrhiza and Arabidopsis thaliana, suggesting that the gene family has undergone expansion in S. bowleyana. Several laccase genes were also highly expressed in roots, where their encoded proteins may catalyze the oxidative reaction from rosmarinic acid to salvianolic acid B. These findings provide an invaluable genomic resource for understanding salvianolic acid B biosynthesis and its regulation, and will be useful for exploring the evolution of the Labiatae.

Topics: Benzofurans; Cinnamates; Depsides; Gene Expression Profiling; Gene Expression Regulation, Plant; Plant Roots; Rosmarinic Acid; Salvia

Fufang Danshen preparation (FDP) is consisted of Salviae Miltiorrhizar Radix et Rhizoma (Danshen), Notoginseng Radix et Rhizoma (Sanqi) and Borneolum Syntheticum (borneol). FDP is usually used to treat myocardial ischemia hypoxia, cerebral ischemia and alzheimer's disease, etc. In the treatment of cerebrovascular diseases, borneol is usually used to promote the absorption and distribution of the bioactive components to proper organs, especially to the brain. The purpose of this study is investigating the effects of borneol on the pharmacokinetics and brain distribution of tanshinone IIA (TS IIA), salvianolic acid B (SAB) and ginsenoside Rg1 in FDP. Male healthy Sprague-Dawley (SD) rats were given Danshen extracts, Sanqi extracts (Panax notoginsengsaponins) or simultaneously administered Danshenextracts, Sanqi extracts and borneol. Plasma and brain samples were collected at different points in time. The concentration of TS IIA, SAB and Rg1 was determined by UPLC-MS/MS method. The main pharmacokinetics parameters of plasma and brain tissue were calculated by using Phoenix WinNolin 6.1 software. In comparison with Danshen and Sanqi alone, there were significant differences in pharmacokinetic parameters of TS IIA, SAB and Rg1, and the brain distribution of SAB and TS IIA when Danshen, Sanqi and borneol were administrated together. Borneol statistically significant shortened t

Topics: Abietanes; Animals; Benzofurans; Brain; Camphanes; Chromatography, Liquid; Drugs, Chinese Herbal; Ginsenosides; Male; Rats; Rats, Sprague-Dawley; Tandem Mass Spectrometry

Cisplatin (CDDP) is an extensively used chemotherapeutic agent but has a high incidence of severe ototoxicity. Although a few molecules have entered clinical trials, none have been approved to prevent or treat CDDP-induced hearing loss by the Food and Drug Administration. In this study, an amphiphilic drug-drug conjugate was synthesized by covalently linking dexamethasone (DEX) and salvianolic acid B (SAL) through an ester or amide bond. The conjugates could self-assemble into nanoparticles (NPs) with ultrahigh drug loading capacity and favorable stability. Compared with DEX, SAL, or their physical mixture at the same concentrations, both conjugates and NPs showed enhanced otoprotection in vitro and in vivo. More importantly, the conjugates and NPs almost completely restored hearing in a guinea pig model with good biocompatibility. Immunohistochemical analyses suggested that conjugates and NPs activated the glucocorticoid receptor, which may work as one of the major mechanisms for their protective effects.

Topics: Animals; Antineoplastic Agents; Benzofurans; Cisplatin; Dexamethasone; Drug Design; Guinea Pigs; Hearing Loss; Humans; Protective Agents

Many Chinese herbs are well known for their neuroprotective and anti-oxidant properties. Extracts of Salvia miltiorrhiza and Anemarrhenae asphodeloides, tanshinone IIA (tanIIA), salvianolic acid B (Sal B) and sarsasapogenin (ML-1), were selected to study their dissociation potential towards Aβ

Topics: Abietanes; Alzheimer Disease; Amyloid beta-Peptides; Anemarrhena; Benzofurans; Cell Line; China; Humans; Neuroprotective Agents; Peptide Fragments; Plant Extracts; Salvia miltiorrhiza; Spirostans

The purpose of this study was to investigate the antifibrotic effect and anticoagulant ability of salvianolic acid B (SAB) inhalation solution on bleomycin (BLM)-induced idiopathic pulmonary fibrosis (IPF) in rats. We investigated how the osmotic pressure and concentration of SAB in an aerosol exerted effects. We also determined the aerodynamic particle size distribution and the uniformity of the delivery dose; these parameters were found to be suitable for inhalation. Compared with BLM group, the levels of hydroxyproline (HYP), collagen-1 (Col-1), tissue factor (TF) / coagulation factor VII (TF-VIIa), activated coagulation factor X (FXa), thrombin-antithrombin complex (TAT), fibrinogen degradation product (FDP) and plasminogen activator inhibitor-1 (PAI-1) decreased in SAB group. The increased expression of coagulation factor Ⅱ (FⅡ), coagulation factor X (FX), tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (u-PA) proved that SAB has obvious antifibrotic and anticoagulant effects. Western blotting and immunofluorescence further showed that compared with the BLM group, the SAB group of rats exhibited significant reductions in the expression levels of protease-activated receptors-1 (PAR-1) and phospho-protein kinase C (p-PKC) and increased expression levels of protein kinase C (PKC) in lung tissue. Furthermore, SAB reduced the infiltration of lymphocytes and neutrophils, protected the basic structure of the lung from destruction, inhibited the proliferation of fibrous tissue. Collectively, our data revealed that SAB may exert its antifibrotic and anticoagulant effects by preventing the expression of PAR-1 and phosphorylation of PKC.

Topics: Administration, Inhalation; Animals; Anticoagulants; Antifibrinolytic Agents; Benzofurans; Bleomycin; Dose-Response Relationship, Drug; Drug Synergism; Drugs, Chinese Herbal; Male; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley

Topics: Actins; Adult; Benzofurans; Cell Differentiation; Cell Proliferation; Cells, Cultured; Extracellular Matrix; Female; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Middle Aged; Myofibroblasts; Nasal Polyps; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Cardiovascular complications have been well documented as the downside to conventional cancer chemotherapy. As a notable side effect of cisplatin (CDDP), cardiotoxicity represents a major obstacle to the successful treatment of cancer. It has been reported that Salvianolic acid B (SalB) possesses cardioprotective quality. However, the effect of SalB on cardiac damage caused by conventional cancer chemotherapy remains unclear. In this study, we clarified the protective effect of SalB on cisplatin-induced heart injury. Furthermore, in H9c2 cells, SalB dramatically reduced cisplatin-induced apoptosis and oxidative stress by modulating the nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway. In conclusion, SalB had great potential in mitigating cisplatin-induced cardiac injury. Furthermore, more attention should be placed on natural active compounds containing SalB with antioxidant effects for the treatment of cardiomyopathy.

Topics: Animals; Antineoplastic Agents; Antioxidants; Benzofurans; Cell Line; Cell Survival; Cisplatin; Creatine Kinase, MB Form; Heart; Heart Diseases; Heme Oxygenase-1; L-Lactate Dehydrogenase; Male; Mice, Inbred C57BL; Myocardium; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Oxidative Stress; Rats; Signal Transduction; Stroke Volume

Drug-loaded hydrogels can improve blood supply and inhibit extracellular matrix degradation after myocardial infarction. However, due to the continual dynamic motion of cardiac tissue, the hydrogel structure cannot be reconstructed in time, causing accelerated degradation and drug burst release. Here, a novel, superior, self-healing elastin-mimic peptide hydrogel (EMH) was fabricated for the local delivery of salvianolic acid B (SaB). The self-healing ability of EMH is enhanced by SaB-loaded polydopamine nanoparticles (SaB-PDA). In vitro, the pre-hydrogel (SaB-PDA/pre-EMH) is endowed with excellent biocompatibility and a low viscosity, making it suitable for intramyocardial injection. Once injected into the myocardial infarction (MI) region, SaB-PDA/pre-EMH can form SaB-PDA/EMH with great mechanical strength under the action of upregulated transglutaminase (TGase) in heart tissue post-MI. The superior self-healing ability of SaB-PDA/EMH allows for an increase in retention time in the beating ventricular wall. Therefore, with long-term release of SaB, SaB-PDA/EMH can inhibit ventricular remodeling and promote angiogenesis for MI treatment.

Topics: Benzofurans; Humans; Hydrogels; Myocardial Infarction; Peptides; Ventricular Remodeling

Maintaining normal conditions in the mitochondria and repressing oxidative stress has emerged as a crucial therapeutic strategy to ameliorate neuron damage in Parkinson's disease. Salvianolic acid B (SalB) is a polyphenolic compound isolated from Salvia miltiorrhiza, which has been prescribed for various biological properties, including antioxidative stress, anti-inflammation and neuroprotection in pathological conditions. Previously, SalB was reported to be of benefit in slowing Parkinson's disease pathology, but whether the neuroprotective role of SalB is associated with a mitochondrial protective action is still elusive. Here we aimed to explore the effects of SalB on mitochondrial function in Parkinson's disease to uncover the underlying cellular mechanisms. The results showed that SalB significantly alleviated 1-methyl-4-phenylpyridinium (MPP+)-induced mitochondrial disruption in line with ameliorated oxidative injury, which is evidenced by inhibited mitochondrial membrane potential collapse, reduced reactive oxygen species (ROS) generation, increased expression of NAD(P)H: quinone oxidoreductase, and enhanced mitochondrial biosynthesis - the upregulation of nuclear respiratory factor 1 and mitochondrial transcription factor A expressions. Mechanistically, SalB not only increased AMP-activated protein kinase (AMPK) activation and sirtuin3 mRNA and protein levels, but also attenuated ROS-triggered neuroinflammation by downregulating the expressions of NOD-like receptor family pyrin domain containing 3, caspase-1 and Interleukin-1β (IL-1β). In conclusion, these in-vitro findings, for the first time, demonstrate that SalB offers protection against MPP+-induced neuronal injury via upregulating sirtuin3 expression and activating the AMPK signaling to restore mitochondrial function.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Apoptosis; Benzofurans; Cell Line, Tumor; Cell Survival; Mice; Mitochondria; Neurons; Neuroprotective Agents; Oxidative Stress; Reactive Oxygen Species

The metabolites of salvianolic acid A and salvianolic acid B in rats were analyzed and compared by ultra-high-perfor-mance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS). After the rats were administrated by gavage, plasma at different time points and urine within 24 hours were collected to be treated by solid phase extraction(SPE), then they were gradient eluted by Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) and 0.1% formic acid solution(A)-acetonitrile(B) mobile phase system, and finally all biological samples of rats were analyzed under negative ion scanning mode. By obtaining the accurate relative molecular mass and multi-level mass spectrometry information of metabolites, combined with the characteristic cleavage law of the reference standard and literature reports, a total of 30 metabolites, including salvianolic acid A and B, were identified. Among them, there were 24 metabolites derived from salvianolic acid A, with the main metabolic pathways including ester bond cleavage, dehydroxylation, decarboxylation, hydrogenation, methylation, hydroxylation, sulfonation, glucuronidation, and their multiple reactions. There were 15 metabolites of salvianolic acid B, and the main biotransformation pathways were five-membered ring cracking, ester bond cleavage, decarboxylation, dehydroxylation, hydrogenation, methylation, sulfonation, glucuronidation, and their compound reactions. In this study, the cross-metabolic profile of salvianolic acid A and B was elucidated completely, which would provide reference for further studies on the basis of pharmacodynamic substances and the exploration of pharmacological mechanism.

Topics: Animals; Benzofurans; Caffeic Acids; Chromatography, High Pressure Liquid; Lactates; Mass Spectrometry; Rats; Technology

Salvianolic acid B (Sal B), the main water-soluble compound in

Topics: Anti-Inflammatory Agents; Benzofurans; Lipopolysaccharides; Macrophages; Proteomics

Salvianolic acid B (SalB) is a polyphenolic compound in Salvia miltiorrhiza Bunge ("Danshen"), which has been largely used in Traditional Chinese Medicine for the treatment of metabolic syndrome, obesity, diabetes, among others.. This study was to investigate the effects of Salvianolic acid B (SalB) on mRNA, lncRNA and circRNA's expression profile in brown adipose tissue (BAT) of obese mice.. High-fat-diet induced obese C57BL/6J mice were treated with SalB (100 mg/kg/day) for 8 weeks. Then, BAT was harvested for RNA-Seq analysis. Differentially expressed mRNAs, lncRNAs and circRNAs were analyzed using the Illumina Hiseq 4000. Following this procedure, bioinformatic tools including Gene ontology (GO), KEGG pathway and lncRNA-mRNA co-network analysis were utilized. Finally, RT-qPCR was performed to validate the differentially expressed RNAs.. Compared with control group, 2532 mRNAs, 774 lncRNAs and 25 circRNAs were differentially expressed in SalB group. Additionally, 40 upregulated and 109 downregulated gene-related pathways were identified in the SalB group. Among them, metabolic pathways showed the highest enrichment coefficient in upregulated genes. Moreover, 54 up-regulated and 626 down-regulated coding mRNAs associated with lncRNA-Hsd11b1 and lncRNA-Vmp1.. SalB may play an anti-obesity role by adjusting the expression of mRNAs correlated with inflammatory response and energy metabolism through regulating the expression of lncRNA-Hsd11b1. The findings of this research provide new directions to study the mechanisms of SalB, and would open therapeutic avenues for the treatment of obesity.

Topics: Adipose Tissue, Brown; Animals; Benzofurans; Computational Biology; Diet, High-Fat; Down-Regulation; Energy Metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; RNA, Circular; RNA, Long Noncoding; RNA, Messenger; Salvia miltiorrhiza; Up-Regulation

Topics: Anisotropy; Antineoplastic Agents; Benzofurans; Binding Sites; Circular Dichroism; Humans; Models, Molecular; Molecular Conformation; Proto-Oncogene Proteins c-myc; Spectrometry, Fluorescence

Danshen water extract (DWE), obtained from the Salvia miltiorrhiza Bunge (Family Lamiaceae) root, is usually employed in Chinese traditional medicine as treatment to cardiovascular ailments and cerebrovascular diseases. Intriguingly, the extract was also found to contain vast beneficial properties in Alzheimer's disease (AD) treatment.. Alzheimer's disease is the most significant type of neurodegenerative disorder plaguing societies globally. Its pathogenesis encompasses the hallmark aggregation of amyloid-beta (Aβ). Of all the Aβ oligomers formed in the brain, Aβ42 is the most toxic and aggressive. Despite this, the mechanism behind this disease remains elusive. In this study, DWE, and its major components, Salvianolic acid A (SalA) and Salvianolic acid B (SalB) were tested for their abilities to attenuate Aβ42's toxic effects.. The composition of DWE was determined via Ultra-Performance Liquid Chromatography (UPLC). DWE, SalA and SalB were first verified for their capability to diminish Aβ42 fibrillation using an in vitro activity assay. Since Aβ42 aggregation results in neuronal degeneration, the potential Aβ42 inhibitors were next evaluated on Aβ42-exposed PC12 neuronal cells. The Drosophila melanogaster AD model was then employed to determine the effects of DWE, SalA and SalB.. DWE, SalA and SalB were shown to be able to reduce fibrillation of Aβ42. When tested on PC12 neuronal cells, DWE, SalA and SalB ameliorated cells from cell death associated with Aβ42 exposure. Next, DWE and its components were tested on the Drosophila melanogaster AD model and their rescue effects were further characterized. The UPLC analysis showed that SalA and SalB were present in the brains and bodies of Drosophila after DWE feeding. When human Aβ42 was expressed, the AD Drosophila exhibited degenerated eye structures known as the rough eye phenotype (REP), reduced lifespan and deteriorated locomotor ability. Administration of DWE, SalA and SalB partially reverted the REP, increased the age of AD Drosophila and improved most of the mobility of AD Drosophila.. Collectively, DWE and its components may have therapeutic potential for AD patients and possibly other forms of brain diseases.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Benzofurans; Caffeic Acids; Cell Survival; Drosophila melanogaster; Female; Lactates; Neurons; PC12 Cells; Peptide Fragments; Phytotherapy; Plant Extracts; Rats; Salvia miltiorrhiza

MicroRNAs (miRNAs) are important regulators of gene expression involved in plant development and abiotic stress responses. Recently, miRNAs have also been reported to be engaged in the regulation of secondary plant metabolism. However, there are few functional studies of miRNAs in medicinal plants. For this study, we obtained

Topics: Benzofurans; Gene Expression Regulation, Plant; MicroRNAs; Plant Proteins; Plant Roots; Salvia miltiorrhiza

Despite a wide range of bactericides and antiseptics, the treatment of chronic or complicated wounds is still a major challenge for modern medicine. Topical medications are the most sought-after new agents for use as treatment. The therapeutic concentration of their active substances is easy to achieve with the lowest possible burden on the patient's body. This study assesses the effect of salvianolic acid B (Sal B) on the proliferation, migration, and production of collagen type III by fibroblasts, which are the most important processes in wound healing. The study was conducted on human gingival fibroblasts obtained from primary cell culture. The results showed that Sal B at a dose of 75 µg/mL increases the cell viability with significant stimulation of the cell migration as demonstrated in the wound healing assay, as well as an increase in the expression of collagen type III, which has great importance in the initial stages of wound scarring. The results obtained in the conducted studies and previous scientific reports on the antibacterial properties and low toxicity of Sal B indicate its high potential in wound healing.

Topics: Benzofurans; Cell Line; Cell Movement; Cell Proliferation; Cell Survival; Fibroblasts; Gingiva; Humans; Models, Theoretical; Wound Healing

Topics: Animals; Antioxidants; Apoptosis; Benzofurans; Cell Line; Disease Models, Animal; Enterocytes; Glycogen Synthase Kinase 3 beta; Humans; Ileum; Intestinal Diseases; Intestinal Mucosa; Jejunum; Male; Mitochondria; Oxidative Stress; Proto-Oncogene Proteins c-akt; Rats; Salvia miltiorrhiza; Signal Transduction

The ability to separate bioactive compounds from herbal medicines, which contain abundant components, is crucial for drug discovery. Conventional Countercurrent chromatography (CCC) methods for separating bioactive compounds are labor intensive and show low efficiency. Here, we present a novel integrative CCC method for separating lysine-specific demethylase 1 (LSD1) inhibitors from the roots of Salvia miltiorrhiza (RSM). The methanol extracts of RSM were separated into hydrosoluble and liposoluble fractions, which were online stored in coils. Subsequently, the targeting LSD1 constituents were isolated using isocratic, gradient, or recycling elution mode. All separation processes could be accomplished using one CCC apparatus. Using our separation strategy, two phenylpropanoids and four tanshinones were isolated, which were determined to be new classes of natural LSD1 inhibitors. Salvianolic acid B, which showed the most potent inhibitory activity with an IC

Topics: Benzofurans; Cell Line, Tumor; Cell Movement; Cinnamates; Countercurrent Distribution; Depsides; Enzyme Inhibitors; Histone Demethylases; Humans; Molecular Docking Simulation; Phenanthrenes; Plant Roots; Protein Binding; Rosmarinic Acid; Salvia miltiorrhiza

The root of Salvia miltiorrhiza f. alba (RSMA) (Lamiaceae) is used for the treatment of patients with thromboangiitis obliterans (TAO) in traditional Chinese medicine. Previously, a mixture of phenolic acids extracted from RSMA has shown significant protective effects on TAO rats.. This study investigates the inhibitory effects of salvianolic acid B on TAO induced by sodium laurate injection in rats to explore the effective constituents of RSMA in TAO treatment.. TAO rats were developed using injected sodium laurate. After treatment with ligustrazine hydrochloride (15 mg/kg) and various doses of salvianolic acid B (10, 20, 40 mg/kg) by tail intravenous injection, levels of thromboxane B. Salvianolic acid B significantly decreased the expressions of TXB. Salvianolic acid B has a protective effect on TAO rats. The mechanism may involve inhibition of thrombosis and TAO-associated inflammatory responses, which may explain the success of RSMA treatment of TAO in humans in traditional Chinese medical practice. Hence, it may be a potential drug for TAO treatment in conventional medicine.

Topics: Animals; Benzofurans; Humans; Inflammation; Lauric Acids; Male; Medicine, Chinese Traditional; Plant Roots; Rats; Rats, Wistar; Salvia miltiorrhiza; Thromboangiitis Obliterans; Thrombosis

Salvianolic acid B (Sal B) has a significant protective effect on myocardial ischaemia-reperfusion (I/R) injury. Therefore, the aims of this study were to determine the effects of Sal B on myocardial ischaemic-reperfusion (I/R) injury in rats and to explore whether its underlying mechanism of cardioprotection occurs through activating the expression of the phosphoinositide 3-kinase/protein, kinase B (PI3K/Akt) and inhibiting the expression of high mobility group protein 1 (HMGB1). Ninety Sprague-Dawley rats were randomized into five groups: group 1 (sham-operated), group 2 (myocardial I/R), group 3 (low dose of Sal B+I/R), group 4 (high dose of Sal B+I/R), and group 5 (high dose of Sal B+I/R+LY294002, which is a specific PI3k inhibitor). All I/R rats received 30 min myocardial ischaemia followed by 24-h reperfusion. Cardiac function, infarct size, myocardial injury marker levels, inflammatory response and cardiomyocyte apoptosis as well as Bcl-2, Bax, P-Akt, HMGB1 and TLR4 expression were measured. In the current study, Sal B significantly ameliorated myocardial I/R injury in a dose-dependent manner, ameliorated cardiac function, reduced myocardial infarction size, decreased myocardial injury marker expression, decreased inflammatory responses, reduced apoptosis, activated PI3K/Akt expression and inhibited HMGB1 expression. However, all effects of Sal B were significantly reversed by LY294002. Overall, the present study indicated that Sal B attenuated myocardial I/R injury by activating PI3K/Akt and inhibiting the release of HMGB1 in rats.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Benzofurans; Disease Models, Animal; HMGB1 Protein; Inflammation Mediators; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Signal Transduction

Pregnancy is a kind of natural immune tolerance. Immune factors play an important role in recurrent spontaneous abortion and repeated implantation failure. Salvianolic acid B (SalB) has anti-tumor, anti-inflammatory, anti-oxidation and immunomodulatory functions. However, there are few reports on the relationship between SalB and maternal-fetal immune tolerance. In this study, CBA/J × DBA/2 J mice as a spontaneous abortion mouse model were given SalB. The results showed that the abortion rate was significantly decreased after SalB treatment. The populations of Nkp46 and cytotoxic CD8

Topics: Abortion, Habitual; Abortion, Spontaneous; Animals; Benzofurans; Chorionic Villi; Disease Models, Animal; Down-Regulation; Embryo Implantation; Female; Humans; Immune Tolerance; Immunologic Factors; Inflammation Mediators; Male; Maternal-Fetal Exchange; Mice; Pregnancy; T-Lymphocytes, Cytotoxic

Macrophages play important roles in the healing and remodeling of cardiac tissues after myocardial ischemia/reperfusion (MI/R) injury. Here we investigated the potential effects of salvianolic acid B (SalB), one of the abundant and bioactive compounds extracted from Chinese herb Salvia Miltiorrhiza (Danshen), on macrophage-mediated inflammation after MI/R and the underlying mechanisms. In primary cultured bone marrow-derived macrophages (BMDMs), SalB attenuated lipopolysaccharide (LPS)-induced M1 biomarkers (IL-6, iNOS, CCL2 and TNF-α) mRNA expression in a concentration-dependent manner. In contrast, M2 biomarkers (Arg1, Clec10a and Mrc) mRNA levels following interleukinin-4 (IL-4) stimulation were significantly upregulated by SalB. In addition, LPS stimulation potently induced transcriptional upregulation of RagD, an important activation factor of mammalian target of rapamycin complex 1 (mTORC1). Interestingly, SalB inhibited RagD upregulation and mTORC1 activation, decreased glycolysis, and reduced inflammatory cytokine production in LPS-stimulated macrophages, all of which were blunted in RagD knockdown macrophages. In mice subjected to MI/R, SalB treatment decreased cardiac M1-macrophages and increased M2-macrophages at 3 days post-MI/R, followed by decreased collagen deposition and ameliorated cardiac dysfunction at 7 days post-MI/R. Collectively, our data have shown that SalB decreases M1-polarized macrophages in MI/R hearts via inhibiting mTORC1-dependent glycolysis, which might contribute to alleviated inflammation and improved cardiac dysfunction afforded by SalB after MI/R.

Topics: Animals; Benzofurans; Glycolysis; Heart; Macrophage Activation; Macrophages; Mechanistic Target of Rapamycin Complex 1; Mice; Myocardial Reperfusion Injury

Diabetes is related to alterations in glucose and lipid metabolism, which are linked to endothelial cell (EC) dysfunction. Salvianolic acid B (Sal B), one of the major ingredient of Danshen (Salvia miltiorrhiza), possesses many of the biological activities. However, protective effect of Sal B against oxLDL induced ECs dysfunction under high glucose condition (high Glu) is not well known. Thus, in this study, we investigated the protective effects of Sal B against EC dysfunction induced by oxLDL and high Glu and examined the associated mechanisms. Our results showed that Sal B significantly and dose-dependently decreased oxLDL- and high Glu-mediated induction of lectin-like oxLDL receptor-1 and significantly decreased oxLDL- and high Glu-induced mitochondrial ROS (mtROS) production and mitochondrial DNA (mtDNA) expression. In addition, oxLDL stimulation under high-Glu conditions activated the intrinsic apoptosis pathway in ECs. These effects were abolished by Sal B through reductions in mtROS and mtDNA. Furthermore, Sal B inhibited oxLDL- and high Glu-induced increases in fission protein (p-DRP 1 and FIS 1) levels. OxLDL and high Glu activated the ROCK1 pathway, which is involved in apoptosis and mitophagy, while Sal B significantly reduced ROCK1 protein levels. The protective effects of Sal B against oxLDL- and high Glu-induced endothelial dysfunction may be mediated by reductions in apoptosis-related proteins and fission proteins through suppression of the ROCK1-mediated pathway.

Topics: Apoptosis; Benzofurans; Cell Survival; Dose-Response Relationship, Drug; Down-Regulation; Drugs, Chinese Herbal; Endothelial Cells; Glucose; Human Umbilical Vein Endothelial Cells; Humans; Lipoproteins, LDL; Mitophagy; rho-Associated Kinases

Salvianolic acid B (Sal B) is the representative component of phenolic acids derived from the dried root and rhizome of Salvia miltiorrhiza Bge. (Labiatae), which has been widely used for the treatment of cardiovascular and cerebrovascular diseases. However, the effect of Sal B on diabetic cardiomyopathy (DCM) is still unclear.. Type 1 diabetes mellitus was induced in C57BL/6 J mice by streptozotocin (STZ) treatment, whereas meanwhile Salvianolic Acid B (Sal B (15 or 30 mg/kg/d) was intraperitoneally injected for 16 weeks. At the end of this period, cardiac function was assessed by echocardiography, and total collagen deposition was evaluated by Masson's trichrome and Picrosirius Red staining. Human umbilical vein endothelial cells exposed to hypoxia were used to investigate the effect of different doses of Sal B on angiogenesis and tube formation in vitro. Transcriptome sequencing was performed to identify potential targets of Sal B.. Sal B ameliorated left ventricular dysfunction and remodeling, and decreased collagen deposition in the heart of diabetic mice. Administration of Sal B increased the expression of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and VEGFA in a dose-dependent manner and promoted angiogenesis both in vivo and in vitro. Furthermore, Sal B reduced HG-induced insulin-like growth factor-binding protein 3 (IGFBP3) expression, induced the phosphorylation of extracellular signal-regulated protein kinase and protein kinase B (AKT) activities, enhanced cell proliferation, and activated VEGFR2/VEGFA signaling in endothelial cells. The underlying mechanisms involve SalB that enhances IGFBP3 promoter DNA methylation and induce nuclear translocation of IGFBP3 in HUVECs under hypoxia.. Sal B promoted angiogenesis and alleviated cardiac fibrosis and cardiac remodeling in DCM by suppressing IGFBP3.

Topics: Animals; Base Sequence; Benzofurans; Cell Hypoxia; CpG Islands; Cytoplasm; Diabetic Cardiomyopathies; DNA Methylation; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Human Umbilical Vein Endothelial Cells; Humans; Hyperglycemia; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Male; Mice, Inbred C57BL; Myocardium; Neovascularization, Physiologic; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-akt; Ventricular Remodeling

Topics: Animals; Benzofurans; Cytokines; Inflammation; Intestines; Male; Oxidative Stress; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Recent studies indicated that seeded fibril formation and toxicity of α-synuclein (α-syn) play a main role in the pathogenesis of certain diseases including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Therefore, examination of compounds that abolish the process of seeding is considered a key step towards therapy of several synucleinopathies.. Using biophysical, biochemical and cell-culture-based assays, assessment of eleven compounds, extracted from Chinese medicinal herbs, was performed in this study for their effect on α-syn fibril formation and toxicity caused by the seeding process.. Salvianolic acid B and dihydromyricetin were the two compounds that strongly inhibited the fibril growth and neurotoxicity of α-syn. In an in-vitro cell model, these compounds decreased the insoluble phosphorylated α-syn and aggregation. Also, in primary neuronal cells, these compounds showed a reduction in α-syn aggregates. Both compounds inhibited the seeded fibril growth with dihydromyricetin having the ability to disaggregate preformed α-syn fibrils. In order to investigate the inhibitory mechanisms of these two compounds towards fibril formation, we demonstrated that salvianolic acid B binds predominantly to monomers, while dihydromyricetin binds to oligomeric species and to a lower extent to monomers. Remarkably, these two compounds stabilized the soluble non-toxic oligomers lacking β-sheet content after subjecting them to proteinase K digestion.. Eleven compounds were tested but only two showed inhibition of α-syn aggregation, seeded fibril formation and toxicity in vitro. These findings highlight an essential beginning for development of new molecules in the field of synucleinopathies treatment.

Topics: alpha-Synuclein; Animals; Benzofurans; Drugs, Chinese Herbal; Flavonols; HEK293 Cells; Humans; Mice; Molecular Structure; Plant Extracts; Protein Aggregation, Pathological; Synucleinopathies

Osteogenic differentiation of human periodontal ligament cells (hPDLCs) is crucial for regenerate periodontal tissues. In this study, we investigated the function of salvianolic acid B (Sal B) in osteogenesis of hPDLCs.. HPDLCs were isolated from healthy third molar roots. HPDLCs at passage 3 were identified by morphological observation and immunohistochemistry of vimentin. The viability of hPDLCs incubated with Sal B at concentrations of 0μM, 0.1μM, 0.5μM, 1μM and 5μM were measured by CCK-8 assay. To evaluate the effect of Sal B on osteogenic differentiation of hPDLCs, the alkaline phosphatase (ALP) activity, osteogenic differentiation markers, and mineralized nodules were determined by ALP kit, qRT-PCR and alizarin red S staining, respectively. To confirm the function of Sal B in hPDLCs involved in Wnt/β-catenin signaling pathway, hPDLCs were incubated with Sal B or co-incubated with Sal B and DKK-1 (a inhibitor of Wnt/β-catenin). The levels of Wnt/β-catenin signaling pathway and osteogenic differentiation-associated indicators were then determined.. HPDLCs showed a typical fibroblast-like and spindle-shaped, with vimentin-positive. The viability of hPDLCs had no obvious change with stimulation of Sal B at various doses. Sal B promoted the increase of ALP activity, osteogenic differentiation markers levels, mineralized nodules and activation of Wnt/β-catenin signaling pathway, and DKK-1 could block those effects of Sal B on hPDLCs.. Sal B promoted osteogenesis of hPDLCs through Wnt/β-catenin signaling pathway, which providing a potential drug for periodontitis treatment.

Topics: Benzofurans; Cell Differentiation; Cells, Cultured; Humans; Osteogenesis; Periodontal Ligament; Wnt Signaling Pathway

Impaired insulin sensitivity of insulin-sensitive tissues plays a major role in the pathogenesis of type 2 diabetes, salvianolic acid B (SalB), a natural antioxidant usually treated various cardiovascular diseases was also reported potential utility on diabetes and dyslipidemia. Based on these, we aimed to explored whether the antioxidant effect of SalB play a pivotal role in the molecular mechanisms leading to insulin resistance. We found that SalB improved glucose tolerance and insulin sensitivity, decreased serum ALT, AST and ALP levels of ob/ob mice. Also, transcription of Bip and CHOP, phosphorylation of PERK and IRE1 for endoplasmic reticulum stress (ER) and phosphorylation of IRS-1 for insulin sensitivity in the liver of ob/ob mice were relieved by SalB. Further, SalB decreased phosphorylation of PERK, IRE1 and IRS1 and transcription of Bip and CHOP stimulated by palmitate of hepatic cells HL7702, but did not reversed phosphorylation of JNK and IRS1 and transcription of Bip and CHOP when ER stress was stimulated by tunicamycin. These data shows that SalB improved insulin resistance of ob/ob mice through suppression of hepatic ER stress.

Topics: Animals; Benzofurans; Diabetes Mellitus, Type 2; Disease Models, Animal; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Hepatocytes; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Liver; Male; Membrane Proteins; Mice; Palmitates; Phosphorylation; Protein Serine-Threonine Kinases; Signal Transduction; Transcription Factor CHOP; Tunicamycin

It has been claimed that salvianolic acid B (Sal B), a natural bioactive antioxidant, exerts protective effects in various types of cells. This study aims to evaluate the antioxidant and anti-apoptosis effects of Sal B in a cultured HEI-OC1 cell line and in transgenic zebrafish (Brn3C: EGFP). A CCK-8 assay, Annexin V Apoptosis Detection Kit, TUNEL and caspase-3/7 staining, respectively, examined apoptosis and cell viability. The levels of reactive oxygen species (ROS) were evaluated by CellROX and MitoSOX Red staining. JC-1 staining was employed to detect the mitochondrial membrane potential (ΔΨm). Western blotting was used to assess expressions of Bax and Bcl-2. The expression pattern of p-PI3K and p-Akt was determined by immunofluorescent staining. We found that Sal B protected against neomycin- and cisplatin-induced apoptotic features, enhanced cell viability and accompanied with decreased caspase-3 activity in the HEI-OC1 cells. Supplementary experiments determined that Sal B reduced ROS production (increased ΔΨm), promoted Bcl-2 expression and down-regulated the expression of Bax, as well as activated PI3K/AKT signalling pathways in neomycin- and cisplatin-injured HEI-OC1 cells. Moreover, Sal B markedly decreased the TUNEL signal and protected against neomycin- and cisplatin-induced neuromast HC loss in the transgenic zebrafish. These results unravel a novel role for Sal B as an otoprotective agent against ototoxic drug-induced HC apoptosis, offering a potential use in the treatment of hearing loss.

Topics: Animals; Apoptosis; Benzofurans; Cell Line; Cell Survival; Cisplatin; Cytoprotection; Lateral Line System; Membrane Potential, Mitochondrial; Mice; Mitochondria; Neomycin; Ototoxicity; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Zebrafish

The emergence of resistance among fungal phytopathogens poses a biggest threat across the world. Streptomyces are a group of spore-forming Gram + ve bacteria and prolific producers of secondary bioactive metabolites which have been used as biocontrol agents against phytopathogens and also known for plant growth promotion. The current study identified a potent isolate M4 from soil with broad spectrum antifungal activity against different fungal phytopathogens. The isolate was identified as a Streptomyces sp. on the basis of cultural, morphological, physiological, biochemical and phylogenetic characteristics. 16S rRNA gene sequence of M4 showed 100 % similarity with three Streptomyces spp. i.e. Streptomyces plicatus NBRC 13071 T (AB184291), Streptomyces rochei NBRC 12908 T AB184237 and Streptomyces enissocaesilis NRRL-B-16365 T (DQ026641). However, phenotypic and phylogenetic analysis concluded that M4 represents a novel sp. within the genus Streptomyces. One of the two antifungal compounds purified from Streptomyces M4 was identified as salvianolic acid B. To our knowledge, the present study is the first work reporting purification and characterization of salvianolic acid B from Streptomyces and its broad spectrum antifungal activity against different fungal phytopathogens viz. Alternaria spp., Fusarium spp., Colletotrichum spp., Cladosporium herbarum and Botrytis cineria. Salvianolic acid B was found to be photostable, thermostable (up to 70 °C) and non-mutagenic in nature and might be developed as safe biofungicide to control phytopathogens.

Topics: Alternaria; Antifungal Agents; Benzofurans; Biological Control Agents; Botrytis; Cladosporium; Colletotrichum; Fungi; Fusarium; Genes, Bacterial; Phylogeny; Plant Diseases; RNA, Ribosomal, 16S; Streptomyces

Renal fibrosis is a kind of progressive kidney disease leading to end-stage renal damage. Epithelial-mesenchymal transition (EMT) is one of the crucial features of renal fibrosis. Salvianolic acid B (SalB), isolated from traditional Chinese medicine Radix Salviae miltiorrhizae, has been proved to be suitable for renal protection. The aims of this study are to investigate the pharmacological effects of SalB on renal fibrosis and explore the underlying mechanisms. In vivo, our study showed that SalB could improve kidney dysfunction and reduce the expression of EMT-related proteins, including fibronectin (FN), α-smooth muscle actin (α-SMA) and transforming growth factor-β (TGF-β). In addition, SalB activated autophagy and up-regulated the expression of Sirt1. In vitro, our study showed that SalB reversed EMT in TGF-β1-induced human kidney proximal tubular epithelial cells (HK-2 cells). Further mechanism studies showed that the inhibition of Sirt1 and autophagy could reverse the protective effect of SalB on the EMT process in TGF-β1-induced HK-2 cells. Taken together, this study demonstrated that SalB attenuates EMT in the process of renal fibrosis through activating Sirt1-mediated autophagy, and Sirt1 could be a key target for treatment of renal fibrosis.

Topics: Actins; Animals; Autophagy; Benzofurans; Cell Line; Disease Models, Animal; Epithelial-Mesenchymal Transition; Fibronectins; Fibrosis; Humans; Kidney; Kidney Diseases; Male; Rats, Sprague-Dawley; Signal Transduction; Sirtuin 1; Transforming Growth Factor beta

Degree of mucosal recovery is an important indicator for evaluating the therapeutic effects of drugs in treatment of inflammatory bowel disease (IBD). Increasing evidences has proved that tight junction (TJ) barrier dysfunction is one of the pathological mechanisms of IBD. The aim of this study was to observe whether enhancement of TJ can decrease colitis recurrence.. Eighty C57BL/6 mice were randomly divided into four groups including normal group, colitis group, sulfasalazine (SASP) treated group, and traditional Chinese drug salvianolic acid B (Sal B) treated group. Colitis was established in mice by free drinking water containing dextran sulfate sodium, after treatments by SASP and Sal B, recombinant human interleukin-1β (IL-1β) was injected intraperitoneally to induce colitis recurrence.. Compared with sham control, cell apoptosis in colitis group was increased from 100.85 ± 3.46% to 162.89 ± 11.45% (P = 0.0038), and TJ dysfunction marker myosin light chain kinase (MLCK) was also significantly increased from 99.70 ± 9.29% to 296.23 ± 30.78% (P = 0.0025). The increased cell apoptosis was reversed by both SASP (125.99 ± 8.45% vs. 162.89 ± 11.45%, P = 0.0059) and Sal B (104.27 ± 6.09% vs. 162.89 ± 11.45%, P = 0.0044). High MLCK expression in colitis group was reversed by Sal B (182.44 ± 89.42% vs. 296.23 ± 30.78%, P = 0.0028) but not influenced by SASP (285.23 ± 41.04% vs. 296.23 ± 30.78%, P > 0.05). The recurrence rate induced by recombinant human IL-1β in Sal B-treated group was significantly lower than that in SASP-treated group.. These results suggested a link between intestinal mucosal barrier dysfunction, especially TJ barrier dysfunction, and colitis recurrence. The TJ barrier dysfunction in remission stage of colitis increased the colitis recurrence. This study might provide potential treatment strategies for IBD recurrence.

Topics: Animals; Benzofurans; Colitis; Dextran Sulfate; Disease Models, Animal; Interleukin-1beta; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Myosin-Light-Chain Kinase

Topics: Animals; Benzofurans; Biomarkers; Carbon Tetrachloride; Cell Movement; Cell Proliferation; Cells, Cultured; Computational Biology; Disease Models, Animal; Disease Progression; Down-Regulation; Hepatic Stellate Cells; Humans; Liver; Liver Cirrhosis; Male; Mice; Mice, Transgenic; MicroRNAs; Primary Cell Culture; RNA-Binding Proteins; RNA, Circular; Transfection

Basic helix-loop-helix (bHLH) transcription factors play essential roles in myriad regulatory processes, including secondary metabolism. In this study with Salvia miltiorrhiza, we isolated and characterized SmbHLH53, which encodes a bHLH family member. Expression of this gene was significantly induced by wounding and multiple hormones, including methyl jasmonic acid; transcript levels were highest in the leaves and roots. Phylogenetic analysis indicated that SmbHLH53 clusters withAtbHLH17 and AtbHLH13, two negative regulators of jasmonate (JA) responses, and is localized in the nucleus and cell membrane. Yeast two-hybrid and bimolecular fluorescent complementation assays indicated that SmbHLH53 forms a homodimer as well as a heterodimer with SmbHLH37. It also interacts with both SmJAZs1/3/8 and SmMYC2, the core members of the JA signal pathway. Unexpectedly, we noted that overexpression of SmbHLH53 did not significantly influence the concentrations of rosmarinic acid and salvianolic acid B in transgenic plants. Results from yeast one-hybrid assays showed that SmbHLH53 binds to the promoters of SmTAT1, SmPAL1, and Sm4CL9, the key genes for enzymes in the pathway for phenolic acid synthesis. Assays of transient transcriptional activity demonstrated that SmbHLH53 represses the promoter of SmTAT1 while activating the promoter of Sm4CL9. Thus, the present work revealed that SmbHLH53 may play dual roles in regulating the genes for enzymes in the pathway for Sal B biosynthesis.

Topics: Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Benzofurans; Biosynthetic Pathways; Cell Nucleus; Cyclopentanes; Oxylipins; Phylogeny; Plant Proteins; Promoter Regions, Genetic; Protein Interaction Maps; Protein Multimerization; Salvia miltiorrhiza; Secondary Metabolism; Signal Transduction

Salvianolic acid B (Sal B) is one of the main water‑soluble components of Salvia miltiorrhiza Bge. Numerous reports have demonstrated that it could exert significant renal‑protective effects, but the underlying mechanism remains unclear. The present study demonstrated that Sal B could alleviate renal injury by regulating the heparanase/syndecan‑1 (HPSE/SDC1) axis. In vivo, the serum creatinine, blood urea nitrogen, transforming growth factor‑β1 (TGF‑β1) and fibroblast growth factor‑2 (FGF‑2) levels, and the histopathological changes of mice kidneys were examined. Sal B could notably reduce the renal injury caused by left ureteral ligation. In vitro, Sal B downregulated the expression levels of HPSE/FGF‑2/TGF‑β1/α‑smooth muscle actin and upregulated the expression levels of SDC1/E‑cadherin in angiotensin II‑stimulated HK‑2 cells in a dose‑dependent manner. In summary, to the best of the authors' knowledge, the present study provided evidence for the first time that Sal B could exert renal‑protective effects via the inhibition of the HPSE/SDC1 axis, and these results suggest that the administration of Sal B may be a novel therapeutic strategy in treating renal interstitial fibrosis.

Topics: Animals; Benzofurans; Cell Line; Fibrosis; Glucuronidase; Humans; Kidney; Male; Mice; Mice, Inbred C57BL; Protective Agents; Renal Insufficiency, Chronic; Syndecan-1

The aim of this paper was to investigate whether the mechanism of salvianolic acid B in protecting H9 c2 cardiomyocytes from hypoxia/reoxygenation injury is related to the regulation of mitochondrial autophagy mediated by NIX. H9 c2 cardiomyocytes were cultured in vitro and divided into normal group, model group and salvianolic acid B group(50 μmol·L~(-1)). Hypoxia/reoxygenation injury model was established by hypoxia for 4 h and reoxygenation for 2 h. In normal group, high glucose DMEM medium was used for culture. Those in model group were cultured with DMEM medium without glucose and oxygen, and no drugs for hypoxia and reoxyge-nation. In salvianolic acid B group, salvianolic acid B prepared by glucose-free DMEM medium was added during hypoxia, and the other process was as same as the model group. The cell viability was evaluated by CCK-8 assay. The leakage of lactate dehydrogenase(LDH) was detected by microplate method. The levels of intracellular reactive oxygen species(ROS) and mitochondrial membrane potential(ΔΨm) were measured by chemical fluorescence method. The level of intracellular adenosine triphosphate(ATP) was mea-sured by fluorescein enzyme method. The autophagy related proteins LC3-Ⅰ, LC3-Ⅱ, apoptosis related protein cleaved caspase-3 and mitochondrial autophagy receptor protein NIX were detected by Western blot. As compared with the normal group, the activity of H9 c2 cardiomyocytes and ATP level were decreased(P<0.05); LDH leakage and ROS production were increased(P<0.01); ΔΨm was decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio, cleaved caspase-3 and NIX protein expression levels were increased(all P<0.05) in the model group. As compared with the model group, the activity of cells and ΔΨm were significantly increased(P<0.01); ATP level was increased(P<0.05); LDH leakage and ROS generation were decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio was decreased(P<0.01); cleaved caspase-3 and NIX expression levels were decreased(P<0.05) in the salvianolic acid B group. The protective effect of salvianolic acid B on hypoxia/reoxygenation injury of H9 c2 cardiomyocytes may be associated with inhibiting mitochondrial auto-phagy. The specific mechanism may be related to inhibiting the activation of mitochondrial autophagy mediated by NIX, increasing ΔΨm, reducing ROS production, reducing the expression of cleaved caspase-3, LC3-Ⅱ, and increasing cell viability.

Topics: Apoptosis; Autophagy; Benzofurans; Cell Hypoxia; Cell Survival; Humans; Hypoxia; Myocytes, Cardiac

Pulmonary fibrosis (PF) is an interstitial lung disease characterized by interstitial inflammation and fibrosis. Salvianolic acid B (SAB) and sodium tanshinone IIA sulfonate (STS) are representative components in Salvia miltiorrhiza, which have been reported using in the treatment of PF. The aim of the study was to explain the role of inflammation in the process of PF and to investigate the effect of SAB and STS on inflammation and fibrosis in vitro. The results clearly indicated that lipopolysaccharide (LPS)-stimulated inflammatory response could induce fibroblast proliferation and fibroblast to myofibroblast transformation (FMT). Both SAB and STS significantly inhibited LPS-induced inflammation in vitro, including down-regulated the protein expression levels of IL-1β and TNF-α and the mRNA expression levels of IL1B and TNFA. Furthermore, both SAB and STS inhibited TGF-β1-induced the proliferation in MRC-5 cells and the overexpression of α-SMA and COL1α1, both the protein and mRNA levels. In conclusion, these results indicate that the inflammatory response is necessary for the development of PF, and the therapeutic effect of SAB and STS on PF may be related to anti-inflammatory and anti-fibrotic effects.

Topics: Actins; Anti-Inflammatory Agents; Benzofurans; Cell Proliferation; Coculture Techniques; Collagen Type I; Collagen Type I, alpha 1 Chain; Cytokines; Fibroblasts; Humans; Inflammation Mediators; Lung; Macrophages; Phenanthrenes; Pneumonia; Pulmonary Fibrosis; THP-1 Cells

Liver fibrosis is a pathological change existing in most chronic liver diseases, which leads to abnormal changes in liver tissue structure and affects the normal physiological function of liver. Without effectively control, liver fibrosis can develop into cirrhosis and increase the risk of liver cancer. Salvianolic acid B (Sal B) is the main active component in the water-soluble extract from Salvia miltiorrhiza, which is a traditional Chinese medicine usually used for treating cardiovascular and liver diseases. It is reported that Sal B shown a good action against liver fibrosis via numerous signaling pathways, which indicate that Sal B is a potential candidate drug for the treatment of liver fibrosis.. We searched the related researches from the following electronic databases: PubMed, EMBASE, Web of science, China National Knowledge Infrastructure (CNKI), China Biology Medicine (CBM), Wan fang Database for Chinese Technical Periodicals and VIP Database. All the databases were searched from inception to December 2019. No restriction of language, publication date, or publication status. PICO of this systematic review are shown as flowing: P, preclinical studies which evaluated the effects of Sal B on the animal models of liver fibrosis with controlled studies; I, received Sal B as only treat in any dose; C, received normal saline, distilled water, or no treatment; O, the primary outcome include measure will be the decrease in liver fibrosis score, and the secondary outcomes include the index of liver fibrosis. All the included data will be analyzed with the software of Review Manager 5.2 and STATA 14.2.. The purpose of this study is to conduct a systematic review and meta-analysis to assess the effects on anti-liver fibrosis of Sal B, and this will be contribute to drug development and pathological mechanisms of clinical research.. INPLASY202050101, registered on 28/5/2020.

Topics: Animals; Benzofurans; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Humans; Liver Cirrhosis; Meta-Analysis as Topic; Research Design; Severity of Illness Index; Systematic Review as Topic

Topics: Acyltransferases; Adaptor Proteins, Signal Transducing; Animals; Apolipoproteins E; Atherosclerosis; Benzofurans; Cell Survival; Endothelial Cells; Gene Expression Regulation; Humans; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mice; Pericytes; Transcription Factors; YAP-Signaling Proteins

Topics: Animals; Anticonvulsants; Benzofurans; Brain-Derived Neurotrophic Factor; Epilepsy; Pentylenetetrazole; Proto-Oncogene Proteins c-akt; Rats

The aim of this paper was to observe the effect of salvianolic acid B(Sal B) on high-glucose induced renal tubular epithelial-mesenchymal transition(EMT) in rats, and to explore its possible mechanisms of prevention and treatment of diabetic nephropathy. The rat renal tubular epithelial NRK-52 E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+10 μmol·L~(-1)Sal B group(Sal B), the above 3 groups were set at 6, 12, 24 and 48 h for dynamic observation; high glucose+Sal B different concentration(1, 5, 10 μmol·L~(-1)) groups, high glucose+5.0 μmol·L~(-1) pioglitazone group, high glucose+10 μmol·L~(-1)Sal B+5 μmol·L~(-1)GW9662 group. The protein expression levels of PPARγ, PTEN, α-SMA, E-cadherin and PI3 K/Akt signaling molecules were determined by Western blot. The mRNA expression of PPARγ and PTEN were detected by Real-time PCR. The viabi-lity of NRK52 E cells was determined by MTT assay. The results showed that as compared with control group, the mRNA and protein expression levels of PPARγ and PTEN in high glucose group gradually reduced, the protein expression levels of α-SMA and p-Akt~((Thr308))gradually increased, and the protein expression of E-cadherin gradually reduced(P<0.05). As compared with high glucose group, when increases in Sal B doses, the mRNA and protein expression levels of PPARγ, PTEN in high glucose + different concentrations of Sal B groups gradually increased, the protein expression levels of α-SMA and p-Akt~((Thr308)) gradually reduced, and the protein expression of E-cadherin gradually increased(P<0.05), however, the effect of 1 μmol·L~(-1)concentration of Sal B on the expression of PPARγ mRNA and protein and PTEN mRNA was not significantly different. As compared with high glucose group, the mRNA and protein expression levels of PPARγ mRNA(except 6 h) and protein(except 6 h), PTEN mRNA(except 6 h) and protein(except 6, 12 h) kept increasing, the protein expression levels of α-SMA and p-Akt~((Thr308))(except 6 h) continued to reduce, the protein expression of E-cadherin kept increasing in high glucose+10 μmol·L~(-1) Sal B dynamic observation group(P<0.05). As compared with high glucose group, Sal B and the pioglitazone(PIO) can greatly enhance the expression of PPARγ, PTEN at mRNA and protein levels, enhance the expression of E-cadherin at protein levels, and reduce the expression of α-SMA, p-Akt~((Thr308))protein level(P<0.05), there was no significant difference be

Topics: Animals; Benzofurans; Epithelial Cells; Epithelial-Mesenchymal Transition; Glucose; Rats; Transforming Growth Factor beta1

Salvianolic acid B (Sal B) exhibits anti-obesity activity, yet the underlying mechanism linking this effect to metabolic endotoxemia remains unexplored. For this purpose, high-fat diet-induced obese mice were orally administered with Sal B for 10 weeks. Hematoxylin/eosin staining, transmission electron microscopy, and immunohistochemical staining were used to evaluate histopathological alterations in the white adipose tissue (WAT) and/or jejunums. The expression levels of genes related to fat and cholesterol synthesis in the WAT were determined by qPCR. The composition of fecal microbiota was profiled by 16S rRNA gene pyrosequencing. Western blotting was employed to evaluate the relative protein expressions involved in lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway in the WAT. Treatment of obese mice with Sal B improves insulin sensitivity, attenuates body weight gain and alleviates serum levels of LPS and tumor necrosis factor alpha, which is associated with an improvement in intestinal epithelial integrity and probiotic composition as well as a reduction in Gram-negative Proteobacteria and Deferribacteres. In addition, Sal B downregulates the expressions of TLR4 and myeloid differential factor-88, as well as the phosphorylation levels of Jun N-terminal kinase, nuclear factor-kappa B p65, and an insulin receptor substrate in the WAT. In summary, Sal B may attenuate body weight gain and insulin resistance through the regulation of gut microbiota abundances and LPS/TLR4 signaling pathway in obese mice, suggesting Sal B could be a promising drug candidate for protection against obesity.

Topics: Administration, Oral; Animals; Anti-Obesity Agents; Benzofurans; Diet, High-Fat; Gastrointestinal Microbiome; Lipopolysaccharides; Mice; Mice, Obese; Obesity; Signal Transduction; Toll-Like Receptor 4; Weight Gain

The purpose of this study is to reveal the impact of the plant hormone salicylic acid (SA) and methyl jasmonate (MeJA) on the growth, effective components accumulation, and related gene expression of the hairy root of Salvia przewalskii Maxim. Various concentrations of SA (0, 25, 50, 100, 200 μM) or MeJA (0, 50, 100, 200, 400, 600 μM) were added to the culture medium of Salvia przewalskii Maxim. Low concentrations of SA promoted the growth of hairy root, while a high concentration inhibited it. 0 to 400 μM MeJA promoted the growth of hairy root, but 600 μM MeJA starts to inhibit its growth. 50 μM SA and 400 μM MeJA significantly enhanced the production of caffeic acid, rosmarinic acid, salvianolic acid B, cryptotanshinone, and tanshinone IIA. In general, 50 μM SA can be used to accumulate of tanshinone in hairy roots of S. przewalskii with 6 days. 400 μM MeJA can be used to accumulate of phenolic acids in hairy roots of S. przewalskii with 3 days. The selected genes in the tanshinone and phenolic acid biosynthetic pathway were upregulated with elicitation. To obtain a higher yield and content of secondary metabolites, it is advisable to use 50 μM SA or 400 μM MeJA as the optimal doses to cultivate the hairy root of S. przewalskii. This study provides, for the first time, an efficient tanshinone and phenolic acid production method for S. przewalskii.

Topics: Abietanes; Acetates; Benzofurans; Caffeic Acids; Cinnamates; Cyclopentanes; Depsides; Dose-Response Relationship, Drug; Gene Expression Regulation, Plant; Hydroxybenzoates; Oxylipins; Phenanthrenes; Plant Growth Regulators; Plant Proteins; Plant Roots; Rosmarinic Acid; Salicylic Acid; Salvia; Time Factors

Increased matrix metalloprotease 9 (MMP9) after myocardial infarction (MI) exacerbates ischemia-induced chronic heart failure (CHF). Autophagy is cardioprotective during CHF; however, whether increased MMP9 suppresses autophagic activity in CHF is unknown. This study aimed to determine whether increased MMP9 suppressed autophagic flux and MMP9 inhibition increased autophagic flux in the heart of rats with post-MI CHF. Sprague-Dawley rats underwent either sham surgery or coronary artery ligation 6-8 wk before being treated with MMP9 inhibitor for 7 days, followed by cardiac autophagic flux measurement with lysosomal inhibitor bafilomycin A1. Furthermore, autophagic flux was measured in vitro by treating H9c2 cardiomyocytes with two independent pharmacological MMP9 inhibitors, salvianolic acid B (SalB) and MMP9 inhibitor-I, and CRISPR/cas9-mediated MMP9 genetic ablation. CHF rats showed cardiac infarct, significantly increased left ventricular end-diastolic pressure (LVEDP), and increased MMP9 activity and fibrosis in the peri-infarct areas of left ventricular myocardium. Measurement of the autophagic markers LC3B-II and p62 with lysosomal inhibition showed decreased autophagic flux in the peri-infarct myocardium. Treatment with SalB for 7 days in CHF rats decreased MMP9 activity and cardiac fibrosis but increased autophagic flux in the peri-infarct myocardium. As an in vitro corollary study, measurement of autophagic flux in H9c2 cardiomyocytes and fibroblasts showed that pharmacological inhibition or genetic ablation of MMP9 upregulates autophagic flux. These data are consistent with our observations that MMP9 inhibition upregulates autophagic flux in the heart of rats with CHF. In conclusion, the results in this study suggest that the beneficial outcome of MMP9 inhibition in pathological cardiac remodeling is in part mediated by improved autophagic flux.

Topics: Animals; Autophagy; Benzofurans; Cell Line; Disease Models, Animal; Fibroblasts; Fibrosis; Heart Failure; Male; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Myocytes, Cardiac; Rats, Sprague-Dawley; Signal Transduction; Ventricular Function, Left; Ventricular Remodeling

Ischemic heart disease is a major cause of mortality and disability worldwide. Salvianolic acid B (Sal B) is one of the main water‑soluble components of Salvia miltiorrhiza Bge. Numerous studies have demonstrated that Sal B could exert significant anti‑inflammatory and cardiovascular protective effects; however, the underlying mechanisms remain unclear. To elucidate the association between myocardial ischemia and inflammation, and to develop effective protective drugs, a rat model of myocardial ischemia was induced using isoproterenol (ISO) and an inflammation model in H9C2 cells was induced with lipopolysaccharide + adenosine triphosphate. Both of these models were treated with different concentrations of Sal B (5, 10 and 15 mg/kg in vivo; 1, 5 and 25 µM in vitro). In vivo, the serum levels of creatine kinase isoenzyme MB, glutamic oxaloacetic transaminase and IL‑1β, the cardiac function and the mRNA expression levels of NLR family pyrin domain‑containing 3 (NLRP3) inflammasome components were evaluated using ELISAs, an electrocardiogram, hematoxylin and eosin staining and reverse transcription‑quantitative PCR, respectively. The results demonstrated that treatment with Sal B markedly alleviated the acute myocardial ischemic injury induced by hypodermic injection of ISO in rats. In vitro, the results of reactive oxygen species (ROS) detection, JC‑1 staining, western blotting and TUNEL assays showed that Sal B treatment significantly inhibited intracellular ROS production, increased the mitochondrial membrane potential, regulated the expression of mitophagy‑related proteins, inhibited the activation of the NLRP3 inflammasome and inhibited apoptosis in H9C2 cells. In conclusion, these findings indicated that Sal B exerted protective effects against myocardial ischemic injury by promoting mitophagy and maintaining mitochondrial function.

Topics: Animals; Apoptosis; Benzofurans; Cell Line; China; Disease Models, Animal; Inflammasomes; Inflammation; Ischemia; Lipopolysaccharides; Male; Mitophagy; Myocardial Ischemia; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Toll-Like Receptor 4

The efficiency of drugs often hinges on drug carriers. To effectively transport therapeutic plant molecules, drug delivery carriers should be able to carry large doses of therapeutic drugs, enable their sustained release, and maintain their biological activity. Here, graphene oxide (GO) is demonstrated to be a valid carrier for delivering therapeutic plant molecules. Salvianolic acid B (SB), which contains a large number of hydroxyl groups, bound to the carboxyl groups of GO by self-assembly. Silk fibroin (SF) substrates were combined with functionalized GO through the freeze-drying method. SF/GO scaffolds could be loaded with large doses of SB, maintain the biological activity of SB while continuously releasing SB, and significantly promote the osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). SF/GO/SB also dramatically enhanced endothelial cell (EA-hy9.26) migration and tubulogenesis in vitro. Eight weeks after implantation of SF/GO/SB scaffolds in a rat cranial defect model, the defect area showed more new bone and angiogenesis than that following SF and SF/GO scaffold implantation. Therefore, GO is an effective sustained-release carrier for therapeutic plant molecules, such as SB, which can repair bone defects by promoting osteogenic differentiation and angiogenesis.

Topics: Animals; Benzofurans; Fibroins; Graphite; Osteogenesis; Rats; Tissue Scaffolds

The lack of inclusion of comorbidities in animal models of stroke may underlie the limited development of therapy in stroke. Previous studies in mice deficient of CD36, an immune receptor, indicated its contribution to stroke-induced inflammation and injury in hyperlipidemic conditions. The current study, therefore, tested whether pharmacological inhibition of CD36 provides neuroprotection in hyperlipidemic stroke. The hyperlipidemic mice subjected to stroke showed an exacerbation of infarct size and profound brain swelling. However, post-stroke treatment with CD36 inhibitors did not reduce, and in some cases worsened, acute stroke outcome, suggesting potential benefits of elevated CD36 in the post-stroke brain in a hyperlipidemic condition. On the other hand, chronic treatment of a CD36 inhibitor prior to stroke significantly reduced stroke-induced brain swelling. There was a trend toward infarct reduction, although it did not reach statistical significance. The observed benefit of preventative CD36 inhibition is in line with previously reported smaller infarct volume and swelling in CD36 KO mice. Thus, the current findings suggest that insights gained from the genetic models should be carefully considered before the implementation of pharmacological interventions, as a potential therapeutic strategy may depend on preventative treatment or a post-stroke acute treatment paradigm.

Topics: Animals; Apolipoproteins E; Benzofurans; Brain Edema; CD36 Antigens; Disease Models, Animal; Drug Administration Schedule; Drugs, Chinese Herbal; Hyperlipidemias; Inflammation; Male; Mice, Inbred C57BL; Mice, Knockout; Protective Agents; Stroke

Spinal cord injury (SCI) is a common type of injury, and about half of patients affected by SCI will suffer from neuropathic pain within a year after injury. However, the treatment effect of neuropathic pain is far from satisfactory. Our study attempted to reveal whether salvianolic acid B (SalB) could relieve the neuropathic pain caused by SCI in mice by inhibiting the Toll-like receptor 4 (TLR4)/Myeloid differentiation factor 88 (MyD88) pathway.. The mice were randomly divided into a sham group, model group, high-dose treatment group, and low-dose treatment group. The high- and low-dose groups received varying doses of SalB after modeling.. The increase of pain sensitivity was evaluated by detecting paw withdrawal mechanical threshold and withdrawal thermal latency. Messenger RNA and protein expression levels of TLR4 and myD88 were detected by using quantitative reverse-transcription polymerase chain reaction and western blot, respectively. Compared with the model group, there was a significant reduction in paw withdrawal mechanical threshold and withdrawal thermal latency after SalB treatment.. SalB reduced the release of tumor necrosis factor-α and substance P by inhibiting the TLR4/MyD88 pathway in the SCI mouse model. This not only resulted in lower pain, but also contributed to long-term relief of mechanical hyperalgesia.

Topics: Animals; Benzofurans; Hyperalgesia; Male; Mice; Myeloid Differentiation Factor 88; Neuralgia; Pain Threshold; Random Allocation; Spinal Cord Injuries; Toll-Like Receptor 4

Glucocorticoids (GCs) are widely used to treat a variety of autoimmune diseases, but long-term use can lead to osteoporosis. To elucidate the mechanism of osteoporosis caused by glucocorticoids and to find effective protective drugs/foods, osteoblasts treated by prednisolone acetate were studied and salvianolic acid B (Sal B) was added to osteoblasts. The results showed that Sal B increased the activity of ALP and stimulated the expression of ALP that had been suppressed by prednisolone acetate. To further study the mechanisms of the protective effect of Sal B on osteoblasts treated with prednisolone acetate, the effects of gene expression involved with bone formation and differentiation were studied. The results show that the mRNA and protein expression of Runx2, Osx, OCN, IGF-I, Col-I and HO-I was up-regulated by Sal B. In conclusion, by stimulating the osteoblast activity and the expression of genes related to bone formation and differentiation, Sal B had a protective effect on osteoblasts that had been treated with prednisolone acetate.

Topics: Animals; Benzofurans; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Insulin-Like Growth Factor I; Osteoblasts; Osteogenesis; Prednisolone; Rats; Rats, Sprague-Dawley; Transcription Factors; Up-Regulation

In order to deliver Salvianolic acid B (Sal B) and Baicalin (BA) to the brain tissue to repair neuron damage and improve cerebral ischemia-reperfusion injury (IRI), in our previous study, a nanostructured lipid carrier (NLC) containing BA and Sal B, and modified by the transferrin receptor monoclonal antibody OX26 (OX26-BA/Sal B-NLC) was constructed. The present study is to evaluate its in vitro release behavior, in vitro and in vivo targeting ability, in vitro pharmacodynamics and brain pharmacokinetics. The results showed that the release mechanism of the formulation was in line with the Weibull model release equation. The in-vitro and in-vivo targeting ability study exhibited that OX26 modified formulations was obviously higher than that of non-modified and solution groups. The results of in vitro preliminary study to investigate the protective effect of OX26-BA/Sal B-NLC on oxygen-glucose deprivation/reperfusion injured cells showed that it could decrease the injury. Furthermore, the results of brain microdialysis study showed that the OX26-modified preparation group could significantly increase the content of BA in the brain. In the solution group and the unmodified group, Sal B can only be detected at few time points, while OX26-modified BA/Sal B-NLC could be detected within 4 h. These results indicating that OX26-modified NLC can promote the brain delivery of Sal B and BA combination.

Topics: Administration, Intravenous; Animals; Antibodies, Monoclonal; Benzofurans; Brain; Cell Line; Disease Models, Animal; Drug Carriers; Drug Combinations; Drug Evaluation, Preclinical; Drug Liberation; Drugs, Chinese Herbal; Flavonoids; Humans; Lipids; Male; Mice; Microdialysis; Nanoparticles; Permeability; Receptors, Transferrin; Reperfusion Injury; Tissue Distribution

Salvianolic acid B has been proven as an effective drug to promote osteogenesis and angiogenesis which could be beneficial for bone repair.. The objective of this study was to construct a salvianolic acid B-loaded chitosan/hydroxyapatite (Sal B-CS/HA) bone scaffold with controlled release and effective bioactivity.. The characteristics, controlled release behavior and bioactivity of Sal B-CS/HA scaffold were evaluated in vitro. The bone repair effect was evaluated in the rabbit radius defect model.. The results showed that chemical and physical characteristics of salvianolic acid B and chitosan/hydroxyapatite (CS/HA) material did not obviously change after the drug loading procedure; the drug release of salvianolic acid B was stable and continuous from the Sal B-CS/HA scaffold for 8 weeks in vitro; the biocompatibility of the Sal B-CS/HA was favorable by evaluation of cell morphology and proliferation; the osteogenic and angiogenic bioactivities of the Sal B-CS/HA scaffold were proved to be effective by in vivo and in vitro tests.. Our results suggest that this salvianolic acid B-loaded bone scaffold has potential to be used for bone defect repair with both osteogenic and angiogenic bioactivities.

Topics: Alkaline Phosphatase; Animals; Benzofurans; Bone and Bones; Cell Line; Cell Proliferation; Chitosan; Drug Liberation; Durapatite; Human Umbilical Vein Endothelial Cells; Humans; Mice; Neovascularization, Physiologic; Osteogenesis; Rabbits; Spectroscopy, Fourier Transform Infrared; Tissue Scaffolds; Tomography, X-Ray Computed; Vascular Endothelial Growth Factor A

Salvianolic acid B is one of the main water-soluble components of Salvia miltiorrhiza Bge. Many reports have shown that it has significant anti-myocardial ischemia effect. However, the underlying mechanism remains unclear. Our present study demonstrated that Sal B could alleviate myocardial ischemic injury by inhibiting the priming phase of NLRP3 inflammasome. In vivo, serum c-troponin I (cTn), lactate dehydrogenase (LDH) levels, the cardiac function and infract size were examined. We found that Sal B could notably reduce the myocardial ischemic injury caused by ligation of the left anterior descending coronary artery. In vitro, Sal B down-regulated the TLR4/NF-κB signaling cascades in lipopolysaccharide (LPS)-stimulated H9C2 cells. Furthermore, Sal B reduced the expression levels of IL-1β and NLRP3 inflammasome in a dose-dependent manner. In short, our study provided evidence that Sal B could attenuate myocardial ischemic injury via inhibition of TLR4/NF-κB/NLRP3 signaling pathway. And in an upstream level, MD-2 may be the potential target.

Topics: Animals; Benzofurans; Cell Line; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Lipopolysaccharides; Lymphocyte Antigen 96; Male; Models, Molecular; Myocardial Ischemia; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Signal Transduction; Toll-Like Receptor 4

Testing and screening of plant-derived molecules on normal human cells in vitro is a widely used approach for discovering their eventual health beneficial effects for human ageing and longevity. As little is known about age-associated differential effects of such molecules, here we report that young (<25% replicative lifespan completed) and near-senescent (>90% replicative lifespan completed) human skin fibroblasts exposed for 1-15 days to a wide range of concentrations (0.1-100 μM) of the three selected phytochemicals, namely α-boswellic acid acetate (ABC), praeruptorin-A (PTA), and salvianolic acid-B (SAB) had age-related differential effects. The parameters studied were the metabolic activity (MTT assay), cellular morphological phenotype, one-step growth characteristics, expression of genes involved in the cell cycle regulation and cytokine network genes, protein levels of p53, cytosolic superoxide dismutase (SOD1) and microtubule-associated protein 1A/1B-light chain 3 (LC3), and the extent of protein carbonylation and protein aggregation as a sign of oxidative stress. All three compounds showed biphasic hormetic dose response by stimulating cell growth, survival and metabolic activity at low doses (up to 1 μM), while showing inhibitory effects at high doses (>10 μM). Furthermore, the response of early passage young cells was different from that of the late passage near-senescent cells, especially with respect to the expression of cell cycle-related and inflammation-related genes. Such studies have importance with respect to the use of low doses of such molecules as health-promoting and/or ageing-interventions through the phenomenon of hormesis.

Topics: Autophagy; Benzofurans; Cell Cycle; Cells, Cultured; Coumarins; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Humans; Molecular Structure; Oxidative Stress; Phytochemicals; Protein Aggregates; Triterpenes

Spinal disorders often require surgical treatment called spinal fusion to restore a stabilized spine where bone grafts are implanted for the fusion of adjacent vertebras. In this study, we developed a bioactive composite scaffold incorporated with salvianolic acid B (SB), an active component extracted from Danshen. This study aimed to evaluate the effects of SB-incorporated porous scaffold on spinal fusion models. The composite scaffolds composed of poly (lactic-co-glycolic acid) and tricalcium phosphate (PLGA/β-TCP) were fabricated with low-temperature rapid prototyping technique, which incorporated SB at low (SB-L), middle (SB-M), high (SB-H) doses, and pure PLGA/β-TCP as blank control (Con). The release profile of SB from the scaffolds was determined by high performance liquid chromatography. Osteoconductive and osteoinductive properties of the scaffolds were reflected by the osteogenic differentiation ability of rat primary mesenchymal stem cells. The angiogenesis was determined by the forming of tube-like structures resembling capillaries using endothelial cell line (EA hy9.26). A well-established spinal fusion model was used to evaluate the in vivo bony fusion. Animals were transplanted with scaffolds, or autografts from iliac crest as positive controls. Micro-computed tomography (CT) analysis, CT-based angiography, manual palpation test, histomorphometry, and histology were performed after 8 weeks of transplantation. Results revealed that incorporated SB was steadily released from the scaffolds. The aliquot of released SB promoted osteogenesis and angiogenesis in vitro in a dose-dependent manner. In animal study, a dose-dependent effect of SB on new bone formation, mineral apposition rate, and vessel density within the scaffold were demonstrated. Manual palpation test showed little numerical improvement in fusion rate when compared with the blank controls. In summary, our results suggested that SB-incorporated PLGA/β-TCP composite scaffold could enhance bony fusion through the promotion of osteogenesis and angiogenesis.

Topics: Animals; Benzofurans; Biocompatible Materials; Calcium Phosphates; Cell Differentiation; Cell Line; Cell Movement; Culture Media, Conditioned; Disease Models, Animal; Lumbar Vertebrae; Male; Mesenchymal Stem Cells; Neovascularization, Physiologic; Osteogenesis; Polyglycolic Acid; Rats, Sprague-Dawley; Spinal Fusion; Tissue Scaffolds; Wound Healing; X-Ray Microtomography

Salvianolic acid B (SAB) has a high concentration in the liver, but the mechanism of its distribution in the liver is unclear. The aim of this study was to investigate the mechanisms of hepatic uptake of SAB. In this study, we first explored the uptake features of SAB in HepG2 cells and the effect of rifampicin on uptake. Then, we explored the effects of SAB on the uptake of pitavastatin in HepG2 cells. Finally, we established an HEK239T-OATP1B1 cell model to confirm whether OATP1B1 mediated the transport of SAB. Results showed that the uptake kinetic parameters Vmax and Km for SAB in HepG2 cells were 21.28 ± 2.06 pmol mg

Topics: Benzofurans; Biological Transport; Humans; Liver; Organic Anion Transporters, Sodium-Independent; Solute Carrier Organic Anion Transporter Family Member 1B3

Salviae miltiorrliza-borneol Jun-Shi coupled-herbs have been widely used for treatment of ischemia stroke. Salvianolic acid B was the most abundant and bioactive compound of Salviae miltiorrliza and used for prevention and treatment of cerebrovascular diseases. However, the scientific intension and compatible mechanism of Salvianolic acid B - borneol combination were still unknown. A metabolomics study approach based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) combined with a pathological study has been applied to study the metabolic disturbances of cerebral ischemia and evaluate the efficacies of Sal B and Sal B/borneol against cerebral ischemia in middle cerebral artery occlusion (MCAO) rats. The neuroprotection of Sal B and Sal B/borneol was reversed through the evaluation of neurological deficits, infarct volume, and neuronal apoptosis in MCAO model. The metabonomic analysis revealed that the MCAO-induced cerebral ischemia could be ameliorated by Sal B through improving the energy metabolism, lipids metabolism, inflammatory responses, and oxidant stress. Borneol could enhance the neuroprotective effects, was associated with the increased concentration of Sal B, and attenuate the function of sphingolipid metabolism pathway in cerebral ischemia rats. These findings perhaps clarify the mechanism of neuroprotective effects of treating ischemia stroke by Sal B or Sal B/borneol preliminarily through metabolomics and push the quality promotion and the composition of borneol/Sal B in secondary development of prescription.

Topics: Animals; Benzofurans; Brain Ischemia; Camphanes; Chromatography, High Pressure Liquid; Male; Malondialdehyde; Mass Spectrometry; Metabolic Networks and Pathways; Metabolome; Metabolomics; Multivariate Analysis; Neuroprotection; Oxidative Stress; Principal Component Analysis; Rats, Sprague-Dawley

Systemic sclerosis (SSc) is a connective tissue disease characterized mainly by fibrosis of skin and internal organs. Our previous study has shown that salvianolic acid B (SAB), a bioactive component extracted from Salvia miltiorrhiza (SM), was one of the essential ingredients in the traditional Chinese medicine Yiqihuoxue formula, which has been used to treat SSc-related dermal and pulmonary fibrosis. The aim of the present study was to evaluate the effect of SAB on skin fibrosis and explore its underlying anti-fibrotic mechanism. We found that SAB was capable of alleviating skin fibrosis in a bleomycin-induced SSc mouse model, alleviating skin thickness and reducing collagen deposition. in vitro studies indicated that SAB reduced SSc skin fibroblast proliferation and downregulated extracellular matrix gene transcription and collagen protein expression. TGF-β/SMAD and MAPK/ERK pathway activation were also shown to be suppressed in SAB treated fibroblasts. Moreover, RNA-seq revealed that the anti-fibrotic effect of SAB might be related to antioxidant activity, the cell cycle, and the p53 signaling pathway. Taken together, our results suggest that SAB has the ability to alleviate SSc-related skin fibrosis both in vivo and in vitro.

Topics: Animals; Benzofurans; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Female; Fibroblasts; Fibrosis; Mice; Mice, Inbred C57BL; Random Allocation; Scleroderma, Systemic; Skin

Salvianolic acid B (SB) is an antioxidant derived from Salvia militarize, and is one of the most widely used herbs in traditional Chinese medicine. SB is a potent antioxidant that has been well documented as a scavenger of oxygen free radicals, and has been used for the prevention and treatment of atherosclerosis‑associated disorders. To explore its potential therapeutic effects in treating radiation damage, in this study, mice were treated with SB at different doses of 5, 12.5 and 20 mg/kg, subsequent to receiving γ‑irradiation. The effects of SB on peripheral blood, bone marrow nucleated cells, spleen and thymus indices, and oxidation resistance were evaluated in both radiated mice and control groups. The results indicated that SB significantly increased the counts of peripheral white blood cells, red blood cells and platelets. The number of nucleated cells in the bone marrow and the level of protein increased as well. In addition, improved spleen and thymus indices in the bone marrow were observed. SB treatment additionally reversed the deterioration of both the thymus and spleen indices, which is associated with increased serum superoxide dismutase activity and decreasing malondialdehyde levels via nuclear factor (erythroid‑derived 2)‑like 2 protein/BTB and CNC homology 1 mediated antioxidant effect. Furthermore, ROS levels and Bax protein expression were also suppressed by SB. The data suggested that SB is effective in protecting mice from γ‑radiation injury, and could potentially be applicable for clinical use. Notably, the present study identified a promising candidate drug for enhancing the hematopoietic and immune systems.

Topics: Animals; Antioxidants; Basic-Leucine Zipper Transcription Factors; Benzofurans; Female; Gamma Rays; Male; Malondialdehyde; Mice; NF-E2-Related Factor 2; Protective Agents; Reactive Oxygen Species; Salvia

The aim of this study was to evaluate the neuroprotective effects of SalB on high glucose (HG)-induced excessive autophagy and apoptosis in vitro.. The proliferation and apoptosis of RSC96 cells were determined using the MTT assay and flow cytometry, respectively. Western blot analysis was performed to examine the expression of autophagy and apoptosis-related proteins. RT-PCR and flow cytometry were manipulated to examine the level of Bcl-2. The signals of autophagy markers were detected using immunofluorescence methods.. We found that HG significantly reduced RSC96 cell's proliferation and induced apoptosis. What's more, HG increased the level of autophagy and apoptosis-related proteins. However, these effects were reversed by SalB. In addition, we also found that 3-MA decreased the expression of LC3A/B and Beclin1, while the JNK inhibitor SP600125 reduced the levels of phosphorylated JNK, LC3A/B and Beclin1.. High glucose not only induced apoptosis but also caused autophagic cell death by activating the JNK pathway. These effects prevented by SalB in an opposite manner.

Topics: Animals; Anthracenes; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Benzofurans; Cell Line; Cell Proliferation; Diabetic Neuropathies; Glucose; MAP Kinase Signaling System; Peripheral Nervous System Diseases; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Rats; Signal Transduction

Danshen (Salvia miltiorrhiza, DS) and Honghua (Carthamus tinctorius, HH) are commonly used traditional Chinese medicines for activating blood and removing stasis, and DS-HH (DH) herbal pair had potential synergistic effects on promoting blood circulation. Therefore, it is essential to make clear the active components of this herbal pair for better understanding their potential synergistic effects.. To comprehensively evaluate the activity of DH herbal pair on physiological coagulation system of rats, and seek their potential active components by spectrum-effect relationship analysis.. The water extracts of DH herbal pair with different proportions (DS: HH = 1:1, 2:1, 3:1, 5:1, 1:5 and 1:3) were prepared. Male Sprague-Dawley rats were randomly divided into eight groups: blank group, model group, model + 1:1 (DH) group, model + 2:1 group, model + 3:1 group, model + 5:1 group, model + 1:5 group and model + 1:3 group. The intragastric administration was performed for eight times with 12 h intervals. SC40 semi-automatic coagulation analyzer was employed to determine coagulation indices. Meanwhile, HPLC and LC-MS were applied for chemical analyses of DH extracts. Finally, the active ingredients were screened by spectrum-effect relationship analysis and the activities of major predicted compounds were validated in vitro.. Different proportions of DH extracts could significantly prolong thrombin time (TT) and activated partial thromboplastin time (APTT), increase prothrombin time (PT) and decrease fibrinogen (FIB) content, reduced whole blood viscosity (WBV) and plasma viscosity (PV), decreased erythrocyte sedimentation rate blood (ESR) compared with model group. Furthermore, fifteen highly related components were screened out by the spectrum-effect relationship and LC-MS analysis, of which caffeic acid, salvianolic acid B, hydroxysafflor yellow A and lithospermate acid had significant blood-activing effect by prolong APTT and decrease FIB content at high (0.6 mM), medium (0.3 mM) and low (0.15 mM) (except lithospermate acid) concentrations in vitro.. DH herbal pair showed strong blood-activating effect on blood stasis rat through regulating the parameters involved in haemorheology and plasma coagulation system. Four active compounds, caffeic acid, salvianolic acid B, hydroxysafflor yellow A and lithospermate acid predicted by spectrum-effect relationship analysis had good blood-activating effect. Therefore, spectrum-effect relationship analysis is an effective approach for seeking active components in herbal pairs.

Topics: Animals; Benzofurans; Blood Coagulation; Blood Sedimentation; Caffeic Acids; Carthamus tinctorius; Chalcone; Chromatography, High Pressure Liquid; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Fibrinogen; Hemorheology; Male; Prothrombin Time; Quinones; Rats, Sprague-Dawley; Salvia miltiorrhiza

The aim of this study was to investigate the role of oxidative stress in cystine crystal formation and whether salvianolic acid B, a natural antioxidant, could prevent cystine-mediated oxidative injury in vivo and in vitro. The levels of oxidative stress and antioxidase activity in cystine stone patients were assessed. Then, the oxidative stress exerted by cystine on human kidney-2 (HK-2) cell viability and biochemical parameters including antioxidase activity and antioxidant protein expression were evaluated, and the protective action of salvianolic acid B was also examined. Finally, salvianolic acid B was tested to determine whether it could prevent or reduce renal crystal formation in Slc7a9 knockout mice. The activity levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were decreased, and the amount of malondialdehyde (MDA) was increased in patients with cystine stones compared with people without cystine stones (p < 0.05). Significant reductions in cell viability, antioxidase activity and antioxidant protein expression levels were found in the cystine group compared with controls. However, such oxidative injuries were prevented by salvianolic acid B. In the animal study, loose crystals with white spots were seen in the renal parenchyma, bilateral renal pelvis and bladders in the Slc7a9 knockout group. In contrast, no renal crystals were seen in the control group, and markedly fewer crystals with significantly higher antioxidase activity and diminished oxidative stress were detected in the salvianolic acid B group. Cystine cytotoxicity in vitro and cystine stone formation in vivo were associated with oxidative stress, and salvianolic acid B could protect against cystine stone-induced injury.

Topics: Animals; Benzofurans; Cells, Cultured; Cystine; Humans; Kidney Calculi; Male; Mice; Mice, Knockout; Oxidative Stress

Acute graft-versus-host disease (aGvHD) remains lethal, even after allogeneic hematopoietic stem cell transplantation. Inflammatory responses play an important role in aGvHD. Salvianolic acid B (Sal B) has been widely reported to have a major effect on the anti-inflammatory response, but these effects in an aGvHD model have never been reported. B6 donor splenocytes were transplanted into unirradiated BDF1 recipients and liver and serum were collected on day 14 after transplantation with or without Sal B administration. We measured the expression of pro-inflammatory cytokines and chemokines and other manifestations in aGvHD mice after Sal B treatment. Sal B ameliorated liver injury in aGvHD and promoted survival in mice. Sal B treatment resulted in decreased expression of pro-inflammatory cytokines and chemokines whose expressions in liver are normally elevated by aGvHD. Furthermore, Sal B treatment also enhanced PGC-1α expression in liver tissue and HO-1 expression in nonparenchymal cells. In addition, HO-1 inhibitor abrogated the improvement of survival rate of mice with aGvHD. These results indicated that the protective effect of Sal B relies on suppressing the inflammatory response phase in the aGvHD model, presumably by inducing HO-1. Taken together our data showed that Sal B ameliorates liver injury in aGvHD by decreasing inflammatory responses via upregulation of HO-1. It may provide a novel way to deal with this disease.

Topics: Acute Disease; Animals; Benzofurans; Disease Models, Animal; Gene Expression Regulation, Enzymologic; Graft vs Host Disease; Heme Oxygenase-1; Inflammation; Liver; Liver Diseases; Male; Membrane Proteins; Mice; Up-Regulation

Salvianolic acid B (Sal B), a major bioactive component of Chinese herb, was identified as a mediator for bone metabolism recently. The aim of this study is to investigate the underlying mechanisms by which Sal B regulates osteogenesis and adipogenesis. We used MC3T3-E1 and 3T3-L1 as the study model to explore the changes of cell differentiation induced by Sal B. The results indicated that Sal B at different concentrations had no obvious toxicity effects on cell proliferation during differentiation. Furthermore, Sal B facilitated osteogenesis but inhibited adipogenesis by increasing the expression of transcriptional co-activator with PDZ-binding motif (TAZ). Accordingly, TAZ knock-down offset the effects of Sal B on cell differentiation into osteoblasts or adipocytes. Notably, the Sal B induced up-expression of TAZ was blocked by U0126 (the MEK-ERK inhibitor), rather than LY294002 (the PI3K-Akt inhibitor). Moreover, Sal B increased the p-ERK/ERK ratio to regulate the TAZ expression as well as the cell differentiation. In summary, this study suggests for the first time that Sal B targets TAZ to facilitate osteogenesis and reduce adipogenesis by activating MEK-ERK signalling pathway, which provides evidence for Sal B to be used as a potential therapeutic agent for the management of bone diseases.

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Amino Acid Motifs; Animals; Benzofurans; Cell Differentiation; Cell Line; Cell Proliferation; Drugs, Chinese Herbal; Gene Expression Regulation; MAP Kinase Signaling System; Mice; Osteoblasts; Osteogenesis; Transcription Factors

A preparation process of salvianolic acid B (SAB) disodium salt from Salvia miltiorrhiza Bunge (Danshen) is provided in this work. A water extract quality standard was also developed to estimate the influences of Danshen quality on SAB disodium salt quality at an early stage of the preparation process. Crude SAB solution was obtained after water extraction, concentration, acidification, 1-butanol extraction, water washing, basification, and water back extraction. Extraction temperature, extraction pH, and back-extraction pH were identified to be key parameters for the preparation of crude SAB solution. These parameters were optimized with Box⁻Behnken designed experiments. Crude SAB solution was further purified with a chromatography process. AMBERCHROW CG161M resin was selected as the best adsorbent. SAB disodium salt could be obtained by drying the eluate. Considering the quality of Danshen may affect the purity and yield of SAB disodium salt, different batches of Danshen were used to prepare SAB disodium salt with the optimized parameters. Water extract indices of phenolic compound purity and phenolic compound yield were measured. By developing models between SAB disodium salt purity and yield with water extract indices, the quality standard of Danshen water extract was obtained. The application of water extract quality standards can improve the quality consistency of SAB disodium salt. The effects of different batches of Danshen raw materials on the final product could be evaluated at the beginning of production stages. The present method could prepare about five grams of high-purity SAB disodium salt (>95%) in one preparation cycle. The method reported in this work can also be used to develop process intermediate quality standards for other natural products.

Topics: Benzofurans; Plant Extracts; Salvia miltiorrhiza; Sodium Chloride; Water

Hypertrophic scar( HS) is a very common skin fibrosis disorder after human skin injury and wound healing. The objective of this study was to investigate the efficacy of cell penetrating peptide TAT-modified liposomes loaded with salvianolic acid B( SAB-TAT-LIP) on proliferation,migration and cell cycle of human skin fibroblasts( HSF),and preliminarily evaluate its effect on prevention and treatment of HS. HSF were cultured in vitro,and MTT assay was used to detect the inhibitory effect of SAB-TAT-LIP on cell proliferation. Cell migration was assessed by Transwell chamber method and scratch method; and cell cycle change was detected by flow cytometry. In vitro cell studies showed that blank liposome basically had no toxic effect on HSF. Different concentrations of SABTAT-LIP inhibited proliferation on HSF in varying degrees after intervention for different periods in a dose and time dependent manner;meanwhile,SAB-TAT-LIP significantly inhibited the migration and invasion of HSF. At the same time,SAB-TAT-LIP could block the cell cycle at G0/G1 phase after intervention for 48 h,P<0.01 as compared with the blank control group. Conclusively,our experimental data quantitatively demonstrate that SAB-TAT-LIP has significant inhibitory effect on cells proliferation,invasion and migration,with blocking effect on G0/G1 phase. This may offer a promising therapeutic strategy for transdermal delivery in prevention and treatment of HS.

Topics: Benzofurans; Cell Cycle; Cell Movement; Cell Proliferation; Cell-Penetrating Peptides; Cells, Cultured; Drug Carriers; Fibroblasts; Humans; Liposomes; Skin

Danshen is a first-line traditional Chinese medicine derived from Salvia miltiorrhiza Bunge consisting mainly of tanshinone IIA, tanshinol, protocatechuic aldehyde, and salvianolic acid B, it is widely used to treat cardiovascular diseases based on the synergistic effect of its multiple active components. Recent studies have indicated that the overall effect of traditional Chinese medicine is closely related to the in vivo coexistence of a variety of active components.. The prolongation of the coexistence of the four active components in Danshen in vivo by regulating their pharmacokinetic processes may contribute to better efficiency.. Individual sustained-release pellets of the four main active components in Danshen were respectively prepared according to the optimised formulations developed in our previous studies to modulate their in vivo processes, in which the desired release profiles of each kind of sustained-release pellets for formulation optimisation were calculated based on the point-area deconvolution and circadian rhythm of variant angina. The four kinds of sustained-release pellets were filled into capsules on the basis of the original weight ratio of the four active components in purified Salvia miltiorrhiza extract for further in vitro release and pharmacokinetic and pharmacodynamic investigations.. The release behaviours of the combined Danshen capsules composed of the four kinds of sustained-release pellets were evaluated in three media with different pH levels (pH 1.2, 6.8, and pure water). The release profiles of each kind of sustained-release pellets in pH 6.8 PBS and pH 1.2 HCl were similar to the release profile of those in pure water (similarity factors f. Sustained-release preparations can markedly prolong the in vivo coexistence of multiple components in Danshen to enhance their overall effects, which provides a potent strategy for developing the combination therapy of traditional Chinese medicine.

Topics: Abietanes; Benzaldehydes; Benzofurans; Caffeic Acids; Capsules; Cardiovascular Diseases; Catechols; Delayed-Action Preparations; Drugs, Chinese Herbal; Humans; Hydrogen-Ion Concentration; Medicine, Chinese Traditional; Salvia miltiorrhiza

Salvianolic acid B (Sal B), a water-soluble compound extracted from Salvia miltiorrhiza that has been widely used to treat cardiovascular diseases for hundreds of years in China, exerts cardiovascular protection by multiple mechanisms. miR-146a is involved in vascular smooth muscle cell (VSMC) phenotypic modulation and proliferation. However, it has yet to be investigated whether the cardiovascular protective effect of Sal B is mediated by miR-146a.. To determine the relationship among the cardiovascular protective effect of Sal B, miR-146a expression, and VSMC proliferation.. MTS assay and cell counting were performed to evaluate the effect of Ang II, Sal B and miR-146a on VSMC proliferation. The neointima hyperplasia was assessed by hematoxylin/eosin staining. qRT-PCR was used to detect the expression of miR-146a, KLF5, cyclin D1 and PCNA. Western blot analysis was used to detect the expressions of KLF5, cyclin D1 and PCNA after miR-20b-5p was knocked down or overexpressed in VSMC.. Sal B suppressed intimal hyperplasia induced by carotid artery ligation and decreased Ang II-induced VSMC proliferation by down-regulating the positive cell-cycle regulators KLF5 and cyclin D1. Further experiments showed that VSMC proliferation and upregulation of KLF5 and cyclin D1 induced by Ang II were accompanied by elevated miR-146a level. Furthermore, overexpression of miR-146a promoted and knockdown of miR-146a reduced Ang II-induced VSMC proliferation and ameliorated intimal hyperplasia induced by carotid artery ligation. Sal B inhibited Ang II-induced VSMC proliferation by suppressing miR-146a expression.. Sal B inhibited Ang II-induced VSMC proliferation in vitro and intimal hyperplasia in vivo by downregulating miR-146a expression.

Topics: Angiotensin II; Animals; Benzofurans; Carotid Arteries; Cell Proliferation; Cells, Cultured; Down-Regulation; Gene Expression Regulation; Hyperplasia; Kruppel-Like Transcription Factors; Male; Mice, Inbred C57BL; MicroRNAs; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Tunica Intima

A sensitive and accurate LC-MS/MS method was established for quantifying salvianolic acid B (Sal B), rosmarinic acid (Ros A) and Danshensu (DA) in rat plasma. Salvia miltiorrhiza polyphenolic acid (SMPA), active water-soluble ingredients isolated and purified from Salvia miltiorrhiza Bge included Sal B, Ros A and DA. The pharmacokinetic analysis of Sal B, Ros A and DA after pulmonary administration of SMPA solution to rat was performed by LC-MS/MS. Results from the pharmacokinetic studies showed that the peak concentration of DA was 21.85 ± 6.43 and 65.39 ± 3.83 ng/mL after pulmonary and intravenous administration, respectively. DA was not detected at 2 h after administration. The absolute bioavailabilities of Sal B and Ros A were respectively 50.37 ± 27.04 and 89.63 ± 12.16% after pulmonary administration of 10 mg/kg SMPA solution in rats. The absolute bioavailability of Sal B increased at least 10-fold after pulmonary administration, compared with oral administration. It was concluded that the newly established LC-MS/MS method was suitable for describing the pharmacokinetic characteristics of Sal B, Ros A and DA in rat after pulmonary administration of SMPA solution. The data from this study will provide a preclinical insight into the feasibility of pulmonary administration of SMPA.

Topics: Administration, Inhalation; Animals; Benzofurans; Biological Availability; Chromatography, Liquid; Cinnamates; Depsides; Drug Stability; Drugs, Chinese Herbal; Lactates; Limit of Detection; Linear Models; Male; Polyphenols; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Rosmarinic Acid; Salvia miltiorrhiza; Tandem Mass Spectrometry

Rheumatoid arthritis (RA) is a common inflammatory disease, which significantly reduces the quality of life and increases the risk of cardiovascular and cerebrovascular diseases. The present work studied the therapeutic potency of Salvianolic acid B (Sal-B) for RA and revealed one of the possible underlying mechanisms.. Human rheumatoid fibroblast-like synoviocytes (MH7 A) were treated with Sal-B before, during or after lipopolysaccharide (LPS) stimulation. CCK-8 assay, Annexin V-FITC/PI double-staining, RT-qPCR, Western blotting and ELISA were carried out to measure the changes of cell viability, apoptosis, and the release of pro-inflammatory cytokines. Next, the involvement of miR-142-3p and related signaling pathways in Sal-B-mediated protection was studied.. Sal-B (10 μM) treatment significantly ameliorated LPS injury to MH7 A cells, as cell viability was increased, expression of p53 and p21 was repressed, apoptosis was inhibited, and the release of MCP-1, IL-6 and TNF-α was reduced. However, Sal-B (10 μM) treated alone has no impacts on MH7 A cells in the abovementioned aspects. miR-142-3p was down-regulated by LPS stimulation, while was up-regulated by treatment of Sal-B. Rescue assay results showed that Sal-B did not remit LPS injury when miR-142-3p was silenced. And also, the inhibitory effects of Sal-B on NF-κB and JNK pathways were abolished by miR-142-3p silence.. Sal-B could protect against and reverse LPS-induced injury in MH7 A cells, showing anti-apoptotic and anti-inflammatory capacities. The anti-RA potential of Sal-B might be via up-regulating miR-142-3p, and subsequently modulating NF-κB and JNK pathways.

Topics: Benzofurans; Caspase 3; Caspase 9; Cell Line; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cytokines; Cytoprotection; Gene Expression Regulation; Humans; Lipopolysaccharides; MicroRNAs; Molecular Structure; RNA, Messenger; Synoviocytes; Tumor Suppressor Protein p53; Up-Regulation

Salvia miltiorrhiza Bunge is a traditional Chinese medicine, and its water-soluble phenolic acid active compounds have very important medicinal value; however, the synthesis pathways of the main active ingredients remain unknown. Here, we employed nuclear magnetic resonance (NMR)-based metabolomics and transcriptomics techniques to study the biosynthesis mechanism of salvianolic acids. High-performance liquid chromatography (HPLC) combined with NMR showed an improvement over traditional techniques, and 54 metabolites were detected. The results of the multivariate statistical analysis showed that salvianolic acid B (SAB), rosmarinic acid (RA), caffeic acid, succinate, and citrate were among the multiple compounds that were increased in the methyl jasmonate (MeJA)-elicited group; the levels of sucrose, fructose, glutamine, and tyrosine were decreased. Combined with the differentially expressed genes (DEGs) found by transcriptome sequencing, we speculate that the synthesis of RA after MeJA treatment mostly occurred through caffeic acid and bypassed 4-hydroxyphenyllactic acid. This provides useful information for the study of salvianolic acids synthesis.

Topics: Acetates; Benzofurans; Biosynthetic Pathways; Cells, Cultured; Chromatography, High Pressure Liquid; Cinnamates; Cyclopentanes; Depsides; Gene Expression Profiling; Metabolomics; Oxylipins; Phenylpropionates; Plant Roots; Proton Magnetic Resonance Spectroscopy; Rosmarinic Acid; Salvia miltiorrhiza

The purpose of this study was to investigate the pharmacological effects of salvianolic acid B (SA-B) on α-naphthylisothiocyanate (ANIT)-induced cholestatic liver injury with the focus on bile acid homeostasis and anti-inflammatory pathways. Rats were randomly assigned into four groups. The control group was given normal saline (i.p.) for 7 consecutive days and on the 5th day was given the vehicle (i.g.). Model group was treated with normal saline (i.p.) for 7 days and administrated with ANIT (75 mg/kg, i.g.) on the 5th day. The SA-B groups were treated with SA-B (15 mg/kg and 30 mg/kg, i.p.) for 7 consecutive days as well as ANIT (75 mg/kg, i.g.) on the 5th day. We found that the serum levels of ALT, γ-GT, TBA, and other liver function indexes were found to be lower in the SA-B treatment groups than in the model group. SA-B also upregulated the transporters and enzymes involved in bile acid homeostasis such as Bsep, Oatp2, and Cyp3a2 in rats and BSEP, CYP3A4, and OATP2 in human cell lines. Moreover, SA-B suppressed NF-κB translocation into the nucleus, inhibited phosphorylation of p38 and JNK, and inhibited inflammation markers including IL-1β, IL-6, TGF-β, TNF-α, and COX-2 to extenuate cholestatic liver injury both in vivo and vitro. Taken together, our findings suggest that anti-cholestatic effects of SA-B may be associated with its ability to regulate NF-κB/IκB and MAPK inflammatory signaling pathways to inhibit inflammation and regulate transporters and enzymes to maintain bile acid homeostasis.

Topics: 1-Naphthylisothiocyanate; Animals; Benzofurans; Carrier Proteins; Cell Line; Cell Survival; Chemical and Drug Induced Liver Injury; Cholestasis; Cytokines; Humans; JNK Mitogen-Activated Protein Kinases; Liver; Male; Membrane Glycoproteins; NF-kappa B; NF-KappaB Inhibitor alpha; p38 Mitogen-Activated Protein Kinases; Protective Agents; Rats, Sprague-Dawley; Signal Transduction

Neuronal apoptosis is a regulated intrinsic cell mechanism and common pathological phenomenon after seizures, which involves the protein kinase B/cAMP response element binding protein / brain derived neurotrophic factor (AKT/CREB/ BDNF) signaling pathway. In this study, we aimed to identify the effects of salvianolic acid B (Sal B), a major water-soluble component of the Chinese herb, Danshen, on rats in which seizures had been induced by pentylenetetrazole (PTZ) and the underlying molecular mechanisms mediating these effects. For this, 60 adult male Sprague-Dawley rats were divided into a control group, a 'PTZ' group and a 'PTZ + Sal B' group. The animals in the control group received an intraperitoneal (i.p.) injection of saline on alternate days for a total of 15 injections and saline orally once a day for 29 days. The animals in the 'PTZ' group received PTZ (40 mg/kg, i.p.) on alternate days for a total of 15 injections and saline orally once a day for 29 days. Similarly, the animals in the 'PTZ + Sal B' group received PTZ (40 mg/kg, i.p.) on alternate days and Sal B (20 mg/kg) orally once a day for 29 days. Neural density was then evaluated using immunofluorescence (IF) staining of microtubule-associated protein 2 (MAP2). Neuronal apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. In addition, the expression of several proteins related to AKT/CREB/BDNF signaling was measured using Western blotting. The results indicated that more severe seizures, decreased neural density, decreased expression of Bcl-2, increased expression of Bax and cleaved caspase-3, and inactivation of AKT/CREB/BDNF signaling occurred in the 'PTZ' group in comparison with the control group. However, those changes were suppressed by Sal B. Thus, these data suggest that Sal B has anticonvulsant and anti-apoptotic effects in a PTZ-induced seizure model through activation of the AKT/CREB/BDNF signaling pathways.

Topics: Animals; Anticonvulsants; Apoptosis; Benzofurans; Brain-Derived Neurotrophic Factor; Cyclic AMP Response Element-Binding Protein; Drugs, Chinese Herbal; Male; Neurons; Pentylenetetrazole; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Seizures; Signal Transduction; Treatment Outcome

Salvianolic acid B (Sal B) is a water‑soluble active component of Danshen and has anti‑atherosclerotic effects. The present study aimed to evaluate the cytoprotective effects of Sal B against hydrogen peroxide (H2O2)‑induced oxidative stress damage in human umbilical vein endothelial cells (HUVECs) and investigate the underlying mechanisms. It was revealed that Sal B protected the cells from H2O2‑induced damage, as indicated by MTT results showing enhanced cell viability and by flow cytometric analysis showing reduced apoptosis of cells challenged with H2O2. Furthermore, as an underlying mechanism, the enhancement of autophagy was indicated to be accountable for the decrease in apoptosis, as Sal B caused the upregulation of light chain 3‑Ⅱ and Beclin‑1, and downregulation of p62 under H2O2‑induced oxidative stress. Finally, Sal B increased the phosphorylation of AMP kinase (AMPK) and decreased the phosphorylation of mammalian target of rapamycin (mTOR), but had no effect on the phosphorylation of AKT. In conclusion, the present study revealed that Sal B protects HUVECs from oxidative stress, at least partially by promoting autophagy via activation of the AMPK pathway and downregulation of the mTOR pathway.

Topics: Antioxidants; Apoptosis; Autophagy; Benzofurans; Cytoprotection; Drugs, Chinese Herbal; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Humans; Hydrogen Peroxide; Oxidative Stress; Salvia miltiorrhiza

The morphological feature of apoptosis is induced by oxygen and glucose deprivation (OGD) in cardiomyocytes H9c2 cells. Salvianolic acid B (Sal-B) has been studied in several pathological progresses, whereas it is still unclear whether maternally expressed gene 3 (MEG3) is an intermediate regulator during this progress. After pre-incubation with Sal-B and stimulation with OGD, viability and apoptosis of were examined in MEG3-overexpressed H9c2 cells. Cyclin D1, apoptosis-correlated proteins and regulators of signalling pathways were quantified with Western blot assay. MEG3 was detected by quantitative reverse transcription PCR (qRT-PCR). Sal-B was implicated in the enhancement of cell viability and suppression of apoptosis in OGD-treated H9c2 cells by repressing MEG3. In addition, MEG3 overexpression exerted an inhibitory effect on murine double minute 2 (MDM2) expression while aggrandized p53 expression in OGD-treated H9c2 cells which were pre-incubated with Sal-B. Furthermore, MEG3 overexpression abolished the up-regulative effect of Sal-B on phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in OGD-treated H9c2 cells. These results indicated that cardio-protective function of Sal-B might be ascribed to its down-regulatory property on MEG3 expression which hence blocks p53 and triggers AMPK activation in OGD-treated cells.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Benzofurans; Cardiotonic Agents; Carrier Proteins; Cell Line; Down-Regulation; Glucose; Mice; Myocytes, Cardiac; Oxygen; RNA, Long Noncoding; Signal Transduction; Tumor Suppressor Protein p53

This study was framed to investigate the molecular mechanism behind the anti-depressant effect of salvianolic acid B (SB) against unpredictable chronic mild stress (CMS) induced depression rat model. Control rats received only saline without CMS exposure, whereas CMS model rats were induced to several stress (CMS) for 6 weeks. Treatment group rats were induced with CMS for 6 weeks but received either 20 or 40 mg/kg of SB or 20 mg/kg imipramine (CMS+IMP) from the 4th week to 6th week. Treatment with SB or IMP significantly ameliorated body weight, sucrose consumption rate with shorter immobility time than the control group. Also, administration with SB or IMP could reverse the hyperactivity of hypothalamic-pituitary-adrenal axis as well as decreased inflammatory cytokines with improved antioxidant status. Furthermore, the protein expression of NLRP3 (inflammasome) was markedly downregulated upon treatment with SB (both 20 and 40 mg) or IMP and thereby confirming its potent anti-depressant activity. PRACTICAL APPLICATIONS: Salvianolic acid B (SB) is a phenolic acid extracted from Salvia militiorrhiza Bunge, a popular Chinese herb, which has been prescribed for various pathological conditions. SB has been previously reported with anti-depressant activity but, the in-depth mechanism behind the anti-depressant effect of SB against CMS is still elusive. Hence, the current study was plotted to explore the in-depth mechanism behind the anti-depressant effect of SB against CMS model of depression in rats. The outcome of the current study has confirmed the anti-depressant activity by abolishing oxidative stress, and neuroinflammatory response in the hippocampus through inhibiting NLRP3 inflammasome activation. Hence, SB can be prescribed to major depression patients with standard anti-depressant agents to abolish oxidative stress, neuro-inflammatory response, and related neurological changes.

Topics: Animals; Benzofurans; Cytokines; Depression; Drugs, Chinese Herbal; Humans; Hypothalamo-Hypophyseal System; Inflammasomes; Male; NLR Family, Pyrin Domain-Containing 3 Protein; Pituitary-Adrenal System; Rats; Salvia miltiorrhiza; Stress, Psychological

The study explored the isolation and characterisation of three compounds of high purity salvianolic acid B freeze-dried powder extracted from Salvia miltiorrhiza Bunge. A new salvianolic acid, salvianolic acid V (2) together with two known compounds (3-4) was identified. The antibacterial activity tests showed that compound 2 combined with clinical antibiotics such as Levofloxacin or Colistin sulphate together exhibited potent effects against MRSA or Acinetobacter baumanii. This report has considerably extended our knowledge about the diversity and bioactivity of caffeic acid derivatives from S. miltiorrhiza.

Topics: Acinetobacter baumannii; Alkenes; Anti-Bacterial Agents; Benzofurans; Caffeic Acids; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Freeze Drying; Methicillin-Resistant Staphylococcus aureus; Molecular Structure; Polyphenols; Powders; Salvia miltiorrhiza

Topics: Animals; Benzaldehydes; Benzofurans; Catechols; Chromatography, Liquid; Cyclooctanes; Drug Stability; Drugs, Chinese Herbal; Flavonoids; Lignans; Limit of Detection; Linear Models; Male; Polycyclic Compounds; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Tandem Mass Spectrometry

Salvianolic acid B (SalB) is one of the most bioactive components extracted from Salvia miltiorrhiza, and its antioxidant capacity corresponds with its protective effects against cell injury from oxidative stress. The aim of the present study was to evaluate the effect of SalB on experimental pulmonary fibrosis and its ability to ameliorate the oxidative/antioxidative imbalance during fibrosis pathogenesis. The anti-fibrotic activity of SalB was first confirmed in Transforming growth factor β1(TGF-β1)-stimulated MRC-5 cells. The protection of SalB against oxidative stress during fibrogenesis in vitro was verified by detecting ROS production, the levels of glutathione (GSH) and malondialdehyde (MDA). The Western blot and PCR results indicated that SalB could up-regulate nuclear factor erythroid-derived 2-like 2 (Nrf2) at both the protein and mRNA levels and induce Nrf2 nuclear translocation in vitro, which may be the mechanism underlying the anti-fibrotic capacity of SalB. Furthermore, the anti-fibrotic and antioxidant capacities of SalB in vivo were confirmed in rats with BLM-induced pulmonary fibrosis. The immunohistochemistry results showed that Nrf2 was absent in fibroblastic foci (FF) areas, while the SalB treatment could increase the expression of Nrf2 in lung tissues, especially in FF areas.

Topics: Animals; Benzofurans; Cell Line; Cell Transdifferentiation; Dose-Response Relationship, Drug; Humans; Male; Myofibroblasts; NF-E2-Related Factor 2; Pulmonary Fibrosis; Rats; Rats, Wistar; Reactive Oxygen Species; Up-Regulation

The purpose of this study was to evaluate the cerebral protection of salvianolic acid B (Sal B) against cerebral I/R injury and investigate the underlying mechanism. As shown by 2,3,5-Triphenyltetrazolium chloride (TTC) staining and magnetic resonance imaging (MRI) analyses, Sal B significantly reduced cerebral infarct size, and accompanied with improved neurobehavioral functions as indicated by the modified Bederson score and Longa five-point scale. Sal B decreased the production of reactive oxygen species (p < .05, n = 10). The data of Western blotting and reverse transcription quantitative real time polymerase chain reaction (qRT-PCR) analyses showed that the expression of GFAP, Iba1, IL-1β, IL-6, TNF-α and Cleaved-caspase 3 was significantly reduced by Sal B in I/R injured brain tissues as compared to corresponding controls (p < .05, n = 10). Over activation of astrocytes and microglia were inhibited by Sal B as shown by immunostaining of GFAP and Iba 1. These data suggest that Sal B has neural protective effects against I/R-induced cerebral injury and could be an effective candidate for further development of clinical therapy.

Topics: Animals; Benzofurans; Brain Injuries; Calcium-Binding Proteins; Caspase 3; Cerebral Infarction; Cytokines; Disease Models, Animal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Infarction, Middle Cerebral Artery; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Neuroprotective Agents; Reperfusion

Hepatocytes differentiated from human embryonic stem cells (ESCs) could provide a powerful tool for enabling cell-based therapies, studying the mechanisms underlying human liver development and disease, and testing the efficacy and safety of pharmaceuticals. However, currently most in vitro protocols yield hepatocytes with low levels of liver function. In this study, we investigated the potential of Salvianolic acid B (Sal B), an active pharmaceutical compound present in Salvia miltiorrhiza, which has been shown to have an antifibrotic effect in previous studies, to enhance hepatocyte differentiation from human ESCs. After treatment with Sal B, albumin expression and secretion were consistently increased, indicating that Sal B could promote hepatocyte differentiation process. Expression of a large number of important phase 1 and 2 metabolizing enzymes and phase 3 transporters was also increased in treated cells, indicating an enhanced biotransformation function. Our investigations further revealed the activation of Wnt pathway in treated cells, as determined by upregulation of Wnts, which increased amounts of nuclear β-catenin. This increased nuclear β-catenin led in turn to the enhanced expression of T cell factor (TCF) 3 and lymphoid enhancer-binding factor (LEF) 1 which upregulated their downstream targets, cyclin D1 and c-Myc. Notch receptors (Notch1, Notch3), Notch ligand (Jagged2), and Notch receptor targets [hairy and enhancer of split (Hes) 1, 5] were downregulated in treated cells, suggesting that Notch pathway was inhibited. Consistent with the inhibition of Notch pathway, expression of cholangiocyte marker, CK7, was significantly reduced by treatment with Sal B. Numb, a direct transcriptional target of Wnt pathway and a negative regulator of Notch pathway, was upregulated, consistent with activation of Wnt signaling and suppression of Notch signaling. In conclusion, our study demonstrated that Sal B enhanced hepatocyte differentiation from human ESCs through activation of Wnt pathway and inhibition of Notch pathway. Therefore, this study suggests that Sal B can be used as a potential agent to generate more mature hepatocytes for cell-based therapeutics and pharmaceutical studies.

Topics: Animals; Benzofurans; Cell Differentiation; Cells, Cultured; Hepatocytes; Human Embryonic Stem Cells; Humans; Liver; Receptors, Notch; Signal Transduction; Up-Regulation; Wnt Signaling Pathway

Salvianolic acid B is one of the key water‑soluble components of Salvia extract. It has been verified that salvianolic acid B possesses multiple pharmacological activities, it protects against myocardial infarction, however additionally improves injury of myocardial ischemia‑reperfusion. The present study, the possible effects of salvianolic acid B on cognitive deficits and angiogenesis in cerebral small vessel disease were investigated. Salvianolic acid B was identified to recover cognitive deficits and neurocytes, reduce inflammation, oxidative stress and neurocyte apoptosis (caspase‑3 and Bax protein expression) in cerebral small vessel disease rats. In addition, salvianolic acid B upregulated signal transducer and activator of transcription 3 (STAT3) phosphorylation protein expression, and induced vascular endothelial growth factor (VEGF) and VEGF receptor 2 protein expression in cerebral small vessel disease rats. In conclusion, the results demonstrated that salvianolic acid B recovers cognitive deficits and angiogenesis in the cerebral small vessel disease rat model via STAT3/VEGF signaling pathway.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Behavior, Animal; Benzofurans; Caspase 3; Cerebral Small Vessel Diseases; Cognitive Dysfunction; Cytokines; Disease Models, Animal; Glutathione; Male; Neovascularization, Physiologic; Oxidative Stress; Rats; Rats, Sprague-Dawley; Signal Transduction; STAT3 Transcription Factor; Superoxide Dismutase; Vascular Endothelial Growth Factor A

Salvianolic acid B (SalB) is one of the main water-soluble composites from Chinese medicine Dansen (Radix miltiorrhiza). It is used for clinical treatment of various diseases including cardiovascular, lung, Liver, renal and cancers. However, the effects of SalB to allergy induced airway mucin hypersecretion, inflammation and hyperresponsiveness (AHR) remains not clear. Overproduction of airway MUC5AC is a central effector of inflammation that is strongly associated with AHR in asthmatic attack. In this study, we investigated the anti-asthmatic activity and mechanism of SalB in a murine model and human epithelial cells by monitoring changes in mucin expression and secretion, airway inflammation, AHR, and signaling pathways. SalB was administered by intragastric administration (i.g) daily for a week, starting at 21 days after sensitization of ovalbumin (OVA). All examinations were performed 24h after the last antigen challenge. We found that treatments with SalB significantly inhibited increase in the tracheobronchial secretion, glycosaminoglycan levels, interleukin (IL)-13, IL-4, and IL-5 cytokines mRNA and protein expression, and decrease in mucociliary clearance in lung tissues. Histological results demonstrated that SalB attenuated OVA-induced eosinophil infiltration, airway goblet cell hyperplasia, and MUC5AC and MUC5B mRNA and protein expression in lung tissues. SalB exhibited protective effects against AHR in OVA-challenged animals. In vitro, SalB significantly inhibited IL-13-induced MUC5AC and MUC5B mRNA and protein expression in human epithelial cells. These effects were blocked by SalB by downregulating the Erk1/2 and P38 signaling pathways. Taken together, these data indicate that treatment with SalB may improve AHR by inhibiting MUC5AC overproduction.

Topics: Animals; Anti-Asthmatic Agents; Asthma; Benzofurans; Bronchi; Cell Line; Female; Gene Expression Regulation; Glycosaminoglycans; Humans; Interleukin-13; Lung; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mucin 5AC; p38 Mitogen-Activated Protein Kinases; Respiratory Hypersensitivity; Trachea

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated) system is a powerful genome editing tool that has been used in many species. In this study, we focused on the phenolic acid metabolic pathway in the traditional Chinese medicinal herb Salvia miltiorrhiza, using the CRISPR/Cas9 system to edit the rosmarinic acid synthase gene (SmRAS) in the water-soluble phenolic acid biosynthetic pathway. The single guide RNA (sgRNA) was designed to precisely edit the most important SmRAS gene, which was selected from 11 family members through a bioinformatics analysis. The sequencing results showed that the genomes of 50% of the transgenic regenerated hairy roots had been successfully edited. Five biallelic mutants, two heterozygous mutants and one homozygous mutant were obtained from 16 independent transgenic hairy root lines when the sgRNA was driven by the Arabidopsis U6 promoter, while no mutants were obtained from 13 independent transgenic hairy root lines when the sgRNA was driven by the rice U3 promoter. Subsequently, expression and metabolomics analysis showed that the contents of phenolic acids, including rosmarinic acid (RA) and lithospermic acid B, and the RAS expression level were decreased in the successfully edited hairy root lines, particularly in the homozygous mutants. In addition, the level of the RA precursor 3,4-dihydroxyphenyllactic acid clearly increased. These results indicated that the CRISPR/Cas9 system can be utilized to identify important genes in a gene family with the assistance of bioinformatics analysis and that this new technology is an efficient and specific tool for genome editing in S. miltiorrhiza. This new system presents a promising potential method to regulate plant metabolic networks and improve the quality of traditional Chinese medicinal herbs.

Topics: Benzofurans; Cinnamates; CRISPR-Cas Systems; Depsides; Genome, Plant; Hydroxybenzoates; Lactates; Mutagenesis; Plants, Genetically Modified; RNA, Guide, Kinetoplastida; Rosmarinic Acid; Salvia miltiorrhiza

Salvianolic acid B (Sal B), a water-soluble component mainly extracted from the traditional Chinese medicine Salvia miltiorrhiza, has potential anti-inflammatory, anti-oxidative and anti-apoptotic actions to protect neural cells. Here, we explore the effects and mechanisms of Sal B on the promotion of differentiation of induced pluripotent stem cells (iPSCs) into neural stem cells (NSCs), then further into neurons. During the processes of neural differentiation of iPSCs, Sal B or a phosphatidylinositide 3 kinase (PI3K) inhibitor (LY294002) were added to the medium. Sal B substantially improved proliferation of iPSC-derived NSCs and neurons. Furthermore, Sal B significantly stimulated PI3K/AKT/GSK3 β/β-catenin pathway. However, LY294002 attenuated the Sal B-induced increase. Therefore, these outcomes suggest that Sal B markedly enhances neural differentiation of iPSCs via the PI3K/AKT/GSK3β/β-catenin pathway.

Topics: Animals; Apoptosis; Benzofurans; beta Catenin; Glycogen Synthase Kinase 3 beta; Induced Pluripotent Stem Cells; Mice; Neurogenesis; Neurons; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Orthosiphon aristatus (Blume) Miq. is a medicinal herb which is traditionally used for the treatment of diabetes and kidney diseases in South East Asia. Previous studies reported higher concentration of antioxidative phytochemicals, especially rosmarinic acid (ester of caffeic acid) and other caffeic acid derivatives in this plant extract than the other herbs such as rosemary and sage which are usually used as raw materials to produce rosmarinic acid supplement in the market.. The phytochemical profile of O. aristatus was investigated at different storage durations for quality comparison.. The phytochemicals were extracted from the leaves and stems of O. aristatus using a reflux reactor. The extracts were examined for total phenolic and flavonoid contents, as well as their antioxidant capacities, in terms of radical scavenging, metal chelating and reducing power. The phytochemical profiles were also analyzed by unsupervised principal component analysis and hierarchical cluster analysis, in relation to the factor of storage at 4 °C for 5 weeks.. The leaf extract was likely to have more phytochemicals than stem extract, particularly caffeic acid derivatives including glycosylated and alkylated caffeic acids. This explains higher ratio of total phenolic content to total flavonoid content with higher antioxidant capacities for the leaf extracts. Rosmarinic acid dimer and salvianolic acid B appeared to be the major constituents, possibly contributing to the previously reported pharmacological properties. However, the phytochemical profiles were found changing, even though the extracts were stored in the refrigerator (4 °C). The change was significantly observed at the fifth week based on the statistical pattern recognition technique.. O. aristatus could be a promising source of rosmarinic acid and its dimer, as well as salvianolic acid B with remarkably antioxidant properties. The phytochemical profile was at least stable for a month stored at 4 °C. It is likely to be a good choice of herbal tea with comparable radical scavenging activity, but lower caffeine content than other tea samples.

Topics: Antioxidants; Benzofurans; Caffeic Acids; Chromatography, Liquid; Cinnamates; Cluster Analysis; Depsides; Drug Evaluation, Preclinical; Drug Storage; Flavonoids; Orthosiphon; Phenols; Phytochemicals; Plant Extracts; Plant Leaves; Plant Stems; Plants, Medicinal; Rosmarinic Acid; Tandem Mass Spectrometry

Ischemic stroke (IS) is characterized by the sudden loss of blood circulation to an area of the brain, resulting in a corresponding loss of neurologic function. It has been a worldwide critical disease threatening to the health and life of human beings. Despite significant progresses achieved, effective treatment still remains a formidable challenge due to the complexity of the disease. Salvianolic acid B (Sal-B) and Puerarin (Pue) are two active neuroprotectants isolated from traditional Chinese herbs, Salvia miltiorrhiza and Kudzu root respectively, which have been used for the prevention and treatment of IS for thousands of years in China. The activities of two compounds against cerebral ischemia reperfusion injury have been confirmed via various pathways. However, the therapeutic efficacy of any of the two components is still unsatisfied. In the present study, the effect of the combination of Sal-B and Pue on IS was evaluated and validated in vitro and in vivo. The ratio of two compounds was firstly optimized based on the results of CoCl₂ damaged PC12 cells model. The co-administration exhibited significantly protective effect in CoCl₂ induced PC12 cells injury model by reducing ROS, inhibiting apoptosis and improving mitochondrial membrane potential in vitro. Moreover, Sal-B + Pue significantly relieved neurological deficit scores and infarct area than Sal-B or Pue alone in vivo. The results indicated that neuroprotection mechanism of Sal-B + Pue was related to TLR4/MyD88 and SIRT1 activation signaling pathway to achieve synergistic effect, due to the inhibition of NF-κB transcriptional activity and expression of pro-inflammatory cytokine (TNF-α, IL-1β, IL-6). In conclusion, the combination of Sal-B and Pue exerted much stronger neuroprotective effect than Sal-B or Pue alone, which provides a potential new drug and has great significance for the treatment of IS.

Topics: Animals; Apoptosis; Benzofurans; Brain Ischemia; Cerebrovascular Disorders; Cobalt; Drug Combinations; Drug Synergism; Gene Expression Regulation; Interleukin-1beta; Interleukin-6; Isoflavones; Middle Cerebral Artery; Myeloid Differentiation Factor 88; Neuroprotective Agents; NF-kappa B; PC12 Cells; Rats; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

The quality of Chinese medicine (CM) has being an active and challenging research area for CM. Prof. Chang-Xiao Liu et al first proposed the concept of quality marker (Q-Marker) for the quality evaluation and control on CM. This article describe the exploratory studies of Q-Marker in salvianolic acids for injection (SAI) based on this new concept.. This study was designed to screen Q-Marker of SAI and establish its quality control method based on the concept of CM Q-Marker.. Based on the concept of CM Q-Marker, the SAI was investigated for the identification of chemical components and their sources. The pharmacological effects on cerebral ischemia and reperfusion induced injury in rats were also investigated. Furthermore, the target cell extracts and pharmacokinetic studies were conducted to screen Q-Markers. Finally, the fingerprints and determination based on Q-Markers were established to assess the quality of SAI more effectively.. Overall, 20 constituents in SAI were identified. It was found that salvianolic acid B (SA-B), rosmarinic acid (RA), lithospermic acid (LA), salvianolic acid D (SA-D) and salvianolic acid Y (SA-Y) are major chemical components of SAI. Based on chemical components identifications, analysis of their sources, target cell extracts and pharmacokinetic studies, four phenolic acids, namely SA-B, RA, LA and SA-D, were screened and determined as effective Q-Markers of SAI.. This study demonstrated that the described method is a powerful approach for detecting Q-Markers, which can be used as control index for the quality assessment of CM.

Topics: Alkenes; Animals; Benzofurans; Biomarkers; Brain; Brain Ischemia; Cell Line; Cinnamates; Depsides; Drugs, Chinese Herbal; Endothelium, Vascular; Injections; Interleukin-1; Interleukin-6; Male; Polyphenols; Quality Control; Rats, Sprague-Dawley; Rosmarinic Acid; Superoxide Dismutase

Mitochondrial dysfunction is a featured pathology underlying synaptic injury and neuronal stress in Alzheimer's disease (AD). In recent years, the vicious cycle between mitochondrial deficits and intra-neuronal Redox state imbalance has received considerable attention. In this regard, it is of great interest to determine whether antioxidants could alleviate mitochondrial dysfunction in AD-related conditions. Salvianolic acid B (SalB), a bioactive component of alvia miltiorrhiza Bge, is a potent antioxidant. Here we have determined the protective effect of SalB against Aβ-induced mitochondrial abnormalities. Our results showed that the application of SalB substantially alleviated intra-neuronal glutathione (GSH) and lipid oxidation and suppressed excess mitochondrial superoxide generation in Aβ-insulted neurons. Moreover, SalB has demonstrated strong protection on mitochondrial bioenergetics against Aβ toxicity evidenced by preserved mitochondrial membrane potential and ATP production, as well as rescued enzymatic activities of cytochrome C oxidase and F1Fo ATP synthase. In addition, Aβ-induced axonal mitochondrial fragmentation and increased dynamin-like protein 1 phosphorylation at Ser 616 were substantially mitigated by SalB. Lastly, the application of SalB restored synaptic density in Aβ-exposed neurons. The most parsimonious interpretation of the results is that intra-neuronal oxidative stress promotes mitochondrial dysfunction in AD-relevant pathological settings, and SalB has the potential to be a promising agent for AD therapy.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Benzofurans; Cells, Cultured; Mice; Mitochondria; Neurons; Oxidative Stress; Protective Agents

Glia-mediated neuroinflammation is related to brain injury exacerbation after cerebral ischemia/reperfusion (I/R) injury. Astrocytic hemichannels or gap junctions, which were mainly formed by connexin-43, have been implicated in I/R damage. However, the exact roles of astrocytic hemichannels and gap junction in neuroinflammatory responses induced by I/R injury remain unknown.. Primary cultured astrocytes were subjected to OGD/R injury, an in vitro model of I/R injury. Salvianolic acid B (SalB) or carbenoxolone (CBX) were applied for those astrocytes. Besides, Cx43 mimetic peptides Gap19 or Gap26 were also applied during OGD/R injury; Cx43 protein levels were determined by western blot and cytoimmunofluorescene staining, hemichannel activities by Ethidium bromide uptake and ATP concentration detection, and gap junction intercellular communication (GJIC) permeability by parachute assay. Further, astrocyte-conditioned medium (ACM) was collected and incubated with microglia. Meanwhile, ATP or apyrase were applied to explore the role of ATP during OGD/R injury. Microglial activation, M1/M2 phenotypes, and M1/M2-related cytokines were detected. Also, microglia-conditioned medium (MEM) was collected and incubated with astrocytes to further investigate its influence on astrocytic hemichannel activity and GJIC permeability. Lastly, effects of ACM and MCM on neuronal viability were detected by flow cytometry.. We found that OGD/R induced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability. WB tests showed decreased astrocytic plasma membrane's Cx43, while showing an increase in cytoplasma. Treating OGD/R-injured microglia with ATP or OGD/R-ACM induced further microglial activation and secondary pro-inflammatory cytokine release, with the M1 phenotype predominating. Conversely, astrocytes incubated with OGD/R-MCM exhibited increased hemichannel opening but reduced GJIC coupling. Both SalB and CBX inhibited abnormal astrocytic hemichannel opening and ATP release and switched the activated microglial phenotype from M1 to M2, thus providing effective neuroprotection. Application of Gap19 or Gap26 showed similar results with CBX. We also found that OGD/R injury caused both plasma membrane p-Cx43(Ser265) and p-Src(Tyr416) significantly upregulated; application of SalB may be inhibiting Src kinase and attenuating Cx43 internalization. Meanwhile, CBX treatment induced obviously downregulation of p-Cx43(Ser368) and p-PKC(Ser729) protein levels in plasma membrane.. We propose a vicious cycle exists between astrocytic hemichannel and microglial activation after OGD/R injury, which would aggravate neuroinflammatory responses and neuronal damage. Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses; treatment differentially targeting astrocytic Cx43, hemichannels, and GJIC may provide novel avenues for therapeutics during cerebral I/R injury.

Topics: Animals; Animals, Newborn; Antigens, CD; Astrocytes; Benzofurans; Carbenoxolone; Cell Communication; Cell Hypoxia; Cell Polarity; Cells, Cultured; Connexin 43; Culture Media, Conditioned; Drugs, Chinese Herbal; Gap Junctions; Gene Expression Regulation; Glucose; Mice; Neurons; Oxygen; Quinolines

TLR4 signaling is known to be involved in podocyte injury. We have previously shown that Salvia przewalskii extract of total phenolic acids (SPE) and its active monomer salvianolic acid B (SalB) and rosmarinic acid (RA) protect podocytes from injury induced by PAN. In the present study, we test whether SPE inhibits TLR4 signaling.. The conditionally immortalized mouse podocytes were treated with SPE, SalB, RA, SalB + RA or tacrolimus for 30 min, followed by PAN (100 μg/mL) for 24 h. The F-actin staining with phalloidin was used to assess cytoskeletal injury in the podocytes. Western blotting and semi-quantitatives RT-PCR were used to assess the changes of the components in the TLR4 signaling pathway.. (1) The F-actin stress fibers of podocytes were almost completely disrupted after PAN treatment for 24 h, and the disruption was significantly alleviated by SPE; (2) the PAN-induced elevation of mRNA levels of TLR4, MyD88 and p65 were inhibited except p65 with high-dose SalB; (3) consistently, the protein levels of TLR4, MyD88 and pp65 were significantly elevated by PAN, and SPE, SalB, RA and admixture, respectively, attenuated the elevations of TLR4 and pp65 proteins; (4) SPE and tacrolimus have a similarly strong effect on inhibition of the expression of TLR4 signaling components.. SPE protects podocytes from PAN-induced injury at least partly through inhibiting TLR4 signaling. SPE is as strong as tacrolimus in inhibiting TLR4 signaling in podocytes.

Topics: Animals; Benzofurans; Cell Line; Cinnamates; Depsides; Drugs, Chinese Herbal; Mice; Podocytes; Puromycin Aminonucleoside; Rosmarinic Acid; Salvia; Signal Transduction; Toll-Like Receptor 4

BACKGROUND Salvianolic acid B (SB) is a major active phyto-component of the plant Radix Salvia miltiorrhiza, which is traditionally used to treat joint pain and arthritis. The present study examined the anti-rheumatoid arthritis efficacy of SB on collagen-induced rheumatoid arthritis (CIA) in a rat model. MATERIAL AND METHODS Forty-eight rats were divided into 4 groups: Control rats treated with saline (Group I), rats subjected to CIA induction by intradermal injection of bovine collagen II type at the tail (Group II), and rats subjected to CIA and supplemented with either 20 or 40 mg/kg of SB for 28 days (group III or IV). RESULTS Paw swelling, edema, arthritis score, thymus and spleen indexes, and neutrophil infiltration were significantly decreased (p<0.01) by treatment with 20 or 40 mg/kg of SB. The levels of inflammatory cytokines (interleukin-1β, -6, and -17, and TNF-α) and anti-collagen II-specific immunoglobulins (IgG1 and IgG2a) were markedly decreased (p<0.01), and those of antioxidant enzymes (SOD, CAT, and GSH) were significantly increased (p<0.01) in SB-treated rats. Administration with SB (20 or 40 mg/kg) resulted in lower phosphorylated IkB-a and NF-κB p65 protein levels and markedly downregulated IκB-a expression. Furthermore, CIA rats revealed the presence of highly diffused polymorphonuclear cells (PMNs) infiltration with eroded cartilage; however, these phenomena were considerably ameliorated by SB. CONCLUSIONS SB alleviates oxidative stress and inflammation in CIA rats, thus verifying its anti-rheumatoid arthritis property.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Antirheumatic Agents; Arthritis, Experimental; Arthritis, Rheumatoid; Benzofurans; Disease Models, Animal; Down-Regulation; Edema; Inflammation; Inflammation Mediators; Male; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidative Stress; Plant Extracts; Rats; Rats, Sprague-Dawley; Transcription Factor RelA

Qiliqiangxin capsule (QLQX), composed of 11 herbs, is an effective traditional Chinese medicine (TCM) that has been widely used for treatment of chronic heart failure (CHF) in China. In the Chinese pharmacopoeia (Ch.P.) only astragaloside was described as the marker component to control the quality of QLQX, which could not reflect the overall effectiveness.. The aim of this work was to investigate the quality markers (Q-markers) of QLQX based on the association of the pharmacodynamics (PD) of inhibitory effect on activated renin-angiotensin-aldosterone system (RAAS) and the pharmacokinetics (PK) of bioactive compounds according to the Q-marker theory.. The contents of astragaloside, calycosin-7-glucoside, sinapine, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rg1, salvianolic acid A, salvianolic acid B, danshensu, rosmarinic acid, formononetin, aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypacoitine were determined by an HPLC-MS/MS method both in QLQX preparation and in the plasma of CHF rats administered intragastrically with QLQX. The effect of lowering angiotensin II (Ang II) production by QLQX was assayed by ELISA. The association between PK and PD was explored and the bioactive compounds with higher content in vitro and better exposure in vivo, which were closely related to the inhibitory effect on the activated RAAS, were identified as Q-markers of QLQX for CHF treatment.. The contents of 17 constituents were in the order of salvianolic acid B > danshensu > ginsenoside Rb1 > sinapine > benzoylmesaconine > astragaloside > benzoylhypacoitine > ginsenoside Rb2 > salvianolic acid A > ginsenoside Rg1 > calycosin-7-glucoside > rosmarinic acid > formononetin > benzoylaconine > hypaconitine > aconitine > mesaconitine in QLQX preparation. PK and PD association study of 14 bioactive compounds of QLQX showed the maximum effect (E. Astragaloside, calycosin-7-glucoside, sinapine and ginsenoside Rg1 are suitable as the Q-markers of QLQX for CHF treatment, which have higher content in vitro, finer exposure in vivo and a direct correlation with the inhibitory effect on activated RAAS.

Topics: Aconitine; Angiotensin II; Animals; Benzofurans; Biomarkers; Chromatography, High Pressure Liquid; Chronic Disease; Drugs, Chinese Herbal; Ginsenosides; Heart Failure; Isoflavones; Male; Medicine, Chinese Traditional; Quality Control; Rats, Sprague-Dawley; Tandem Mass Spectrometry

The effect of light-emitting diodes (LEDs) on the production of secondary metabolites in medicinal plants and hairy roots is receiving much attention. The roots and rhizomes of the traditional Chinese medicinal plant Salvia miltiorrhiza Bunge are widely used for treating cardiovascular and cerebrovascular diseases. The main components are liposoluble tanshinones and hydrophilic phenolic acids. Moreover, hairy root culture of S. miltiorrhiza has been used in research of valuable plant-derived secondary metabolites. In this study, we examined the effect of LEDs with different combinations of wavelengths on the content of the main components in hairy roots of S. miltiorrhiza. Tanshinone IIA (TSIIA) content in hairy roots was significantly decreased with all light treatments containing blue light by >60% and was 9 times lower with LED treatment duration changed from 1 week to 3 weeks. HMGR, DXS2, DXR, GGPPS, CPS and CYP76AH1 genes involved in the tanshinone biosynthesis pathway were downregulated by blue light. Furthermore, light quality treatments have different effect on the accumulation of phenolic acids in hairy roots of S. miltiorrhiza. The light treatments 6R3B, 6B3IR, 7RGB and 2R6BUV for 3 weeks could increase rosmarinic acid (RA) content slightly but not salvianolic acid B (SAB) content. Different secondary metabolite contents could be regulated by different wavelength combinations of LEDs. Blue light could reduce TSIIA content in hairy roots of S. miltiorrhiza via gene regulation.

Topics: Abietanes; Alkyl and Aryl Transferases; Benzofurans; Biomass; Chromatography, High Pressure Liquid; Farnesyltranstransferase; Gene Expression Regulation, Plant; Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; Light; Plant Proteins; Plant Roots; Salvia miltiorrhiza

Studies have shown that salvianolic acid B (SAB), which is derived from Chinese salvia ( Salvia miltiorrhiza), a plant used in traditional Chinese medicine, can promote the proliferation and migration of endothelial cells. The inner layer of an artificial vascular graft was fabricated using the coaxial electrospinning method and was loaded with the anticoagulant heparin and SAB. The release of heparin and SAB was sustained for almost 30 days and without an initial burst release of SAB. Furthermore, the combined effect of SAB and heparin contributed to promoting human umbilical vein endothelial cell (HUVEC) growth and improved the blood compatibility of the graft. In addition, upregulation of GRP78 by SAB protected human endothelial cells from oxidative stress-induced cellular damage. In vivo evaluation through Masson's trichrome and H&E staining was performed after the graft was subcutaneously embedded in SD rats for 2 weeks and indicated that the graft possessed satisfactory biocompatibility and did not cause a significant immune response. Hence, the functional inner layer is promising for preventing acute thrombosis and promotes rapid endothelialization of artificial vascular grafts.

Topics: Animals; Benzofurans; Endoplasmic Reticulum Chaperone BiP; Heparin; Humans; Rats; Rats, Sprague-Dawley; Vascular Grafting

Our previous study demonstrated a progressive glycolytic perturbation during the course of DMBA-induced hamster oral carcinogenesis, which was attenuated by salvianolic acid B (Sal-B) treatment along with decreased incidences of oral squamous cell carcinoma (OSCC) formation. It was proposed that metabolic modulation should be an additional mode of action attributable to Sal-B's anti-carcinogenic activity. However, the molecular mechanisms underlying Sal-B-induced metabolic modulation function remained elusive. In the present study, we performed next-generation sequencing (NGS) profiling in the same animal model and found Sal-B treatment evoked a general downregulation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and hypoxia inducible factor 1α subunit (HIF-1α) signaling pathways, which might contribute to Sal-B's metabolic modulation activity. The inhibitory effects of Sal-B on aerobic glycolysis, as well as PI3K/AKT and HIF-1α signaling pathways, were validated in two well-characterized OSCC cell lines (Cal27 and HN4), and premalignant oral Leuk1 cells and Sal-B treatment led to elevation of the loss of mitochondrial membrane potential (MMP), increased cell apoptosis, and reduced abilities of colony formation. Rescue assays suggested that compared with Sal-B treatment group, Akt or hif-1a overexpression attenuated the inhibitory effect of Sal-B on glucose uptake and intracellular lactate level. Taken together, our results suggested that Sal-B modulated aberrant glucose metabolism via the PI3K/AKT/HIF-1α signaling pathways, which might contribute to the anti-carcinogenic activity of Sal-B.

Topics: Animals; Apoptosis; Benzofurans; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Clone Cells; Disease Models, Animal; Glucose; Glycolysis; Hypoxia-Inducible Factor 1, alpha Subunit; Lactates; Male; Membrane Potential, Mitochondrial; Mesocricetus; Models, Biological; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction

Salvianolic acid B (SalB) is a water‑soluble phenolic compound, extractable from Salvia miltiorrhiza, and has previously been demonstrated to reverse tumor multidrug resistance (MDR) in colon cancer cells. Cancer stem cells (CSCs) are closely associated with drug resistance. Therefore, establishing a nude mouse model bearing human colon CSCs is important for the study of the mechanisms underlying colon cancer drug resistance as well as the reversal of drug resistance. The present study aimed to establish a nude mouse model bearing human colon CSCs and to investigate the effects of SalB on the drug resistance exhibited by the nude mouse model as well as determine its underlying mechanism. Cells from two colon cancer cell lines (LoVo and HCT‑116) were cultured in serum‑free medium to obtain CSCs‑enriched spheroid cells. Following this, nude mice were transplanted with LoVo and HCT‑116 colon CSCs to establish the CSC nude mouse model, which was subsequently demonstrated to exhibit MDR. The results of the present study revealed that following treatment with SalB, the chemotherapeutic drug resistance of xenografts was reversed to a certain extent. Western blot analysis was performed to investigate the expression levels of cluster of differentiation (CD)44, CD133, transcription factor sox‑2 (SOX2) and ATP‑binding cassette sub‑family G member 2 (ABCG2) proteins, and the results demonstrated that treatment with SalB downregulated the expression of CD44, SOX2 and ABCG2 proteins in both LoVo and HCT‑116 colon CSCs xenografts. In conclusion, the results of the present study revealed that a serum‑free suspension method can be performed to successfully isolate colon CSCs. In addition, a nude mice bearing colon CSCs animal model was successfully established, and associated tumors were confirmed to exhibit MDR. Furthermore, SalB was demonstrated to successfully reverse MDR in nude mice bearing LoVo and HCT‑116 colon CSCs, as well as suppress the expression of CD44, SOX2 and ABCG2 proteins.

Topics: Animals; Benzofurans; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Xenograft Model Antitumor Assays

The major loss of myocardial tissue extracellular matrix after infarction is a serious complication that leads to heart failure. Regeneration and integration of damaged cardiac tissue is challenging since the functional restoration of the injured myocardium is an incredible task. The injured micro environment of myocardium fails to regenerate spontaneously. The emergence of nano-biomaterials would be a promising approach to regenerate such a damaged cardiomyocytes tissue. Here, we have fabricated a dual bioactive embedded nanofibrous cardiac patch via coaxial electrospinning technique, to mimic the topographical and chemical cues of the natural cardiac tissue. The proportion and the concentration of the polymers were optimized for tailored delivery of bioactives from a spatio-temporally designed scaffold. The functionalization of polymeric core shell nanofibrous scaffold with dual bioactives enhanced the physico-chemical and bio-mechanical properties of the scaffolds that has resulted in a 3-dimensional topography mimicking the natural cardiac like extracellular matrix. The sustained delivery of bioactive signals, improved cell adhesion, proliferation, migration and differentiation could be attributed to its highly interconnected nanofibrous matrix with good extended morphology. Further, the expression of cardiac specific markers were found to increase on investigation of mRNA by real time PCR studies and proteins by immunofluorescence and western blotting techniques, confirming cell - biomaterial interactions. Flow cytometry analysis authenticated a potent mitochondrial membrane potential of cells treated with nanocomposite. In addition, in ovo studies in chicken chorioallantoic membrane assay confirm the efficacy of the developed scaffold in inducing angiogenesis required for maintaining its viability after transplantation onto the infarcted zone. These promising results demonstrate the potential of the composite nanofibrous scaffold as an effective biomaterial substrate for cardiac regeneration providing cues for development of novel cardiac therapeutics.

Topics: Animals; Ascorbic Acid; Benzofurans; Blotting, Western; Cell Adhesion; Cell Differentiation; Cell Line; Cell Movement; Chick Embryo; Chorioallantoic Membrane; Humans; Magnesium; Membrane Potential, Mitochondrial; Microscopy, Atomic Force; Myoblasts; Nanofibers; Tissue Engineering; Tissue Scaffolds

Obesity-induced metabolic dysfunctions increase the risk for vascular diseases, including type II diabetes and stroke. Managing obesity is of interest to address the worldwide health problem; however, the role of genetic variability in human obesity development and specific targets for obesity-related metabolic disease have not been thoroughly studied. A SNP in the brain-derived neurotropic factor (

Topics: Animals; Benzofurans; Brain-Derived Neurotrophic Factor; CD36 Antigens; Cell Differentiation; Diet, High-Fat; Insulin Resistance; Intra-Abdominal Fat; Male; Methionine; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Obese; Mutation; Obesity; Valine

Salvianolic acid B (SalB), a natural polyphenolic compound extracted from the herb of Salvia miltiorrhiza, possesses antioxidant and neuroprotective properties and has been shown to be beneficial for diseases that affect vasculature and cognitive function. Here we investigated the protective effects of SalB against subarachnoid hemorrhage (SAH)-induced oxidative damage, and the involvement of underlying molecular mechanisms. In a rat model of SAH, SalB inhibited SAH-induced oxidative damage. The reduction in oxidative damage was associated with suppressed reactive oxygen species generation; decreased lipid peroxidation; and increased glutathione peroxidase, glutathione, and superoxide dismutase activities. Concomitant with the suppressed oxidative stress, SalB significantly reduced neurologic impairment, brain edema, and neural cell apoptosis after SAH. Moreover, SalB dramatically induced nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and increased expression of heme oxygenase-1 and NADPH: quinine oxidoreductase-1. In a mouse model of SAH, Nrf2 knockout significantly reversed the antioxidant effects of SalB against SAH. Additionally, SalB activated sirtuin 1 (SIRT1) expression, whereas SIRT1-specific inhibitor sirtinol pretreatment significantly suppressed SalB-induced SIRT1 activation and Nrf2 expression. Sirtinol pretreatment also reversed the antioxidant and neuroprotective effects of SalB. In primary cultured cortical neurons, SalB suppressed oxidative damage, alleviated neuronal degeneration, and improved cell viability. These beneficial effects were associated with activation of the SIRT1 and Nrf2 signaling pathway and were reversed by sirtinol treatment. Taken together, these in vivo and in vitro findings suggest that SalB provides protection against SAH-triggered oxidative damage by upregulating the Nrf2 antioxidant signaling pathway, which may be modulated by SIRT1 activation.

Topics: Animals; Antioxidants; Apoptosis; Benzofurans; Cell Survival; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidative Stress; Rats; Rats, Sprague-Dawley; Sirtuin 1; Subarachnoid Hemorrhage

To determine the contents of salvianolic acid B and borneol in compound Danshen tablet (composed of Salviae Miltiorrhizae Radix et Rhizome, Notoginseng Radix et Rhizome and Borneolum Syntheticum) by near-infrared spectroscopy (NIR) and to establish a dependency model for rapid quantitative analysis. NIR data of 74 batches of compound Danshen tablet from different companies were collected; the contents of salvianolic acid B and borneol were determined by using high performanceliquid chromatography (HPLC) and gas chromatography (GC) respectively to establish the dependency model for salvianolic acid B and borneol. The results showed that the best waveband for salvianolic acid B was 10 846.2-10 013, 9 195.3-8 362.2, 6 719.1-4 242.8 cm⁻¹; root-mean-squares error of cross-validation (RMSECV), coefficient of determination

Topics: Benzofurans; Camphanes; Salvia miltiorrhiza; Spectroscopy, Near-Infrared; Tablets

Emphysema progressively destroys alveolar structures, leading to disability and death, yet remains irreversible and incurable to date. Impaired vascular endothelial growth factor (VEGF) signaling is an emerging pathogenic mechanism, thereby proposing a hypothesis that VEGF stimulation/elevation enables recovery from alveolar structural destruction and loss of emphysema. Our previous in vitro study identified that salvianolic acid B (Sal-B), a polyphenol of traditional Chinese herbal danshen, stimulated lung cell proliferation and migration, and protected against induced lung cell death, by virtue of signal transducer and activator of transcription 3 (STAT3) activation and VEGF stimulation/elevation. Thus, this study examined Sal-B for in vivo therapeutic reversal of established emphysema in two rat models. Emphysema was induced with porcine pancreatic elastase (PPE) and cigarette smoke extract (CSE), and established by day 21. Sal-B was then spray-dosed to the lung three times weekly for three weeks. Functional treadmill exercise endurance; morphological airspace enlargement and alveolar destruction; apoptosis, cell proliferation and tissue matrix proteins; phosphorylated STAT3 (pSTAT3) and VEGF expressions; neutrophil accumulation; and lipid peroxidation were determined. In both models, Sal-B at 0.2 mg/kg significantly reversed impaired exercise endurance by 80 and 64%; airspace enlargement [mean linear intercept (MLI)] by 56 and 67%; and alveolar destructive index (%DI) by 63 and 66%, respectively. Induced apoptosis activity [cleaved caspase-3] was normalized by 94 and 82%; and cell proliferation activity [proliferative cell nuclear antigen (PCNA)] was stimulated by 1.6 and 2.1-fold. In the PPE-induced model, Sal-B reduced induction of lung's matrix metalloproteinase (MMP)-9 and MMP-2 activities by 59 and 94%, respectively, and restored pSTAT3 and VEGF expressions to the healthy lung levels, while leaving neutrophil accumulation unchecked [myeloperoxidase (MPO) activity]. In the CSE-induced model, Sal-B elevated pSTAT3 and VEGF expressions both by 1.8-fold over the healthy lung levels, and normalized induced lipid peroxidation [malondialdehyde (MDA) activity] by 68%. These results provide an in vivo proof-of-concept for Sal-B as one of the first anti-emphysema agents enabling reversal of alveolar structural destruction and loss via local lung treatment by virtue of its STAT3 activation and VEGF stimulation.

Topics: Animals; Benzofurans; Disease Models, Animal; Disease Progression; Lipid Peroxidation; Male; Malondialdehyde; Pancreatic Elastase; Pulmonary Alveoli; Pulmonary Emphysema; Rats; Rats, Sprague-Dawley; Smoke; STAT3 Transcription Factor; Swine; Vascular Endothelial Growth Factor A

Glioma remains the leading cause of brain tumor-related death worldwide, and radiation is a standard adjuvant therapy with proven efficacy. Salvianolic acid B (SalB), a bioactive compound isolated from Radix Salviae, has been shown to exert anti-cancer effects in many cancer cell lines, including glioma. This study aimed to investigate whether SalB could affect response to radiation in human glioma cells. We found that SalB decreased cell viability of U87 cells in a-dose-dependent manner. A subthreshold dose of SalB at 0.5 μM, which had no effect on cell viability and apoptosis, significantly increased radiation sensitivity of U87 cells in a dose- and time-dependent manner, but had no effect on sensitivity to temozolomide (TMZ). Similar results were also observed in human glioma U373 cells. In addition, SalB aggravated the radiation-induced apoptosis and mitochondrial dysfunction, as measured by mitochondrial Ca

Topics: Apoptosis; Benzofurans; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Drugs, Chinese Herbal; Gene Knockdown Techniques; Glioma; Humans; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Neurons; Radiation-Sensitizing Agents; Radiation, Ionizing; RNA, Small Interfering

Endothelial cell injury and subsequent inflammation play pivotal roles in the pathogenesis of pulmonary fibrosis, a progressive and fatal disorder. We found previously that salvianolic acid B (SAB) attenuated experimental pulmonary fibrosis. Pulmonary fibrosis is driven by inflammation, but the anti-inflammatory role and mechanism of SAB on the treatment of pulmonary fibrosis is still unknown. Here, our in vivo studies showed that SAB had a strong anti-inflammatory effect on bleomycin-instilled mice by inhibiting inflammatory cell infiltration and inflammatory cytokine production. Moreover, SAB protected endothelial cells against oxidative stress injury and inhibited endothelial cell apoptosis in bleomycin-treated mice. The in vitro studies also showed that SAB decreased the H

Topics: Animals; Benzofurans; Cell Line; Cytokines; Cytoprotection; Disease Models, Animal; Endothelial Cells; Humans; Hydrogen Peroxide; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; NF-kappa B; Oxidative Stress; Permeability; Pneumonia; Signal Transduction; Tight Junctions

Topics: A549 Cells; Animals; Benzofurans; Cell Death; Cell Line; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Humans; Hydrogen Peroxide; Lung; Male; Pulmonary Emphysema; Rats, Sprague-Dawley; Receptors, Vascular Endothelial Growth Factor; STAT3 Transcription Factor; Vascular Endothelial Growth Factor A

BACKGROUND Salvianolic acid B (SalB) is the representative component of phenolic acids derived from the roots and rhizomes of Salvia miltiorrhiza Bge (Labiatae), which has been used widely in Asian countries for clinical therapy of various cardiovascular dysfunction-related diseases. However, cardiac protection effects and the underlying mechanism for clinical application are still poorly understood. Here, we investigated the potential anti-myocardial fibrosis effect and mechanism of SalB on Angiotensin II (Ang II)-induced cardiac fibrosis in vitro. MATERIAL AND METHODS The proliferation and migration capacity of cardiac fibroblasts (CFBs) were measured by MTT assay and scratch analysis, respectively. The colorimetric assay determined the hydroxyproline content in medium. Western blotting detected the protein expressions of nuclear transcription factor-kappa B (NF-κB) pathway-associated proteins, fibronectin (FN), collagen type I (Coll I), α-smooth muscle actin (α-SMA), and connective tissue growth factor (CTGF). The expression of α-SMA protein was observed by immunofluorescence staining. qRT-PCR detected the mRNA expression of NF-κB. RESULTS SalB attenuated Ang II-induced the proliferation and the migration ability of CFBs. Ang II-induced the extracellular matrix protein Coll I, FN, and α-SMA, the pro-fibrotic cytokine CTGF protein expression was inhibited, and the nuclear translocation of NF-κB p65 subunit was reduced by SalB. Western blotting and qRT-PCR confirmed that SalB blocked the activation of NF-κB induced by Ang II. PDTC (the NF-κB inhibitor) also inhibited proliferation of CFBs and reduced α-SMA and Coll I expression induced by Ang II. CONCLUSIONS SalB can alleviate Ang II-induced cardiac fibrosis via suppressing the NF-κB pathway in vitro.

Topics: Angiotensin II; Animals; Benzofurans; Cell Movement; Cell Proliferation; Collagen Type I; Fibroblasts; Fibrosis; Heart; I-kappa B Proteins; Myocardium; NF-kappa B; Rats; Rats, Sprague-Dawley

To fabricate stable sized and shaped controlled release delivery systems for salvianolic acid B (Sal B), different food gums were individually added to short-chain glucan solution to prepare starch nanoparticles (StNPs)/gum nanocomposites by self-assembly, and Sal B was embedded in situ. The results showed that size of StNPs was reduced to ca. 45nm with the addition of chitosan and rosin, which decreased by over 50% than that of StNPs without the gum. The StNPs/guar gum nanocomposites had the largest size (109.2nm) among samples of StNPs with gums. The StNPs with chitosan and gum arabic exhibited an obvious core-shell structure. The loading capacities of Sal B in StNPs, StNPs/chitosan, and StNPs/gum arabic nanocomposites were 5.2, 8.26 and 8.08%, respectively. The in vitro release of Sal B from StNPs/gum nanocomposites were sustained and prolonged for over 12h, indicating that StNPs/gum nanocomposites are good candidates to control Sal B release.

Topics: Benzofurans; Nanoparticles; Plant Gums; Starch

It is known that the activation of hepatic stellate cells (HSCs) is a pivotal step in the initiation and progression of liver fibrosis. Aberrant activated Wnt/β-catenin pathway is known to accelerate the development of liver fibrosis. microRNAs (miRNAs)-mediated Wnt/β-catenin pathway has been reported to be involved in HSC activation during liver fibrosis. However, whether long noncoding RNAs (lncRNAs) regulate Wnt/β-catenin pathway during HSC activation still remains unclear.. Long intergenic noncoding RNA-p21 (lincRNA-p21) expression was detected in Salvianolic acid B (Sal B)-treated cells. Effects of lincRNA-p21 knockdown on HSC activation and Wnt/β-catenin pathway activity were analyzed in Sal B-treated cells. In lincRNA-p21-overexpressing cells, effects of miR-17-5p on HSC activation and Wnt/β-catenin pathway activity were examined.. LincRNA-p21 expression was up-regulated in HSCs after Sal B treatment. In primary HSCs, lincRNA-p21 expression was down-regulated at Day 5 relative to Day 2. Sal B-inhibited HSC activation including the reduction of cell proliferation, α-smooth muscle actin (α-SMA) and type I collagen was inhibited by lincRNA-p21 knockdown. Sal B-induced Wnt/β-catenin pathway inactivation was blocked down by loss of lincRNA-p21. Notably, lincRNA-p21, confirmed as a target of miR-17-5p, suppresses miR-17-5p level. Lack of the miR-17-5p binding site in lincRNA-p21 prevents the suppression of miR-17-5p expression. In addition, the suppression of HSC activation and Wnt/β-catenin pathway induced by lincRNA-p21 overexpression was almost inhibited by miR-17-5p.. We demonstrate that lincRNA-p21-inhibited Wnt/β-catenin pathway is involved in the effects of Sal B on HSC activation and lincRNA-p21 suppresses HSC activation, at least in part, via miR-17-5p-mediated-Wnt/β-catenin pathway.

Topics: Animals; Benzofurans; beta Catenin; Cell Proliferation; Cells, Cultured; Hepatic Stellate Cells; Male; Mice; MicroRNAs; RNA, Long Noncoding; Wnt Signaling Pathway

Salvianolic acid B (Sal B) is one of the most abundant phenolic acids derived from the root of Danshen with potent anti-oxidative properties. The present study examined the vasoprotective effect of Sal B in hypertensive mice induced by angiotensin II (Ang II). Sal B (25mg/kg/day) was administered via oral gavage for 11days to Ang II (1.2mg/kg/day)-infused C57BL/6J mice (8-10weeks old). The vascular reactivity (both endothelium-dependent relaxations and contractions) in mouse arteries was examined by wire myography. The production of reactive oxygen species (ROS), protein level and localization of angiotensin AT

Topics: Angiotensin II; Animals; Benzofurans; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Endothelium, Vascular; Human Umbilical Vein Endothelial Cells; Humans; Hypertension; Male; Mice; Mice, Inbred C57BL; Organ Culture Techniques; Treatment Outcome

Generation and accumulation of the amyloid-β (Aβ) peptide after proteolytic processing of the full length amyloid precursor protein (FL-APP) by β-secretase (β-site APP cleaving enzyme or BACE1) and γ-secretase are the main causal factors of Alzheimer's disease (AD). Thus, inhibition of BACE1, a rate-limiting enzyme in the production of Aβ, is an attractive therapeutic approach for the treatment of AD. Recent studies suggest that salvianolic acid B (Sal B) is isolated from the radix of Salvia miltiorrhiza Bunge, a Chinese herbal medicine commonly used for the treatment of cardiovascular, cerebrovascular and liver diseases in China.. In this study, we discovered that Sal B acted as a BACE1 modulator and reduced the level of secreted Aβ in two different Swedish APP (SwedAPP) mutant cell lines. Using N2a-mouse and H4- human neuroglioma cell lines expressing SwedAPP, it was demonstrated that Sal B significantly and dose-dependently decreased the generation of extracellular Aβ, soluble APPβ (by-product of APP cleaved by BACE1), and intracellular C-terminal fragment β from APP without influencing α-secretase and γ-secretase activity and the levels of FL-APP. In addition, using protein-docking, we determined the potential conformation of Sal B on BACE1 docking and revealed the interactions of Sal B with the BACE1 catalytic center.. The docking provides a feasible explanation for the experimental results, especially in terms of the molecular basis of Sal B's action. Our results indicate that Sal B is a BACE1 inhibitor and, as such, is a promising candidate for the treatment of AD.

Topics: Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Benzofurans; Catalysis; Catalytic Domain; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Space; Humans; Intracellular Space; Mice; Molecular Docking Simulation; Molecular Structure; Neuroprotective Agents

Salvianolic acid B (Sal B), one of the chief water-soluble constituents in Radix Salviae Milthiorrhizae, has often been reported to possess considerable cardiovascular regulatory effects. However, the underlying biochemical and cellular mechanisms of its cardioprotection remain unclear. This study was designed to evaluate the role of Sal B regulation in L-type Ca

Topics: Animals; Benzofurans; Calcium; Calcium Channels, L-Type; Cardiotonic Agents; Ether-A-Go-Go Potassium Channels; Heart Ventricles; HEK293 Cells; Humans; Myocardial Contraction; Myocytes, Cardiac; Rats, Sprague-Dawley

Doxorubicin (DOX)-induced cardiotoxicity is a clinically complex syndrome that leads to significant pain to cancer survivors. Endoplasmic reticulum (ER) stress has been suggested to be an important contributor to myocardium dysfunction during this phenomenon. Our previous study proved that Salvianolic acid B (Sal B) protected against doxorubicin induced cardiac dysfunction by inhibiting ER stress and cardiomyocyte apoptosis. However, the underlying molecular mechanism is not yet clearly. In this study, we investigated the protective effect and mechanisms of Sal B againest DOX-induced cardiac injury and ER stress in vivo and in vitro. After pretreatment with Sal B (0.25, 0.5, 1mg/kg i.v.) for 7 days, male SD rats were intraperitoneally injected with a single dose of DOX (3mg/kg) every 2 days for three injections. The cardioprotective effect of Sal B was observed 2 weeks after the first administration. Adult rat ventricular myocytes were isolated and treated with Sal B (20μg/ml) for 6h and then exposed in DOX (1μm) for 4h. The cardiomyocyte contractility and the level of intracellular Ca

Topics: Animals; Apoptosis; Benzofurans; Calcium Signaling; Cardiotoxicity; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Doxorubicin; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Heart Diseases; Male; Membrane Potential, Mitochondrial; Myocardial Contraction; Myocytes, Cardiac; Protective Agents; Rats, Sprague-Dawley; Time Factors; TRPC Cation Channels

Salvianolic acid B (SAB) is one the major phytocomponents of Radix Salvia miltiorrhiza and exhibit numerous health promoting properties. The objective of the current study was to examine whether SAB exerts a renoprotective effect by attenuating oxidative stress and inflammatory response through activating phosphatidylinositol 3-kinase/serine-threonine kinase B (PI3K/Akt) signaling pathway in a renal ischemic reperfusion rat model. Forty Sprague-Dawley male rats (250-300 g) were obtained and split into four groups with ten rats in each group. The right kidney of all rats was removed (nephrectomy). The rats of the Control group received only saline (occlusion) and served as a sham control group, whereas rats subjected to ischemic reperfusion (IR) insult by clamping the left renal artery served as a postitive control group. The other 2 groups of rats were pretreated with SAB (20 and 40 mg·kg-1·day-1) for 7 days prior IR induction and served as treatment groups (SAB 20+IR; SAB 40+IR). Renal markers creatinine (Cr) and blood urea nitrogen (BUN) were significantly lower in the groups that received SAB. Pretreatment with SAB appears to attenuate oxidative stress by suppressing the production of lipid peroxidation products like malondialdehyde as well as elevating antioxidant activity. The concentration of inflammatory markers and neutrophil infiltration (myeloperoxidase) were significantly decreased. Meanwhile, PI3K protein expression and pAkt/Akt ratio were significantly upregulated upon supplementation with SAB, indicating its renoprotective activity. Taken together, these results indicate that SAB can therapeutically alleviate oxidative stress and inflammatory process via modulating PI3K/Akt signaling pathway and probably ameliorate renal function and thus act as a renoprotective agent.

Topics: Animals; Benzofurans; Blood Urea Nitrogen; Creatinine; Drugs, Chinese Herbal; Inflammation; Kidney; Lipid Peroxidation; Male; Oxidative Stress; Peroxidase; Phosphatidylinositol 3-Kinases; Protective Agents; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction

The physiological and pathological complexity of the wound healing process makes it more challenging to design an ideal tissue regeneration scaffold. Precise scaffolding with high drug loading efficiency, efficient intracellular efficacy for therapeutic delivery, minimal nonspecific cellular and blood protein binding, and maximum biocompatibility forms the basis for an ideal delivery system. This paper describes a combinational multiphasic delivery system, where biomolecules are delivered through the fabrication of coaxial electrospinning of different biocompatible polymers. The ratio and specificity of polymers for specific biofunction are optimized and the delivery system is completely characterized with reference to the mechanical property and structural integrity of bromelain (debridement enzyme) and salvianolic acid B (pro-angiogenesis and re-epithelialization). The in vitro release profile illustrated the sustained release of debriding protease and bioactive component in a timely fashion. The fabricated scaffold showed angiogenic potential through in vitro migration of endothelial cells and increased new capillaries from the existing blood vessel in response to an in ovo chicken chorioallantoic membrane assay. In addition, in vivo studies confirm the efficacy of the fabricated scaffold. Our results therefore open up a new avenue for designing a bioactive combinational multiphasic delivery system to enhance wound healing.

Topics: Absorption, Physicochemical; Administration, Cutaneous; Animals; Benzofurans; Bromelains; Delayed-Action Preparations; Diffusion; Drug Combinations; Electroplating; Female; Lacerations; Nanocapsules; Nanofibers; Rats; Rats, Wistar; Regeneration; Skin; Treatment Outcome; Wound Healing

Topics: Benzaldehydes; Benzofurans; Bilirubin; Caffeic Acids; Catechols; Cinnamates; Depsides; Glucuronosyltransferase; Humans; Kinetics; Lactates; Microsomes, Liver; Polyphenols; Rosmarinic Acid; Salvia miltiorrhiza

Phytochemical investigation of the roots of Salvia miltiorrhiza led to isolations of two new depsides (1-2), along with thirteen known compounds (3-15). Their structures and relative stereochemistry were elucidated by NMR spectral (

Topics: Benzofurans; Caffeic Acids; Cell Line; Depsides; Free Radical Scavengers; Humans; Hydrogen Peroxide; Lactates; Molecular Structure; Plant Roots; Salvia miltiorrhiza

Although accumulating evidence suggests that activated microglia are associated with deficits in neurogenesis and contribute to the physiopathology of major depressive disorder, the role of microglia in treating depression remains poorly understood. Our previous study showed that salvianolic acid (SalB) has the regulation of neuroinflammatory responses and antidepressant-like effects. Here, we hypothesized that SalB's therapeutic effects occur because it modulates microglial phenotypes that are associated with neurogenesis. To test this hypothesis, we treated CMS-exposed C57BL/6 mice with SalB (20mg/kg, intraperitoneally, once daily) for 3weeks and investigated microglial phenotypic profiles and hippocampal neurogenesis. The results showed that the SalB treatment skewed M1 microglial polarization toward M2 activation in the hippocampus and cortex and remedied CMS-induced deficits in hippocampal neurogenesis. SalB (40µM) inhibited LPS-stimulated microglial M1 activation as well as induced M2 activation in vitro, and the cultured microglia with the SalB treatment showed enhanced neural precursor cell proliferation, differentiation, and survival. SalB treatment also ameliorated the depressive-like behaviors of the CMS-treated mice in sucrose preference, forced swimming, and tail suspension tests. These findings suggest a possible antidepressive mechanism for anti-inflammatory agents that is correlated with microglial polarization and hippocampal neurogenesis and which may provide a new microglia-targeted strategy for depression therapy.

Topics: Animals; Benzofurans; Cerebral Cortex; Depression; Depressive Disorder; Hippocampus; Inflammation; Inflammation Mediators; Male; Mice, Inbred C57BL; Microglia; Neurogenesis; Phenotype; Stress, Psychological

Topics: Animals; Benzofurans; Cyclooxygenase 2; Diet, High-Fat; Dietary Supplements; Humans; Inflammation; Interleukin-10; Interleukin-6; Male; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Plant Extracts; Salvia miltiorrhiza; Tumor Necrosis Factor-alpha

Blood glucose fluctuations, also referred to as intermittent high glucose, have been validated to be more harmful than sustained high glucose in exacerbating pancreatic dysfunction by inducing β cell apoptosis. Salvianolic acid B (Sal B), an aqueous component of Salvia miltiorrhiza, has been proved beneficial to pancreatic islet function in diabetes, but the underlying mechanisms remain to be elucidated. The present study investigated the protective effect of Sal B on INS-1 cells exposed to intermittent high glucose and the possible mechanisms implicated. The results indicated that Sal B was able to restore cell viability and suppress INS-1 cell apoptosis induced by intermittent high glucose. Preincubation with Sal B led to a significant decrease of caspase-9 and caspase-3 activity and poly ADP-ribose polymerase (PARP) cleavage. Exposure to intermittent high glucose induced significant up-regulation of proapoptotic proteins, down-regulation of antiapoptotic protein and depolarization of mitochondrial membrane potential (MMP) in INS-1 cells, while these changes were reversed effectively in Sal B treated groups. In addition, Sal B markedly attenuated intermittent high glucose-induced oxidative stress as manifested by notably decreased levels of intracellular reactive oxygen species and malondialdehyde (MDA). Taken together, these results indicate that Sal B is able to suppress intermittent high glucose-induced INS-1 cell apoptosis, which might be ascribed to regulation of Bcl-2 family protein expression and preservation of mitochondrial membrane potential.

Topics: Apoptosis; Benzofurans; Caspase 3; Caspase 9; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Glucose; Humans; Intracellular Space; Membrane Potential, Mitochondrial; Poly(ADP-ribose) Polymerases; Proteolysis; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

The aim of this study was to explore the possible antibacterial components of Salvia miltiorrhizae on Pseudomonas aeruginosa using a combination of chemical fingerprint and bioactivity evaluation. The chemical fingerprints of 32 batches of S. miltiorrhizae samples from different sources were developed using high-performance liquid chromatography with diode array detection, and then were evaluated by similarity analysis and hierarchical clustering analysis. Anti-P. aeruginosa activity was determined by microcalorimetry. Some crucial thermokinetic parameters obtained from the heat-flow power-time curves of P. aeruginosa growth in the absence or presence of these S. miltiorrhizae samples were evaluated using principal component analysis. Thereafter, multiple linear regression analysis was used to analyze the fingerprint-activity relationship between the chemical fingerprints and anti-P. aeruginosa activity. This established the related equation between the inhibition ratio (I, %) of S. miltiorrhizae samples on P. aeruginosa and the peak areas of the common peaks. The results showed that the 32S. miltiorrhizae samples could be grouped into three clusters according to their chemical fingerprints and anti-P. aeruginosa activities. Protocatechualdehyde, salvianolic acid B, together with three unidentified compounds might be the major components that contributed largely to the antibacterial properties of S. miltiorrhizae and should be the focus of S. miltiorrhizae quality control. Thus, this study provided a preferred way for exploring the bioactive components of medicinal plants.

Topics: Anti-Bacterial Agents; Benzaldehydes; Benzofurans; Catechols; Drugs, Chinese Herbal; Multivariate Analysis; Plant Extracts; Plants, Medicinal; Principal Component Analysis; Pseudomonas aeruginosa; Salvia miltiorrhiza

To introduce the methods of triamcinolone acetonide (TA) and salvianolic acid B(SA-B) intralesional injection in the treatment of oral submucous fibrosis and evaluate the treatment effects.. TA combined with SA-B were consecutively applied intralesionally 1 time weekly for 30 times. The technique of intralesional injection was that syringe needle be emptied into submucous tissues 1 cm posterior to the corner of oral cavity running from the front to retromolar area by pushing the injection, then the syringe needle retained to the original point and upward from the front to retromolar area by pushing the injection, finally the syringe needle returned to the starting point and downward from the front to retromolar area by pushing the injection. Mouth opening, the color change of the buccal mucosa and the increase of capillary vessels as determined by degree I-IV visual analog scale were evaluated at 12, 24, and 36 month. The data were analyzed with SPSS 21.0 software package.. One hundred and fourteen subjects fulfilled the study without obvious adverse reactions. After treatment for 36 months , the net gain in mouth opening of the early stage group，middle stage group and advanced stage group was (12.0±1.2) mm, (14.5±2.4) mm and (15.5±1.5) mm, respectively. The response rate of color change of the buccal mucosa and the increase of capillary vessels in early stage group, middle stage group and advanced stage group after treatment for 36 months was 100%, 94.7% and 90.7%, respectively.. Triamcinolone acetonide and salvianolic acid B intralesional injection is effective in the treatment of oral submucous fibrosis.

Topics: Benzofurans; Glucocorticoids; Humans; Injections, Intralesional; Mouth Mucosa; Oral Submucous Fibrosis; Pain Measurement; Treatment Outcome; Triamcinolone Acetonide

A sensitive and specific ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed for analysis of tanshinone ⅡA(TSⅡA), salvianolic acid B(SAB) and ginsenoside Rg₁ (GRg₁) in rat plasma and brain tissues. Male healthy Sprague-Dawley(SD) rats were orally given single dose of Fufang Danshen preparation (TS ⅡA 60 mg•kg⁻¹, SAB 300 mg•kg⁻¹, GRg₁ 150 mg•kg⁻¹, borneol 300 mg•kg⁻¹), and their blood samples and brain tissues were collected at different time points. The drug plasma and brain tissue concentrations of the three analytes were determined by UPLC-MS/MS method. Subsequently, the main pharmacokinetics parameters of plasma and brain tissues were calculated by using Phoenix WinNolin 6.1 software. The methodological test showed that all of analytes in both plasma and brain homogenate exhibited a good linearity within the concentration range(r>0.992 2). Their mean recoveries were between 58.86% and 112.1%. Intra-day and inter-day precisions of the investigated components exhibited RSD≤9.7%, and the accuracy(RE) ranged from -9.68% to 8.20% at all quality control levels. The results of accuracy and stability meet the requirements for biopharmaceutical analysis. For TSⅡA, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (1.58±0.081) h, (725.4±88.20) μg•L⁻¹, (2 101.3±124.85) μg•h•L⁻¹ and (3.66±0.05) h, respectively. For SAB, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (1.29±0.21) h, (307.9±46.75) μg•L⁻¹, (537.4±88.24) μg•h•L⁻¹ and (2.08±0.11) h, respectively. For GRg₁, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (1.42±0.20) h, (460.38±154.60) μg•L⁻¹, (383.4±88.16) μg•h•L⁻¹ and (1.87±0.046) h, respectively. For TSⅡA, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the brain tissue were (0.75±0.22) h, (1.41±0.42) ng•g⁻¹, (4.34±2.48) ng•h•g⁻¹ and (4.00±1.90) h, respectively. For SAB, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (1.08±0.20) h, (21.09±4.850) ng•g⁻¹, (14.83±3.160) ng•h•g⁻¹ and (0.99±0.08) h, respectively. For GRg₁, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (0.50±0.16) h, (130.96±54.220) ng•g⁻¹, (136.24±34.350) ng•h•g⁻¹ and (2.87±0.33) h, respectively. The developed method was successfully applied in pharmacokinetic studies on content of TS ⅡA, SAB and GRg₁ in rat plasma and brain tissues.

Topics: Abietanes; Animals; Benzofurans; Brain; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Ginsenosides; Male; Plasma; Rats; Rats, Sprague-Dawley; Tandem Mass Spectrometry

To optimize the ethanol extraction process for Shenlian formula. On the basis of the pharmacodynamics index for different extraction process routes, the contents of salvianolic acid B, tanshinone ⅡA and berberine, as well as the extraction ratio in different experimental schemes were used as the ethanol extraction examining indexes, and multi-criterion synthesizing grading method was used for data analysis to optimize and verify the ethanol-extraction process conditions in orthogonal experiment. The optimum ethanol extraction process was as follows: adding 60% ethanol, 10 times amount, extracting for 2.0 h each time for a total of 2 times. This extraction process showed good stability and availability.

Topics: Abietanes; Benzofurans; Berberine; Drugs, Chinese Herbal; Ethanol; Plants, Medicinal; Technology, Pharmaceutical

The present study tested the hypotheses that bone morphogenetic protein 2(BMP-2) combined with salvianolic acid B(Sal-B) enhance the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) towards cardiomyocytes in vitro. BMSCs were treated with BMP-2 and Sal-B, alone or in combination, for 72 h, then added new media (excluding inductive substance) and cultured for 4 weeks. The morphologic characteristics, surface antigens and mRNA expression of several transcription factors were also assessed. We found that they could all be identified by the positive staining for cardiomyocyte-specific proteins, desmin and cardiac troponin T, in these cells. Furthermore, the mRNA expression of GATA-4 and Nkx2.5 genes was slightly increased on day 7, enhanced on day 14 and decreased on day 28 while α-MHC gene was not expressed on day 7, but expressed slightly on day 14 and enhanced on day 28. The expression of these cardiac-specific markers in treatment groups were all significantly higher than those in the control group, respectively (P < 0.05). Transmission electron microscopy showed that BMSCs in treatment groups all had myofilaments, z line-like substances and mitochondria. Taken together, these results indicate that BMP-2 combined with Sal-B promotes myocardial differentiation of BMSCs, which may represent a potential therapeutic strategy for the treatment of ischemic heart disease.

Topics: Animals; Benzofurans; Biomarkers; Bone Marrow Cells; Bone Morphogenetic Protein 2; Cell Differentiation; Desmin; Femur; GATA4 Transcription Factor; Gene Expression; Homeobox Protein Nkx-2.5; Male; Mesenchymal Stem Cells; Myocytes, Cardiac; Myofibrils; Myosin Heavy Chains; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Tibia; Troponin T

In this study, we analyzed danshen (Salvia miltiorrhiza) constituents using biopartitioning and microemulsion high-performance liquid chromatography (MELC). The quantitative retention-activity relationships (QRARs) of the constituents were established to model their pharmacokinetic (PK) parameters and chromatographic retention data, and generate their biological effectiveness fingerprints. A high-performance liquid chromatography (HPLC) method was established to determine the abundance of the extracted danshen constituents, such as sodium danshensu, rosmarinic acid, salvianolic acid B, protocatechuic aldehyde, cryptotanshinone, and tanshinone IIA. And another HPLC protocol was established to determine the abundance of those constituents in rat plasma samples. An experimental model was built in Sprague Dawley (SD) rats, and calculated the corresponding PK parameterst with 3P97 software package. Thirty-five model drugs were selected to test the PK parameter prediction capacities of the various MELC systems and to optimize the chromatographic protocols. QRARs and generated PK fingerprints were established. The test included water/oil-soluble danshen constituents and the prediction capacity of the regression model was validated. The results showed that the model had good predictability.

Topics: Abietanes; Animals; Area Under Curve; Benzofurans; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Drugs, Chinese Herbal; Emulsions; Male; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Rosmarinic Acid; Salvia miltiorrhiza; Surface-Active Agents

BackgroundAims: Salvianolic acid B (SalB) is a natural polyphenolic compound enriched in Salvia miltiorrhiza Bunge. Our study was designed to explore the role of Sal B on cognitive impairment in vascular dementia (VD) model rats, as well as its possible molecular mechanisms.. Rats were randomly divided into four groups (n = 15 for each group): Control group, Sal B group (normal Sprague Dawley rats treated with Sal B), VD group and VD + Sal B group. The VD group rats were established by permanent bilateral common carotid artery occlusion (BCCAO). Animals in the Control and Sal B group received the same operation without bilateral common carotid arteries occlusion. The animals in Sal B group and VD + Sal B group received Sal B (20 mg/kg) orally once a day for consecutive 6 weeks. We investigated the effects of SalB on BCCAO-induced cognitive deficits rats models via the Morris water maze experiment. To explore the mechanisms of Sal B on cognitive function, we detected the expression of IGF-1, Akt and p-Akt, and the rate of cell apoptosis in CA1 region.. Our results observed that hippocampal IGF-1 was decreased in VD model rats, while SalB reversed the alteration of IGF-1 levels. The expression of hippocampal Akt showed no significant difference between control and VD group, however, p-Akt level was significantly decreased in VD group. After 6 weeks of SalB treatment, p-Akt level was significantly increased. A large number of apoptotic neurons were found in VD model rats, while SalB prevented apoptosis of hippocampal neurons in CA1 region in VD model rats.. SalB significantly ameliorated cognitive deficits in BCCAO-induced VD model rats. The potential mechanism underlying the protective effects may be mediated through IGF-1/Akt pathway.

Topics: Animals; Benzofurans; Cognition; Dementia, Vascular; Drugs, Chinese Herbal; Hippocampus; Insulin-Like Growth Factor I; Male; Maze Learning; Neurons; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Signal Transduction

The quality of Danshen extract granules on market is largely different from each other mainly due to the heterogeneous quality of raw materials of Salvia miltiorrhiza, various producing procedures and lack of good quality evaluation method. Formula granule and "standard decoction" have the same quality. In this paper, a systematic evaluation method for the quality of Danshen decoction was established from the perspective of "standard decoction", in order to explore the main factors affecting the quality uniformity of Danshen extract granules. Danshen standard decoction was prepared; then the fingerprint method was developed to determine the content of salvianolic acid B; and the main peaks in the fingerprint were identified with UPLC-QTOF/MS to clarify the chemical compositions of Danshen decoction. Three indexes were calculated to evaluate the stability of whole process, including the extraction ratio; transfer rate of index components and pH value. The results showed that the main components of Danshen decoction were phenolic acids, while the extraction rate, the transfer rate of salvianolic acid B and pH value were in a relatively stable level, and the similarity in the fingerprint of standard decoction was high, indicating that the preparation procedure was stable. The level of salvianolic acid B in the standard decoction was in a large range, which was mainly due to the difference in the quality of Salviae Miltiorrhizae Radix et Rhizoma.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Mass Spectrometry; Plant Extracts; Plant Roots; Quality Control; Rhizome; Salvia miltiorrhiza

To achieve the combination therapy of acute myocardial ischemia, arginyl-glycyl-aspartic acid (RGD) conjugated lipid was synthesized and RGD modified, salvianolic acid B (Sal B) and panax notoginsenoside (PNS) co-loaded lipid-polymer hybrid nanoparticles (RGD-S/P-LPNs) was fabricated an evaluated.. The LPNs are generally spherical in shape with uniform size distribution, have sizes of 100-200nm and zeta potentials range from -30.7∼ -39.8. In vitro release behaviors of drugs loaded LPNs are in a sustained release manner, which does not exhibit obviously cytotoxicity against H9c2 cardiomyocytes. RGD-S/P-LPNs group shows the most significant cardiac distribution and infarct therapy effect in vivo.. The results illustrated that RGD modified dual drugs co-loaded LPNs are stable, sustained release carriers. Cardiac distribution, pharmacokinetics, and infarct therapy effect results suggested that the RGD-S/P-LPNs could improve the in vivo therapeutic efficacy of the double drugs.

Topics: Animals; Benzofurans; Cell Line; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Therapy, Combination; Ginsenosides; Integrin alphaVbeta3; Lipids; Male; Myocardial Ischemia; Nanomedicine; Nanoparticles; Oligopeptides; Panax; Peptides; Phosphatidylethanolamines; Polyethylene Glycols; Polymers; Rats; Rats, Sprague-Dawley

Kidney-specific delivery is critically important for the treatment of renal fibrosis with drugs such as salvianolic acid B (Sal B). Here we report a kidney-specific nanocomplex formed by the coordination-driven assembly of catechol-modified low molecular weight chitosan (HCA-Chi), calcium ions and Sal B. The prepared HCA-Chi-Ca-Sal B (HChi-Ca-Sal B) nanocomplex reversed the TGF-β1-induced epithelial-mesenchymal transition (EMT) in HK-2 cells. In vivo imaging demonstrated a kidney-specific biodistribution of the nanocomplex. The anti-fibrosis effect of HChi-Ca-Sal B was tested in a mouse model of unilateral ureteral obstruction (UUO). Significant attenuation of the morphological lesions and the levels of extracellular matrix (ECM) proteins in the tubulointerstitium was observed in mice treated with HChi-Ca-Sal B, suggesting that the nanocomplex was able to prevent fibrosis better than the treatment with free Sal B. It was concluded that the HChi-Ca-Sal B nanocomplex showed a specific renal targeting capacity and could be utilized to enhance Sal B delivery for treating renal fibrosis.

Topics: Animals; Benzofurans; Catechols; Cell Line; Chitosan; Epithelial-Mesenchymal Transition; Kidney; Mice

The NLRP3 inflammasome activation and neuroinflammation are known to be involved in the pathology of depression, whereas autophagy has multiple effects on immunity, which is partly mediated by the regulation of inflammasome and clearance of proinflammatory cytokines. Given the emerging evidence that autophagy dysfunction plays an essential role in depression, it is very likely that autophagy may interact with the inflammatory process in the development and treatment of depression. Salvianolic acid B (SalB), a naturally occurring compound extracted from Salvia miltiorrhiza, contains anti-inflammatory and antidepression properties and has recently been proven to modulate autophagy. In this study, we sought to investigate whether autophagy is involved in the inflammation-induced depression and the antidepressant effects of SalB.. The effects of prolonged lipopolysaccharide (LPS) treatment and SalB administration on behavioral changes, neuroinflammation, autophagic markers and NLRP3 activation in rat hippocampus were determined by using behavioral tests, real-time PCR analysis, western blot, and immunostaining.. Our data showed that periphery immune challenge by LPS for 2 weeks successfully induced the rats to a depression-like state, accompanied with enhanced expression of pro-inflammatory cytokines and NLRP3 inflammasome activation. Interestingly, autophagic markers, including Beclin-1, and the ratio of LC3II to LC3I were suppressed following prolonged LPS exposure. Meanwhile, co-treatment with SalB showed robust antidepressant effects and ameliorated the LPS-induced neuroinflammation. Additionally, SalB restored the compromised autophagy and overactivated NLRP3 inflammasome in LPS-treated rats.. Collectively, these data suggest that autophagy may interact with NLRP3 activation to contribute to the development of depression, whereas SalB can promote autophagy and induce the clearance of NLRP3, thereby resulting in neuroprotective and antidepressant actions.

Topics: Animals; Anti-Inflammatory Agents; Autophagy; Behavior, Animal; Benzofurans; Depression; Inflammasomes; Inflammation; Lipopolysaccharides; Male; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Rats, Sprague-Dawley

To explore the effect of salvianolic acid B (Sal B) on regulation of SIRT3/FOXO1 signaling pathway in rats with non-alcoholic steatohepatitis (NASH).. Sixty Sprague Dawley (SD) rats were randomly divided into control, model and treatment groups. After 12 weeks of successful model establishment with high fat diet, treatment group was given Sal B by intragastric administration. After 12 weeks of treatment, rats were sacrificed and livers were taken to test indicators such as liver index, TG, TC, ALT, AST, reactive oxygen species (ROS) by DCFH-DA probe, SOD2 activity by WST-8 test. mRNA and protein expression of SIRT3, SOD2, catalase were detected by real time PCR and western blot, respectively. The acetylation level of FOXO1 and SOD2 was detected by immuno-precipitation (IP).. Liver index, ALT, AST, TG, TC, and ROS of model group were higher than those of control and treatment groups, which the difference was statistically significant (p <0.01). SOD2 activity of model group was lower than that of control and treatment groups. In treatment group, HE staining and electron microscopy showed hepatic tissue pathological change and mitochondrial structure damage alleviate. mRNA and protein expression of SIRT3, SOD2, catalase were lower in model group and the difference was statistically significant (p <0.05), which was opposite in the acetylation level of FOXO1 and SOD2 by IP.. Sal B can decrease oxidative stress reaction by regulating SIRT3/FOXO1 signaling pathway and play a therapeutic role in the treatment of NASH in rats.

Topics: Animals; Benzofurans; Diet, High-Fat; Disease Models, Animal; Drugs, Chinese Herbal; Liver; Male; Nerve Tissue Proteins; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Sirtuins; Superoxide Dismutase

Based on the research of active liver targeting liposomes mediated by glycyrrhetinic acid ligand at home and abroad, this paper focuses on the liver targeting effect of liposomes mediated with 18-GA-Gly, a kind of glycyrrhetinic acid ligand. salvianolic acid B(Sal B)-tanshinone ⅡA (TSN)liposomes mediated by 18-GA-Gly as well as the liposomes with unmodified ligands were prepared by film dispersion-high pressure homogenization method, and then the particle size, potential, encapsulation efficiency and ligand binding rate were detected. Plasma samples of the heart, liver, spleen, lung and kidney tissues were taken at different time points after tail vein injections. The contents of Sal B and TSN in each sample were determined with UPLC methods and the liver targeting effect of 18-GA-Gly ligands was evaluated. The results showed that the particle size, potential, encapsulation efficiency and ligand binding rate met the basic requirements; in vivo targeting investigation results showed no difference between GLY-TS-Lip group and TS-Lip group. The liposomes mediated by glycyrrhetinic acid derivative ligand 18-GA-Gly can increase the peak concentration of Sal B and TSN in liver, but showed no significant liver targeting effect.

Topics: Abietanes; Animals; Benzofurans; Drug Carriers; Glycyrrhetinic Acid; Ligands; Liposomes; Liver

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA

Topics: Amino Acid Sequence; Benzofurans; Biosynthetic Pathways; Cinnamates; Depsides; Gene Expression Regulation, Plant; Oxidoreductases; Phenylpropionates; Phenylpyruvic Acids; Phylogeny; Plant Proteins; Plant Roots; Plants, Genetically Modified; Recombinant Proteins; Rosmarinic Acid; Salvia miltiorrhiza; Sequence Alignment

Salvianolic acid B (SalB), a water-soluble polyphenol extracted from Radix Salvia miltiorrhiza, has been reported to possess many pharmacological activities. This study investigated the hepatoprotective effects of SalB in chronic alcoholic liver disease (ALD) and explored the related signaling mechanisms. In vivo, SalB treatment significantly attenuated ethanol-induced liver injury by blocking the elevation of serum aminotransferase activities and markedly decreased hepatic lipid accumulation by reducing serum and liver triglyceride (TG) and total cholesterol (TC) levels. Moreover, SalB treatment ameliorated ethanol-induced hepatic inflammation by decreasing the levels of hepatotoxic cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Importantly, SalB pretreatment significantly increased the expression of SIRT1 and downregulated the expression of inflammatory mediator C-reactive protein (CRP) and lipoprotein carbohydrate response element-binding protein (ChREBP). In vitro, SalB significantly reversed ethanol-induced down-regulation of SIRT1 and increased CRP and ChREBP expression. Interestingly, the effects of SalB on SIRT1, CRP and ChREBP were mostly abolished by treatment with either SIRT1 siRNA or EX527, a specific inhibitor of SIRT1, indicating that SalB decreased CRP and ChREBP expression by activating SIRT1. SalB exerted anti-steatotic and anti-inflammatory effects against alcoholic liver injury by inducing SIRT1-mediated inhibition of CRP and ChREBP expression.

Topics: Animals; Anti-Inflammatory Agents; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Benzofurans; Biomarkers; Carbazoles; Carrier Proteins; Chronic Disease; Cytokines; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Hep G2 Cells; Hepatocyte Nuclear Factor 1-alpha; Histone Deacetylase Inhibitors; Humans; Inflammation Mediators; Liver; Liver Diseases, Alcoholic; Male; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirtuin 1; Transfection

Salvianolic acid B (SalB) a water‑soluble phenolic compound, extracted from Salvia miltiorrhiza, has previously been demonstrated to reverse tumor multidrug resistance (MDR), including in colorectal cancer. Reactive oxygen species (ROS) are oxygen radicals generated during aerobic metabolism (superoxide and hydroxyl radicals) and superoxide easily generating free radicals (H2O2). The concept that increased ROS levels can lead to augmented tumor cell‑sensitivity to chemotherapy drugs has become notable. The aim of the present study was to elucidate the role of ROS in mediating the effect of SalB on drug resistance and the correlation with drug resistance‑associated protein, P‑glycoprotein (P‑gp), and apoptosis‑associated proteins, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X (Bax). In the current study, through utilizing the multidrug resistant colorectal cancer cell line, HCT‑8/VCR, it was demonstrate that SalB reversed MDR in HCT‑8/VCR. In addition, SalB significantly increased ROS levels, which may have accelerated the apoptosis of HCT‑8/VCR cells by downregulating Bcl‑2 and increasing Bax protein expression. Furthermore the increased intracellular ROS levels may have inhibited P‑gp expression at the gene and protein levels. In conclusion, the data of the current study demonstrate that SalB reversed MDR in HCT‑8/VCR cells, and the effect is associated with increased ROS levels, which may downregulate P‑gp expression and promote tumor cell apoptosis, which in turn increases the sensitivity of drug‑resistant cells to chemotherapy drugs.

Topics: Acetylcysteine; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; bcl-2-Associated X Protein; Benzofurans; Cell Line, Tumor; Colorectal Neoplasms; Drug Resistance, Neoplasm; Drugs, Chinese Herbal; Humans; Hydrogen Peroxide; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

Shen-Shuai-Ning (SSN) granule, a traditional Chinese medicine formula, is widely used in clinical practice for treating chronic renal failure. However, its detailed chemical profile is unknown. Here, HPLC-ESI-QTOF-MS was employed for the systematic chemical analysis of SSN. A total of 52 compounds were identified and the characteristic ions of the compounds were described. Furthermore, chemical consistency between the combined decoction and the separated decoction of SSN was evaluated using HPLC-DAD. A chemical comparison between two preparations of SSN granule (combined decoction and separated decoction of Coptides Rhizoma) indicated a significant difference in the content of many compounds, including salvianolic acid A, salvianolic acid B, berberine, palmatine and epiberberine. As a result, separated decoction of Coptides Rhizoma would lead to a significantly decrease in depsides in Salviae Miltiorrhizae Radix et Rhizoma and an increase in alkaloids in Coptidis Rhizoma.

Topics: Benzofurans; Berberine Alkaloids; Caffeic Acids; Chromatography, High Pressure Liquid; Drug Compounding; Drugs, Chinese Herbal; Flavones; Lactates; Saponins; Spectrometry, Mass, Electrospray Ionization

Neuroinflammation plays a critical role in the pathogenesis of ischemia/reperfusion (I/R) injury. Activated platelets are increasingly regarded as initiators and/or amplifiers of inflammatory processes in cerebral I/R injury. Salvianolic acid B (SAB) is the most abundant bioactive compound of Salviae miltiorrhizae, a well-known Chinese herb used to promote blood circulation and eliminating blood stasis. S. miltiorrhizae has been used clinically in Asia for the treatment of ischemic cerebrovascular diseases. In the present study, a rat model of transient middle cerebral artery occlusion (tMCAO) was established to investigate the neuroprotective effects and mechanisms of SAB treatment against focal cerebral I/R insult. The results showed that SAB treatment (3mg/kg, 6mg/kg and 12mg/kg, i.p.) dose-dependently decreased I/R-induced neurological deficits at 24, 48, and 72h after reperfusion and decreased plasma-soluble P-selectin and soluble CD40 ligand as early as 6h after onset of I/R insult. At 24h after reperfusion, SAB treatment significantly reduced neuronal and DNA damage in the hippocampal CA1 region and decreased neural cell loss in the ischemic core. The I/R-induced pro-inflammatory mediator mRNA and protein overexpression in the penumbra cortex, including ICAM-1, IL-1β, IL-6, IL-8, and MCP-1, were significantly inhibited by SAB in a dose-dependent manner. Further studies suggested SAB treatment attenuated CD40 expression and NF-κB activation, which involved NF-κB/p65 phosphorylation and IκBα phosphorylation and degradation. In conclusion, our findings indicated that the neuroprotective effects of SAB post cerebral I/R injury are associated with the inhibition of both platelets activation and production of pro-inflammatory mediators and the downregulation of the CD40/NF-κB pathway.

Topics: Animals; Benzofurans; Blood Platelets; Brain Ischemia; CA1 Region, Hippocampal; CD40 Antigens; Dose-Response Relationship, Drug; Infarction, Middle Cerebral Artery; Inflammation; Male; Neuroimmunomodulation; Neuroprotective Agents; NF-kappa B; Platelet Activation; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Signal Transduction; Transcription Factor RelA

Articular chondrocytes reside in lacunae distributed in cartilage responsible for the remodelling of the tissue with limited ability of damage repairing. The in vitro expanded chondrocytes enhanced by factors/agents to obtain large numbers of cells with strengthened phenotype are essential for successful repair of cartilage lesions by clinical cell implantation therapies. Because the salvianolic acid B (Sal B), a major hydrophilic therapeutic agent isolated from Salvia miltiorrhiza, has been widely used to treat diseases and able to stimulate activity of cells, this study examines the effects of Sal B on passaged chondrocytes. Chondrocytes were treated with various concentrations of Sal B in monolayer culture, their morphological properties and changes, and mitochondrial membrane potential were analysed using microscopic analyses, including cellular biochemical staining and confocal laser scanning microscopy. The proteins were quantified by BCA and Western blotting, and the transcription of genes was detected by qRT-PCR. The passaged chondrocytes treated with Sal B showed strengthened cellular synthesis and stabilized mitochondrial membrane potential with upregulated expression of the marker genes for chondrocyte phenotype, Col2-α1, Acan and Sox9, the key Wnt signalling molecule β-catenin and paracrine cytokine Cytl-1. The treatments using CYTL-1 protein significantly increased expression of Col2-α1 and Acan with no effect on Sox9, indicating the paracrine cytokine acts on chondrocytes independent of SOX9. Sal B has ultimately promoted cell growth and enhanced chondrocyte phenotype. The chondrocytes treated with pharmaceutical agent and cytokine in the formulated medium for generating large number of differentiated chondrocytes would facilitate the cell-based therapies for cartilage repair.

Topics: Aggrecans; Animals; Benzofurans; Cell Proliferation; Cell Shape; Cell Survival; Cells, Cultured; Chondrocytes; Collagen Type II; Gene Expression Regulation; Humans; Membrane Potential, Mitochondrial; Nucleic Acids; Rabbits; Receptors, Cytokine; SOX9 Transcription Factor; Transcription, Genetic

Salvianolic acid B is an antioxidative ingredient derived from Radix Salviae miltiorrhizae that has been widely used to treat liver diseases. However, the therapeutic mechanism underlying Salvianolic acid B has remained largely unknown. Our studies verified that Salvianolic acid B efficiently blocked mitochondrial deformation and dysfunction induced by H

Topics: Benzofurans; Cell Line; Gene Expression Regulation; Hepatocytes; HSP70 Heat-Shock Proteins; Humans; Mitochondria; Oxidative Stress; Protective Agents; Up-Regulation

Osteoarthritis (OA) is a chronic progressive disease that has complicated mechanisms that involve inflammation and cartilage degradation. In this study, we investigated the anti-inflammatory action of Salvianolic acid B (Sal B) in both human OA chondrocytes and a mouse OA model that was induced by destabilization of the medial meniscus. In vitro, chondrocytes were pretreated with Sal B (0, 25, 50, 100μM) for 2h, then incubated with IL-1β (10ng/mL) for 24h. NO production was determined by Griess method and PGE2 was assessed by ELISA. The expression of INOS, COX-2, MMP-13, ADAMTS-5 and NF-κB-related signaling molecules were tested by Western blotting. Immunofluorescence staining was used to detect P65 nuclear translocation. In vivo, the mouse OA model received intraperitoneal-injection of either Sal B (25mg/kg) or saline every other day. Hematoxylin and Eosin, as well as Safranin-O-Fast green staining, were utilized to evaluate the severity of cartilage lesions up to 8weeks following the surgery. Sal B inhibited the over-production of NO and PGE2, while the elevated expression of INOS, COX-2, MMP-13 and ADAMTS-5 were reversed by Sal B in IL-1β-induced chondrocytes. In addition, IL-1β significantly induced phosphorylation of NF-κB signaling, and this phosphorylation response was blocked by Sal B. Immunofluorescence staining demonstrated that Sal B could suppress IL-1β-induced p65 nuclear translocation. In vivo, the cartilage in Sal B-treated mice exhibited less cartilage degradation and lower OARSI scores. Taken together, Sal B possesses great potential value as a therapeutic agent for OA treatment.

Topics: Animals; Anti-Inflammatory Agents; Benzofurans; Cartilage; Chondrocytes; Cytokines; Disease Models, Animal; Humans; Inflammation; Interleukin-1beta; Male; Matrix Metalloproteinase 13; Menisci, Tibial; Mice; Mice, Inbred C57BL; NF-kappa B; Nitric Oxide; Osteoarthritis; Phosphorylation; Signal Transduction

This study investigated the effects of salvianolic acid B (Sal B), a major bioactive component of the Chinese medicine salvia miltiorrhiza, on osteogenic differentiation of human periodontal ligament cells (hPDLCs).. Third passage PDLCs were used in this experiment. Methyl thiazolyl tetrazolium (MTT) method was employed to observe the effects of different Sal B concentrations on proliferation activity of hPDLCs. Alkaline phosphatase (ALP) activity and mineralization capability were measured, and mRNA expression of osteocalcin (OCN) was detected to investigate the effects of Sal B on osteogenesis of hPDLCs.. Sal B did not influence the viability of hPDLCs. The ALP activity and OCN mRNA expression levels of hPDLCs were both significantly improved (P<0.05) under treatment with different Sal B concentrations (0.5, 1, and 5 μmol·L⁻¹) compared with those in OIM group. Moreover, the number of mineralized nodules formed by hPDLCs were considerably higher under treatment with different Sal B concentrations (0.5, 1, and 5 μmol·L⁻¹) than that in the OIM group.. Appropriate Sal B concentration can improve the osteogenic differentiation of hPDLCs.. 目的 探讨中药丹参主要活性成分丹酚酸B（Sal B）对人牙周膜细胞（hPDLCs）成骨分化的影响。方法 取第3代hPDLCs进行实验，运用甲基噻唑基四唑（MTT）法检测不同浓度丹酚酸B对hPDLCs增殖活性影响，通过检测碱性磷酸酶（ALP）活性、矿化结节染色、骨钙素（OCN）mRNA表达来探讨丹酚酸B对hPDLCs成骨分化的影响。结果 丹酚酸B对hPDLCs增殖活性无影响。当丹酚酸B浓度为0.5、1、5 μmol·L⁻¹时均能增加hPDLCs的ALP活性和OCN mRNA表达，与OIM组比较差异有统计学意义（P<0.05）；且当丹酚酸B浓度为0.5、1、5 μmol·L⁻¹时hPDLCs形成矿化结节数量明显高于OIM组。结论 适宜浓度的丹酚酸B能有效促进hPDLCs成骨分化。.

Topics: Benzofurans; Cell Differentiation; Cells, Cultured; Humans; Osteocalcin; Osteogenesis; Periodontal Ligament

Salvia miltiorrhiza (SM) Bunge is one of the widely-used Chinese medicinal herbs. Salvianolic acid B (Sal B), a bioactive compound isolated from the Chinese herb Radix Salviae Miltiorrhizae, has been reported to exhibit anti-inflammatory and anti-oxidantive effects.. To study the cardioprotective effects of salvianolic acid B (Sal B) on acute myocardial ischemia reperfusion (MIR) injury rats, on the basis of this investigation, the possible mechanism of salvianolic acid B was elucidated. Male Sprague- Dawley rats (200-220 g) were randomly divided into five groups: sham-operated, MIR, MIR + Sal B (10 mg/kg/day, orally), MIR + Sal B (20 mg/kg/ day, orally) and MIR + Sal B (30 mg/kg/ day, orally). Before operation, the foregoing groups were pretreated with homologous drug once a day for 7 days, respectively. After twelve hours in MIR, the cardioprotective effects of SPJ were evaluated by infarct size, biochemical values, and the antioxidative and antiapoptotic relative gene expressions.. Sal B significantly improved heart function and decreased infarct size; remarkably decreased levels of serum TNF-α and IL-Ιβ levels, increased contents of myocardium antioxidant enzymes activities; western blot results showed that Sal B ameliorate the increased Bax and caspase-3 protins expressions and decreased Bcl-2 proteins expression and ratios of Bcl-2 to Bax.. In ischemic myocardium, oxidative stress caused the overgeneration and accumulation of reactive oxygen species (ROS), which was central of cardiac ischemic injury. Sal B exerted beneficially cardioprotective effects on myocardial ischemia injury rats, mainly scavenging oxidative stress-triggered overgeneration and accumulation of ROS, alleviating myocardial ischemia injury and cardiac cell death. List of abbreviations: salvianolic acid B (Sal B); myocardial ischemia reperfusion (MIR); reactive oxygen species (ROS); Left ventricular end-diastolic pressure (LVEDP); left ventricular end-diastolic volume (LVEDV); Malondialdehyde (MDA); superoxide dismutase (SOD); catalase (CAT); Glutathione peroxidase (GSH-Px); glutathione reductase (GR).

Topics: Animals; bcl-2-Associated X Protein; Benzofurans; Humans; Interleukin-1beta; Ischemia; Male; Myocardial Reperfusion Injury; Myocardium; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Salvia; Tumor Necrosis Factor-alpha

This experiment focused on the effect of salvianolic acid B's nasal absorption characteristics in rats. In the study, HPLC determination of salvianolic acid B(SalB) in perfusion liquid was established to examine the SalB nasal irritation in different pH buffers and stability in nasal perfusion solution, and systematically study in vivo nasal absorption characteristics of SalB. Improved rats were adopted to establish the in situ nasal perfusion model to measure the release of total protein and lactate dehydrogenase in perfusion fluid, quantitatively evaluate the nasal irritation and the stability in perfusion liquid of pH 4.0, 5.0, 6.0 SalB phosphate buffer, compare the absorption of SalB in pH 5.0 buffer solution with low, medium and high concentrations (200, 400, 800 mg•L⁻¹). According to the results, nasal irritation: pH 4.0>pH 5.0>pH 6.0, RSD of pH 6.0 SalB buffer solution within 24 h was 3.1%, stability was poor. PH 5.0 SalB buffer solution had a smaller irritation and good stability. According to the nose perfusion test in rats, the nasal absorption of SalB fitted the first-order process and could be considered as passive absorption based on concentration gradient. SalB buffer solution of pH 5.0 had also a small nasal irritation and good stability, with a good absorption in rat nasal perfusion test, which therefore had a certain significance for the development of SalB nasal formulation.

Topics: Administration, Intranasal; Animals; Benzofurans; Chromatography, High Pressure Liquid; Hydrogen-Ion Concentration; Male; Nasal Mucosa; Rats; Rats, Sprague-Dawley

An in vitro anti-thrombin bioassay was developed to investigate the chemical constituents which have anti-thrombin effect from the water soluble components of Salvia miltiorrhiza. Using Chromozym TH as a probe combined with ethyl acetate Semi-micro extraction was applied to measure p-nitroaniline by HPLC. According to the results, the inactivationrate of thrombin by sodium danshensu, salvianolic acid A and salvianolic acid B under a given set of conditions were 3.06%, 77.77% and 2.35%, respectively. In the water-soluble components, salvianolic acid A has a direct inhibition of thrombin, while sodium danshensu and salvianolic acid B have no significant effect on thrombin. The method is sensitive and low consumption. It can eliminate the interference absorbed for the sample itself which can be used for screening single or multiple direct antithrombin active ingredient of herbal extract.

Topics: Aniline Compounds; Benzofurans; Caffeic Acids; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Fibrinolytic Agents; Lactates; Salvia miltiorrhiza; Thrombin

The root yield and active constituent contents were analyzed from four Salvia miltiorrhiza cultivars grown at three different locations( Zhuyang, Changqing, and Taian, Shandong Province) to determine the influence of environmental conditions and cultivars. .. Phenolic acids and tanshinones were analyzed by HPLC method. Total phenolic acids content were analyzed by FolinCiocalteu method. Klason method was used to determine the content of lignin.. The root yield and the active constituent contents were significantly affected by different environments and cultivars of Salvia miltiorrhiza. The root yield was negatively correlated with active constituent contents. Salvia miltiorrhiza of Zhuyang location had the highest active constituent content, but it had the lowest root yield. Salvia miltiorrhiza of Taian location had the lowest active constituent contents, while it had the highest root yield. Salvia miltiorrhiza of Changqing location had relatively higher bio-yields of phenolic acids and tanshinones, which made it suitable for Salvia miltiorrhiza cultivation. Furthermore, compared with three other cultivars,105 cultivar could remain the salvianolic acid B stable, which indicating that 105 cultivar was possible related to resistances.. The research provides a theoretical basis for the selecting of the optimal cultivar and the optimal environmental condition.

Topics: Abietanes; Benzofurans; Chromatography, High Pressure Liquid; Genotype; Hydroxybenzoates; Plant Roots; Salvia miltiorrhiza

The aim of this study was to investigate the pharmacokinetic interaction between tanshinones and polyphenolics which act as the main bioactive compounds in Saliva miltiorrhiza Bunge (SMB). Thus, a rapid and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine the concentrations of Tanshinone IIA (TSIIA), Tanshinone I (TI), Cryptotanshinone (CT), Salvianolic acid B (Sal B), Protocatechuic aldehyde (PAL), Rosmarinic acid (RA), and Danshensu (DSS) in rat plasma. The Sprague-Dawley rats were allocated to three groups which orally administered tanshinones (DST), polyphenolics (DFS), and a mixture of tanshinones and polyphenolics (DTF). These samples were processed by a simple liquid-liquid extraction (LLE) method with ethyl acetate. Chromatographic separation was achieved on an Acquity BEH C18 column (100 mm × 2. 1 mm, 1.7 µm) with the mobile phase consisting of 0.1% (v/v) formic acid and acetonitrile by gradient elution at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole-tandem mass spectrometer TQ-MS/MS equipped with negative and positive electrospray ionization (ESI) interface in multiple reaction monitoring (MRM) mode. The statistical analysis was performed by the Student's t-test with P ≤ 0.05 as the level of significance. The method showed good precision, accuracy, recovery, sensitivity, linearity, and stability. The pharmacokinetic profiles and parameters of these polyphenolics changed when co-administrated with tanshinones. The tanshinones improved the bioavailability of DSS, accelerated the eliminating rate of RA and Sal B and promoted their distribution in vivo. They also contributed to promoting the biotransformation of Sal B to DSS. The polyphenolics could affect the pharmacokinetic of tanshinones, especially CT and TSIIA. Furthermore, the biotransformation of CT to TSIIA and the bioavailability of TSIIA were both improved. This study may provide useful information to avoid unexpected increase of the plasma drug concentration in the clinical practice. Copyright © 2015 John Wiley & Sons, Ltd.

Topics: Abietanes; Animals; Benzaldehydes; Benzofurans; Catechols; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Lactates; Limit of Detection; Liquid-Liquid Extraction; Male; Phenanthrenes; Polyphenols; Rats, Sprague-Dawley; Rosmarinic Acid; Tandem Mass Spectrometry

Aberrant Wnt/β-catenin pathway contributes to the development of liver fibrosis. MicroRNAs (MiRNAs) are found to act as regulators of the activation of hepatic stellate cell (HSC) in liver fibrosis. However, whether miRNAs activate Wnt/β-catenin pathway in activated HSCs during liver fibrosis is largely unknown. In this study, we found that Salvianolic acid B (Sal B) treatment significantly inhibited liver fibrosis in CCl4-treated rats, HSC-T6 cells and rat primary HSCs, resulting in the suppression of type I… collagen and alpha-smooth muscle actin. Also, Sal B suppressed HSC activation and cell proliferation in vitro. Interestingly, Sal B treatment induced the inactivation of Wnt/β-catenin pathway, with an increase in P-β-catenin and Wnt inhibitory factor 1 (WIF1). We demonstrated that the anti-fibrotic effects caused by Sal B were, at least in part, via WIF1. Moreover, our study revealed that miR-17-5p was reduced in vivo and in vitro after Sal B treatment. As confirmed by luciferase activity assays, WIF1 was a direct target of miR-17-5p. Notably, the suppression of HSCs induced by Sal B was almost inhibited by miR-17-5p mimics. Collectively, we demonstrated that miR-17-5p activates Wnt/β-catenin pathway to result in HSC activation through inhibiting WIF1 expression.

Topics: 3' Untranslated Regions; Animals; Benzofurans; Blotting, Western; Carbon Tetrachloride; Cell Line; Cells, Cultured; Disease Progression; Drugs, Chinese Herbal; Extracellular Matrix Proteins; Gene Expression Regulation; Hepatic Stellate Cells; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Liver Cirrhosis; Male; MicroRNAs; Microscopy, Fluorescence; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Wnt Signaling Pathway

Salvianolic acid B (Sal B) is regarded as a potent antidiabetic agent and has been reported to possess cardioprotective effect in vivo.. This study investigated the cardioprotective effects of Sal B on H9c2 cells injury caused by high glucose in vitro, and clarified the possible mechanisms.. Di ferent concentrations of Sal B were incubated with cells for 12 h prior being exposed to high glucose for 24 h. Cardioprotective effects of Sal B were evaluated using CCK-8 assay, ELISA, Hoechst 33258 nucleus staining, and western blot.. Following a 24 h exposure of H9c2 to high glucose, obvious reduction was found in cell viability (45%), GSH (54.8 ± 9.4 ng/mg protein), catalase (1.22 ± 0.12 U/mg protein), and GPX level (67.9 ± 9.4 U/mg protein), which were associated with the increases of GSSG (1.99 ± 0.28 ng/mg protein) and ROS (2.00 ± 0.19 RFU/mg protein) production. High glucose also elevated IL-6 (1.8-fold), IL-1β (1.9-fold), and TNF-α (1.6-fold) level, as well as induced cell apoptosis and NF-κB (6.1-fold) activation. However, Sal B (25 and 50 μM) elevated cell viability (28% and 44%), ameliorated oxidative stress (GSH, 1.3- and 1.6-fold; catalase, 1.9- and 2.0-fold; GPX, 1.1- and 1.4-fold; GSSG, 0.9- and 0.8-fold; ROS, 0.6- and 0.5-fold), and inflammatory response (IL-6, 0.9- and 0.7-fold; IL-1β, 0.8- and 0.6-fold; TNF-α, 0.9- and 0.8-fold), and inhibited cell apoptosis and NF-κB (0.5- and 0.2-fold) expression.. Sal B attenuated high glucose-induced injury and cytotoxicity through inhibiting inflammatory cytokine production in H9c2 cardiac cells.

Topics: Animals; Benzofurans; Cardiotonic Agents; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Inflammation Mediators; Myocytes, Cardiac; Rats

Epidemics of meningococcal meningitis cause significant health problems especially in Sub-Saharan Africa. Novel anti-infective candidates are needed. In modern anti-adhesion therapy initial attachment of bacteria to host cells is prevented. Our unique studies have revealed anti-adhesive candidates from natural products, namely milk and berries, against Neisseria meningitidis adhesion. In the present study against N. meningitidis adhesion, a novel binding inhibitor was found; salvianolic acid B (SA-B), a polyphenol from the radix Salviae miltiorrhizae, an important part of Chinese folk medicine.. In order to test inhibition of meningococcal pili binding and anti-adhesion activity of SA-B, bovine thyroglobulin, a reference glycoprotein for meningococcal receptor was used in a microtiter plate assay. Inhibitory activity was tested by using serial dilutions of SA-B extracts of 98 and 70% purity. Results were confirmed in a HEC-1B cell dot assay and antimicrobial activity was measured by using a microbroth dilution assay.. Almost total (93%) inhibition of pili binding, anti-adhesion, was achieved with the 70% extract of SA-B at the concentration of 0.3 mg/mL in the bovine thyroglobulin reference model. 50% binding inhibition activity was achieved with 0.6 µg/mL of the SA-B extract. Total inhibition of the pili binding to HEC-1B cells was found at the tested concentration of 0.5 mg/mL. The 98% pure SA-B resulted in weaker inhibition. At the concentration of 0.3 mg/mL 78% inhibition was achieved in the thyroglobulin model. For 50% inhibition 2.4 μg/mL of pure SA-B was needed. The difference between the binding inhibition activities (70 and 98% pure SA-B) was statistically significant (P = 0.03). Antimicrobial activity of 70% SA-B, when investigated against N. meningitidis, was detected only in relatively high concentrations.. Our results indicate that plant SA-B may prevent meningococcal infections by inhibiting meningococcal binding and may thus have an impact on the amount of nasopharyngeal carriers of N. meningitidis. This may prevent the spreading of meningococcal infections between humans. One could conclude that SA-B and its source dried radix S. miltiorrhizae, which is an important part of Chinese folk medicine, could be valuable candidates for further research in meningococcal disease prevention.

Topics: Animals; Anti-Bacterial Agents; Bacterial Adhesion; Benzofurans; Cattle; Cell Line, Tumor; Dose-Response Relationship, Drug; Epithelial Cells; Fimbriae, Bacterial; Humans; Neisseria meningitidis; Plant Extracts; Protein Binding; Salvia miltiorrhiza; Thyroglobulin

Experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS), is a Th1 and Th17 cell-mediated CNS autoimmune disease. Therefore, immune regulation is a key target for therapy. Salvianolic acid B (Sal B) is a major water-soluble bioactive component of the famous traditional Chinese medicine Salvia miltiorrhiza, which is notable for its anti-oxidative and anti-inflammatory effects. Thus Sal B, by impairing Th1 or Th17 responses in EAE/MS, might ameliorate the crippling symptoms. Here we show that the intraperitoneal administration of 30mg/kg Sal B daily for 14 days after the onset of MOG-induced EAE in mice effectively reduced its severity. Additionally, Sal B treatment downgraded the infiltration of inflammatory cells, limited astrogliosis and blocked Th1 responses other than that of Th17. These results indicated that Sal B may serve as an effective therapeutic agent for MS/EAE by inhibiting Th1 cell responses.

Topics: Animals; Autoimmunity; Benzofurans; Encephalomyelitis, Autoimmune, Experimental; Female; Mice, Inbred C57BL; Neuroprotective Agents; Spinal Cord; Th1 Cells

Losartan (LST) is a common chemical drug used to treat high blood pressure and reduce the risk of stroke in certain people with heart disease. Danshen, prepared from the dried root and rhizome of Salvia miltiorrhiza Bunge, has been widely used for prevention and treatment of various cardiovascular and cerebrovascular diseases. There are more than 35 formulations containing Danshen indexed in the 2010 Chinese Pharmacopoeia, which are often combined with LST to treat cardiovascular and cerebrovascular diseases in the clinic. The effects of the two major components of Danshen, salvianolic acid B (SA-B) and tanshinone IIA (Tan IIA), on the pharmacokinetics of losartan and its metabolite, EXP3174, in rats were investigated by liquid chromatography coupled with mass spectrometry (LC-MS). Male Sprague-Dawley rats were randomly assigned to 3 groups: LST, LST+SA-B and LST+Tan IIA, and the main pharmacokinetic parameters were estimated after oral administration of LST, LST+SA-B and LST+Tan IIA. It was found that there are significant differences in the pharmacokinetic parameters among the three groups: Cmax, t1/2, AUC, AUMC in the LST+SA-B group was smaller than those in group LST, while larger in group LST+Tan IIA. Further, the effects of SA-B and Tan IIA on the metabolism of losartan was also investigated using rat liver microsomes in vitro. The results indicated that SA-B can induce the metabolism of LST, while Tan IIA can inhibit the metabolism of LST in rat liver microsomes in vitro by regulating activities of CYP450 enzymes. In addition, the effect of SA-B and Tan IIA on CYP3A4 and CYP2C9 expression was studied in Chang liver cells by western-blotting and Real-time PCR. It was concluded that the two components of Danshen, SA-B and Tan IIA have different influences on the metabolism of LST: SA-B can obviously speed up the metabolism of LST by inducing CYP3A4/CYP2C9 activities and expression, however, Tan IIA can slow down the metabolism of LST by inhibiting CYP3A4/CYP2C9 activities.

Topics: Abietanes; Animals; Antihypertensive Agents; Benzofurans; Cell Line; Cell Survival; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2C9; Cytochrome P-450 CYP3A; Drug Interactions; Drugs, Chinese Herbal; Losartan; Male; Mass Spectrometry; Microsomes, Liver; Rats, Sprague-Dawley; RNA, Messenger

Tumor vessels are known to be abnormal, with typically aberrant, leaky and disordered vessels. Here, we investigated whether polarized macrophage phenotypes are involved in tumor abnormal angiogenesis and what is its mechanism. We found that there was no difference in chemotaxis of polarized M1 and M2 macrophages to lewis lung carcinoma (LLC) cells and that either M1 or M2 macrophage-conditioned media had no effect on LLC cell proliferation. Unexpectedly, the M2 but not M1 macrophage-conditioned media promoted the proliferation of human umbilical vein endothelial cells (HUVECs) and simultaneously increased endothelial cell permeability in vitro and angiogenic index in the chick embryo chorioallantoic membrane (CAM). The treatment with M2 but not M1 macrophage-conditioned media increased autophagosomes as well as microtubule-associated protein light chain 3B (LC3-B) expression (a robust marker of autophagosomes) but decreased p62 protein expression (a selective autophagy substrate) in HUVECs, the treatment with chloroquine that blocked autophagy abrogated the abnormal angiogenic efficacy of M2 macrophage-conditioned media. These results were confirmed in urethane-induced lung carcinogenic progression. Urethane-induced lung carcinogenesis led to more M2 macrophage phenotype and increased abnormal angiogenesis concomitant with the upregulation of LC3-B and the downregulation of p62. Clodronate liposome-induced macrophage depletion, chloroquine-induced autophagic prevention or salvianolic acid B-induced vascular protection decreased abnormal angiogenesis and lung carcinogenesis. In addition, we found that the tendency of age-related M2 macrophage polarization also promoted vascular permeability and carcinogenesis in urethane carcinogenic progression. These findings indicate that the M2 macrophages induce autophagic vascular disorder to promote lung cancer progression, and the autophagy improvement represents an efficacious strategy for abnormal angiogenesis and cancer prevention.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Apoptosis; Autophagy; Benzofurans; Capillary Permeability; Carcinogenesis; Carcinoma, Lewis Lung; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chick Embryo; Chloroquine; Clodronic Acid; Culture Media, Conditioned; Endothelial Cells; Female; Human Umbilical Vein Endothelial Cells; Humans; Lung Neoplasms; Macrophages; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Neovascularization, Pathologic; Phosphoproteins; Trans-Activators; Urethane

Salvia miltiorrhiza (SM) Bunge is one of the widely-used Chinese medicinal herbs. In this study, the chemical constituents and anticancer potential of SM stems and leaves were examined with those of respective callus cultures. The callus culture for stem and leaf explants was initiated in modified Murashige and Skoog (MS) medium. Active constituents of respective extracts were analyzed by high performance liquid chromatography coupled with DAD and MS (HPLC-DAD-MS). Rosmarinic acid (RA) and salvianolic acid B (Sal B) were determined to be the main phenolic compounds. Quantitative analyses revealed that callus stem extracts produced higher amount of RA and Sal B (stem RA: 1.27±0.38%; stem Sal B: 0.87±0.20%) than callus leaf did (leaf RA: 0.28±0.02%; leaf Sal B: 0.07±0.03%). Stem and leaf callus extracts exerted cytotoxic effects towards CCRF-CEM cells (stem: 13.1±0.90 μg/ml; leaf: 18.1±0.33 μg/ml). As expected, stem extract with higher amount of RA and Sal B showed lower IC50 value than leaf extract. These findings suggest the possibility to isolate bioactive constituents with anticancer properties from in vitro callus cultures of stems and leaves of SM.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Drugs, Chinese Herbal; Plant Extracts; Plant Leaves; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Rosmarinic Acid; Salvia miltiorrhiza

7'(Z)-(8″S, 8‴S)-epi-Salvianolic acid E (compound 1) and (7'R, 8'R, 8″S, 8‴S)-epi-salvianolic acid B (compound 2), two novel analogs of salvianolic acid B (Sal B), have been recently isolated from Salvianolic acid for injection. They both show powerful antioxidant effects, including inducing NQO1 activity and scavenging DPPH free radical, and potential protecting effects for cerebral ischemia. However, no reports have been described the pharmacokinetic study of them. In this study, an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed and validated for the determination of compound 1, compound 2 and Sal B in rat plasma, respectively. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters Acquity UPLC(®) HSS T3 column (1.7μm particles, 2.1mm i.d.×100mm) with the mobile phase of 0.1% aqueous formic acid (A)-acetonitrile (B) (65:35, v/v). Quantification was performed on a triple quadruple tandem mass spectrometry with electrospray ionization (ESI) by multiple reaction monitoring (MRM) in the negative ion mode. Monitored transitions were set at m/z 717.0→519.0, 717.1→519.1, 717.2→518.9 and 320.9→152.1 for compound 1, compound 2, Sal B and chloramphenicol (internal standard, IS), respectively. Linear calibration curves were acquired over the concentration range of 2.0-1000ng/mL for the three analytes in rat plasma. The extraction recoveries, matrix effects, intra- and inter-day precisions and accuracies of the three analytes were all within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of compound 1, compound 2 and Sal B after intravenous administration of 6.0mg/kg in rats, respectively. The results indicated that compound 1 and compound 2 were both eliminated more slowly than Sal B. Exposure levels of both compound 1 and Sal B were higher than compound 2 in the same dosage range. This study provided critical reference for the pharmacokinetic study of compound 1 and compound 2.

Topics: Alkenes; Animals; Benzofurans; Chromatography, High Pressure Liquid; Drug Stability; Linear Models; Male; Polyphenols; Rats; Rats, Wistar; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry

The aim of this study was to investigate the cardioprotective effect of salvianolic acid B (Sal B) on acute myocardial infarction (AMI) in rats and its potential mechanisms.. The AMI model was established in rats to study the effect of Sal B on AMI. Haematoxylin-eosin (HE) staining was used to evaluate the pathological change in AMI rats. Immunofluorescence and TUNEL staining were used to detect autophagy and apoptosis of myocardial cells in hearts of AMI rats, respectively. Protein expression of apoptosis-related, autophagy-related and angiogenesis-related proteins were examined by Western blot.. Sal B attenuated myocardial infarction significantly compared with that of the model group. Rats administered with Sal B showed higher inhibition rate of infarction and lower infarct size than those of the model group. Moreover, Sal B decreased the serum levels of creatine kinase, lactate dehydrogenase and malondialdehyde, while increased such level of superoxide dismutase significantly compared with those of the model group. Sal B inhibited the expression of Bax, cleaved caspase-9 and cleaved PARP, while promoted the expression of Bcl-2, LC3-II, Beclin1 and VEGF.. Sal B has cardioprotective effect on AMI and Sal B may be a promising candidate for AMI treatment.

Topics: Angiogenesis Inducing Agents; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Benzofurans; Cardiotonic Agents; Creatine Kinase; L-Lactate Dehydrogenase; Male; Malondialdehyde; Microtubule-Associated Proteins; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Rats; Superoxide Dismutase; Vascular Endothelial Growth Factor A

To investigate the effects of salvianolic acid B (SAB) on tumor necrosis factor a (TNF-α) induced alterations of cerebral microcirculation with a bone-abrading model.. The influences of craniotomy model and bone-abrading model on cerebral microcirculation were compared. The bone-abrading method was used to detect the effects of intracerebroventricular application of 40 μg/kg·bw TNF-α on cerebral venular leakage of fluorescein isothiocyanate (FITC)- albulmin and the rolling and adhesion of leukocytes on venules with fluorescence tracer rhodamine 6G. The therapeutical effects of SAB on TNF-α induced microcirculatory alteration were observed, with continuous intravenous injection of 5 mg/kg·h SAB starting at 20 min before or 20 min after TNF-α administration, respectively. The expressions of CD11b/CD18 and CD62L in leukocytes were measured with flow cytometry. Immunohistochemical staining was also used to detect E-selectin and ICAM-1 expression in endothelial cells.. Compared with craniotomy method, the bone-abrading method preserved a higher erythrocyte velocity in cerebral venules and more opening capillaries. TNF-α intervention only caused responses of vascular hyperpermeability and leukocyte rolling on venular walls, without leukocyte adhesion and other hemodynamic changes. Pre- or post-SAB treatment attenuated those responses and suppressed the enhanced expressions of CD11b/CD18 and CD62L in leukocytes and E-selectin and ICAM-1 in endothelial cells induced by TNF-α.. The pre- and post-applications of SAB during TNF-α stimulation could suppress adhesive molecular expression and subsequently attenuate the increase of cerebral vascular permeability and leukocyte rolling.

Topics: Animals; Benzofurans; Blood Flow Velocity; Cerebrovascular Circulation; Craniotomy; Disease Models, Animal; E-Selectin; Intercellular Adhesion Molecule-1; Mice; Mice, Inbred C57BL; Microcirculation; Random Allocation; Reference Values; Tumor Necrosis Factor-alpha

The antioxidant activities and total phenolic content of fermented Salvia miltiorrhiza with fungus Geomyces luteus were investigated. The results revealed that G. luteus fermentation could significantly improve the antioxidant activity and total phenolic content of S. miltiorrhiza. The main antioxidant constituents were characterized by spectroscopic analysis as salvianolic acids. High-performance liquid chromatography (HPLC) quantification also showed the enhanced content of salvianolic acid B after fermentation. The present study suggests that G. luteus fermentations are effective in the S. miltiorrhiza salvianolic acids' enrichment process.

Topics: Alkenes; Antioxidants; Ascomycota; Benzofurans; Biphenyl Compounds; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Fermentation; Phenols; Picrates; Polyphenols; Salvia miltiorrhiza; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared

Ischemic stroke can activate multiple transcription factors and cause inflammatory reactions, which involve pattern recognition receptors with immunostimulatory effects. Toll-like receptor 4 (TLR4) is one of the receptors related to innate immunity and several inflammatory reactions. The promising anti- inflammatory activity of salvianolic acid B (SAB) had been previously reported, but its effect on ischemic stroke remains unknown. An oxygen-glucose deprivation and reoxygenation (OGD/R) model in vitro and a middle cerebral artery occlusion (MCAO) model in vivo were used in this paper, and the results showned that SAB remarkably increased the viabilities of PC12 cells and primary cortical neurons after OGD/R injury and notably prevented cerebral ischemia/reperfusion (I/R) injury. SAB also significantly ameliorated NeuN release from primary cortical neurons. Further research indicated that the neuroprotection of SAB was completed through inhibiting the TLR4/MyD88/TRAF6 signaling pathway. The blocking of TLR4 by SAB also restrained NF-kB transcriptional activity and pro-inflammatory cytokine responses (IL-1β, IL-6, and TNF-α). These findings supply a new insight that will aid in clarifying the effect of SAB against cerebral I/R injury and provide the development of SAB as a potential candidate for treating ischemic stroke.

Topics: Animals; Anti-Inflammatory Agents; Benzofurans; Brain Ischemia; Cells, Cultured; Cytokines; Disease Models, Animal; Infarction, Middle Cerebral Artery; Myeloid Differentiation Factor 88; Neurons; Neuroprotective Agents; PC12 Cells; Rats; Reperfusion Injury; Signal Transduction; TNF Receptor-Associated Factor 6; Toll-Like Receptor 4

Alzheimer's disease (AD) is a neurodegenerative disease in humans. The accumulation of amyloid-β (Aβ) plays a critical role in the pathogenesis of AD. Previous studies indicated that Salvianolic acid B (SalB) could ameliorate Aβ-induced memory impairment. However, whether SalB could influence the generation of Aβ is unclear. Here, we show that SalB (25, 50, or 100 µM) reduces the generation of Aβ40 and Aβ42 in culture media by decreasing the protein expressions of BACE1 and sAPPβ in SH-SY5Y-APPsw cells. Meanwhile, SalB increases the levels of ADAM10 and sAPPα in the cells. However, SalB has no impact on the protein expressions of APP and PS1. Moreover, SalB attenuates oxidative stress and inhibits the activity of GSK3β, which might be related to the suppression of BACE1 expression and amyloidogenesis. Our study suggests that SalB is a promising therapeutic agent for AD by targeting Aβ generation.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Benzofurans; Cell Line; Gene Expression Regulation, Enzymologic; Humans; Molecular Structure; Salvia miltiorrhiza

Pulmonary fibrosis is a progressive and fatal disorder. In our previous study, we found that the Yiqihuoxue formula (YQHX), a prescription of Traditional Chinese Medicine, had a curative effect on scleroderma, a typical fibrotic disease. The aim of this study was to determine the key ingredient mediating the therapeutic effects of YQHX and to examine its effect on pulmonary fibrosis, including its mechanism. Luciferase reporter assays showed that the most important anti-fibrotic component of the YQHX was Salviae miltiorrhiza (SM). Experiments performed using a bleomycin-instilled mouse model of pulmonary fibrosis showed that Salvianolic acid B (SAB), the major ingredient of SM, had strong anti-inflammatory and anti-fibrotic effects through its inhibition of inflammatory cell infiltration, alveolar structure disruption, and collagen deposition. Furthermore, SAB suppressed TGF-β-induced myofibroblastic differentiation of MRC-5 fibroblasts and TGF-β-mediated epithelial-to-mesenchymal transition of A549 cells by inhibiting both Smad-dependent signaling and the Smad-independent MAPK pathway. Taken together, our results suggest that SM is the key anti-fibrotic component of the YQHX and that SAB, the major ingredient of SM, alleviates experimental pulmonary fibrosis both in vivo and in vitro by inhibiting the TGF-β signaling pathway. Together, these results suggest that SAB potently inhibits pulmonary fibrosis.

Topics: A549 Cells; Animals; Benzofurans; Bleomycin; Bronchoalveolar Lavage Fluid; Cell Differentiation; Cell Proliferation; Disease Models, Animal; Drugs, Chinese Herbal; Fibroblasts; Humans; Medicine, Chinese Traditional; Mice; NIH 3T3 Cells; Pulmonary Fibrosis; Salvia; Signal Transduction; Transforming Growth Factor beta

Contrast-induced acute renal injury (CI-AKI) has become a common cause of hospital-acquired renal failure. However, the development of prophylaxis strategies and approved therapies for CI-AKI is limited. Salvianolic acid B (SB) can treat cardiovascular-related diseases. The aim of the present study was to assess the effect of SB on prevention of CI-AKI and explore its underlying mechanisms. We examined its effectiveness of preventing renal injury in a novel CI-AKI rat model. Compared with saline, intravenous SB pretreatment significantly attenuated elevations in serum creatinine and the histological changes of renal tubular injuries, reduced the number of apoptosis-positive tubular cells, activated Nrf2, and lowered the levels of renal oxidative stress induced by iodinated contrast media. The above renoprotection of SB was abolished by the PI3K inhibitor (wortmannin). In HK-2 cells, SB activated Nrf2 and decreased the levels of oxidative stress induced by hydrogen peroxide and subsequently improved cell viability. The above cytoprotection of SB was blocked by the PI3K inhibitor (wortmannin) or siNrf2. Thus, our results demonstrate that, due to its antioxidant properties, SB has the potential to effectively prevent CI-AKI via the PI3K/Akt/Nrf2 pathway.

Topics: Acute Kidney Injury; Animals; Antioxidants; Apoptosis; Benzofurans; Biomarkers; Cell Line, Tumor; Contrast Media; Creatinine; Cytoprotection; Disease Models, Animal; Iohexol; Kidney Tubules, Proximal; Male; NF-E2-Related Factor 2; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; RNA Interference; Signal Transduction; Transfection

To investigate roles of nitric oxide (NO) signal in accumulations of phenolic acids in abscisic.acid (ABA)-induced Salvia miltiorrhiza hairy roots, S. miltiorrhiza hairy roots were treated with different concentrations of sodium nitroprusside (SNP)-an exogenous NO donor, for 6 days, and contents of phenolic acids in the hairy roots are determined. Then with treatment of ABA and NO scavenger (2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide, c-PTIO) or NO synthase inhibitor (NG-nitro-L-arginine methyl ester, L-NAME), contents of phenolic acids and expression levels of three key genes involved in phenolic acids biosynthesis were detected. Phenolic acids production in S. miltiorrhiza hairy roots was most significantly improved by 100 µmoL/L SNP. Contents of RA and salvianolic acid B increased by 3 and 4 folds. ABA significantly improved transcript levels of PAL (phenylalanine ammonia lyase), TAT (tyrosine aminotransferase) and RAS (rosmarinic acid synthase), and increased phenolic acids accumulations. However, with treatments of ABA+c-PTIO or ABA+L-NAME, accumulations of phenolic acids and expression levels of the three key genes were significantly inhibited. Both NO and ABA can increase accumulations of phenolic acids in S. miltiorrhiza hairy roots. NO signal probably mediates the ABA-induced phenolic acids production.

Topics: Abscisic Acid; Benzofurans; Free Radical Scavengers; Hydroxybenzoates; Nitric Oxide; Phenylalanine Ammonia-Lyase; Plant Roots; Salvia miltiorrhiza; Tyrosine Transaminase

Major depressive disorder (MDD) is a debilitating mental disorder associated with dysfunction of the neurotransmitter-neuroendocrine system and neuroinflammatory responses. Salvianolic acid B (SalB) has shown a variety of pharmacological activities, including anti-inflammatory, antioxidant and neuroprotective effects. In this study, we examined whether SalB produced antidepressant-like actions in a chronic mild stress (CMS) mouse model, and explored the mechanisms underlying the antidepressant-like actions of SalB.. Mice were subjected to a CMS paradigm for 6 weeks. In the last 3 weeks the mice were daily administered SalB (20 mg·kg(-1)·d(-1), ip) or a positive control drug imipramine (20 mg·kg(-1)·d(-1), ip). The depressant-like behaviors were evaluated using the sucrose preference test, the forced swimming test (FST), and the tail suspension test (TST). The gene expression of cytokines in the hippocampus and cortex was analyzed with RT-PCR. Plasma corticosterone (CORT) and cerebral cytokines levels were assayed with an ELISA kit. Neural apoptosis and microglial activation in brain tissues were detected using immunofluorescence staining.. Administration of SalB or imipramine reversed the reduced sucrose preference ratio of CMS-treated mice, and significantly decreased their immobility time in the FST and TST. Administration of SalB significantly decreased the expression of pro-inflammatory cytokines IL-1β and TNF-α, and markedly increased the expression of anti-inflammatory cytokines IL-10 and TGF-β in the hippocampus and cortex of CMS-treated mice, and normalized their elevated plasma CORT levels, whereas administration of imipramine did not significantly affect the imbalance between pro- and anti-inflammatory cytokines in the hippocampus and cortex of CMS-treated mice. Finally, administration of SalB significantly decreased CMS-induced apoptosis and microglia activation in the hippocampus and cortex, whereas administration of imipramine had no significant effect on CMS-induced apoptosis and microglia activation in the hippocampus and cortex.. SalB exerts potent antidepressant-like effects in CMS-induced mouse model of depression, which is associated with the inhibiting microglia-related apoptosis in the hippocampus and the cortex.

Topics: Animals; Apoptosis; Behavior, Animal; Benzofurans; Cerebral Cortex; Corticosterone; Cytokines; Depressive Disorder, Major; Drugs, Chinese Herbal; Gene Expression; Hippocampus; Male; Mice, Inbred C57BL; Microglia; Neuroimmunomodulation; Neurons; Stress, Psychological

To evaluate the stability of salvianolic acid B (Sal B) under ultrasound-assisted extraction in the pharmaceutical industry, degradation of Sal B under ultrasonic irradiation was investigated as the function of buffer concentration, pH, and temperature. With regard to Sal-B concentration, a first-order degradation process was determined, with 10% change in assay from its initial concentration as t90=4.81h, under maximum stability acidic conditions (pH 2.0) and at 25°C. The logkpH-pH profile described by specific acid-base catalysis and water molecules supported the experimental results. Liquid chromatography-mass spectrometry (LC-MS) analyses revealed 7 major degradation products whose structures were characterized by electrospray ionization/mass spectrometry. A primary degradation pathway involved cleavage of the ester bond and ring-opening of benzofuran in Sal B was proposed. The complete degradation pathway of Sal B was also proposed. Results showed that ultrasonic irradiation leads to degradation of Sal B in aqueous solution.

Topics: Benzofurans; Chromatography, Liquid; Drug Stability; Hydrogen-Ion Concentration; Solutions; Spectrometry, Mass, Electrospray Ionization; Temperature; Ultrasonics; Water

A rapid and effective method integrating separation and purification of lithospermic acid B from Salvia miltiorrhiza Bunge was developed by combining an aqueous two-phase system extraction with preparative chromatography. An aqueous two-phase system of n-butyl alcohol/KH2 PO4 was chosen from seven systems. The influence of parameters including concentration of KH2 PO4 , n-butyl alcohol concentration, pH, and the ratio of an aqueous two-phase system to crude extract were investigated using a single factor design. Response surface methodology was subsequently used to find the optimal compositions of an aqueous two-phase system. Keeping a solvent-to-solid ratio of 10, the final optimized composition of an aqueous two-phase system was 39.1% w/w n-butyl alcohol and 22.6% w/w KH2 PO4 . Under these conditions a recovery yield of 99.8% and a high partition coefficient of 310.4 were obtained. In a pilot-scale experiment using optimized conditions, 18.79 g of lithospermic acid B with a purity of 70.5% and in a yield of 99.8% was separated from 0.5 kg of crude extract. Subsequently, 9.94 g lithospermic acid B with a purity of 99.3% and recovery yield of 70.3% was obtained with a preparative chromatographic process, and the two-step total recovery was 70.1%.

Topics: Benzofurans; Chemical Fractionation; Chromatography, High Pressure Liquid; Depsides; Drugs, Chinese Herbal; Salvia miltiorrhiza

Salvianolic acid B (SalB) was demonstrated to be a promising chemopreventive agent for head and neck squamous cell carcinoma (HNSCC) in the previous studies by our and other research institution, but the properties like low efficacy, poor systemic delivery, and low bioavailability has hampered its clinical applications. To continue our research program focused on the use of natural compounds on cancer chemoprevention, we propose a first example of phospholipid complex loaded nanoparticles (PLC-NPs) encapsulating SalB as a potential carrier for intervention of HNSCC (HN13, HN30) cells and precancer Leuk1 cells in this study. Qualitative and quantitive studies of cellular uptake showed that intracellular accumulation of SalB was significantly higher when HN13, HN30 and Leuk1 cells were incubated with SalB-PLC-NPs complex (nano-SalB) as against free-SalB. Cell viability assay revealed that the cell growth of HN13 and HN30 cells was significantly inhibited of 56.1% and 29.3%, respectively, for nano-SalB compared to an equivalent amount of free-SalB (P<0.001). Moreover, cell cycle and apoptosis assay showed that a clear trend of cell cycle arrest and induction of apoptosis was also observed within the HNSCC cells treated with nano-SalB. Collectively, this study demonstrated that nano-SalB was significantly more potent had an anticancer effect against HNSCC cells, which serves as the first step toward establishing SalB nano-formulations as promising cancer chemopreventive agents. The current study could pave a new way for the development of drugs that target HNSCC in the future.

Topics: Antineoplastic Agents; Apoptosis; Benzofurans; Carcinoma, Squamous Cell; Cell Cycle; Cell Proliferation; Drugs, Chinese Herbal; Head and Neck Neoplasms; Humans; Nanoparticles; Precancerous Conditions; Tumor Cells, Cultured

The study investigated the roles and mechanisms of Salvianolic acid B (Sal B) on permeability of rat brain microvascular endothelial cells (RBMECs) exposed to high glucose. The results demonstrated that Sal B greatly up-regulated the expression of tight junction (TJ) proteins and decreased the permeability of RBMECs compared with the control group. And the increase of reactive oxidative species (ROS) production, the upregulation of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) protein induced by high glucose were antagonized by Sal B. In addition, a great decrease of microRNA-200b (miR-200b) was observed in the RBMECs under high-glucose condition, which was significantly increased by Sal B pretreatment. And overexpression of miR-200b markedly attenuated the RBMECs permeability and inhibited the expression of VEGF protein by targeting with 3'-UTR of its mRNA. This led to the conclusion that Sal B-mediated improvement of blood-brain barrier dysfunction induced by high-glucose is related to the ROS/HIF-1α/VEGF and miR-200b/VEGF signaling pathways.

Topics: Animals; Benzofurans; Blood-Brain Barrier; Cells, Cultured; Endothelial Cells; Glucose; Hypoxia-Inducible Factor 1, alpha Subunit; MicroRNAs; Microvessels; Occludin; Rats; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; Up-Regulation; Vascular Endothelial Growth Factor A; Zonula Occludens-1 Protein

The present study was aimed at exploring the protective effects of Salvianolic acid B (SalB) against paraquat (PQ)-induced lung injury in mice. Lung fibrotic injuries were induced in mice by a single intragastrical administration of 300mg/kg PQ, then the mice were administrated with 200mg/kg, 400mg/kg SalB, 100mg/kg vitamin C (Vit C) and dexamethasone (DXM) for 14days. PQ-triggered structure distortion, collagen overproduction, excessive inflammatory infiltration, pro-inflammatory cytokine release, and oxidative stress damages in lung tissues and mortality of mice were attenuated by SalB in a dose-dependent manner. Furthermore, SalB was noted to enhance the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and reduce expression of the reactive oxygen species-generating enzyme Nox4 [NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-4]. SalB also inhibited the increasing expression of transforming growth factor (TGF)-β1 and the phosphorylation of its downstream target Smad3 which were enhanced by PQ. These results suggest that SalB may exert protective effects against PQ-induced lung injury and pulmonary fibrosis. Its mechanisms involve the mediation of Nrf2/Nox4 redox balance and TGF-β1/Smad3 signaling.

Topics: Animals; Benzofurans; Herbicides; Inflammation Mediators; Lung; Male; Mice; NADPH Oxidase 4; NADPH Oxidases; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Paraquat; Smad3 Protein; Transforming Growth Factor beta1

Caffeic acid derivatives (CADs) are well-known phytochemicals with multiple physiological and pharmacological activities. This study aimed to investigate the combined protective effects of CADs on PCB126-induced liver damages and oxidative stress in mice. Here, we used chemiluminescence and chose chlorogenic acid (CGA), salvianolic acid B (Sal B) as the best antioxidants. Then, mice were intragastrically administered with 60mg/kg/d CGA, Sal B, and CGA plus Sal B (1:1) for 3 weeks before exposing to 0.05mg/kg/d PCB126 for 2 weeks. We found that pretreatment with CGA, Sal B, and CGA plus Sal B effectively attenuated liver injury and cytotoxicity caused by PCB126, but improved the expressions of superoxide dismutase (SOD), glutathione reduced (GSH), heme oxygenase-1 (HO-1) and nuclear factor E2-related factor 2 (Nrf2), CGA plus Sal B especially, was found to have the best effects that indicated a synergetic protective effect. Taken together, as the Nrf2 regulates the cyto-protective response by up-regulating the expression of antioxidant genes, we suggested that CGA plus Sal B had a combined protection on PCB126-induced tissue damages and that the Nrf2 signaling might be involved.

Topics: Animals; Antioxidants; Benzofurans; Biomarkers; Chlorogenic Acid; DNA Damage; Drug Synergism; Environmental Pollutants; Heme Oxygenase-1; Liver; Male; Membrane Proteins; Mice, Inbred ICR; Molecular Structure; Oxidative Stress; Polychlorinated Biphenyls

Salvianolic Acid B (Sal B), an active compound extracted from the Chinese herb Salvia miltiorrhiza, is attracting more and more attention due to its biological activities, including antioxidant, anticoagulant and antitumor effects. However, autophagy induction in cancer cells by Sal B has never been recognized. In this study, we demonstrated that Sal B induced cell death and triggered autophagy in HCT116 and HT29 cells in a dose-dependent manner. Specific inhibition of autophagy by 3-MA or shRNA targeting Atg5 rescued Sal B-induced cell death in vitro and in vivo, suggesting that Sal B-induced autophagy may play a pro-death role and contribute to the cell death of colorectal cancer cell lines. Furthermore, AKT/mTOR signaling pathway was demonstrated to be a critical mediator in regulating Sal B-induced cell death. Overexpression of AKT by the transfection with AKT plasmid or pretreatment with insulin decreased Sal B-induced autophagy and cell death. Inversely, inhibition of AKT by LY294002 treatment markedly enhanced Sal B-induced autophagy and cell death. Taken together, our results demonstrate, for the first time, that Sal B is a novel autophagy inducer and exerts its antitumor activity as a single agent in colorectal cancer cells through the suppression of AKT/mTOR pathway.

Topics: Adenine; Animals; Antineoplastic Agents; Autophagosomes; Autophagy; Autophagy-Related Protein 5; Benzofurans; Chromones; Colorectal Neoplasms; Drugs, Chinese Herbal; Female; HCT116 Cells; HT29 Cells; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Morpholines; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Salvia miltiorrhiza; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Our previous studies show that salvianolic acid B (Sal B) promotes osteoblast differentiation and matrix mineralization. In this study, we evaluated the protective effects of Sal B on the osteogenesis in dexamethasone (Dex)-treated larval zebrafish, and elucidated the underlying mechanisms.. At 3 d post fertilization, wild-type AB zebrafish larvae or bone transgenic tg (sp7:egfp) zebrafish larvae were exposed to Sal B, Dex, or a mixture of Dex+Sal B for 6 d. Bone mineralization in AB strain larval zebrafish was assessed with alizarin red staining, and osteoblast differentiation in tg (sp7:egfp) larval zebrafish was examined with fluorescence scanning. The expression of osteoblast-specific genes in the larvae was detected using qRT-PCR assay. The levels of oxidative stress markers (ROS and MDA) in the larvae were also measured.. Exposure to Dex (5-20 μmol/L) dose-dependently decreased the bone mineralization area and integral optical density (IOD) in wild-type AB zebrafish larvae and the osteoblast fluorescence area and IOD in tg (sp7:egfp) zebrafish larvae. Exposure to Dex (10 μmol/L) significantly reduced the expression of osteoblast-specific genes, including runx2a, osteocalcin (OC), alkaline phosphatase (ALP) and osterix (sp7), and increased the accumulation of ROS and MDA in the larvae. Co-exposure to Sal B (0.2-2 μmol/L) dose-dependently increased the bone mineralization area and IOD in AB zebafish larvae and osteoblast fluorescence in tg (sp7:egfp) zebrafish larvae. Co-exposure to Sal B (2 μmol/L) significantly attenuated deleterious alterations in bony tissue and oxidative stress in both Dex-treated AB zebafish larvae and tg (sp7:egfp) zebrafish larvae.. Sal B stimulates bone formation and rescues GC-caused inhibition on osteogenesis in larval zebrafish by counteracting oxidative stress and increasing the expression of osteoblast-specific genes. Thus, Sal B may have protective effects on bone loss trigged by GC.

Topics: Animals; Benzofurans; Dexamethasone; Glucocorticoids; Osteogenesis; Oxidative Stress; Protective Agents; Zebrafish

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. This study was designed to ascertain if miR-30a is involved in the cardioprotective actions of salvianolic acid B (Sal B) against myocardial ischemia-reperfusion (I-R) injury through suppression of autophagy.. Murine myocardial cells that had undergone primary culture were induced by I-R and incubated with Sal B (25, 50, 100 μM) in the presence of a miR-30a mimic or miR-30a inhibitor. Expression of miR-30a, beclin-1, LC3-II and p-Akt protein, cell viability, and lactic acid dehydrogenase (LDH) release were assessed.. miR-30a expression was down-regulated remarkably in I-R cells, and this suppression could be reversed by Sal B in a dose-dependent manner. Sal B repressed autophagy in I-R myocardial cells. Sal B improved cell viability and reduced the rate of LDH leakage, which suggested that autophagy suppression was beneficial for cell survival. Knockdown of miR-30a with a miR-30a inhibitor could reverse the anti-autophagy effect of Sal B against I-R injury. Furthermore, we confirmed that Sal B has a protective role in miR-30a-mediated autophagy through the PI3K/Akt signaling pathway, which was abrogated by the PI3K inhibitor LY294002.. These data suggest that miR-30a is involved in Sal B-mediated cardioprotection against I-R injury through the PI3K/Akt signaling pathway.

Topics: Animals; Autophagy; Benzofurans; Cell Survival; Cells, Cultured; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Myocytes, Cardiac; Phosphatidylinositol 3-Kinases; Reperfusion Injury; Up-Regulation

Topics: Benzofurans; Catechin; Drug Stability; Drugs, Chinese Herbal; Lactates; Proton Magnetic Resonance Spectroscopy; Quality Control; Reproducibility of Results; Salvia miltiorrhiza

Vascular endothelial cell injury is an initial event in atherosclerosis. Salvianolic acid B (Sal B), a main bioactive component in the root of Salvia miltiorrhiza, has vascular protective effect in diabetes, but the underlying mechanisms remain unclear. The present study investigated the effect of Sal B on vascular endothelial function in diabetic rats with blood glucose fluctuations and the possible mechanisms implicated. The results showed that diabetic rats developed marked endothelial dysfunction as exhibited by impaired acetylcholine induced vasodilation. Supplementation with Sal B resulted in an evident improvement of endothelial function. Phosphorylation (Ser 1177) of endothelial nitric oxide synthase (eNOS) was significantly restored in Sal B treated diabetic rats, accompanied by an evident recovery of NO metabolites. Sal B effectively reduced vascular endothelial cell apoptosis, with Bcl-2 protein up-regulated and Bax protein down-regulated markedly. Treatment with Sal B led to an evident amelioration of oxidative stress in diabetic rats as manifested by enhanced antioxidant capacity and decreased contents of malondialdehyde in aortas. Protein levels of NOX2 and NOX4, two main isoforms of NADPH oxidase known as the major source of reactive oxygen species in the vasculature, were markedly decreased in Sal B treated groups. In addition, treatment with Sal B led to an evident decrease of serum lipids. Taken together, this study indicates that Sal B is capable of improving endothelial function in diabetic rats with blood glucose fluctuations, of which the underlying mechanisms might be related to suppression of endothelial cell apoptosis and stimulation of eNOS phosphorylation (Ser 1177).

Topics: Animals; Antioxidants; Aorta; Apoptosis; bcl-2-Associated X Protein; Benzofurans; Blood Glucose; Diabetes Mellitus, Experimental; Endothelial Cells; Gene Expression Regulation; Lipids; Male; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Vasodilation

Porous hydroxyapatite (HA) scaffolds combined with a drug delivery system have attracted much attention for bone tissue engineering. In this study, an easy and highly efficient method was developed to immobilize salvianolic acid B (Sal B)-loaded chitosan (CS) microspheres three dimensionally and homogeneously on the surface of HA scaffolds pre-coated with alginate. Porous HA scaffolds were prepared via a template-leaching process and CS microspheres (used as drug carriers) were fabricated by an emulsion method. To improve adhesion between the microspheres and HA scaffolds, alginate was used to pre-coat the porous surface of the HA scaffolds. Various concentrations of alginate were used to optimize the adhesion of Sal B-loaded CS microspheres to the scaffold surface. During the adherence process, coated HA scaffolds were immersed in an aqueous solution containing Sal B-loaded CS microspheres, followed by standing or shaking at 37 °C for a certain time. The results showed that the microspheres were solidly and homogeneously distributed on the porous surface of the alginate pre-coated HA scaffolds via electrostatic interactions. Few microspheres detached from the porous surface, even after the HA scaffolds with microspheres were treated by shaking in distilled water for as long as 7 d. Compared with the static condition, the distribution of Sal B-loaded CS microspheres on the porous surface of pre-coated HA scaffolds in the shaken condition was more homogeneous and almost unaggregated. Additionally, the compressive strength of the scaffolds coated with alginate was obviously improved. The optimal alginate coating concentration was 1% (i.e. the microstructure of the porous surfaces of the HA scaffolds was almost unchanged). The release profile of Sal B over a 30 d immersion found an initial burst release followed by a sustained release. The result of cell culture in vitro was that 1% alginate-coated scaffolds with Sal B-loaded CS microspheres obviously promoted cell proliferation after cell culture for 3 and 7 d, and cells were attached and uniformly distributed on the porous surface of the scaffolds. The strategy of incorporating drug-loaded microspheres with porous HA scaffolds could provide an excellent bone substitute for repair of bone tissue defects.

Topics: Alginates; Animals; Benzofurans; Bone and Bones; Cell Proliferation; Chitosan; Compressive Strength; Drug Carriers; Durapatite; Glucuronic Acid; Hexuronic Acids; Microspheres; Porosity; Powders; Rats; Tissue Engineering; Tissue Scaffolds

Chinese medicines are emerging as an attractive new generation of anticancer drugs. Here, we explored the impact of salvianolic acid B (Sal B), the major water-soluble compounds of Danshen, on apoptosis and autophagy of human hepatocellular carcinoma cells (HCC). We also investigated the related molecular mechanisms. We found that Sal B exhibits potent ability to inhibit HCC cells viability in a concentration-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, Sal B could also induce autophagy. Furthermore, pretreatment with the autophagy inhibitor chloroquine or 3-methyladenine showed the potential in attenuating the apoptosis rate induced by Sal B. Mechanistically, Sal B treatment inhibited the AKT/mTOR signaling cascade in vitro. Overexpression of AKT abolished the effects of Sal B on HCC cells, suggesting a critical role of the AKT/mTOR signaling pathway in Sal B-induced biological effects. Our results indicated that the mitochondrial pathway was involved in Sal B-induced apoptosis of HCC cells. Moreover, the AKT/mTOR signaling pathway was involved in Sal B-induced autophagy, which promoted apoptosis. This study may provide a promising strategy for using Sal B as a chemotherapeutic agent for patients with HCC.

Topics: Adenine; Antineoplastic Agents; Apoptosis; Autophagy; Beclin-1; Benzofurans; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chloroquine; Drugs, Chinese Herbal; Humans; Liver Neoplasms; Mitochondria; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Salvia miltiorrhiza; Signal Transduction; TOR Serine-Threonine Kinases

Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H₂O₂-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of

Topics: Antioxidants; Benzofurans; Disulfides; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Enzyme Activation; Epithelial Cells; Fetus; Gene Expression Regulation; Glutaredoxins; Glutathione; Humans; Hydrogen Peroxide; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Primary Cell Culture; Retinal Pigment Epithelium; RNA, Small Interfering; Signal Transduction; Time Factors

Salvianolic acid B (Sal B) was newly reported to be able to attenuate fibrosis in the animal model. The aim of the present study was to investigate the effect of the intragastric application of Sal B on the prevention of epidural fibrosis (EF).. Forty healthy adult male Wistar rats were divided into four treatment groups (n = 10 per group): (1) 10 mg/kg Sal B, (2) 30 mg/kg Sal B, (3) 50 mg/kg Sal B and (4) Saline (vehicle treatment, control group). All animals underwent a laminectomy at the lumbar 1-2 (L 1-2) level. After intragastric treatment, all rats were sacrificed at post-operative week 8. The extent of the epidural scar, the regeneration of the vasculature and the expression levels of vascular endothelial growth factor (VEGF) were analysed.. The animals' recovery was uneventful during the experimental period. The extent of the epidural scar, the regeneration of the vasculature and the expression levels of VEGF suggested better outcomes in the Sal B-treated groups. Sal B exerted the ability to prevent the formation of an epidural scar and vascularization at the laminectomy sites. The effects of Sal B were dose-dependent, with the 50 mg/kg Sal B group showing the best outcomes compared with the other groups.. Post-operative intragastric application of Sal B can prevent the formation of epidural scarring. Sal B exerted these effects in a dose-dependent manner, and 50 mg/kg dose was shown to be the best effect in the present study. The results of this study reveal that Sal B could be a potential therapy for EF and valuable for further research.

Topics: Animals; Benzofurans; Cell Proliferation; Disease Models, Animal; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Epidural Space; Fibrosis; Male; Rats; Rats, Wistar

Heart failure(HF) is a dangerous disease that affects millions of patients. Radix Salvia is widely used in Chinese clinics to treat heart diseases. Salvianolic acid B(SalB) is the major active component of Radix Salvia. This study investigated the mechanisms of action and effects of SalB on HF in an experimental mouse model of HF.. We created a mouse model of HF by inducing pressure overload with transverse aortic constriction(TAC) surgery for 2 weeks and compared among 4 study groups: SHAM group (n = 10), TAC group (n = 9), TAC+MET group (metprolol, positive drug treatment, n = 9) and TAC+SalB group (SalB, 240 mg•kg-1•day-1, n = 9). Echocardiography was used to evaluate the dynamic changes in cardiac structure and function in vivo. Plasma brain natriuretic peptide (BNP) concentration was detected by Elisa method. In addition, H9C2 rat cardiomyocytes were cultured and Western blot were implemented to evaluate the phosphorylation of ERK1/2, AKT, and protein expression of GATA4.. SalB significantly inhibited the phosphorylation of Thr202/Tyr204 sites of ERK1/2, but not Ser473 site of AKT, subsequently inhibited protein expression of GATA4 and plasma BNP(P < 0.001), and then inhibited HF at 2 weeks after TAC surgery.. Our data provide a mechanism of inactivating the ERK1/2/GATA4 signaling pathway for SalB inhibition of the TAC-induced HF.

Topics: Animals; Aorta, Thoracic; Benzofurans; Blood Pressure; Cell Line; Disease Models, Animal; Drugs, Chinese Herbal; GATA4 Transcription Factor; Heart Failure; Heart Ventricles; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardium; Myocytes, Cardiac; Natriuretic Peptide, Brain; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

Salvianolic acid B (Sal B), a major polyphenolic compound of Salvia miltiorrhiza Bunge, has been shown to possess potential antidiabetic activities. However, the action mechanism of SalB in type 2 diabetes has not been investigated extensively. The present study was designed to investigate the effects of Sal B on diabetes-related metabolic changes in a spontaneous model of type 2 diabetes, as well as its potential molecular mechanism.. Male C57BL/KsJ-db/db mice were orally treated with Sal B (50 and 100 mg/kg) or metformin (positive drug, 300 mg/kg) for 6 weeks.. Both doses of Sal B significantly decreased fasting blood glucose, serum insulin, triglyceride and free fatty acid levels, reduced hepatic gluconeogenic gene expression and improved insulin intolerance in db/db mice. High dose Sal B also significantly improved glucose intolerance, increased hepatic glycolytic gene expression and muscle glycogen content, and ameliorated histopathological alterations of pancreas, similar to metformin. Sal B treatment resulted in increased phosphorylated AMP-activated protein kinase (p-AMPK) protein expression in skeletal muscle and liver, increased glucose transporter 4 (GLUT4) and glycogen synthase protein expressions in skeletal muscle, and increased peroxisome proliferator-activated receptor alpha (PPARα) and phosphorylated acetyl CoA carboxylase (p-ACC) protein expressions in liver.. Our data suggest that Sal B displays beneficial effects in the prevention and treatment of type 2 diabetes at least in part via modulation of the AMPK pathway.

Topics: AMP-Activated Protein Kinases; Animals; Benzofurans; Body Weight; Dyslipidemias; Gene Expression Regulation; Gluconeogenesis; Glucose; Glucose Intolerance; Glucose Transporter Type 4; Glycogen; Glycogen Synthase; Glycolysis; Hyperglycemia; Hyperinsulinism; Lipids; Liver; Male; Mice, Inbred C57BL; Muscle, Skeletal; Pancreas; Phosphorylation; PPAR alpha; RNA, Messenger; Signal Transduction

The aim of the work was to determine the interactions of a set of anti-cancer compounds with bovine serum albumin (BSA) using a ProteOn XPR36 array biosensor and molecular docking studies. The results revealed that a total of six anti-cancer compounds: gallic acid, doxorubicin, acteoside, salvianolic acid B, echinacoside, and vincristine were able to reversibly bind to the immobilized BSA. The sensorgrams of these six compounds were globally fit to a Langmuir 1:1 interaction model for binding kinetics analysis. There were significant differences in their affinity for BSA, with doxorubicin, the weakest binding compound having 1000-fold less affinity than salvianolic acid B, the strongest binding compound. However, compounds with a similar KD often exhibited markedly different kinetics due to the differences in

Topics: Animals; Antineoplastic Agents; Benzofurans; Binding Sites; Biosensing Techniques; Cattle; Doxorubicin; Gallic Acid; Glucosides; Glycosides; Molecular Docking Simulation; Phenols; Protein Array Analysis; Protein Binding; Serum Albumin, Bovine; Surface Plasmon Resonance; Vincristine

Salvianolic acid B micro-porous osmotic pump controlled release pellets (SalB-CRPs) with suitable in vitro release profiles and good in vitro and in vivo correlation (IVIVC) were developed.. Extrusion-spheronization was used to prepare the starter cores containing SalB/MCC/Kollidon®CL-SF/Flowlac®100 of 30:40:15:15 [w/w, The formulation composition of SalB immediate-release pellets (SalB-IRPs)] and complexed with lactose. The pellets were subsequently coated with Surelease aqueous dispersion to achieve controlled-release properties. Furthermore, a single-dose pharmacokinetics study was carried out in New Zealand White (NZW) rabbits.. In the starter cores, the lactose content was 25% based on the SalB-IRPs constituent. The optimal coating polymer ratio of Surelease aqueous dispersion and polyvinyl alcohol-polyethylene glycol (PVA-PEG) graft copolymer (EC/PVA-PEG) was found to be 70:30 (w/w, %) with a coating weight of 5%. The prepared SalB-CRPs had similar in vitro release under three different pH release mediums. A good IVIVC was characterized by a high coefficient of determination (r=0.9801). The in vivo study indicated that the maximum plasma concentration (Cmax) of SalB-CRPs was decreased, peak concentration time (Tmax) and mean residence time (MRT) were all prolonged, as that of SalB-IRPs. In addition, the area under concentration-time curve from 0 to 24 h (AUC0-24 h) and 0 to infinity (AUC0-∞) were significantly higher, compared with those of SalB-IRPs.. Collectively, these results manifested that SalB-CRPs were likely to be a more suitable formulation in treating cardiovascular disease with improved in vivo retention, decreased plasma drug concentration fluctuation.

Topics: Animals; Benzofurans; Chemistry, Pharmaceutical; Drug Implants; Male; Porosity; Rabbits

To investigate the neuroprotective effects and underlying mechanisms of salvianolic acid B (Sal B) extracted from Salvia miltiorrhiza on hippocampal CA1 neurons in mice with cerebral ischemia reperfusion injury.. Forty male National Institute of Health (NIH) mice were randomly divided into 4 groups with 10 animals each, including the sham group, the model group, the SalB group (SalB 22.5 mg/kg) and the nimodipine (Nim) group (Nim 1 mg/kg). A mouse model of cerebral ischemia and reperfusion injury was established by bilateral carotid artery occlusion for 30 min followed by 24-h reperfusion. The malondialdehyde (MDA) content, the nitric oxide synthase (NOS) activity, the superoxide dismutase (SOD) activity and total antioxidant capability (T-AOC) of the pallium were determined by biochemistry methods. The morphologic changes and Bcl-2 and Bax protein expression in hippocampal CA1 neurons were observed by using hematoxylineosin staining and immunohistochemistry staining, respectively.. In the SalB group, the MDA content and the NOS activity of the pallium in cerebral ischemia-reperfusion mice significantly decreased and the SOD activity and the T-AOC significantly increased, as compared with the model group (P<0.05 or P<0.01). The SalB treatment also rescued neuronal loss (P<0.01) in the hippocampal CA1 region, strongly promoted Bcl-2 protein expression (P<0.01) and inhibited Bax protein expression (P<0.05).. SalB increases the level of antioxidant substances and decreases free radicals production. Moreover, it also improves Bcl-2 expression and reduces Bax expression. SalB may exert the neuroprotective effect through mitochondria-dependent pathway on hippocampal CA1 neurons in mice with cerebral ischemia and reperfusion injury and suggested that SalB represents a promising candidate for the prevention and treatment of ischemic cerebrovascular disease.

Topics: Animals; Antioxidants; bcl-2-Associated X Protein; Benzofurans; Brain Ischemia; CA1 Region, Hippocampal; Cell Count; Immunohistochemistry; Male; Malondialdehyde; Mice; Neurons; Nitric Oxide Synthase; Reperfusion Injury; Superoxide Dismutase

Salvianolic acid B (SAB) is a hydrophilic component isolated from the Chinese herb Salviae miltiorrhizae, which has been used clinically for the treatment of ischemic cardiovascular and cerebrovascular diseases. Platelets-mediated vascular inflammatory response contributes to the initiation and progression of atherosclerosis. In this paper, we focus on the modulating effects of SAB on the inflammatory reaction of endothelial cells triggered by activated platelets. Human umbilical vein endothelial cells (EA.hy926) were pretreated with SAB followed by co-culture with ADP-activated platelets. Adhesion of platelets to endothelial cells was observed by amorphological method. The activation of nuclear factor-kappa B was evaluated by NF-κB p65 nuclear translocation and the protein phosphorylation. A determination of the pro-inflammatory mediators (ICAM-1, IL-1β, IL-6, IL-8, MCP-1) mRNA and protein were also conducted. In addition, the inhibitory effects of SAB on platelets activation were also evaluated using a platelet aggregation assay and assessing the release level of soluble P-selectin. The results showed that SAB dose-dependently inhibited ADP- or α-thrombin-induced human platelets aggregation in platelet rich plasma (PRP) samples, and significantly decreased soluble P-selectin release from both agonists stimulated washed platelets. It was also found that pre-treatment with SAB reduced adhesion of ADP-activated platelets to EA.hy926 cells and inhibited NF-κB activation. In addition, SAB significantly suppressed pro-inflammatory mediators mRNA and protein in EA.hy926 cells in a dose-dependent manner. These results indicated that, in addition to its inhibitory effects on platelets activation, SAB was able to attenuate platelets-mediated inflammatory responses in endothelial cells even if the platelets had already been activated. This anti-inflammatory effect was related to the inhibition of NF-κB activation. Our findings suggest that SAB may be a potential candidate for the treatment of various atherosclerotic diseases.

Topics: Benzofurans; Blood Platelets; Cell Adhesion; Cells, Cultured; Coculture Techniques; Disease Progression; Drugs, Chinese Herbal; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Microscopy, Fluorescence; NF-kappa B; P-Selectin; Plant Preparations; Platelet Aggregation; RNA; Salvia

The herb pair of Salvia miltiorrhiza and Panax notoginseng has widely been used for improving coronary and cerebral circulation in China. However, the exact contribution of the major active components of S. miltiorrhiza and P. notoginseng to cardioprotection is far from clear. In the present study, three representative ingredients, salvianolic acid B (SalB) from S. miltiorrhiza and ginsenoside Rg1 (Rg1) and ginsenoside Rb1 (Rb1) from P. notoginseng, were selected to elucidate the mechanism of the herb pair at the ingredient level.. The purity of SalB, Rg1, and Rb1 was >99%, as detected by high-performance liquid chromatography. Acute myocardial infarction was introduced by ligation of the left anterior descending coronary artery near the main pulmonary artery. Cardiac contractility was detected through a Mikro-tipped catheter, and cardiac infarct size was determined using triphenyltetrazolium chloride stain.. The combination of SalB and Rg1, and not the combination of SalB and Rb1, improved heart contractility in rats with myocardial infarction. The different contributions of Rg1 and Rb1, in combination with SalB, to cardioprotection provides further direction to optimize and modernize the herbal medicines containing S. miltiorrhiza and P. notoginseng.. The combination of SalB and Rg1 may provide potential protection against myocardial infarction.

Topics: Animals; Benzofurans; Cardiotonic Agents; Drug Combinations; Ginsenosides; Male; Myocardial Infarction; Rats; Rats, Wistar

In targeting delivery system research on salvianolic acid B, it's vital but hard to evaluate the tissue distribution for its low concentrations in tissues. So the simple, rapid, selective and sensitive UPLC-MS/MS method was provided hereby to determine the concentration of salvianolic acid B in mice tissues after intravenous administration of salvianolic acid B injections, conventional liposomes and long-circulating liposomes. The UPLC was conducted by a C(18) column with a gradient mobile phase consisting of acetonitrile and water containing 0.1% formic acid. The tandem mass spectrometry was operated in negative-electrospray ionization selected-reaction-monitoring mode, and the optimized characteristic precursor to product ion transition m/z 717.3→519.1 was selected. The biosamples were homogenized and treated with a protein precipitation, which led to an acceptable matrix effect and extraction recovery. The linear calibration curves were plotted in the given concentration ranges. The intra-day and inter-day precisions were less than 13.9% and the accuracies were in the range of 86.3-109.2%. The tissue distribution results determined by UPLC-MS/MS we developed showed that the conventional and long-circulating liposomes we made had succeeded in prolonging the retention time and increasing the level of salvianolic acid B in certain distribution tissues such as liver, kidney and brain.

Topics: Animals; Benzofurans; Calibration; Chromatography, High Pressure Liquid; Injections, Intravenous; Linear Models; Liposomes; Male; Mice; Reference Standards; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tissue Distribution

Salvianolic acid Y (TSL 1), a new phenolic acid with the same planar structure as salvianolic acid B, was isolated from Salvia officinalis. The structural elucidation and stereochemistry determination were achieved by spectroscopic and chemical methods, including 1D, 2D-NMR (1H-1H COSY, HMQC and HMBC) and circular dichroism (CD) experiments. The biosynthesis pathway of salvianolic acid B and salvianolic acid Y (TSL 1) was proposed based on structural analysis. The protection of PC12 cells from injury induced by H2O2 was assessed in vitro using a cell viability assay. Salvianolic acid Y (TSL 1) protected cells from injury by 54.2%, which was significantly higher than salvianolic acid B (35.2%).

Topics: Alkenes; Animals; Benzofurans; Cytoprotection; Drugs, Chinese Herbal; Hydrogen Peroxide; Neuroprotective Agents; PC12 Cells; Polyphenols; Proton Magnetic Resonance Spectroscopy; Rats; Salvia officinalis

Salvia miltiorrhiza Bge. (Labiatae) has been widely used for treating diabetes for centuries. Salvianolic acid B (SalB) is the main bioactive component in Salvia miltiorrhiza; however, its antidiabetic activity and possible mechanism are not yet clear.. To investigate the effects of SalB on glycometabolism, lipid metabolism, insulin resistance, oxidative stress, and glycogen synthesis in type 2 diabetic rat model.. High-fat diet (HFD) and streptozotocin-induced diabetic rats were randomly divided into model group, SalB subgroups (50, 100, and 200 mg/kg), and rosiglitazone group.. Compared with the model group, SalB (100 and 200 mg/kg) significantly decreased blood glucose (by 23.8 and 21.7%; p < 0.05 and p < 0.01) and insulin (by 31.3 and 26.6%; p < 0.05), and increased insulin sensitivity index (by 10.9 and 9.3%; p < 0.05). They also significantly decreased total cholesterol (by 24.9 and 27.9%; p < 0.01), low-density lipoprotein cholesterol (by 56.2 and 64.6%; p < 0.01), non-esterified fatty acids (by 32.1 and 37.9%; p < 0.01), hepatic glycogen (by 41.3 and 60.5%; p < 0.01), and muscle glycogen (by 33.2 and 38.6%; p < 0.05), and increased high-density lipoprotein cholesterol (by 50.0 and 61.4%; p < 0.05 and p < 0.01), which were originally altered by HFD and streptozotocin. In addition, SalB (200 mg/kg) markedly decreased triglyceride and malondialdehyde (by 31.5 and 29.0%; p < 0.05 and p < 0.01), and increased superoxide dismutase (by 56.6%; p < 0.01), which were originally altered by HFD and streptozotocin.. The results indicate that SalB can inhibit symptoms of diabetes mellitus in rats and these effects may partially be correlated with its insulin sensitivity, glycogen synthesis and antioxidant activities.

Topics: Animals; Benzofurans; Blood Glucose; Diabetes Mellitus, Type 2; Diet, High-Fat; Drugs, Chinese Herbal; Male; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Streptozocin

This study aimed to investigate the protective effects of Danshen (Salvia miltiorrhiza) water extract (DSE) and its major phenolic acid components against CYP2E1-mediated paracetamol (APAP)-induced hepatic toxicity.. The protection and underlying mechanisms were detected in CYP2E1 overexpression primary rat hepatocytes by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alamar blue assay, CYP2E1 inhibition assay and glutathione assay.. After APAP treatment, DSE (0.06-1 mg/ml) significantly increased cell viability in MTT assay. Two major components danshensu (8.2-130.5 μm) and salvianolic acid B (Sal B; 3.3-53.5 μm) mainly contributed to this protection, but rosmarinic acid, protocatechuic aldehyde and Sal A did not. Alamar blue assay showed that DSE, danshensu and Sal B maintained mitochondrial metabolic activity. DSE inhibited CYP2E1 (Ki = 1.46 mg/ml) in a mixed mode in rat liver microsomes in vitro; DSE decreased APAP-induced total glutathione depletion and preserved redox status (GSH/GSSG ratio) in hepatocytes. Danshensu and Sal B did not inhibit CYP2E1 or decrease total glutathione depletion, but preserved redox status.. DSE protected hepatocytes against APAP-induced injury via maintenance of mitochondrial metabolic activity, CYP2E1 inhibition, reduction of total glutathione depletion and preservation of redox status. Danshensu and Sal B were mainly responsible for this protection.

Topics: Acetaminophen; Animals; Benzofurans; Cell Survival; Cytochrome P-450 CYP2E1; Drugs, Chinese Herbal; Glutathione; Hepatocytes; Hydroxybenzoates; Lactates; Male; Microsomes, Liver; Oxidative Stress; Plant Extracts; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Water

Cardiovascular diseases are related to many risk factors, such as diabetes, high blood pressure, smoking, and obesity. Myocardial infarction (MI), a cardiovascular disease, is the most common cause of cardiomyocyte death. In MI, hypoxia induces cardiomyocyte apoptosis; in particular, diabetes combined with MI has a synergistic effect that exacerbates cardiomyocyte death. The hypoxia-inducible factor-1α (HIF1α) transcriptional factor and a BH-3 only protein, Bcl-2 adenovirus E1B 19-kDa interacting protein 3 (BNIP3), are known to play fundamental roles in both adaptive and cell death processes in response to hypoxia. In addition, most cardioprotective studies used H9c2 cells that were not beating, so H9c2 cells may not be the best model for testing cardioprotective effects. Embryonic stem cells (ESCs) are pluripotent stem cells that are able to differentiate into several types of cells, including cardiomyocytes. In this study, we reveal a simple method to differentiate ESCs into cardiomyocytes by using poly-d-lysine-coated plates combined with ITS and N2-containing medium and characterized the ESC-derived cardiomyocytes by cardiomyocyte marker staining. The ESC-derived cardiomyocytes were used to investigate the protective effect of salvianolic acid B (Sal-B) in high glucose combined with hypoxic conditions to mimic diabetes patients with ischemia. The results of MTT and TUNEL assays indicate that Sal-B suppresses the apoptotic effect of treatment with high glucose combined with hypoxia in ESC-derived cardiomyocytes. In particular, Sal-B inhibited HIF1α, BNIP3, and cleavage caspase 3 expression levels, thereby suppressing apoptosis. This is the first study to mention the correlation between BNIP3 and Sal-B for cardioprotective effects. In conclusion, we suggest that Sal-B may be suitable for use as a future cardioprotective medicine.

Topics: Animals; Apoptosis; Benzofurans; Caspase 3; Cell Differentiation; Cell Hypoxia; Cells, Cultured; Down-Regulation; Embryonic Stem Cells; Glucose; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Proteins; Mice; Mice, Inbred C57BL; Mitochondrial Proteins; Myocytes, Cardiac; Polylysine; Protective Agents; Rats; Signal Transduction

An ultra high performance liquid chromatography with photodiode array detection method is developed for the simultaneous quantitative determination of five water-soluble compounds including danshensu, protocatechualdehyde, rosmarinic acid, salvianolic acid B, and salvianolic acid A in Salvia miltiorrhiza Bge.. Through method optimization, the five compounds all expressed good linearity (R(2) > 0.9990) in a wide concentration range together with satisfactory accuracy, precision, and stability. Moreover, through qualitative analysis of the chemical fingerprint combined with similarity analysis, hierarchical cluster analysis, principle component analysis, and partial least-squares discriminate analysis, we determined that the 13 batches of Salvia miltiorrhiza Bge. were similar in internal quality and the differences resulted from various cultivation environments, recovery elements, and others. Seen from the results of hierarchical cluster analysis and principle component analysis, the classification of 13 batches was in accordance, and partial least-squares discriminate analysis technique was more suitable than the principle component analysis model to provide a distinct classification of test samples on the basis of their different components. Moreover, a permutation test verified the rationality of partial least-squares discriminate analysis and variable importance plot showed that peaks 37 and 38 were the most significant variables in distinguishing the Salvia miltiorrhiza Bge.. The idea of the quantitative and qualitative analysis of Salvia miltiorrhiza Bge. was convenient, sensitive, and comprehensive, which could be applied to evaluate the quality of more traditional Chinese medicines.

Topics: Benzaldehydes; Benzofurans; Caffeic Acids; Catechols; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Lactates; Least-Squares Analysis; Principal Component Analysis; Rosmarinic Acid; Salvia miltiorrhiza

Acetaminophen (APAP) is used drugs worldwide for treating pain and fever. However, APAP overdose is the principal cause of acute liver failure in Western countries. Salvianolic acid B (SalB), a major water-soluble compound extracted from Radix Salvia miltiorrhiza, has well-known antioxidant and anti-inflammatory actions. We aimed to evaluate the ability of SalB to protect against APAP-induced acute hepatotoxicity by inducing nuclear factor-erythroid-2-related factor 2 (Nrf2) expression. SalB pretreatment ameliorated acute liver injury caused by APAP, as indicated by blood aspartate transaminase levels and histological findings. Moreover, SalB pretreatment increased the expression of Nrf2, Heme oxygenase-1 (HO-1) and glutamate-l-cysteine ligase catalytic subunit (GCLC). Furthermore, the HO-1 inhibitor zinc protoporphyrin and the GCLC inhibitor buthionine sulfoximine reversed the protective effect of SalB. Additionally, siRNA-mediated depletion of Nrf2 reduced the induction of HO-1 and GCLC by SalB, and SalB pretreatment activated the phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC) signaling pathways. Both inhibitors (PI3K and PKC) blocked the protective effect of SalB against APAP-induced cell death, abolishing the SalB-induced Nrf2 activation and decreasing HO-1 and GCLC expression. These results indicated that SalB induces Nrf2, HO-1 and GCLC expression via activation of the PI3K and PKC pathways, thereby protecting against APAP-induced liver injury.

Topics: Acetaminophen; Animals; Anti-Inflammatory Agents; Antioxidants; Benzofurans; Cell Death; Chemical and Drug Induced Liver Injury; Gene Expression; Glutamate-Cysteine Ligase; Heme Oxygenase-1; Hep G2 Cells; Humans; Male; Metabolic Detoxication, Phase II; Mice, Inbred Strains; NF-E2-Related Factor 2; Phosphatidylinositol 3-Kinases; Protein Kinase C; Salvia miltiorrhiza; Signal Transduction; Up-Regulation

Induced pluripotent stem cells (iPSCs) have the potential to differentiate into neural lineages. Salvianolic acid B (Sal B) is a commonly used, traditional Chinese medicine for enhancing neuroprotective effects, and has antioxidant, anti-inflammatory, and antiapoptotic properties. Here, we explore the potential mechanism of Sal B in protecting iPSC-derived neural stem cells (NSCs) against H2O2-induced injury. iPSCs were induced into NSCs, iPSC-derived NSCs were treated with 50 μM Sal B for 24.5 h and 500 μM H2O2 for 24 h. The resulting effects were examined by flow cytometry analysis, quantitative reverse-transcription polymerase chain reaction, and western blotting. Upon H2O2 exposure, Sal B significantly promoted cell viability and stabilization of the mitochondrial membrane potential. Sal B also visibly decreased the cell apoptotic ratio. In addition, Sal B markedly reduced expression of matrix metalloproteinase (MMP)-2 and -9, and phosphospecific signal transducer and activator of transcription 3 (p-STAT3), and increased the level of tissue inhibitor of metalloproteinase (TIMP)-2 in iPSC-derived NSCs induced by H2O2. These results suggest that Sal B protects iPSC-derived NSCs against H2O2-induced oxidative stress. The mechanisms of this stress tolerance may be attributed to modulation of the MMP/TIMP system and inhibition of the STAT3 signaling pathway.

Topics: Antioxidants; Benzofurans; Cell Survival; Down-Regulation; Flow Cytometry; Free Radicals; Humans; Hydrogen Peroxide; Matrix Metalloproteinases; Membrane Potential, Mitochondrial; Neural Stem Cells; Oxidants; Oxidative Stress; Pluripotent Stem Cells; STAT3 Transcription Factor

Salvianolic acid B and rosmarinic acid are two main water-soluble active ingredients from Salvia miltiorrhiza with important pharmacological activities and clinical applications. The interactions between salvianolic acid B (or rosmarinic acid) and bovine serum albumin (BSA) in the presence and absence of gold nanoparticles (Au NPs) with three different sizes were investigated by using biophysical methods for the first time. Experimental results proved that two components quenched the fluorescence of BSA mainly through a static mechanism irrespective of the absence or presence of Au NPs. The presence of Au NPs decreased the binding constants of salvianolic acid B with BSA from 27.82% to 10.08%, while Au NPs increased the affinities of rosmarinic acid for BSA from 0.4% to 14.32%. The conformational change of BSA in the presence of Au NPs (caused by a noncompetitive binding between Au NPs and drugs at different albumin sites) induced changeable affinity and binding distance between drugs and BSA compared with no Au NPs. The competitive experiments revealed that the site I (subdomain IIA) of BSA was the primary binding site for salvianolic acid B and rosmarinic acid. Additionally, two compounds may induce conformational and micro-environmental changes of BSA. The results would provide valuable binding information between salvianolic acid B (or rosmarinic acid) and BSA, and also indicated that the Au NPs could alter the interaction mechanism and binding capability of drugs to BSA, which might be beneficial to understanding the pharmacokinetics and biological activities of the two drugs.

Topics: Animals; Benzofurans; Binding Sites; Cattle; Cinnamates; Depsides; Energy Transfer; Gold; In Vitro Techniques; Metal Nanoparticles; Particle Size; Protein Binding; Protein Conformation; Rosmarinic Acid; Serum Albumin, Bovine; Spectrometry, Fluorescence; Spectrophotometry

1. The aim of this study was to investigate the biotransformation of salvianolic acid B (SAB) by catechol-O-methyltransferase (COMT) and its interaction with levodopa (l-DOPA) methylation in rats. 2. The enzyme kinetics of SAB were studied after incubation with rat COMT. The in vivo SAB and 3-monomethyl-SAB (3-MMS) levels were determined after a single dose of tolcapone with or without SAB administration. For l-DOPA, the effect of SAB inhibition on l-DOPA methylation was studied in vitro. The l-DOPA and 3-O-methyldopa (3-OMD) levels were determined after single and multiple doses of SAB with or without l-DOPA administration. 3. After incubation, we found that SAB was methylated mainly by rat liver and kidney COMT. Tolcapone strongly inhibited the formation of 3-MMS in vitro and in vivo, without any change in the plasma concentration of SAB. Moreover, tolcapone significantly increased the cumulative bile excretion of SAB from 3% to 40% in the rat. SAB inhibited the methylation of l-DOPA with an IC50 value of 2.08 μM in vitro. In vivo, a single intravenous dose of SAB decreased the plasma concentration of 3-OMD, with no obvious effect on the pharmacokinetics of l-DOPA. Multiple doses of SAB given to rats also decreased the plasma concentration of 3-OMD, while SAB increased the plasma concentration of l-DOPA.

Topics: Animals; Benzofurans; Benzophenones; Catechol O-Methyltransferase; Catechol O-Methyltransferase Inhibitors; Dihydroxyphenylalanine; Kinetics; Levodopa; Male; Metabolome; Methylation; Nitrophenols; Organ Specificity; Rats, Sprague-Dawley; Reproducibility of Results; Substrate Specificity; Tolcapone; Tyrosine

Salvia miltiorrhiza Bunge has been reported to possess excellent antifibrotic activity. In this study, we have investigated the effect and mechanism of tanshinone IIA (Tan-IIA), salvianolic acid A (Sal-A) and salvianolic acid B (Sal-B), the important active compounds of Salvia miltiorrhiza Bunge, on areca nut extract (ANE)-induced oral submucous fibrosis (OSF) in vitro. Through human procollagen gene promoter luciferase reporter plasmid assay, hydroxyproline assay, gelatin zymography assay, qRT-PCR, ELISA and Western blot assay, the influence of these three compounds on ANE-stimulated cell viability, collagen accumulation, procollagen gene transcription, MMP-2/-9 activity, MMP-1/-13 and TIMP-1/-2 expression, cytokine secretion and the activation of PI3K/AKT, ERK/JNK/p38 MAPK and TGF-β/Smads pathways were detected. The results showed that Tan-IIA, Sal-A and Sal-B could significantly inhibit the ANE-stimulated abnormal viability and collagen accumulation of mice oral mucosal fibroblasts (MOMFs), inhibit the transcription of procollagen gene COL1A1 and COL3A1, increase MMP-2/-9 activity, decrease TIMP-1/-2 expression and inhibit the transcription and release of CTGF, TGF-β1, IL-6 and TNF-α; Tan-IIA, Sal-A and Sal-B also inhibited the ANE-induced activation of AKT and ERK MAPK pathways in MOMFs and the activation of TGF-β/Smads pathway in HaCaT cells. In conclusion, Tan-IIA, Sal-A and Sal-B possess excellent antifibrotic activity in vitro and can possibly be used to promote the rehabilitation of OSF patients.

Topics: Abietanes; Animals; Areca; Benzofurans; Caffeic Acids; Cell Line; Cell Survival; Collagen; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression Regulation; Humans; In Vitro Techniques; Lactates; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Nuts; Oral Submucous Fibrosis; Plant Exudates; Proto-Oncogene Proteins c-akt; Signal Transduction; Smad Proteins; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta

Salvianolic acids are the most abundant water-soluble compounds extracted from the herb Salvia miltiorrhiza L. (Lamiaceae) with antioxidant and protective effects.. This study evaluates the antidiabetic effect of salvianolic acid B (Sal B) in multiple low-dose streptozotocin (MLDS)-induced diabetes in rat.. Rats were divided into control, Sal B40-treated control, diabetic, Sal B20-, and Sal B40-treated diabetic groups. Sal B was daily administered at doses of 20 or 40 mg/kg (i.p.), started on third day post-STZ injection for 3 weeks. Serum glucose and insulin level and some oxidative stress markers in pancreas were measured in addition to the oral glucose tolerance test (OGTT), histological assessment, and apoptosis determination.. After 3 weeks, treatment of diabetic rats with Sal B20 and Sal B40 caused a significant decrease of the serum glucose (p < 0.05-0.01) and improvement of OGTT. Meanwhile, serum insulin was significantly higher in Sal B20- and Sal B40-treated diabetics (p < 0.01) and treatment of diabetics with Sal B40 significantly lowered malondialdehyde (MDA) (p < 0.05), raised glutathione (GSH) (p < 0.05), and activity of catalase (p < 0.01) with no significant change of nitrite. Furthermore, the number of pancreatic islets (p < 0.05) and their area (p < 0.01) was significantly higher and apoptosis reactivity was significantly lower (p < 0.05) in the Sal B40-treated diabetic group versus diabetics.. Three-week treatment of diabetic rats with Sal B exhibited antidiabetic activity which is partly exerted via attenuation of oxidative stress and apoptosis and augmentation of antioxidant system.

Topics: Animals; Benzofurans; Blood Glucose; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Hypoglycemic Agents; Male; Rats; Rats, Wistar

Acute lung injury (ALI) is among the most common causes of mortality in intensive care units. Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMSCs) may attenuate pulmonary edema. In addition, alveolar epithelial cells type I (ATI) are involved in reducing the alveolar edema in response to ALI. However, the mechanism involved in improving the efficiency of differentiation of MSCs into ATI remains to be elucidated. In the present study, the effect of salvianolic acid B (Sal B) on the differentiation of BMSCs into ATI and the activities of the Wnt signaling pathways were investigated. The BMSCs were supplemented with conditioned medium (CM). The groups were as follows: i) CM group: BMSCs were supplemented with CM; ii) lithium chloride (LiCl) group: BMSCs were supplemented with CM and 5 mM LiCl; iii) Sal B group: BMSCs were supplemented with CM and 10 mM Sal B. The samples were collected and assessed on days 7 and 14. It was revealed that aquaporin (AQP)-5 and T1α were expressed in BMSCs, and induction with LiCl or Sal B increased the expression of AQP-5 and T1α. Furthermore, the Wnt-1 and Wnt-3a signaling pathways were activated during the differentiation of BMSCs into ATI. In conclusion, it was suggested that the promotive effects of Sal B on the differentiation of BMSCs into ATI occurred through the activation of Wnt signaling pathways.

Topics: Animals; Benzofurans; beta Catenin; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Culture Media, Conditioned; Drugs, Chinese Herbal; Male; Mesenchymal Stem Cells; Pulmonary Alveoli; Rats, Sprague-Dawley; Respiratory Mucosa; Salvia; Wnt Proteins; Wnt Signaling Pathway

Cholesterol efflux has been thought to be the main and basic mechanism by which free cholesterol is transferred from extra hepatic cells to the liver or intestine for excretion. Salvianolic acid B (Sal B) has been widely used for the prevention and treatment of atherosclerotic diseases. Here, we sought to investigate the effects of Sal B on the cholesterol efflux in THP-1 macrophages.. After PMA-stimulated THP-1 cells were exposed to 50 mg/L of oxLDL and [(3)H] cholesterol (1.0 μCi/mL) for another 24 h, the effect of Sal B on cholesterol efflux was evaluated in the presence of apoA-1, HDL2 or HDL3. The expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-gamma (PPAR-γ), and liver X receptor-alpha (LXRα) was detected both at protein and mRNA levels in THP-1 cells after the stimulation of Sal B. Meanwhile, specific inhibition of PPAR-γ and LXRα were performed to investigate the mechanism.. The results showed that Sal B significantly accelerated apoA-I- and HDL-mediated cholesterol efflux in both dose- and time-dependent manners. Meanwhile, Sal B treatment also enhanced the expression of ABCA1 at both mRNA and protein levels. Then the data demonstrated that Sal B increased the expression of PPAR-γ and LXRα. And the application of specific agonists and inhibitors of further confirmed that Sal exert the function through PPAR-γ and LXRα.. These results demonstrate that Sal B promotes cholesterol efflux in THP-1 macrophages through ABCA1/PPAR-γ/LXRα pathway.

Topics: Apolipoprotein A-I; ATP Binding Cassette Transporter 1; Benzofurans; Biological Transport, Active; Cell Line; Cholesterol; Drugs, Chinese Herbal; Humans; Lipoproteins, HDL2; Lipoproteins, HDL3; Liver X Receptors; Macrophages; Orphan Nuclear Receptors; PPAR gamma; RNA, Messenger; Up-Regulation

To elucidate the interaction between hydrophilic lithospermic acid B and lipophilic tanshinone II A in rats.. A reliable high-performance liquid chromatography method was adopted for simultaneous determination of lithospermic acid B and tanshinone II A in rat plasma, through which the pharmacokinetic interaction between lithospermic acid B and tanshinone II A by intravenous injection was investigated.. The simultaneous intravenous injection of tanshinone II A and lithospermic acid B significantly altered the pharmacokinetic parameters of both compounds when compared with the individual intravenous administration of each compound. The area under the concentration-time curve of tanshinone II A and lithospermic acid B increased by 18.35 and 59.31%, respectively. The mean retention time of tanshinone II A and lithospermic acid B increased, respectively, from 9.3 to 32.8 h and 20.2 to 49.1 h. The concomitant use of tanshinone II A magnified the volume of distribution at steady state (Vss) and time for the drug in the plasma to reduce the highest concentration by half (t½) of lithospermic acid B, while at the same time the Vss and t½ of tanshinone II A changed significantly in the presence of lithospermic acid B.. Lithospermic acid B and tanshinone II A interact with each other following simultaneous intravenous injection in rats and this observation may expand the clinical use of Danshen (Radix Salviae Miltiorrhizae).

Topics: Abietanes; Animals; Benzofurans; Chromatography, High Pressure Liquid; Depsides; Drug Interactions; Drugs, Chinese Herbal; Male; Rats; Rats, Wistar

Salvianolic acid B (Sal B), a bioactive compound isolated from the Chinese herb Radix Salviae Miltiorrhizae, has been reported to exhibit anti-inflammatory and anti-oxidantive effects. The aim of this study was to investigate the protective effects of Sal B on cigarette smoke (CS)-induced acute lung inflammation. Sal B was given intraperitoneally (i.p.) to mice 1h before CS exposure daily for four consecutive days. Bronchoalveolar lavage fluid (BALF) was collected to assess the levels of inflammatory cytokines and cell counts. Lung tissues were used to analysis pathological changes, total glutathione (GSH), nuclear factor erythroid-2 related factor 2 (Nrf-2), and nuclear factor-kappa B (NF-κB) expression. The results showed that Sal B inhibited CS-induced lung pathological changes, the infiltration of inflammatory cells, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and monocyte chemoattractant protein 1 (MCP-1) productions. Sal B also up-regulated CS-induced total glutathione (GSH) production. Furthermore, Sal B was found to up-regulate Nrf-2, hemeoxygenase1 (HO1) expression and suppress CS-induced NF-κB activation. In conclusion, the current study demonstrated that Sal B exhibited a protective effect on CS-induced lung injury and the possible mechanism was involved in activating Nrf-2 and inhibiting NF-κB activation.

Topics: Animals; Anti-Inflammatory Agents; Benzofurans; Bronchoalveolar Lavage Fluid; Cytokines; Cytoprotection; Disease Models, Animal; Glutathione; Heme Oxygenase-1; Lung; Male; Membrane Proteins; Mice, Inbred C57BL; NF-E2-Related Factor 2; NF-kappa B; Pneumonia; Smoke; Tobacco Smoke Pollution

Silent information regulator 1 (SIRT1), a histone deacetylase, has been suggested to be effective in ischemic brain diseases. Salvianolic acid B (SalB) is a polyphenolic and one of the active components of Salvia miltiorrhiza Bunge. Previous studies suggested that SalB is protective against ischemic stroke. However, the role of SIRT1 in the protective effect of SalB against cerebral ischemia has not been explored. In this study, the rat brain was subjected to middle cerebral artery occlusion (MCAO). Before this surgery, rats were intraperitoneally administrated SalB with or without EX527, a specific SIRT1 inhibitor. The infarct volume, neurological score and brain water content were assessed. In addition, levels of TNF-α and IL-1β in the brain tissues were detected by commercial ELISA kits. And the expression levels of SIRT, Ac-FOXO1, Bcl-2 and Bax were detected by Western blot. The results suggested that SalB exerted a cerebral-protective effect, as shown by reduced infarct volume, lowered brain edema and increased neurological scores. SalB also exerted anti-inflammatory effects as indicated by the decreased TNF-α and IL-1β levels in the brain tissue. Moreover, SalB upregulated the expression of SIRT1 and Bcl-2 and downregulated the expression of Ac-FOXO1 and Bax. These effects of SalB were abolished by EX527 treatment. In summary, our results demonstrate that SalB treatment attenuates brain injury induced by ischemic stoke via reducing apoptosis and inflammation through the activation of SIRT1 signaling.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Benzofurans; Brain; Brain Edema; Brain Ischemia; Carbazoles; Central Nervous System Agents; Disease Models, Animal; Infarction, Middle Cerebral Artery; Inflammation; Male; Neuroprotective Agents; Random Allocation; Rats, Sprague-Dawley; Severity of Illness Index; Sirtuin 1; Stroke; Treatment Outcome

Lipopolysaccharide (LPS) causes microvascular barrier disruption, leading to albumin leakage from microvessels resulting in a range of disastrous sequels. Salvianolic acid B (SalB) is a major water-soluble component derived from Salvia miltiorrhiza. Previous studies showed its potential to attenuate microvascular barrier dysfunction, but the underlying mechanism is not fully understood. The present study was intended to investigate the impact of SalB on endothelial cell barrier in vivo in rat mesenteric venules as well as in vitro in human umbilical vein endothelial cells (HUVECs), aiming at disclosing the mechanism thereof, particularly the role of Src in its action. Male Wistar rats were challenged by infusion of LPS (2 mg/kg/h) through left femoral vein for 90 min. SalB (5 mg/kg/h) was administrated either simultaneously with LPS or 30 min after LPS infusion through the left jugular vein. Vesicles in venular walls were observed by electron microscopy. HUVECs were incubated with LPS with or without SalB. The expression of Zonula occluden-1 (ZO-1), VE-cadherin, caveolin-1 and Src in HUVECs was assessed by Western blot and confocal microscopy, binding of SalB to Src was measured using Surface Plasmon Resonance and BioLayer Interferometry. Treatment with SalB inhibited albumin leakage from rat mesenteric venules and inhibited the increase of vesicle number in venular endothelial cells induced by LPS. In addition, SalB inhibited the degradation of ZO-1, the phosphorylation and redistribution of VE-cadherin, the expression and phosphorylation of caveolin-1, and phosphoirylation of Src in HUVECs exposed to LPS. Furthermore, SalB was found able to bind to Src. This study demonstrates that protection of SalB against microvascular barrier disruption is a process involving both para- and trans-endothelial cell pathway, and highly suggests Src as the key enzyme for SalB to work.

Topics: Albumins; Animals; Benzofurans; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Male; Mesenteric Veins; Phosphorylation; Proto-Oncogene Proteins pp60(c-src); Rats; Rats, Sprague-Dawley; Venules

Danhong injection (DHI) is a widely used Chinese Materia Medica standardized product for the clinical treatment of ischemic encephalopathy and coronary heart disease. The bindings of eight natural components in DHI between bovine serum albumin (BSA) were studied by fluorescence spectroscopy technology and molecular docking. According to the results, the quenching process of salvianolic acid B and hydroxysafflor yellow A was a static quenching procedure through the analysis of quenching data by the Stern-Volmer equation, the modified Stern-Volmer equation, and the modified Scatchard equation. Meanwhile, syringin (Syr) enhanced the fluorescence of BSA, and the data were analyzed using the Lineweaver-Burk equation. Molecular docking suggested that all of these natural components bind to serum albumin at the site I location. Further competitive experiments of SaB confirmed the result of molecular docking studies duo to the displacement of warfarin by SaB. Base on these studies, we selected SaB as a research target because it presented the strongest binding ability to BSA and investigated the influence of the multi-components coexisting in DHI on the interaction between the components of the SaB-BSA binding system. The participation of these natural components in DHI affected the interaction between the components of the SaB-BSA system. Therefore, when DHI is used in mammals, SaB is released from serum albumin more quickly than it is used alone. This work would provide a new experiment basis for revealing the scientific principle of compatibility for Traditional Chinese Medicine.

Topics: Animals; Benzofurans; Cattle; Chalcone; Drugs, Chinese Herbal; Medicine, Chinese Traditional; Molecular Docking Simulation; Quinones; Serum Albumin, Bovine; Spectrometry, Fluorescence

Fuzheng Huayu recipe (FZHY) is a herbal product for the treatment of liver fibrosis approved by the Chinese State Food and Drug Administration (SFDA), but its pharmacokinetics and tissue distribution had not been investigated. In this study, the liver fibrotic model was induced with intraperitoneal injection of dimethylnitrosamine (DMN), and FZHY was given orally to the model and normal rats. The plasma pharmacokinetics and tissue distribution profiles of four major bioactive components from FZHY were analyzed in the normal and fibrotic rat groups using an ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. Results revealed that the bioavailabilities of danshensu (DSS), salvianolic acid B (SAB) and rosmarinic acid (ROS) in liver fibrotic rats increased 1.49, 3.31 and 2.37-fold, respectively, compared to normal rats. There was no obvious difference in the pharmacokinetics of amygdalin (AMY) between the normal and fibrotic rats. The tissue distribution of DSS, SAB, and AMY trended to be mostly in the kidney and lung. The distribution of DSS, SAB, and AMY in liver tissue of the model rats was significantly decreased compared to the normal rats. Significant differences in the pharmacokinetics and tissue distribution profiles of DSS, ROS, SAB and AMY were observed in rats with hepatic fibrosis after oral administration of FZHY. These results provide a meaningful basis for developing a clinical dosage regimen in the treatment of hepatic fibrosis by FZHY.

Topics: Administration, Oral; Amygdalin; Animals; Area Under Curve; Benzofurans; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Drugs, Chinese Herbal; Fibrosis; Kidney; Lactates; Liver Cirrhosis; Lung; Male; Rats; Rats, Wistar; Rosmarinic Acid; Tandem Mass Spectrometry; Tissue Distribution

The difference between three representative components of total salvianolic acids in pharmacodynamic activity were compared by three different pharmacological experiments: HUVECs oxidative damage experiment, 4 items of blood coagulation in vitro experiment in rabbits and experimental myocardial ischemia in rats. And the effects of contribution rate of each component were calculated by multi index comprehensive evaluation method based on CRITIC weights. The contribution rates of salvianolic acid B, rosmarinic acid and Danshensu were 28.85%, 30.11%, 41.04%. Apparent oil/water partition coefficient of each representative components of total salvianolic acids in n-octyl alcohol-buffer was tested and the total salvianolic acid components were characterized based on a combination of the approach of self-defined weighting coefficient with effects of contribution rate. Apparent oil/water partition coefficient of total salvianolic acids was 0.32, 1.06, 0.89, 0.98, 0.90, 0.13, 0.02, 0.20, 0.56 when in octanol-water/pH 1.2 dilute hydrochloric acid solution/ pH 2.0, 2.5, 5.0, 5.8, 6.8, 7.4, 7.8 phosphate buffer solution. It provides a certain reference for the characterization of components.

Topics: Animals; Benzofurans; Cinnamates; Depsides; Lactates; Male; Rabbits; Rats; Rats, Sprague-Dawley; Rosmarinic Acid; Solubility

To investigate the effect of Salvianolic acid B (Sal B) on the disease progress of NASH and change of intestinal barrier function.. Sixty Sprague-Dawley (SD) rats were randomly divided into control group, model group and treated group, with the former given normal diet and the latter 2 groups rats fed high-fat diet. In treated group, rats were infused through the stomach with 1 mg/ml Sal B every day at a dose of 20 mL/kg body weight. All animals were killed at the 24th week and plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), endotoxin (ET) and diamine oxdase (DAO) were analyzed using the blood samples. The histopathology of liver was observed by H&E staining. The expression changes of tight junction protein occludin and ZO-1 were analyzed by immunocytochemistry. Ultrastructural morphology of small intestinal tissues was investigated by transmission electron microscopy.. Plasma levels of ALT, AST, TG, TC, ET and DAO were significantly higher in model group than those in both control group and group treated with Sal B. In model group, vacuolated swelling of the cytoplasm with aggregates of chronic inflammatory cells was observed in the liver tissue but not in Sal B-treated group. NAFLD Activity Score in the treated group was significantly lower than that in model group. Immunohistochemical staining showed that Sal B administration recovered the expression of occludin and ZO-1, which was downregulated in the model group. Transmission electron microscopy analysis demonstrated that cell surface microvilli and major intercellular junctional complex including tight junction, gap junction and adherens junction were restored in Sal B-treated group.. Sal B exerted protective function against high-fat diet-induced liver damage by restoring healthy barrier function of intestine in NASH rat model.

Topics: Animals; Benzofurans; Biomarkers; Cytoprotection; Diet, High-Fat; Disease Models, Animal; Disease Progression; Immunohistochemistry; Intestinal Mucosa; Intestine, Small; Liver; Microscopy, Electron, Transmission; Non-alcoholic Fatty Liver Disease; Occludin; Permeability; Rats, Sprague-Dawley; Tight Junctions; Time Factors; Zonula Occludens-1 Protein

Salvia miltiorrhiza Bge. var. alba C.Y. Wu and H.W. Li has wide prospects in clinical practice. A useful comprehensive method was developed for the quality evaluation of S. miltiorrhiza var. alba by three quantitative parameters: high-performance liquid chromatography fingerprint, ten-component contents, and antioxidant activity. The established method was validated for linearity, precision, repeatability, stability, and recovery. Principal components analysis and hierarchical clustering analysis were both used to evaluate the quality of the samples from different origins. The results showed that there were category discrepancies in quality of S. miltiorrhiza var. alba samples according to the three quantitative parameters. Multivariate linear regression was adopted to explore the relationship between components and antioxidant activity. Three constituents, namely, danshensu, rosmarinic acid, and salvianolic acid B, significantly correlated with antioxidant activity, and were successfully elucidated by the optimized multivariate linear regression model. The combined use of high-performance liquid chromatography fingerprint analysis, simultaneous multicomponent quantitative analysis, and antioxidant activity for the quality evaluation of S. miltiorrhiza var. alba is a reliable, comprehensive, and promising approach, which might provide a valuable reference for other herbal products in general to improve their quality control.

Topics: Antioxidants; Benzofurans; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Drugs, Chinese Herbal; Humans; Lactates; Principal Component Analysis; Quality Control; Rosmarinic Acid; Salvia miltiorrhiza

Epithelial-mesenchymal transition (EMT) was reported to be involved in the activation of hepatic stellate cells (HSCs), contributing to the development of liver fibrosis. Epithelial-mesenchymal transition can be promoted by the Hedgehog (Hh) pathway. Patched1 (PTCH1), a negative regulatory factor of the Hh signalling pathway, was down-regulated during liver fibrosis and associated with its hypermethylation status. MicroRNAs (miRNAs) are reported to play a critical role in the control of various HSCs functions. However, miRNA-mediated epigenetic regulations in EMT during liver fibrosis are seldom studied. In this study, Salvianolic acid B (Sal B) suppressed the activation of HSCs in CCl4 -treated mice and mouse primary HSCs, leading to inhibition of cell proliferation, type I collagen and alpha-smooth muscle actin. We demonstrated that the antifibrotic effects caused by Sal B were, at least in part, via inhibition of EMT and the Hh pathway. In particular, up-regulation of PTCH1 was associated with decreased DNA methylation level after Sal B treatment. Accordingly, DNA methyltransferase 1 (DNMT1) was attenuated by Sal B in vivo and in vitro. The knockdown of DNMT1 in Sal B-treated HSCs enhanced PTCH1 expression and its demethylation level. Interestingly, increased miR-152 in Sal B-treated cells was responsible for the hypomethylation of PTCH1 by Sal B. As confirmed by the luciferase activity assay, DNMT1 was a direct target of miR-152. Further studies showed that the miR-152 inhibitor reversed Sal B-mediated PTCH1 up-regulation and DNMT1 down-regulation. Collectively, miR-152 induced by Sal B, contributed to DNMT1 down-regulation and epigenetically regulated PTCH1, resulting in the inhibition of EMT in liver fibrosis.

Topics: Animals; Benzofurans; Epigenesis, Genetic; Liver Cirrhosis; Methylation; Mice; MicroRNAs; Patched-1 Receptor; Repressor Proteins

Lack of pharmacological strategies in clinics restricts the patient prognosis with myocardial ischemia/reperfusion (I/R) injury. The aim of this study was to evaluate the cardioprotection of combined salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1) against myocardial I/R injury and further investigate the underlying mechanism. I/R injury was induced by coronary artery ligation for Wistar male rats and hypoxia/reoxygenation injury was induced on H9c2 cells. Firstly, the best ratio between SalB and Rg1was set as 2:5 based on their effects on heart function detected by hemodynamic measurement. Then SalB-Rg1 (2:5) was found to maintain mitochondrial membrane potential and resist apoptosis and necrosis in H9c2 cell with hypoxia/reoxygenation injury. Companying with same dose of SalB or Rg1 only, SalB-Rg1 showed more significant effects on down-regulation of myocardial infarct size, maintenance of myocardium structure, improvement on cardiac function, decrease of cytokine secretion including TNF-α, IL-1β, RANTES and sVCAM-1. Finally, the SalB-Rg1 improved the viability of cardiac myocytes other than cardiac fibroblasts in rats with I/R injury using flow cytometry. Our results revealed that SalB-Rg1 was a promising strategy to prevent myocardial I/R injury.

Topics: Animals; Apoptosis; Benzofurans; Cardiotonic Agents; Chemokine CCL5; Down-Regulation; Drug Therapy, Combination; Fibroblasts; Ginsenosides; Interleukin-1beta; Male; Membrane Potential, Mitochondrial; Myocardial Reperfusion Injury; Myocytes, Cardiac; Necrosis; Rats; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

A study was made on the pharmacokinetic regularity of effective components salvianolic acid B and ferulic acid in Salviae Miltiorrhizae Radix et Rhizoma (SMRR) and Chuanxiong Rhizoma(CR) in rats, so as to discuss the compatibility mechanism of Salviae Miltiorrhizae Radix et Rhizoma and Chuanxiong Rhizoma. Rats were randomly divided into three groups and intravenously injected with 50 mg x kg(-1) salvianolic acid B for the single SMRR extracts group, 0.5 mg x kg(-1) ferulic acid for the single CR extracts group and 50 mg x kg(-1) salvianolic acid B + 0.5 mg x kg(-1) ferulic acid for the SMRR and CR combination group. The blood samples were collected at different time points and purified by liquid-liquid extraction with ethyl acetate. With chloramphenicol as internal standard (IS), UPLC was adopted to determine concentrations of salvianolic acid B and ferulic acid. The pharmacokinetic parameters of salvianolic acid B and ferulic acid were calculated with WinNonlin 6.2 software and analyzed by SPSS 19.0 statistical software. The UPLC analysis method was adopted to determine salvianolic acid B and ferulic acid in rat plasma, including linear equation, stability, repeatability, precision and recovery. The established sample processing and analysis methods were stable and reliable, with significant differences in major pharmacokinetic parameters, e.g., area under the curve (AUC), mean residence time (MRT) and terminal half-life (t(1/2)). According to the experimental results, the combined application of SMRR and CR can significantly impact the pharmacokinetic process of their effective components in rats and promote the wide distribution, shorten the action time and prolong the in vivo action time of salvianolic acid B and increase the blood drug concentration and accelerate the clearance of ferulic acid in vivo.

Topics: Animals; Apiaceae; Benzofurans; Coumaric Acids; Drug Interactions; Drugs, Chinese Herbal; Male; Rats; Rats, Sprague-Dawley; Rhizome; Salvia miltiorrhiza

To investigate the effects of salvianolic acid B (Sal B) on the morphological characteristics and functions of liver mitochondria of rats with nonalcoholic steatohepatitis (NASH).. A total of 60 male Sprague-Dawley rats were randomly divided into three groups: (1) a normal group fed a normal diet; (2) an NASH model group; and (3) a Sal B-treated group fed a high-fat diet. Two rats from each group were executed at the end of the 12th week to detect pathological changes. The rats in the Sal B-treated group were gavaged with 20 mL/kg Sal B (1 mg/mL) daily. The model group received an equal volume of distilled water as a control. At the end of the 24th weekend, the remaining rats were executed. Serum biochemical parameters and liver histological characteristics were observed. Malondialdehyde (MDA) and superoxide dismutase (SOD) in the liver were determined. Protein expression of CytC and caspase-3 was determined by immunohistochemistry. The mRNA transcripts of mitofusin-2 (Mfn2) and NF-κB in the liver tissue were detected by real-time PCR. Mitochondrial membrane potential was detected using a fluorescence spectrophotometer. Mitochondrial respiratory function was detected using a Clark oxygen electrode.. The model group showed significantly higher ALT, AST, TG, TC and MDA but significantly lower SOD than the normal group. In the model group, the histological characteristics of inflammation and steatosis were also evident; mitochondrial swelling and crest were shortened or even disappeared. CytC (18.46 ± 1.21 vs 60.01 ± 3.43, P < 0.01) and caspase-3 protein expression (30.26 ± 2.56 vs 83.31 ± 5.12, P < 0.01) increased significantly. The mRNA expression of NF-κB increased (0.81 ± 0.02 vs 0.91 ± 0.03, P < 0.05), whereas the mRNA expression of Mfn2 decreased (1.65 ± 0.31 vs 0.83 ± 0.16, P < 0.05). Mitochondrial membrane potential also decreased and breathing of rats was weakened. Steatosis and inflammation degrees in the treatment group were significantly alleviated compared with those of the model group. In the treatment group, mitochondrial swelling was alleviated. CytC (60.01 ± 3.43 vs 30.52 ± 2.01, P < 0.01) and caspase-3 protein expression (83.31 ± 5.12 vs 40.15 ± 3.26, P < 0.01) significantly decreased. The mRNA expression of NF-κB also decreased (0.91 ± 0.03 vs 0.74 ± 0.02, P < 0.01), whereas the mRNA expression of Mfn2 increased (0.83 ± 0.16 vs 1.35 ± 0.23, P < 0.01). Mitochondrial membrane potential increased and respiratory function was enhanced.. Sal B can treat NASH by protecting the morphological characteristics and functions of liver mitochondria, regulating lipid metabolism, controlling oxidative stress and lipid peroxidation and inhibiting apoptosis.

Topics: Animals; Apoptosis; Benzofurans; Biomarkers; Caspase 3; Cytochromes c; Diet, High-Fat; Disease Models, Animal; GTP Phosphohydrolases; Lipid Peroxidation; Liver; Male; Malondialdehyde; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria, Liver; Mitochondrial Proteins; Mitochondrial Swelling; NF-kappa B; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Rats, Sprague-Dawley; Superoxide Dismutase

Salvianolic acid B (Sal B) is a bioactive water-soluble compound of Salviae miltiorrhizae, a traditional herbal medicine that has been used clinically for the treatment of cardiovascular diseases. This study sought to evaluate the effect of Sal B on matrix metalloproteinase-9 (MMP-9) and on the underlying mechanisms in tumor necrosis factor-α± (TNF-α±)-activated human coronary artery endothelial cells (HCAECs), a cell model of Kawasaki disease.. HCAECs were pretreated with 1-10 αμmol/L of Sal B, and then stimulated by TNF-α± at different time points. The protein expression and activity of MMP-9 were determined by Western blot assay and gelatin zymogram assay, respectively. Nuclear factor-κB (NF-κB) activation was detected with immunofluorescence, electrophoretic mobility shift assay, and Western blot assay. Protein expression levels of mitogen-activated protein kinase (c-Jun N-terminal kinase [JNK], extra-cellular signal-regulated kinase [ERK], and p38) were determined by Western blot assay.. After HCAECs were exposed to TNF-α±, 1-10 αμmol/L Sal B significantly inhibited TNF-α±-induced MMP-9 expression and activity. Furthermore, Sal B significantly decreased IκBα± phosphorylation and p65 nuclear translocation in HCAECs stimulated with TNF-α± for 30 min. In addition, Sal B decreased the phosphorylation of JNK and ERK1/2 proteins in cells treated with TNF-α± for 10 min.. The data suggested that Sal B suppressed TNF-α±-induced MMP-9 expression and activity by blocking the activation of NF-κB, JNK, and ERK1/2 signaling pathways.

Topics: Benzofurans; Blotting, Western; Cell Line; Cell Survival; Coronary Vessels; Endothelial Cells; Humans; Matrix Metalloproteinase 9; NF-kappa B; Tumor Necrosis Factor-alpha

To establish the HPLC fingerprint of water-soluble components of Salvia miltiorrhiza in Songtao, Guizhou, and to perform simultaneous determination of six components in it, so as to provide analytical method for its quality control.. The analyses were performed on a Phenomenex Luna C18 (2) (250 mm x 4. 6 mm, 5µm) column eluted with 0. 4% formic acid(A) - acetonitrile(B) in a gradient mode. The flow rate was 1. 0 mL/min, column temperature was set at 30 °C.. Eleven common peaks were identified form the HPLC fingerprint of Salvia miltiorrhiza from 10 batches, the HPLC fingerprint similarities of 10 batches were not less than 0. 999. The linear ranges of danshensu, protocatechuic aldehyde, caffeic acid, rosmarinic acid, lithospermic acid and salvianolic acid B were 0. 0680 ~ 1. 3583 mg/mL, 0. 0008 ~ 0. 3967 mg/mL, 0. 0005 ~ 0. 2660 mg/mL, 0. 0020 ~ 0. 3992 mg/mL, 0. 0063 ~ 0. 6311 mg/mL and 0. 0097 ~ 1. 9306 mg/mL with r ≥ 0. 9999, respectively. The recovery rates were 100. 84%,102. 44%, 100. 53% ,100. 63%, 100. 83% and 100. 35% with RSD <2. 3%, respectively.. The established method is simple, accurate and can provide reference for quality control of Salvia miltiorrhiza.

Topics: Benzaldehydes; Benzofurans; Caffeic Acids; Catechols; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Drugs, Chinese Herbal; Lactates; Phytochemicals; Quality Control; Rosmarinic Acid; Salvia miltiorrhiza; Water

The inflammatory mediator high-mobility group box 1 (HMGB1) plays a critical role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the regulation of HMGB1 in NAFLD, particularly through sirtuin 1 (SIRT1), remains unclear. In this study, we investigated the role of SIRT1-mediated inhibition of HMGB1 release in NAFLD and the effect of salvianolic acid B (SalB), which is a water-soluble phenolic acid extracted from Radix Salvia miltiorrhiza, on NAFLD through SIRT1/HMGB1 signaling. In vivo, SalB treatment significantly attenuated high-fat diet (HFD)-induced liver damage, hepatic steatosis, and inflammation. Importantly, SalB significantly inhibited HMGB1 nuclear translocation and release, accompanied by SIRT1 elevation. In HepG2 cells, palmitic acid (PA)-induced pro-inflammatory cytokines release were blocked by HMGB1 small interfering RNA (siRNA) transfection. Moreover, pharmacological SIRT1 inhibition by Ex527 induced HMGB1 translocation and release, whereas SIRT1 activation by resveratrol or SalB reversed this trend. SIRT1 siRNA abrogated the SalB-mediated inhibition of HMGB1 acetylation and release, suggesting that SalB-mediated protection occurs by SIRT1 targeting HMGB1 for deacetylation. We are the first to demonstrate that the SIRT1/HMGB1 pathway is a key therapeutic target for controlling NAFLD inflammation and that SalB confers protection against HFD- and PA-induced hepatic steatosis and inflammation through SIRT1-mediated HMGB1 deacetylation.

Topics: Animals; Benzofurans; Cytokines; Diet, High-Fat; Hep G2 Cells; HMGB1 Protein; Humans; Liver; Male; Non-alcoholic Fatty Liver Disease; Palmitic Acid; Protective Agents; Rats; Rats, Sprague-Dawley; Resveratrol; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirtuin 1; Stilbenes; Up-Regulation

To optimize the method in the Chinese Pharmacopoeia for determining Salviae Miltiorrhizae Radix et Rhizoma.. Tanshinone II(A) and salvianolic acid B were selected as the index in optimization of the sample preparation method of Salviae Miltiorrhizae Radix et Rhizoma in Chinese Pharmacopoeia. Orthogonal test was used to optimize the extraction process of Salviae Miltiorrhizae Radix et Rhizoma, and concentration of contents were detected by high performance liquid chromatography method. A detection of using methanol-water (85: 15) at wavelength of 270 nm was employed for tanshinone II(A) and a detection of using methanol-acetonitrile-formic acid-water (30:10:1: 59) at wavelength of 286 nm was employed for salvianolic acid B.. The optimized extraction process of tanshinone II(A) and salvianolic acid B was: extracted by 90% methanol and reflux twice (0.5 h each time) at 75 degrees C, extracted by 70% methanol and reflux twice (1.5 h each time) at 75 degrees C, respectively.. Optimized extraction and determination methods could be used to reflect the content of tanshinone II(A) and salvianolic acid B in Salviae Miltiorrhizae Radix et Rhizoma more accurately and efficiently.

Topics: Abietanes; Benzofurans; Chromatography, High Pressure Liquid; Rhizome; Salvia miltiorrhiza; Temperature

To develop an ophthalmic preparation of Shedan, an in situ forming gel was prepared with the formulation containing 18% of poloxamer 407 and 5% of poloxamer 188 by response surface designs plus central composite designs. The rheology results showed that LVE range gamma should limited within 0.5%, Shedan high-frequency region, and the thixotropy recovery time is less than 5 seconds. The phase transition temperature was 33.25 °C according to curve of storage modulus and loss modulus determined by temperature scanning. Surface tension and osmometer of it determined by surface tension meter and dew point osmometer were 36.43 mN · m(-1), and 320.6 mOsm · kg(-1), respectively. Fluorescein sodium was selected as the marker to monitor the corneal residence time, and the results showed that Shedan gel could prolong drug residence for 180 min. In line with zero-order kinetics, releases of muscone and salvianolic acid B in vitro depends on gels erosion. The results of rabbit ocular irritation experiments suggested that Shedan in situ forming gel was biocompatible and nonirritant. In conclusion, a novel Shedan in situ forming gel was developed and characterized for potential drug treatment of retinal vein occlusion.

Topics: Animals; Benzofurans; Cycloparaffins; Female; Gels; Male; Ophthalmic Solutions; Poloxamer; Rabbits; Retinal Vein Occlusion; Viscosity

Our previous study indicated that the combination of salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1), the main components of Salvia miltiorrhizae and Panax notoginseng, improves myocardium structure and ventricular function in rats with ischemia/reperfusion injury. The present study aimed to determine the safety of the combined SalB and Rg1 (SalB-Rg1) in mice. The safety of SalB-Rg1 was evaluated through acute toxicity and repeated-dose toxicity. In the acute toxicity study, the up and down procedure was carried out firstly, and then, the Bliss method was applied. In the toxicity study for seven-day repeated treatment of SalB-Rg1, forty Kunming mice were randomly divided into four groups. The intravenous median lethal dose (LD50) of the SalB-Rg1 combination was 1747 mg/kg using the Bliss method. For both the acute toxicity study and the seven-day repeated toxicity study, SalB-Rg1 did not induce significant abnormality on brain, heart, kidney, liver and lung structure at any dose based on H&E stain. There were no significant changes related to the SalB-Rg1 toxicity detected on biochemical parameters for two kinds of toxicity studies. The LD50 in mice was 1747 mg/kg, which was more than one hundred times higher than the effective dose. Both studies of acute toxicity and seven-day repeated dose toxicity indicated the safety of the SalB-Rg1 combination.

Topics: Animals; Benzofurans; Cardiovascular Agents; Drug Combinations; Drug Evaluation, Preclinical; Female; Ginsenosides; Lethal Dose 50; Male; Mice

The change of yield and contents. of active compositions were studied while the fibrous roots were decayed naturally. HPLC method was used to detect the contents of active composition. The results show that fibrousroots could decrease the production of plant by 38.60% (20 g) and 30.99% (40 g), respectively. Treatment 1 could increase the contents of dihydrotanshinone and cryptotanshinone of Salvia miltiorrhiza f. alba by 26.08% and 22.64%, respectively. Compared with the comparison, treatment 2 decreased the contents of ihydrotanshinone, cryptotanshinone, tanshinone I and tanshinone II(A) of S. miltiorrhiza f. alba by 60.87%, 79.24%, 84.61% and 88.99%, respectively. Meanwhile, the total contents of the liposoluble constituents reduced by 86.27%. The different concentration of fibrousroots could increase the content of salvianolic acid B by 4.98% (20 g) and 23.64% (40 g), respectively. Meanwhile, the content of rosemary acid was increased by 4.98% (20 g) and 23.64% (40 g), respectively. The content of water-soluble constituents positively correlated to the mount of added fibrousroots, and the change was significantly. The result indicted that the decay of fibrousroots has a significant impact on the growth and the content of the active composition of S. miltiorrhiza f. alba under the condition of continuous cropping. Fibrousroots could decrease the content of biomass and liposoluble constituents significantly, which maybe one of the main factors to S. miltiorrhiza f. alba continuous cropping obstacle formation.

Topics: Abietanes; Benzofurans; Biomass; Chromatography, High Pressure Liquid; Plant Roots; Salvia miltiorrhiza

A simple and reliable method for distinguishing Danshen is important to evaluate the quality and clinical efficiency of these species. An HPLC method was developed for the determination of protocatechuic aldehyde, salvianolic acid A, salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA in 23 samples of Salvia. The analytes were separated on an Agilent XDB C18 reversed-phase column coupled with a Phenomenex C18 guard column using a gradient elution of acetonitile-0.1% aqueous phosphoric acid as the mobile phase at a flow rate 0.8 mL/min and UV detection at 280 nm. The method allowing the simultaneous quantification of six major active compositions was optimized and validated for linearity, precision, accuracy and limits of detection (LOD) and quantification. The LOD ranged from 0.019 to 0.850 µg/mL (R(2) ≥ 0.9998). Accuracy, precision and reproducibility were all within the required limits. The average recovery between 96.49 and 102.16% and the relative standard deviations were <3.01%. Based on the six compositions content and clustering result, this research results suggest that these six major active compositions could be distinguishing markers for Danshen and non-Danshen.

Topics: Abietanes; Benzofurans; Caffeic Acids; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Lactates; Limit of Detection; Phenanthrenes; Reproducibility of Results; Salvia; Salvia miltiorrhiza

Salvianolic Acid B (Sal B) is one of the main medicinal ingredients of Radix Salvia miltiorrhiza (Danshen) and possesses a variety of pharmacological effects. The purpose of this study was to discover the new mechanism of action of Sal B based on the protein interaction network (PIN) analysis. A PIN of Sal B was constructed with 852 nodes and 8,626 interactions. By fast agglomerate algorithm based on the edge clustering coefficients (FAG-EC), 11 modules were detected from the network. Gene ontology (GO) enrichment analysis of the modules demonstrated that the roles of Sal B played in cardiovascular disease were related to multiple biological processes, which could represent the characteristics of Chinese Material Medica (CMM) as a whole to regulate the disease. The most interesting finding of this work was that the anti-inflammatory effect of Sal B was due to the immune response of T lymphocytes by regulating IL-2 family, CD3E, CD79A, MAP3K7 and PRKCQ. Therefore, the module-based network analysis will be an effective method for better understanding CMM.

Topics: Algorithms; Anti-Inflammatory Agents; Benzofurans; Cardiovascular Diseases; Cluster Analysis; Computational Biology; Databases, Factual; Drugs, Chinese Herbal; Humans; Models, Biological; Plant Extracts; Protein Interaction Mapping; Salvia miltiorrhiza; T-Lymphocytes; Technology, Pharmaceutical

Rosmarinic acid and salvianolic acid B are two important phenolic compounds with therapeutic properties in Salvia miltiorrhiza Bunge. The biosynthesis of rosmarinic acid is initiated by two parallel pathways, namely the phenylpropanoid pathway and the tyrosine-derived pathway. Salvianolic acid B is a structural dimer of rosmarinic acid and is believed to be derived from rosmarinic acid. In the current study, methyl jasmonate (MeJA) and hyphal extracts from fungi were used as elicitors to examine the relationship between enzymes in the two parallel pathways and accumulation of phenolic compounds in S. miltiorrhiza hairy root cultures. The results showed that accumulations of rosmarinic acid, salvianolic acid B and total phenolics were enhanced by MeJA while suppressed by fugal extracts. Responses of enzymes in the tyrosine-derived pathway, at both the gene transcript and enzyme activity levels, showed a better consistency with alterations of phenolic compounds content after the two elicitors treated. Our study implied that compared with enzymes in the phenylpropanoid pathway, enzymes in the tyrosine-derived pathway are more correlated to rosmarinic acid and salvianolic acid B biosynthesis in S. miltiorrhiza hairy roots.

Topics: Acetates; Benzofurans; Biomass; Biosynthetic Pathways; Cinnamates; Cyclopentanes; Depsides; Gene Expression Regulation, Plant; Hyphae; Oxylipins; Phenols; Phenylalanine Ammonia-Lyase; Plant Roots; Rosmarinic Acid; Salvia miltiorrhiza; Time Factors; Trans-Cinnamate 4-Monooxygenase; Tyrosine; Tyrosine Transaminase

To produce beneficial phenolic acids for medical and commercial purposes, researchers are interested in improving the normally low levels of salvianolic acid B (Sal B) produced by Salvia miltiorrhiza. Here, we present a strategy of combinational genetic manipulation to enrich the precursors available for Sal B biosynthesis. This approach, involving the lignin pathway, requires simultaneous, ectopic expression of an Arabidopsis Production of Anthocyanin Pigment 1 transcription factor (AtPAP1) plus co-suppression of two endogenous, key enzyme genes: cinnamoyl-CoA reductase (SmCCR) and caffeic acid O-methyltransferase (SmCOMT). Compared with the untransformed control, we achieved a greater accumulation of Sal B (up to 3-fold higher) along with a reduced lignin concentration. This high-Sal B phenotype was stable in roots during vegetative growth and was closely correlated with increased antioxidant capacity for the corresponding plant extracts. Although no outward change in phenotype was apparent, we characterized the molecular phenotype through integrated analysis of transcriptome and metabolome profiling. Our results demonstrated the far-reaching consequences of phenolic pathway perturbations on carbohydrate metabolism, respiration, photo-respiration, and stress responses. This report is the first to describe the production of valuable end products through combinational genetic manipulation in S. miltiorrhiza plants. Our strategy will be effective in efforts to metabolically engineer multi-branch pathway(s), such as the phenylpropanoid pathway, in economically significant medicinal plants.

Topics: Aldehyde Oxidoreductases; Arabidopsis; Arabidopsis Proteins; Benzofurans; Metabolic Engineering; Methyltransferases; Plants, Genetically Modified; Salvia miltiorrhiza; Transcription Factors

The aim of this study was to systematically optimize and characterize the co-encapsulation process of Salvianolic acid B (Sal B), Tanshinone II A (TSN) and Glycyrrhetinic acid (GA) into liposomes. The liposomes (GTS-lip) were prepared using film hydration method combined with probe sonication to encapsulate two hydrophobic components (TSN and GA), and using pH gradient method to load hydrophilic component Sal B. The concentration of encapsulated drugs was measured by a reversed phase high performance liquid chromatography (RP-HPLC) method. Systematic optimization of encapsulation process was performed using single factor test, orthogonal test in combination with Box-Behnken Design. Optimum conditions are as follows: ratio of GA to lipid (w/w)=0.08, ratio of Sal B to lipid (w/w)=0.12 and pH of buffer=3.3. Based on the conditions mentioned above, encapsulation efficiency of Sal B, TSN and GA reached target levels: (96.03 ± 0.28)%, (80.63 ± 0.91)% and (88.56 ± 0.17)%, respectively. The GTS-lip had a unimodal size-distribution and a mean diameter of 191.3 ± 6.31 nm. Morphology determination of the GTS-lip indicated that the liposomes were spherical, and there was no free drug crystal in the visual field of transmission electron microscopy. Also, the ζ potential of GTS-lip was detected to be -11.6 ± 0.35 mV. In vitro release investigation of GTS-lip suggested that the release rate of GTS-lip significantly decreased compared to drug solution. The accumulative release percentage of TSN, GA and Sal B were 10% in 36 h, 4% in 36 h and 77% in 24 h. Meanwhile, GTS-lip exhibited definite activity on proliferative inhibition of hepatic stellate cells (HSC). GTS-lip decreased the viability of the HSC to higher than 75% at two high drug concentration groups in 24h. At the same time, GTS-lip of two low drug concentration groups increased the inhibition rates by 2.3 folds and 1.9 folds separately at 48 h compared to 24h. By contrast, inhibition activity of G-T-S solution group showed less change between 48 h and 24 h. The prolonged and enhanced activity in 48 h which GTS-lip group manifested might contribute to its sustained release effect.

Topics: Abietanes; Benzofurans; Cell Line; Cell Proliferation; Cell Survival; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Delayed-Action Preparations; Glycyrrhetinic Acid; Hepatic Stellate Cells; Humans; Hydrophobic and Hydrophilic Interactions; Liposomes; Microscopy, Electron, Transmission; Particle Size; Time Factors

Salvianolic acid B (SalB), the main bioactive compound isolated from the traditional Chinese medicinal herb broad Radix Salviae Miltiorrhizae exerts a spectrum of pharmacologic activities. We investigated the effects of SalB treatment in a rat model of spinal cord ischemia and reperfusion (I/R) injury and the underlying mechanism.. SalB was administered at 1, 10, or 50 mg/kg after spinal cord ischemia. The potential protective effects on spinal cord injury were determined by spinal cord edema, infarct volume, and motor function assessment of the hind limbs.. SalB treatment significantly decreased spinal cord edema and infarct volume and preserved motor function of the hind limbs in a dose-dependent manner. SalB administration ameliorated the generation of oxidative products and preserved antioxidant defense activities in the injured spinal cord at both 4 and 24 h after I/R injury. Moreover, SalB prolonged the I/R injury-induced activation of extracellular signal-regulated kinase (ERK), and blocking ERK activation with PD98059 partially prevented the neuroprotective effects of SalB.. These findings demonstrate the neuroprotective effects of SalB in a spinal cord I/R injury model and suggest that SalB-induced neuroprotection was mediated by ERK activation.

Topics: Animals; Antioxidants; Benzofurans; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Hemodynamics; Locomotion; Male; MAP Kinase Signaling System; Oxidative Stress; Phytotherapy; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Spinal Cord Injuries

Chlorogenic acid (1), salvianolic acid B (2), and tanshinone IIA (3) are commonly used as chemoprotective agents for chemotherapy in cancer patients. The present study deals with the effect of these three compounds on cytotoxicity of doxorubicin in HepG2 cells. The results showed that 1 and 2 reduced the cytotoxicity of doxorubicin through scavenging ROS generated by doxorubicin in HepG2 cells. The findings suggest that 1 and 2 could enhance the expression of SOD and decrease that of NADPH oxidase, which resulted in the elimination of ROS. On the contrary, 3 enhanced the cytotoxicity of doxorubicin in HepG2 cells. Furthermore, drug interactions between doxorubicin and 3 produce synergistic effects in HepG2 cells.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Benzofurans; Caspase 3; Chlorogenic Acid; Doxorubicin; Hep G2 Cells; Humans; L-Lactate Dehydrogenase; NADPH Oxidase 4; NADPH Oxidases; Reactive Oxygen Species

Salvianolic acid B (SalB), one of the major bioactive components in Salviamiltiorrhiza, has plenty of cardioprotective effects. The present study was designed to investigate the effect of SalB on angiotensin II (AngII)-induced hypertrophy in neonatal rat cardiomyocytes, and to find out whether or not this effect is attributed to inhibition of poly (ADP-ribose) polymerase-1 (PARP-1), which plays a key role in cardiac hypertrophy. Our results showed that SalB prevented the cardiomyocytes from AngII-induced hypertrophy, associated with attenuation of the mRNA expressions of atrial natriuretic factor and brain natriuretic peptide, and reduction in the cell surface area. SalB inhibited the activity of PARP-1. The inhibitory effect was comparable to that of the PARP-1 inhibitor 3-Aminobenzamide (3-AB). In addition, SalB reversed the depletion of cellular NAD(+) induced by AngII. Moreover, overexpression of PARP-1 attenuated the anti-hypertrophic effect of SalB. These observations suggested that SalB prevented the cardiomyocytes from AngII-induced hypertrophy, at least partially through inhibition of PARP-1. Moreover, SalB attenuated the generation of oxidative stress via suppression of NADPH oxidase 2 and 4, which might probably contribute to the inhibition of PARP-1. These present findings may shed new light on the understanding of the cardioprotective effect of SalB.

Topics: Angiotensin II; Animals; Benzofurans; Cardiomegaly; Cells, Cultured; Myocytes, Cardiac; NAD; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction

To investigate the effect of salvianolic acid B on rats with myocardial ischemia-reperfusion injury.. SD rats were randomly divided into five groups (n=10 in each group): A sham operation group, B ischemic reperfusion group model group, C low dose salvianolic acid B group, D median dose salvianolic acid B group, E high dose salvianolic acid B group. One hour after establishment of the myocardial ischemia-reperfusion model, the concentration and the apoptotic index of the plasma level of myocardial enzymes (CTn I, CK-MB), SOD, MDA, NO, ET were measured. Heart tissues were obtained and micro-structural changes were observed.. Compared the model group, the plasma CTn, CK-MB, MDA and ET contents were significantly increased, NO, T-SOD contents were decreased in the treatment group (group C, D, and E) (P<0.05); compared with group E, the plasma CTn I, CK-MB, MDA and ET levels were increased, the NO, T-SOD levels were decreased in groups C and D (P<0.05). Infarct size was significantly reduced, and the myocardial ultrastructural changes were improved significantly in treatment group.. Salvianolic acid B has a significant protective effect on myocardial ischemia-reperfusion injury. It can alleviate oxidative stress, reduce calcium overload, improve endothelial function and so on.

Topics: Animals; Apoptosis; Benzofurans; Creatine Kinase, MB Form; Heart; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Nitric Oxide; Oxidoreductases; Protective Agents; Rats; Rats, Sprague-Dawley; Troponin I

Danshensu (DSU) and salvianolic acid B (SAB) are the primary water-soluble compounds of Salvia miltiorrhiza Bunge (Lamiaceae). In this study, we analyzed the effects of DSU, SAB and a S. miltiorrhiza extract (SME) on cell proliferation. Additionally, the effects of DSU and SAB on collagen synthesis in Detroit 551 human normal fibroblast cells and on melanin production in B16 melanoma cells were verified. The results demonstrated that SME can enhance the proliferation of Detroit 551 cells and that this boost may be caused by DSU and SAB. This research showed that SME, DSU and SAB all have the ability to increase the production of collagen in Detroit 551 cells. The results also confirmed that DSU and SAB can attenuate the α-MSH-stimulated melanin production of B16 cells by inhibiting tyrosinase activity. Therefore, SME, DSU and SAB each have the potential to be utilized as active ingredients in wound healing or cosmetic treatments. In the future, DSU and SAB could also be used as functional components for treating hyperpigmentation.

Topics: Benzofurans; Cell Line; Cell Proliferation; Collagen; Drugs, Chinese Herbal; Fibroblasts; Humans; Lactates; Melanins; Salvia miltiorrhiza

Salvianolic acid A (Sal A), an important constituent of Radix Salviae Miltiorrhizae (RSM), is effective for the treatment of myocardial infarction (MI) and coronary heart disease due to its potential in the improvement of acute myocardial ischemia. However, its content is very low in RSM. So it is obvious to find a rich source of Sal A or to improve its content by conversion of other related components into Sal A modifying reaction conditions. In this research we focused on the conversion of Sal B into Sal A in aqueous solutions of RSM by using different reaction conditions including pH, temperature, pressure and humidity. During the reactions, the contents of Sal A, Sal B and danshensu in the RSM were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS). The results indicated that the conversion of Sal B into Sal A in RSM tissues under the conditions of a high temperature, high pressure and high humidity was efficient and thereby, was readily utilized to prepare rich Sal A materials in practice.

Topics: Benzofurans; Caffeic Acids; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Hot Temperature; Humidity; Lactates; Mass Spectrometry; Myocardial Ischemia; Phytotherapy; Plant Roots; Pressure; Salvia miltiorrhiza

Przewalskinic acid A is a rare, water-soluble, and highly biologically active ingredient found, thus far, only in the Salvia przewalskii Maxim herb; however, the content in S. przewalskii herb is very low. In order to obtain useful quantities of przewalskinic acid A, the biotransformatin of salvianolic acid B from Salvia miltiorrhiza root (danshen in Chinese) into przewalskinic acid A was studied using a crude enzyme produced from Aspergillus oryzae D30s strain. The crude enzyme from the A. oryzae strain hydrolyzed salvianolic acid B into przewalskinic acid A and danshensu. The preparation afforded 31.3 g przewalskinic acid A (91.0 % purity) and 13.1 g danshensu (95 % purity) from 75 g salvianolic acid B. The preparation of przewalskinic acid A was therefore very successful with a yield of over 86 %, but the yield of danshensu was only 33 %. The product przewalskinic acid A was identified using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and NMR.

Topics: Aspergillus oryzae; Benzofurans; Biotransformation; Lactates

Salvianolic acid B, a major bioactive component of Chinese medicine herb, Salvia miltiorrhiza, is widely used for treatment of cardiovascular diseases. Our recent studies have shown that Salvianolic acid B can prevent development of osteoporosis. However, the underlying mechanisms are still not clarified clearly. In the present study, we aim to investigate the effects of Salvianolic acid B on viability and osteogenic differentiation of human mesenchymal stem cells (hMSCs). The results showed Salvianolic acid B (Sal B) had no obvious toxic effects on hMSCs, whereas Sal B supplementation (5μM) increased the alkaline phosphatase activity, osteopontin, Runx2 and osterix expression in hMSCs. Under osteogenic induction condition, Sal B (5μM) significantly promoted mineralization; and when the extracellular-signal-regulated kinases signaling (ERK) pathway was blocked, the anabolic effects of Sal B were diminished, indicating that Sal B promoted osteogenesis of hMSCs through activating ERK signaling pathway. The current study confirms that Sal B promotes osteogenesis of hMSCs with no cytotoxicity, and it may be used as a potential therapeutic agent for the management of osteoporosis.

Topics: Benzofurans; Cell Differentiation; Cell Survival; Drugs, Chinese Herbal; Humans; MAP Kinase Signaling System; Mesenchymal Stem Cells; Osteogenesis; Osteoporosis; Transfection

Salvianolic acid B (Sal B), one of the major water-soluble compounds of Danshen (a popular Chinese herb), possesses many of the biological activities, such as antifibrogenic effect in liver and renal diseases. Transforming growth factor-β1 (TGF-β1) plays a central role in the development of pulmonary fibrosis by stimulating extracellular matrix (ECM) accumulation and activating fibroblasts. Here, we investigated the effects of Sal B on cell proliferation, collagen synthesis, endogenous TGF-β1 production, and α-smooth muscle actin (α-SMA, a marker of myofibroblasts) expression in human lung fibroblasts stimulated by TGF-β1 in vitro. The cell proliferation rates were analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. The expression of TGF-β1 and type I collagen at both the mRNA and protein levels was detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay, respectively. The α-SMA expression was detected by Western blot. TGF-β1 treatment of lung fibroblasts increased cell proliferation rates, and enhanced the expression level of type I collagen, endogenous TGF-β1 production, and α-SMA expression (P < .05). The treatment with only Sal B did not affect the proliferation and differentiation of lung fibroblasts. Interestingly, Sal B was found to inhibit TGF-β1-induced cell proliferation, expression of type I collagen, endogenous TGF-β1 production, and α-SMA expression in lung fibroblasts. Moreover, the inhibitory effect of Sal B on TGF-β1-induced proliferation and differentiation in lung fibroblasts was more significant when treated with high-dose Sal B (1 μmol/L versus 10 μmol/L, P < .05). These data demonstrate that Sal B inhibits TGF-β1-induced cell proliferation and differentiation in vitro experiment.

Topics: Actins; Benzofurans; Cell Differentiation; Cell Line; Cell Proliferation; Collagen Type I; Down-Regulation; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Fibroblasts; Humans; Phytotherapy; Pulmonary Fibrosis; Salvia miltiorrhiza; Transforming Growth Factor beta1

Salvianolic acid A (Sal A) was considered to be the compound with highest activity in Salvia miltiorrhiza (danshen). Due to its low content in raw materials, many studies reported its preparation from salvianolic acid B (Sal B). However, the process of this transformation is still unknown. Our objective was to find the chemical change of the transformation from Sal B to Sal A. The results showed that Sal B was hydrolyzed to lithospermic acid (LA) first, and the latter was transformed into Sal A in thermal aqueous solution. The radical scavenging ability of Sal A, Sal B, and LA was tested through DPPH, and Sal A showed higher radical elimination ability compared to Sal B and LA. In vitro liver damage was induced by CCl4 in human hepatic WRL68 cell line. Sal A, Sal B, and LA showed liver protective ability in a dose-dependent manner, while Sal A possessed a much higher ability compared to Sal B and LA.

Topics: Antioxidants; Benzofurans; Biphenyl Compounds; Caffeic Acids; Carbon Tetrachloride; Cell Line; Chemical and Drug Induced Liver Injury; Depsides; Drugs, Chinese Herbal; Hot Temperature; Humans; In Vitro Techniques; Lactates; Liver; Phytotherapy; Picrates; Salvia miltiorrhiza; Water

The aim of the present study was to investigate the effects of salvianolic acid B on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) in rabbits. Continuous infusion of LPS was used to induce a DIC model in rabbits. Treatment with salvianolic acid B (1, 3 or 6 mg/kg) was started simultaneously with LPS infusion (0.5 mg/kg LPS in 60 mL saline; 10 mL/h over a period of 6 h) through the contralateral marginal ear vein. Activated partial thromboplastin time (APTT), prothrombin time (PT), platelet count and fibrinogen concentration were determined, as were plasma levels of fibrin-fibrinogen degradation products (FDP), alanine aminotransferase (ALT), blood urea nitrogen (BUN), protein C activity, antithrombin III (ATIII) and tumour necrosis factor (TNF)-α concentration. The gradual impairment of haemostatic parameters was induced by continuous infusion of LPS. There were marked increases in APTT, PT, BUN, ALT and plasma TNF-α and marked decreases in the platelet count, fibrinogen, FDP, protein C and ATIII. The intravenous administration of 1, 3 or 6 mg/kg salvianolic acid B attenuated the increases in APTT, PT, BUN, ALT and plasma TNF-α and the decreases in fibrinogen, platelet, FDP, protein C and ATIII induced by LPS infusion. These observations indicate that salvianolic acid B has an effect against LPS-induced DIC in rabbits.

Topics: Animals; Benzofurans; Disseminated Intravascular Coagulation; Gene Expression Regulation; Kidney Diseases; Lipopolysaccharides; Liver Diseases; Male; Rabbits; Tumor Necrosis Factor-alpha

Salvianolic acid B (SalB) is isolated from the traditional Chinese medical herb salvia miltiorrhiza. It has many biological and pharmaceutical activities. This study aimed to investigate the effect of SalB on acute ethanol-induced hepatic injury in rats and to explore the role of SIRT1 in this process. The results showed that pretreatment with SalB significantly reduced ethanol-induced elevation in aminotransferase activities, decreased hepatotoxic cytokine levels such as Interleukin-6 (IL-6), and increased the antioxidant enzyme activity. Moreover, SalB pretreatment reversed the increase in NF-κB, cleaved caspase-3 and decrease in B-cell lymphoma-extra large (Bcl-xL) caused by ethanol exposure. Importantly, SalB pretreatment significantly increased the expression of SIRT1, a NAD(+)-dependent deacetylase, whereas the increase in SIRT1 was accompanied by decreased acetyl-p53 expression. In HepG2 cells, SalB pretreatment increased SIRT1 expression in a time and dose-dependent manner and such an increase was abrogated by siRNA knockdown of SIRT1. Additionally, inhibition of SIRT1 significantly increased the acetylation of p53, and blocked SalB-induced acetylation of p53 down-regulation. Collectively, this study indicated that SalB can alleviate acute ethanol-induced hepatocyte apoptosis through SIRT1-mediated deacetylation of p53 pathway.

Topics: Acetylation; Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Benzofurans; Blotting, Western; Cell Survival; Central Nervous System Depressants; Chemical and Drug Induced Liver Injury; Cytokines; Ethanol; Glutathione; Immunoprecipitation; Liver; Male; Malondialdehyde; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Sirtuin 1; Tumor Suppressor Protein p53

The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Protection against reactive oxygen species (ROS)-mediated oxidative damage via antioxidant mechanisms is essential for tissue maintenance and shows therapeutic potential for patients suffering from cardiovascular and metabolic disorders. Salvianolic acid B (SalB), a natural bioactive component known from Traditional Chinese Medicine, has been reported to exert cellular protection in various types of cells. However, the underlying mechanisms involved are not fully understood. Here, we showed that SalB significantly promoted the migratory and tube formation abilities of human bone marrow derived-endothelial progenitor cells (BM-EPCs) in vitro, and substantially abrogated hydrogen peroxide (H2O2)-induced cell damage. SalB down-regulated Nox4 and eNOS, as well as nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase expression upon H2O2 induction that in turn prevents oxidative-induced endothelial dysfunction. Moreover, SalB suppressed the Bax/Bcl-xL ratio and caspase-3 activation after H2O2 induction. Furthermore, our results provide mechanistic evidence that activation of the mTOR/p70S6K/4EBP1 pathways is required for both SalB-mediated angiogenic and protective effects against oxidative stress-induced cell injury in BM-EPCs. Suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways by SalB significantly protected BM-EPCs against cell injury caused by oxidative stress via reduction of intracellular ROS levels and apoptosis. Taken together, by providing a mechanistic insight into the modulation of redox states in BM-EPCs by SalB, we suggest that SalB has a strong potential of being a new proangiogenic and cytoprotective therapeutic agent with applications in the field of endothelial injury-mediated vascular diseases.

Topics: Activating Transcription Factor 2; Adaptor Proteins, Signal Transducing; Adult; Benzofurans; Blotting, Western; Bone Marrow Cells; Cell Cycle Proteins; Cell Movement; Cell Survival; Cells, Cultured; Endothelial Cells; Humans; Hydrogen Peroxide; Microscopy, Fluorescence; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oxidants; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphoproteins; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; RNA Interference; Signal Transduction; TOR Serine-Threonine Kinases; Young Adult

Hydrogen peroxide (H2O2) and nitric oxide (NO) are key signaling molecules in cells whose levels are increased in response to various stimuli and are involved in plant secondary metabolite synthesis. In this paper, the roles of H2O2 and NO on salvianolic acid B (Sal B) production in salicylic acid (SA)-induced Salvia miltiorrhiza cell cultures were investigated. The results showed that H2O2 could be significantly elicited by SA, even though IMD (an inhibitor of NADPH oxidase) or DMTU (a quencher of H2O2) were employed to inhibit or quench intracellular H2O2. These elicited H2O2 levels significantly increased NO production by 1.6- and 1.46 fold in IMD+SA and DMTU+SA treatments, respectively, and induced 4.58- and 4.85-fold Sal B accumulation, respectively. NO was also markedly elicited by SA, in which L-NNA (an inhibitor of NO synthase) and cPTIO (a quencher of NO) were used to inhibit or quench NO within cells, and the induced NO could significantly enhance H2O2 production by 1.92- and 1.37-fold in L-NNA+SA and cPTIO+SA treatments, respectively, and 3.27- and 1.50-fold for Sal B accumulation, respectively. These results indicate that elicitation of SA for either H2O2 or NO was independent, and the elicited H2O2 or NO could act independently or synergistically to induce Sal B accumulation in SA-elicited cells.

Topics: Benzofurans; Cell Culture Techniques; Hydrogen Peroxide; Nitric Oxide; Nitric Oxide Synthase; Salicylic Acid; Salvia miltiorrhiza; Signal Transduction

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are considered the most powerful in terms of differentiating into three-germ-layer cells. However, maintaining self-renewing ESCs and iPSCs in vitro requires leukemia-induced factor (LIF), an expensive reagent. Here we describe a less expensive compound that may serve as a LIF substitute-salvianolic acid B (Sal B), a Salvia miltiorrhiza extract. We found that Sal B is capable of upregulating Oct4 and Sox2, two genes considered important for the maintenance of ESC pluripotency. Our MTT data indicate that instead of triggering cell death, Sal B induced cell proliferation, especially at optimum concentrations of 0.01 nM and 0.1 nM. Other results indicate that compared to non-LIF controls, Sal B-treated ESCs expressed higher levels of several stem cell markers while still maintaining differentiation into three-germ-layer cells after six passages. Further, we found that Sal B triggers the Jak2-Stat3 and EGFR-ERK1/2 signaling pathways. Following Sal B treatment, (a) levels of phosphorylated (p)-Jak2, p-Stat3, p-EGFR, and p-ERK proteins all increased; (b) these increases were suppressed by AG490 (a Jak2 inhibitor) and ZD1839 (an EGFR inhibitor); and (c) cytokines associated with the Jak2-Stat3 signaling pathway were upregulated. Our findings suggest that Sal B can be used as a LIF replacement for maintaining ESC pluripotency while increasing cell proliferation.

Topics: Animals; Benzofurans; Cell Proliferation; Cell Survival; Cells, Cultured; Embryonic Stem Cells; ErbB Receptors; Janus Kinase 2; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Octamer Transcription Factor-3; Phosphorylation; Salvia miltiorrhiza; Signal Transduction; SOXB1 Transcription Factors; STAT3 Transcription Factor

Salvianolic acid B (Sal B), one of the most active components of Danshen extracts, has beneficial roles in the prevention and treatment of cardiovascular diseases. However, the precise mechanism by which Sal B exerts its effects on vascular cells is unclear. We aimed to elucidate the effects of Sal B on vascular cells and the underlying mechanisms.. Treatment of vascular smooth muscle cells with Sal B effectively inhibited platelet-derived growth factor (PDGF)-induced cell proliferation and migration, and markedly increased heme oxygenase-1 (HO-1) expression. These changes were accompanied by antioxidant effects, including decreases in the generation of reactive oxygen species and the NADP/NADPH ratio. In human umbilical vein endothelial cells, Sal B also strongly induced HO-1 and effectively inhibited tumor necrosis factor-α-induced NF-κB activation. Knockdown of HO-1 expression by siRNA abolished the effects of Sal B in vascular cells and prevented the inhibition of proliferation, migration, and inflammation in HO-1-deficient cells. In ex vivo culture of arterial rings isolated from nuclear factor-E2-related factor 2 (Nrf2)-knockout mice, Sal B neither induce HO-1 expression and nor inhibit PDGF-induced neointimal hyperplasia in arteries, suggesting that Nrf2 plays a crucial role in the induction of HO-1 expression.. We conclude that Sal B exerts antiatherogenic effects by inhibiting the proliferation, migration, and inflammation of vascular cells through induction of HO-1 via Nrf2 activation.

Topics: Animals; Benzofurans; Cells, Cultured; Heme Oxygenase-1; Humans; Myocytes, Smooth Muscle; NF-E2-Related Factor 2; Rats, Sprague-Dawley; Tissue Culture Techniques

Salvianolic acid B (SalB), a bioactive compound isolated from the plant-derived medicinal herb Danshen, has been shown to exert various anti-oxidative and anti-inflammatory activities in several neurological disorders. In this study, we sought to investigate the potential protective effects and associated molecular mechanisms of SalB in Parkinson's disease (PD) models. To determine the neuroprotective effects of SalB in vitro, MPP+- or lipopolysaccharide (LPS)-induced neuronal injury was achieved using primary cultures with different compositions of neurons, microglia and astrocytes. Our results showed that SalB reduced both LPS- and MPP+-induced toxicity of dopamine neurons in a dose-dependent manner. Additionally, SalB treatment inhibited the release of microglial pro-inflammatory cytokines and resulted in an increase in the expression and release of glial cell line-derived neurotrophic factor (GDNF) from astrocytes. Western blot analysis illustrated that SalB increased the expression and nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The knockdown of Nrf2 using specific small interfering RNA (siRNA) partially reversed the SalB-induced GDNF expression and anti-inflammatory activity. Moreover, SalB treatment significantly attenuated dopaminergic (DA) neuronal loss, inhibited neuroinflammation, increased GDNF expression and improved the neurological function in MPTP-treated mice. Collectively, these findings demonstrated that SalB protects DA neurons by an Nrf-2 -mediated dual action: reducing microglia activation-mediated neuroinflammation and inducing astrocyte activation-dependent GDNF expression. Importantly the present study also highlights critical roles of glial cells as targets for developing new strategies to alter the progression of neurodegenerative disorders.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Astrocytes; Benzofurans; Cells, Cultured; Cytokines; Dopaminergic Neurons; Gene Expression Regulation; Glial Cell Line-Derived Neurotrophic Factor; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Microglia; Models, Biological; NF-E2-Related Factor 2; Parkinson Disease

Atherosclerosis is the common pathological basis of cardiovascular and cerebrovascular disease. This study aimed to investigate the effects of vascular endothelial growth factor (VEGF) and salvianolic acid B (SalB) on the permeability of the rabbit aortary endothelial cells (RAECs) and to figure out the possible underlying molecular mechanisms. The extravasation of (125)I-low density lipoprotein ((125)I-LDL) through the RAECs was significantly increased by VEGF and decreased by SalB. Meanwhile, the tight junction-associated proteins occludin and claudin-5 were found downregulated by VEGF and the caveolae structure proteins caveolin-1 and caveolin-2 upregulated, which were abolished by the infusion of SalB. In addition, a marked increase in levels of cGMP and protein kinase G-1 (PKG-1) as well as activation of nuclear factor-κB (NF-κB) p65 were found after VEGF infusion, which were attenuated by SalB. This study demonstrates that VEGF and SalB can alter the LDL permeability of the RAECs by a paracellular pathway (downregulation of occludin and claudin-5) and a transcellular pathway (upregulation of caveolin-1 and caveolin-2), in which the cGMP/PKG/NF-κB signal pathway is possibly involved. The experimental results provide a new method and basic knowledge of prevention and treatment for cardiovascular and cerebrovascular disease.

Topics: Animals; Aorta; Benzofurans; Cell Membrane Permeability; Cells, Cultured; Drug Interactions; Drugs, Chinese Herbal; Endothelial Cells; Iodine Radioisotopes; Lipoproteins, LDL; Rabbits; Radiopharmaceuticals; Vascular Endothelial Growth Factor A

Obesity contributes to the development of cardiometabolic disorders such as type 2 diabetes, fatty liver disease and cardiovascular disease. Salvianolic acid B (Sal B) is a molecule derived from the root of Salvia miltiorrhiza (Danshen), which is a traditional Chinese medicine that is widely used to treat cardiovascular diseases. However, the role of Sal B in obesity and obesity-related metabolic disorders is unknown. In this study, we aimed to investigate the effects of Sal B on high-fat diet-induced obesity and determine the possible mechanisms involved.. Male C57BL/6J mice fed a high-fat diet for 12 weeks received a supplement of Sal B (100 mg/kg/day) by gavage for a further 8 weeks. These mice were compared to control mice fed an un-supplemented high-fat diet. 3T3-L1 preadipocytes were used in vitro studies.. Sal B administration significantly decreased body weight, white adipose tissue weight, adipocyte size and lipid (triglyceride and total cholesterol) levels in obese mice. Eight weeks of Sal B administration also improved the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) scores in high-fat diet-induced obese mice. In 3T3-L1 preadipocytes that were cultured in vitro and induced to differentiate, Sal B reduced the accumulation of lipid droplets and lipid content in a dose-dependent manner. Immunoblotting indicated that Sal B decreased peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) expression but increased the expression of GATA binding protein 2 and 3 (GATA 2, GATA 3) both in vivo and in vitro.. Our data suggest that Sal B may reduce obesity and obesity-related metabolic disorders by suppressing adipogenesis. The effects of Sal B in adipose tissue may be related to its action on PPARγ, C/EBPα, GATA-2 and GATA-3.

Topics: 3T3-L1 Cells; Animals; Benzofurans; Diet, High-Fat; Hyperlipidemias; Male; Mice; Mice, Inbred C57BL; Obesity; PPAR gamma; Weight Gain

Salviae miltiorrhiza (Danshen) has been used for thousands of years in China and some other Asian countries to treat atherothrombotic diseases. Salvianolate which consists of three water-soluble ingredients purified from Salviae miltiorrhiza, has been approved by Chinese SFDA to treat coronary artery disease. So far, there is no evidence clearly showing the clinical efficiency of salvianolate and the underlying mechanism. This study is to evaluate the effects of salvianolate on platelets in patients with acute coronary syndrome and explore the underlying mechanism. We evaluated the effects of salvianolate on platelets in patients with acute coronary syndrome by measuring ADP-induced PAC-1 binding and P-selectin expression on platelets. Salvianolate significantly potentiated the antiplatelet effects of standard dual antiplatelet therapy. We also investigated the antiplatelet effects of salvianolatic acid B (Sal-B), the major component which composes 85% of salvianolate. Sal-B inhibits human platelet activation induced by multiple agonists in vitro by inhibiting phosphodiesterase (PDE) and antagonizing P2Y12 receptor. For the first time, we show the antiplatelet efficiency of salvianolate in ACS patients undergoing treatment with clopidogrel plus aspirin, and demonstrate that Sal-B, the major component of salvianolate inhibits human platelet activation via PDE inhibition and P2Y12 antagonism which may account for the clinical antiplatelet effects of salvianolate. Our results suggest that Sal-B may substitute salvianolate for clinical use.

Topics: Acute Coronary Syndrome; Aged; Aspirin; Benzofurans; Blood Platelets; Clopidogrel; Drugs, Chinese Herbal; Humans; Male; Middle Aged; Phosphodiesterase Inhibitors; Platelet Activation; Platelet Aggregation Inhibitors; Purinergic P2Y Receptor Antagonists; Salvia miltiorrhiza; Ticlopidine

Epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in renal fibrosis. Previous studies have shown that transforming growth factor (TGF)-β1 induces EMT in human kidney proximal tubular epithelial cells (HK-2 cells), whereas salvianolic acid B (Sal B) has a protective effect against EMT. The molecular pathogenesis of such processes is currently not well understood. In this study, a miRCURYTM LNA Array was used to screen HK-2 cells for expression changes of microRNAs (miRNAs) implicated in EMT. After validation by real-time PCR, all three members of the miR-106b-25 cluster (miR-106b, miR-93, and miR-25) were found to be markedly down-regulated during EMT in response to TGF-β1, whereas these miRNAs were up-regulated by Sal B treatment in a dose-dependent manner. Moreover, enhanced expression of miR-106b attenuated EMT by retaining the epithelial morphology of HK-2 cells, reducing the levels of α-smooth muscle actin (α-SMA), and increasing the levels of E-cadherin. To explore the molecular basis underlying the inhibitive effect of the miR-106b-25 cluster against EMT, bioinformatics analysis revealed that TGF-β type II receptor, a regulator of TGF-β signaling, might be a direct target of the miR-106b-25 cluster. In turn, low levels of TGF-β type II receptor in EMT of HK-2 cells were shown under the increase of miR-106b. In conclusion, our data suggest that the miR-106b-25 cluster may contribute to EMT in the kidney, and is involved in the protective effect of Sal B. Targeting of specific miRNAs may be a novel therapeutic approach to treat renal fibrosis.

Topics: Benzofurans; Cell Line; Cell Proliferation; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Humans; Kidney Tubules, Proximal; MicroRNAs

The present study aimed to investigate the effect of Glomus versiforme and Trichodema harzianum on the growth and quality of Salvia miltiorrhiza continuous cropping under field conditions. The field plot experiment was conducted, these active components in the plant were analyzed by HPLC, the root diseases incidence rate of S. miltiorrhiza determined by observation and counting, and relative parameters were measured. The data was statistically processed. The result showed that inoculation of G. versiforme and combined inoculation of G. versiforme with T. harzianum significantly decreased the root diseases incidence rate of S. miltiorrhiza, and combined inoculation of G. versiforme with T. harzianum was better than other treatments. All treatments improved accumulation of active ingredients in root. Inoculation of G. versiforme and combined inoculation of G. versiforme with T. harzianum significantly increased the content of salvianolic acid B and cryptotanshinone of root (P < 0.05), Inoculation of G. versiforme, T. harzianum and combined inoculation of G. versiforme with T. harzianum significantly enhanced the content of tanshinone I and tanshinone II(A) of the root (P < 0.05). It may conclude that inoculation of G. versiforme and combined inoculation of G. versiforme with T. harzianum can effectively reduce the root diseases incidence of continuous cropping S. miltiorrhiza, and improve the quality of S. miltiorrhiza.

Topics: Abietanes; Antibiosis; Benzofurans; Glomeromycota; Host-Pathogen Interactions; Phenanthrenes; Plant Diseases; Plant Roots; Salvia miltiorrhiza; Trichoderma

To develop a LC-MS/MS method for the determination of protocatechuic acid, protocatechuic aldehyde, salvianolic acid A, salvianolic acid B, cryptotanshinone and tanshinone II(A) in rat plasma and brain. The plasma and brain samples were precipitated with ethyl acetate, then were separated on an Agilent eclipse plus-C18 column (2.1 mm x 50 mm, 3.5 microm) using acetonitrile (consisting of 0.1% formic acid) and water (consisting of 0.1% formic acid) as mobile phase in gradient elution mode. The mass spectrometer was operated under both positive and negative ion mode with the ESI source, and the detection was performed by MRM. The transition of 154.3/153.1 m/z for protocatechuic acid, 137.3/108 m/z for protocatechuic aldehyde, 493.0/295.2 m/z for Salvianolic acid A, 718.0/520.0 m/z for salvianolic acid B, 321.4/152.3 m/z for chloramphenicol, 297.4/254.3 m/z for cryptotanshinone, 295.5/249.3 m/z for tanshinone II(A) and 285.2/154.0 m/z for Diazepam. The calibration curves in the range of 0.625-1 000 microg x L(-1) for protocatechuic acid and protocatechuic aldehyde, 1.25-1 000 microg x L(-1) for salvianolic acid A, 2.5-1 000 microg x L(-1) for salvianolic acid B, 0.15-1 000 microg x L(-1) for cryptotanshinone, 0.625-1 000 microg x L(-1) for tanshinone II(A) are with good linearityin rat plasma and brain. The analysis method is sensitive, simple, and suitable enough to be applied in the pharmacokinetic study of the 6 main components. Animal testing gives the lgBB of the drugs and further studies of the 6 components cross the blood-brain barrier can be carried out.

Topics: Abietanes; Animals; Benzaldehydes; Benzofurans; Blood-Brain Barrier; Brain; Caffeic Acids; Catechols; Chromatography, Liquid; Hydroxybenzoates; Injections, Intravenous; Lactates; Phenanthrenes; Plant Preparations; Rats; Reproducibility of Results; Salvia miltiorrhiza; Tandem Mass Spectrometry

Salvia miltiorrhiza is a significant source of bioactive compounds providing human health effects. Here, we surveyed root yield and the active constituents' divergences of second generation S. miltiorrhiza (SP2) responding to a spaceflight environment. High-performance liquid chromatography was conducted for the comprehensive constituents' characterizations of 28 SP2 lines (224 individuals) and the ground control (eight individuals). The results showed that the mean fresh and dry weight of roots ranged from 116 to 172 g and 25 to 119 g, respectively, in SP2 lines. In addition, the mean contents of four tanshinone compounds (tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I) of 28 SP2 lines varied from 0.32 to 1.04 mg · g(-1), 0.47 to 2.39 mg · g(-1), 0.25 to 1.60 mg · g(-1), and 0.53 to 1.67 mg · g(-1), respectively. Except for salvianolic acid B, which varied drastically from 72 % to 201 % of the ground control treatment, the other six phenolic acid contents of the 28 SP2 lines all increased after spaceflight. Principal component analysis was performed to obtain an overview of the distribution of all samples, and score plots clearly separated the SP2 accessions from ground controls. Moreover, a positive relationship was observed between tanshinone I and tanshinone IIA (r = 0.790, p < 0.01), and rosmarinic acid was positively correlated with salvianolic acid B (r = 0.728, p < 0.01). In conclusion, this study demonstrated that a spaceflight environment induced SP2 accessions remarkably in the variation of root yield and active constituent content.

Topics: Abietanes; Benzofurans; Biomass; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Environment; Humans; Phenanthrenes; Plant Extracts; Plant Roots; Principal Component Analysis; Rosmarinic Acid; Salvia miltiorrhiza; Space Flight

To investigate the effects of salvianolic acid B (Sal B) on endothelin-1 (ET1)-induced contraction and cytoskeleton reorganization of rat hepatic stellate cells (HSCs).. HSCs were collected from Sprague-Dawley rats by in situ perfusion with pronase E and isolated by density-gradient centrifugation with Nycodenz. Cells were treated with ET-1, with or without Sal B or Y-27632 (a specific inhibitor of rho-associated protein kinases) pretreatment. HSC contraction was evaluated by collagen gel contraction assay. Cytoskeletal reorganization in response to ET-1 was evaluated by detecting changes in phosphorylation of myosin light chain 2 (MLC2) using glycerol-urea PAGE and the Odyssey Infrared Imaging System. Changes in actin stress fiber polymerization were detected by FITC-labeled phalloidin. Differences between the various cell treatment/pretreatment groups were statistically analyzed.. Compared to the untreated control cells, the lattice area of ET-1-treated cells showed significant shrinkage (76.89% ± 3.84% vs. 37.10% ± 5.10%; P less than 0.01). Pretreatment with 105 M Sal B or 105 M Y-27632 significantly reduced ET-1-induced contraction (67.01% ± 4.14% and 77.28% ± 2.00%, respectively; bothP less than 0.01 vs. the ET-1-treated cells). The untreated control cells showed a basal MLC2 phosphorylation of (0.35 ± 0.05) mol PO4/mol MLC2. In contrast, ET-1 treatment elicited a rapid and sustained MLC2 phosphorylation, which was (0.87 ± 0.04) mol PO₄/mol MLC2 at 5 min post-treatment and with the maximal level of (0.96 ± 0.04) mol PO₄/mol MLC2 detected at 30 min post-treatment. The Sal B pretreatment led to a significant decrease in ET-1-induced MLC2 phosphorylation (by 63.1%) and an obvious disassembly of actin stress fibers.. Sal B effectively inhibits ET-1-induced rat HSC contraction, through its suppressive effects on MLC2 phosphorylation and promotion of the disassembly of actin stress fibers.

Topics: Actins; Animals; Benzofurans; Cardiac Myosins; Cell Shape; Cells, Cultured; Cytoskeleton; Endothelin-1; Hepatic Stellate Cells; Male; Myosin Light Chains; Phosphorylation; Rats; Rats, Sprague-Dawley

Topics: Animals; Animals, Newborn; Benzofurans; Cells, Cultured; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Myocardial Infarction; Neovascularization, Physiologic; Paracrine Communication; Rats; Rats, Sprague-Dawley

Salvia miltiorrhiza Bunge (Labiatae sp. plant, Chinese name Danshen) and Panax notoginseng (Burk.) F. H. Chen (Araliaceae plant, Chinese name Sanqi) have a long history in treating coronary heart disease, cerebrovascular disease and inner ear disorders in traditional Chinese medicine. To provide a rational basis for the use of these herbs in clinical practice, we investigated the in vivo distribution and pharmacokinetics of marker agents in Danshen and Sanqi via intravenous and inner ear administration and explored the potential interactions of these agents in compound prescription.. Guinea pigs were given Danshen extracts (salvianolic acid B, tanshinone IIA), Sanqi extracts (Panax notoginseng saponins) and combination of the two extracts via intravenous and intratympanic administration (IT). Samples from the brain, inner ear perilymph (PL), cerebrospinal fluid (CSF) and plasma were collected at different time points. The concentration of salvianolic acid B (Sal B), tanshinone IIA (Ts IIA), notoginsenoside R₁ (R₁), ginsenoside Rg₁ (Rg₁) and ginsenoside Rb₁ (Rb₁) was determined by high-performance liquid chromatography coupled with a diode array detector (DAD). Pharmacokinetic parameters were estimated using non-compartmental methods.. Local drug application via inner ear greatly improved drug distribution within the PL, CSF and brain tissues compared with intravenous administration (IV). The values of Cmax and AUC(0-t) after IT were significantly higher than IV. In comparison with IT of Danshen and Sanqi alone, the pharmacokinetic parameters for R₁, Rg₁, Rb₁, Sal B and Ts IIA were markedly different in the compound prescription. The compound compatibility enhanced the transport of Danshen components into the brain through the inner ear and apparently prolonged the retention time in CSF while decreasing the distribution of Sanqi components in the inner ear and brain.. The results indicated that local drug application to the inner ear was a more effective delivery route than systemic administration. Co-administration of Danshen and Sanqi could cause significant pharmacokinetic herb-herb interactions in guinea pigs. The multiple active components via inner ear administration might be promising candidates for the treatment of inner ear and brain diseases.

Topics: Abietanes; Animals; Benzofurans; Cerebrospinal Fluid; Drugs, Chinese Herbal; Ear, Inner; Ginsenosides; Guinea Pigs; Herb-Drug Interactions; Medicine, Chinese Traditional; Panax notoginseng; Salvia miltiorrhiza

Epidural fibrosis (EF) is a common complication after laminectomy. Salvianolic acid B (Sal B) is a major bioactive component of a traditional Chinese medical agent, Salvia miltiorrhiza, which has shown anti-inflammatory, anti-fibrotic and anti-proliferative properties. The object of this study was to investigate the effect of Sal B on the prevention of epidural fibrosis in laminectomy rats.. A controlled double-blinded study was conducted in sixty healthy adult Wistar rats that underwent laminectomy at the L1-L2 levels. The rats were randomly divided into 3 groups of 20: (1) Sal B treatment group; (2) Vehicle group; (3) Sham group (laminectomy without treatment). All rats were sacrificed 4 weeks post-operatively. The extent of epidural fibrosis, fibroblast proliferation and the expression of vascular endothelial growth factor (VEGF) and inflammatory factors were analyzed.. The recovery of all rats was uneventful. In the laminectomy sites treated with Sal B, the dura mater showed no adhesion. Collagen deposition was significantly lower in the Sal B group than the other two groups. In addition, both fibroblast and inflammatory cell counting in the laminectomy sites treated with Sal B showed better grades than the other two groups. The expression of VEGF and inflammatory factors in operative sites also suggested better results in the Sal B group than the other two groups.. Sal B inhibits fibroblast proliferation, blood vessel regeneration, and inflammatory factor expression. Thus, Sal B is able to prevent epidural scar adhesion in post-laminectomy rats.

Topics: Animals; Anti-Inflammatory Agents; Benzofurans; Cell Proliferation; Cicatrix; Double-Blind Method; Drugs, Chinese Herbal; Epidural Space; Fibroblasts; Fibrosis; Hydroxyproline; Interleukin-6; Laminectomy; Male; Rats, Wistar; Tissue Adhesions; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

To establish induction and liquid culture system for hairy roots of Danshen (Salvia miltiorrhiza), Agrobacterium rhizogenes A4, LBA9402, 15834 as test bacterium were used to infect aseptic leaves of Danshen. The hairy roots were induced and positive transgenic hairy roots were selected with PCR using rolB and rolC as the target gene. Then hairy roots of S. miltiorrhiza were harvested and salvianolic acids were extracted with 70% methanol containing 1% formic acid. The content of salvianolic acid B (SalB) and rosmarinic acid (RA) were determined by HPLC. According to the above research results, the Danshen hairy roots induced by A. rhizogenes LBA9402 were inoculated into the following group of culture media: MSOH, MS, B5, and 6,7-V liquid media. Then the same methods of extraction and determination for the content of Danshen hairy roots were adopted. Last, the hairy roots of S. miltiorrhiza induced by A. rhizogenes LBA9402 were inoculated into the MSOH liquid media with different pH values. The content of salvianolic acid were extracted with 70% methanol containing 1% formic acid and determined by HPLC. As a result, three kinds of A. rhizogenes A4, LBA9402, 15834 could induce hairy roots and Ri plasmids were integrated into the genome of S. miltiorrhiza by PCR. Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid, which were (3.27 ± 0.37)% [including (1.04 ±0.36)% of RA and (2.22 ± 0.29)% of SalB] and (3.17 ± 0.20)% [including (0.92 ± 0.31)% of RA and (2.25 ± 0.26)% of SalB], respectively. Hairy roots induced by A. rhizogenes LBA9402 when they were cultured in MSOH liquid media produced much more salvianolic acid, which was (4.56 ± 0.36)%, including (1.12 ± 0.26)% of RA and (3.44 ± 0.23)% of SalB. Hairy roots induced by A. rhizogenes LBA9402 produced the most salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81, which was 4.85%, including 1.16% of RA and 3.69% of SalB. So Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81. The research had established the foundation on genetic engineering to improve the quality of S. miltiorrhiza.

Topics: Agrobacterium; Benzofurans; Cell Culture Techniques; Cinnamates; Culture Media; Depsides; Drugs, Chinese Herbal; Plant Roots; Rosmarinic Acid; Salvia miltiorrhiza

Radix Salviae miltiorrhizae is a herb frequently used within traditional Chinese medicine for the treatment of cardiovascular- and trauma-related diseases. Danshen is the dried root of Salviae miltiorrhizae, from which the polyphenolic compound Salvianolic acid B (Sal B) can be obtained. Sal B is a key component of Danshen. The aim of this study was to determine the effect of Sal B on the healing of long bones following trauma in a rat tibia fracture model.. Tibia fractures were created in 20 male Sprague Dawley rats. The animals were divided into two groups: (1) experimental group (n = 10); and (2) control group (n = 10). Rats in the experimental group were intraperitoneally administered with Sal B (40 mg/kg/d) for 3 weeks, while rats in the control group received an identical volume of physiological saline solution, administered in the same way. X-ray photographs were taken of all animals at the time points. Rats were euthanized at weeks 1, 3, 8 and 12 post-fracture. Fracture calluses were measured and callus sections were obtained and stained using hematoxylin and eosin (HE) and the calcium cobalt method. HE stained sections were observed and evaluated according to different grades of bone remodeling. Sections stained using the calcium cobalt method were analyzed with an imagine analysis system.. Data showed that callus growth was significantly greater in the experimental group compared with the control group (P < 0.05). Furthermore, histological scores in the Sal B-treated group were statistically higher than in the saline treated group at weeks 1, 3 and 8 post-fracture (P < 0.05). Alkaline phosphatase (ALP) activity was enhanced in the experimental group at weeks 1 and 3 post-fracture (P < 0.05).. Our results suggest that Sal B may accelerate early-stage fracture healing. Increased activity of ALP may be one factor which promotes the healing process. This pilot study provides brief insight into the effect of Sal B in fracture healing. These findings will contribute to the development of more and enhanced treatment options for trauma fracture patients.

Topics: Alkaline Phosphatase; Animals; Benzofurans; Bony Callus; Drugs, Chinese Herbal; Fracture Healing; Fractures, Bone; Humans; Male; Osteogenesis; Phytotherapy; Pilot Projects; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Tibia

A pot experiment was conducted to investigate the effects of UV-B radiation on sensitive index (SI) synthetically formed by the height, leaf area and biomass, and on the accumulation of rosmarinic acid (RA) and salvianolic acid B (SAB) of Salvia miltiorrhiza in two growth periods. The results showed the SI in the shoot vigorous growth and harvesting periods both decreased with the increasing UV-B radiation, but the SI in the latter period was even less. The RA and SAB contents in the leaves increased with the increasing UV-B radiation, and the increment was greater in the harvesting period than in the shoot vigorous growth period. The RA and SAB contents decreased in the roots, and decreased with the increasing UV-B intensity and duration. Total contents of RA and SAB in roots decreased to 10.0% and 6.3% of the control under the high UV-B intensity in the harvesting period.

Topics: Benzofurans; Biomass; Cinnamates; Depsides; Plant Leaves; Plant Roots; Radiation Tolerance; Rosmarinic Acid; Salvia miltiorrhiza; Ultraviolet Rays

Salvianolic acid B (SalB) is a polyphenolic compound found in Salvia miltiorrhiza Bunge that has several anti-oxidative and anti-inflammatory effects. In the present study, we investigated whether SalB has neuroprotective effects in an amyloid β (Aβ) peptide-induced Alzheimer's disease mouse model. Mice were injected with Aβ25-35 peptide intracerebroventricularly and were subsequently administered SalB once daily for 7 days. Subchronic SalB administration (10mg/kg) significantly ameliorated the Aβ25-35 peptide-induced memory impairment in the passive avoidance task (P<0.05). SalB treatment also reduced the number of activated microglia and astrocytes that were observed during the inflammatory reaction after the administration of the Aβ25-35 peptide. Moreover, SalB markedly reduced inducible nitric oxide synthase and cyclooxygenase-2 expression levels and thiobarbituric acid reactive substances, which were increased by the administration of the Aβ25-35 peptide. Furthermore, SalB administration significantly rescued the Aβ25-35 peptide-induced decrease of choline acetyltransferase and brain-derived neurotrophic factor protein levels. These results suggest that SalB exerts neuroprotective activity via anti-inflammatory and anti-oxidative effects and that SalB may be a potential candidate for Alzheimer's disease therapy.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Anti-Inflammatory Agents; Avoidance Learning; Behavior, Animal; Benzofurans; Brain-Derived Neurotrophic Factor; Choline O-Acetyltransferase; Cognition Disorders; Cyclooxygenase 2; Disease Models, Animal; Hippocampus; Lipid Peroxidation; Male; Mice; Mice, Inbred ICR; Neuroglia; Neuroprotective Agents; Nitric Oxide Synthase Type II; Peptide Fragments

The effects of salvianolic acid B (Sal B) decoction on antioxidative activities were evaluated. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, Fe(2+)-chelating activity, reducing power, and total phenolic content of the Sal B-decocted solutions did not change significantly after decoction in an aqueous solution. However, the formation of cholesteryl ester hydroperoxide (CE-OOH) in rat blood plasma containing the Sal B-decocted solutions was more effectively inhibited than that of plasma containing the Sal B solution, regardless of the decoction time. In addition, the accumulation of CE-OOH in rat plasma after oral administration of the Sal B-decocted solutions was more effectively suppressed than when the Sal B solution was administered, considering the lag time. It is likely that the decoction was partly responsible for the increased antioxidant activity in blood plasma. Therefore, the Sal B-decocted solution may contribute more to antioxidant defense in blood than a Sal B solution that is not decocted.

Topics: Administration, Oral; Animals; Antioxidants; Benzofurans; Biphenyl Compounds; Chelating Agents; Cholesterol Esters; Copper; Ions; Male; Oxidation-Reduction; Phenols; Picrates; Plant Extracts; Plant Preparations; Rats; Rats, Sprague-Dawley; Salvia

Rosmarinic acid (RA) and lithospermic acid B (LAB) are two typical phenolic acids with significant bioactivities that may contribute to the therapeutic effects of Salvia miltiorrhiza. Precise knowledge of the biosynthetic pathway leading to RA and LAB is a necessary prerequisite to optimize the production of important phenolic compounds in S. miltiorrhiza. In vivo isotopic labeling experiments using [ring-(13)C]-phenylalanine, combined with dynamic measurements of metabolite levels by UPLC/Q-TOF, were used to investigate the metabolic origin of phenolic acids in S. miltiorrhiza. These data indicate the in vivo phenolic biosynthetic pathway: two intermediates from the general phenylpropanoid pathway and the tyrosine-derived pathway, 4-coumaroyl-CoA and 3,4-dihydroxyphenyllactic acid (DHPL), are coupled by the ester-forming enzyme rosmarinic acid synthase (SmRAS) to form 4-coumaroyl-3',4'-dihydroxyphenyllactic acid (4C-DHPL). The 3-hydroxyl group is introduced late in the pathway by a cytochrome P450-dependent monooxygenase (SmCYP98A14) to form RA. Subsequently, RA is transformed to a phenoxyl radical by oxidation, and two phenoxyl radicals unite spontaneously to form LAB. The results indicate aspects of the complexity of phenolic acid biosynthesis in S. miltiorrhiza and expand an understanding of phenylpropanoid-derived metabolic pathways. The candidate genes for the key enzymes that were revealed provide a substantial foundation for follow-up research on improving the production of important phenolic acids through metabolic engineering in the future.

Topics: Acyltransferases; Amino Acid Sequence; Benzofurans; Carbon Isotopes; Cells, Cultured; Chromosomal Proteins, Non-Histone; Cinnamates; Depsides; Hydroxybenzoates; Molecular Structure; Phylogeny; Plant Roots; Rosmarinic Acid; Salvia miltiorrhiza; Sequence Alignment

The traditional Chinese medicinal herb Danshen (Salvia miltiorrhiza), first recorded in the "Shen Nong's Herbal Classic", has long been used to treat cardiovascular conditions, although the mechanism(s) underlying its effects remain unclear. Here, an iron dextran injection (50 mg · kg⁻¹ per day) was delivered intraperitoneally to establish a mouse model for investigating the ameliorative effects of Danshen injection (low dose at 3 g · kg⁻¹ per day or high dose at 6 g · kg⁻¹ per day) on iron overload-induced cardiac damage. The iron-chelating agent deferoxamine (100 mg · kg⁻¹ per day) was administered as a positive control. The main constituents of Danshen injection, salvianic acid A (danshensu), protocatechuic aldehyde, and salvianolic acid B, were quantified at concentrations of 2.15, 0.44, and 1.01 mg · mL⁻¹, respectively, using HPLC with UV detection. Danshen injection significantly lowered cardiac iron deposition and the concentration of the lipid oxidation product malondialdehyde, as well as improved cardiac superoxide dismutase and glutathione peroxidase levels in iron-overloaded mice. Serum levels of creatine kinase, creatine kinase isoenzyme, and lactate dehydrogenase in the iron-overloaded mice were significantly elevated (up to ~ 160 %), whereas their activities were downregulated by Danshen injection by 25 ~ 35 % at the high dose and by ~ 20 % at the low dose. Morphological changes of cardiac tissue analyzed by hematoxylin and eosin staining indicated that lesions induced by iron overload could be ameliorated by Danshen injection dose-dependently. Altogether, these results illustrated that the protective effects of Danshen injection were at least in part due to decreased iron deposition and inhibition of lipid peroxidation.

Topics: Animals; Benzaldehydes; Benzofurans; Catechols; Chromatography, High Pressure Liquid; Creatine Kinase; Disease Models, Animal; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Glutathione Peroxidase; Heart; Injections, Intraperitoneal; Iron Overload; L-Lactate Dehydrogenase; Lactates; Male; Medicine, Chinese Traditional; Mice; Myocardium; Phenanthrolines; Salvia miltiorrhiza; Superoxide Dismutase

Fuzheng Huayu recipe (FZHY) was formulated on the basis of Chinese medicine theory in treating liver fibrosis. It has a significant efficacy against liver fibrosis caused by chronic hepatitis B, with the action mechanisms of inhibition of hepatic stellate cell activation, protection of hepatocyte oxidative injury and regulations of hepatic matrix remodeling etc.. To identify the absorbed components and metabolites of Danshen in FZHY in rat serum, and find their active components for anti-liver fibrosis.. A valid high performance liquid chromatography-electrospray ionization ion trap mass spectrometry (HPLC-ESI/MS(n)) method was established to investigate the absorbed and metabolized compounds of Danshen in FZHY in rat serum after oral administration. Mass spectra were acquired in both negative and positive modes. Otherwise, to evaluate the anti-hepatic fibrosis efficacies of absorbed and metabolized compounds, the LX-2 cell line of hepatic stellate cell (HSC), which was crucial cellular basis of fibrogenesis, was cultured and incubated with absorbed compounds, the cytotoxicity was determined with the cellomics Multiparameter Cytotoxicity Kit 1 by High Content Screening (HCS), the cell proliferation was assayed with EdU-DNA incorporation, and the cell activation was analyzed through α-smooth muscle actin (α-SMA) expression with high content screening technology.. More than 11 compounds and 2 metabolites from Danshen were identified in the serum after oral administration of FZHY by comparing their mass spectra and retention behavior with reference compounds or literature data. Among these compounds, there were no obvious changes in nuclear morphology, membrane permeability with blow 96 μM of six polar compounds treatment in comparison with control cells, respectively. And the salvianolic acid B (6 μM, 48 μM), caffeic acid (6 μM, 48 μM) and rosmarinic acid (48 μM) could obviously inhibit LX-2 cells proliferation, down-regulate α-SMA expression.. The results proved that the established method could be applied to analyze the absorbed into blood compounds of Danshen after oral administration FZHY. These absorbed compounds included 11 compounds and 2 metabolites of Danshen. Among them, the salvianolic acid B, caffeic acid and rosmarinic acid were the effective components of FZHY to anti-hepatic fibrosis effects.

Topics: Animals; Benzofurans; Caffeic Acids; Cell Membrane Permeability; Cell Proliferation; Cells, Cultured; Cinnamates; Depsides; Drugs, Chinese Herbal; Humans; Liver Cirrhosis; Male; Phenanthrolines; Rats; Rats, Wistar; Rosmarinic Acid; Salvia miltiorrhiza

To prepare self-emulsifying delivery system (SEDDS) of Salvianolic acid B phospholipid complex (SalB-PC) and evaluate its quality.. The best formulation was optimized using single-factor and pseudo-ternary phase diagrams, according to the emulsifying efficiency, the characteristics and partical size of the emulsion and other indicators. The morphology, particle size, zeta-potential and the release in artificial intestinal fluid of self-emulsifying formulation were evaluated.. The weight ratio of SalB-PC: Lauroglycol FCC:Cremophor EL:Transcutol P in the best formulation was 9:45:40:15. SalB-PC loaded self-emulsifying formulation is yellow and transparent solution, with partical size about (187.2 +/- 7.1) nm, polydispersity index (PDI) about 0.267 +/- 0.008 and zeta-potential about (-35.6 +/- 2.7) mV after diluted about 100-fold. The self-emulsifying formulation released slower than the solution with only SalB or SalB-PC.. Water-soluble drug Salvianolic acid B can be prepared to SEDDS, and this formulation can slow down the release of SalB in artificial intestinal fluid.

Topics: Administration, Oral; Benzofurans; Drug Carriers; Drug Compounding; Drug Delivery Systems; Emulsifying Agents; Emulsions; Phospholipids; Solubility

SMND-309 is a novel derivative of salvianolic acid B, and has shown protective effects against rat cortical neuron damage in vitro and in vivo. However the molecular mechanisms through which SMND-309 affords this protection are unclear. The present study aimed to investigate the mechanisms associated with the protective activities of SMND-309 in a cerebral ischemia and reperfusion injury rat model. In this study, we used AG490, a specific inhibitor of the signaling pathway involving the Janus Kinase 2 (JAK2)/Signal Transducers and Activators of Transcription 3 (STAT3) signaling molecules and suramin, a potent inhibitor of vascular endothelial growth factor (VEGF), to investigate the mechanisms of SMND-309. The cerebral ischemia and reperfusion injury model was induced by performing middle cerebral artery occlusion (MCAO) in the rats. SMND-309 mitigated the effects of ischemia and reperfusion injury on brain by decreasing the infract volume, improving neurological function, increasing the survival of neurons and promoting angiogenesis by increasing the levels of erythropoietin (EPO), erythropoietin receptor (EPOR), phosphorylated JAK2 (P-JAK2), phosphorylated STAT3 (P-STAT3), VEGF and VEGF receptor 2 (Flk-1) in the brain. Our results suggest that SMND-309 provides significant neuroprotective effects against cerebral ischemia and reperfusion injury. The mechanisms of this protection may be attributed to the increased VEGF expression occurring from the JAK2/STAT3 pathway, activated by the increased EPO/EPOR expression in the brain.

Topics: Animals; Axons; Benzofurans; Brain; Brain Ischemia; Caffeic Acids; Cerebral Infarction; Dendrites; Erythropoietin; Gene Expression Regulation; Infarction, Middle Cerebral Artery; Janus Kinase 2; Male; Neovascularization, Physiologic; Neuroprotective Agents; Phosphoproteins; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Sprague-Dawley; Receptors, Erythropoietin; Recovery of Function; Reperfusion Injury; Signal Transduction; STAT3 Transcription Factor; Survival Analysis; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2; Water

Salvianolic acid B (SalB), the main water-soluble bioactive compounds isolated from the traditional Chinese medical herb Danshen, has been shown to exert anti-cancer effect in several cancer cell lines. The aim of our study was to investigate the potential anti-cancer effect of SalB in human glioma U87 cells. We found that treatment with SalB significantly decreased cell viability of U87 cells in a dose- and time-dependent manner. SalB also enhanced the intracellular ROS generation and induced apoptotic cell death in U87 cells. Western blot analysis suggested that SalB increased the phosphorylation of p38 MAPK and p53 in a dose-dependent manner. Moreover, blocking p38 activation by specific inhibitor SB203580 or p38 specific siRNA partly reversed the anti-proliferative and pro-apoptotic effects, and ROS production induced by SalB treatment. The anti-tumor activity of SalB in vivo was also demonstrated in U87 xenograft glioma model. All of these findings extended the anti-cancer effect of SalB in human glioma cell lines, and suggested that these inhibitory effects of SalB on U87 glioma cell growth might be associated with p38 activation mediated ROS generation. Thus, SalB might be concerned as an effective and safe natural anticancer agent for glioma prevention and treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzofurans; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Glioma; Humans; Mice; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Tumor Suppressor Protein p53

This work presents an exploratory study for monitoring the hydrolytic process of salvianolic acid B (Sal B) in low oxygen condition using a simple quantitative (1)H NMR (Q-NMR) method. The quantity of the compounds was calculated by the relative ratio of the integral values of the target peak for each compound to the known amount of the internal standard trimethylsilyl propionic acid (TSP). Kinetic runs have been carried out on different initial concentrations of Sal B (5.00, 10.0, 20.0mg/mL) and temperatures of 70, 80, 90°C. The effect of these two factors during the transformation process of Sal B was investigated. The hydrolysis followed pseudo-first-order kinetics and the apparent degradation kinetic constant at 80°C decreased when concentration of Sal B increased. Under the given conditions, the rate constant of overall hydrolysis as a function of temperature obeyed the Arrhenius equation. Six degradation products were identified by NMR and mass spectrometric analysis. Four of these degradation products, i.e. danshensu (DSS), protocatechuic aldehyde (PRO), salvianolic acid D (Sal D) and lithospermic acid (LA) were further identified by comparing the retention times with standard compounds. The advantage of this Q-NMR method was that no reference compounds were required for calibration curves, the quantification could be directly realized on hydrolyzed samples. It was proved to be simple, convenient and accurate for hydrolytic kinetic study of Sal B.

Topics: Benzofurans; Drug Stability; Hydrolysis; Kinetics; Magnetic Resonance Spectroscopy; Temperature

Monardic acids A (1) and B (2), which are (7R,8R) diastereomers of lithospermic acid (LA) and lithospermic acid B, respectively, were isolated from Monarda fistulosa. A (7S,8R) isomer (3) of LA was also isolated from this plant, and a (7R,8S) isomer (7) of LA was obtained from Lithospermum erythrorhizon. The absolute configuration of 1 was confirmed by analysis of its hydrolysates, 7-epiblechnic acid and 2R-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid. The configuration in the dihydrobenzofuran moieties of 2, 3, and 7 was extrapolated by using the phenylglycine methyl ester method and a Cotton effect at approximately 250-260 nm in their electronic circular dichroism spectra. Diastereomers (1-3 and 7) displayed moderate hyaluronidase inhibitory and histamine release inhibitory activities.

Topics: Animals; Benzofurans; Depsides; Enzyme Inhibitors; Histamine Antagonists; Humans; Hyaluronoglucosaminidase; Isomerism; Lithospermum; Molecular Structure; Monarda; Plant Extracts

Salvianolic acid B (Sal B) is a major water soluble component extracted from Radix Salviae miltiorrhizae, a traditional Chinese herb widely used for treating cardiovascular and hepatic diseases. Sal B has been reported to inhibit transforming growth factor (TGF)-β1-stimulated hepatic stellate cells (HSCs) activation and collagen type I expression. In this study, we further investigated the mechanisms of Sal B on liver fibrosis relating to TGF-β/Smads signalling pathway, especially to TGF-β1 receptors. Liver fibrosis model was induced by intraperitoneal injection of dimethylnitrosamine (DMN) for four weeks. Rats were randomly divided into three groups: normal, model, and Sal B groups. Rats in Sal B group were treated by oral administration of Sal B for four weeks from the first day of DMN exposure. Hydroxyproline (Hyp) content in liver tissue was assayed using Jamall's method and collagen deposition was visualized using Sirius red staining. HSCs were isolated from normal rats, and were cultured primarily in uncoated plastics. At day 4 after isolation, cells were stimulated with 2.5 ng/mL TGF-β1, and treated with 1 and 10 µmol/L Sal B and 10 µmol/L SB-431542 (TβR-I inhibitor) for 24 h, respectively. Cell proliferation was examined with 5-ethynyl-2'-deoxyuridine assay. The expressions of alpha smooth muscle actin (α-SMA) and Smad3 were assayed by immunofluorescent stain and Western blotting. The expression of TβR-I was analysed by Western blotting and real-time polymerase chain reaction. The activity of TβR-I kinase was measured by ADP-Glo kinase assay. The results showed that Sal B could inhibit collagen deposition and reduce Hyp content significantly, and decrease expressions of TGF-β1 and TβR-I in fibrotic liver in vivo. Also, Sal B decreased the expressions of α-SMA and TβR-I, inhibited Smad3 nuclear translocation and down-regulated TβR-I kinase activity in vitro. These findings suggested that Sal B could prevent HSCs activation through TGF-β signalling pathway, i.e. inhibiting TGF-β1 expression, activity of TβR-I kinase and Smads phosphorylation.

Topics: Animals; Benzofurans; Cell Proliferation; Collagen Type I; Disease Models, Animal; Hepatic Stellate Cells; Hydroxyproline; Liver Cirrhosis; Male; Phosphorylation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1

To develop an HPLC method to determine the contents of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, salvianolic acid B in the water extract of mixed Salviae Miltiorrhizae Radix et Rhizoma and Carthami Flos simultaneously.. The separation were carried out at 30 degrees C on a ZORBAX Eclipse Plus C18 column (4.6 mm x 100 mm, 1.8 microm) with formic acid-500 mmol x L(-1) ammonium formate-water solution (0.5:10:90) as mobile phase A and acetonitrile-formic acid solution (100: 0.5) as mobile phase B in gradient mode at a flow rate of 0.5 mL x min(-1). Detection wavelengths were 280 nm for danshensu, rosmarinic acid, lithospermic acid, salvianolic acid B, and 380 nm for hydroxysafflor yellow A.. The 5 components were separated well with a good linearity (R2 > 0.999 3) in the range of the test concentration. The average recoveries of danshensu, hydroxysafflor yellow A, rosmarinic acid, lithospermic acid, and salvianolic acid B were 99.1%, 102%, 102%, 98.5% and 101%, respectively.. This method is simple, accurate, and repeatable.

Topics: Benzofurans; Chalcone; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Lactates; Quinones; Rhizome; Rosmarinic Acid; Salvia miltiorrhiza

Inflammation plays a crucial role in the pathophysiology of atherosclerosis. Besides cytokines, chemokines and cell adhesion molecules, CD40 and P-selectin play important roles as key regulators of the inflammatory process in atherosclerosis. Danshen (DS) is commonly used in traditional Chinese medicine for therapy of cardiovascular diseases such as coronary artery disease. The aim of the present study was to evaluate the protective effects of DS with respect to possible anti-inflammatory effects. Human umbilical vein endothelial cells as well as platelets were incubated with an extract of DS or one of its major ingredients salvianolic acid B (Sal B), tanshinone IIA (Tansh) and protocatechuic acid (Protoc) under tumor necrosis factor (TNF)-α or ADP stimulation. Expression of CD40 and cellular adhesion molecules (VCAM-1/ICAM-1) were assessed via flow cytometry. Levels of interleukin (IL)-6, IL-8, monocyte-chemoattractant-protein (MCP)-1 as well as soluble VCAM1 and ICAM-1 in the supernatants were examined via luminex based analysis. Treatment with DS attenuated TNF-α induced expression of CD40. Furthermore, the expression of VCAM-1 and ICAM-1 as well as the release of soluble VCAM-1 and ICAM-1 were downregulated. In the cell supernatants we also observed a significant reduction of IL-6, IL8 and MCP-1. DS and its major ingredients, Sal B and Protoc, significantly inhibited TNF-induced expression and release of adhesion molecules, cytokines and chemokines as well as ADP-induced expression of platelet P-selectin. Because of the key roles of inflammatory mediators in the etiology of atherosclerosis, this work provides useful insight in understanding the pharmacological efficacy of Chinese herbal medicine.

Topics: Abietanes; Adenosine Diphosphate; Anti-Inflammatory Agents; Atherosclerosis; Benzofurans; Blood Platelets; CD40 Antigens; Cells, Cultured; Chemokine CCL2; Down-Regulation; Drugs, Chinese Herbal; Human Umbilical Vein Endothelial Cells; Humans; Hydroxybenzoates; Inflammation Mediators; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; P-Selectin; Salvia miltiorrhiza; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Biotic elicitors can be used to stimulate the production of secondary metabolites in plants. However, limited information is available on the effects of biotic elicitors from endophytic fungi on their host plant. Trichoderma atroviride D16 is an endophytic fungus isolated from the root of Salvia miltiorrhiza and previously reported to produce tanshinone I (T-I) and tanshinone IIA (T-IIA). Here, the effects of extract of mycelium (EM) and the polysaccharide fraction (PSF), produced by T. atroviride D16, on the growth and secondary metabolism of S. miltiorrhiza hairy roots are reported. The results indicated that both EM and PSF promoted hairy root growth and stimulated the biosynthesis of tanshinones in hairy roots. EM slightly suppressed the accumulation of phenolic acids, while PSF had no significant influence on the accumulation of these compounds. When comparing the effects of EM versus PSF, it was concluded that PSF is one of the main active constituents responsible for promoting hairy root growth, as well as stimulating biosynthesis of tanshinones in the hairy root cultures. Moreover, the transcriptional activity of genes involved in the tanshinone biosynthetic pathway increased significantly with PSF treatment. Thus, PSF from endophytic T. atroviride D16 affected the chemical composition of the host plant by influencing the expression of genes related to the secondary metabolite biosynthetic pathway. Furthermore, treatment with PSF can be effectively utilized for large-scale production of tanshinones in the S. miltiorrhiza hairy root culture system.

Topics: Abietanes; Benzofurans; Biomass; Cinnamates; Depsides; Endophytes; Gene Expression Regulation, Plant; Mycelium; Phenols; Plant Roots; Polysaccharides; Rosmarinic Acid; Salvia miltiorrhiza; Trichoderma

To identify and quantify the major metabolites of salvianolic acid B (SAB) after intravenous injection in rats.. LC-IT/TOF-MS was used to identify the metabolites in rat bile, plasma, and urine; LC-MS/MS was used to quantify the two major metabolites.. In rat bile, plasma, and urine, nine metabolites were identified, including methylated metabolites of SAB, lithospermic acid (LSA), the decarboxylation and methylation metabolites of LSA, salvianolic acid S (SAS), and dehydrated-SAS. The t1/2 of monomethyl-SAB and LSA were both very short, and monomethyl-SAB had a larger AUC than LSA in rats.. Nine metabolites were found, the metabolic pathway was described, and the pharmacokinetic profiles of LSA and monomethyl-SAB were studied, thereby clarifying that methylation was the dominant metabolic pathway for SAB in rats.

Topics: Administration, Intravenous; Animals; Benzofurans; Bile; Male; Molecular Structure; Plasma; Rats; Rats, Sprague-Dawley; Urine

To investigate the chemical constituents of Euphorbia pekinensis roots. METHODS Column and liquid chromatography were used for the isolation of chemical constituents, and their chemical structures were determined using a spectroscopic method.. Four compounds were isolated and identified as ziyu glycoside I (1), 3β-α-L-arabinopyranosyloxyurs-12,19(29)-dien-28-oic acid 28-β-D-glucopyranosyl ester (2), lithospermic acid B (3), and senarguine B (4), which were obtained for the first time from the roots of Euphorbia pekinensis.

Topics: Benzofurans; Depsides; Euphorbia; Glycosides; Plant Roots

In order to study the effects of Ca2+ in the biosynthesis of salvianolic acid B (Sal B) induced by salicylic acid (SA) in the young seedlings of Salvia miltiorrhiza, we used confocal laser scanning microscopy and high performance liquid chromatography to measure the change of relative fluorescence intensity of Ca2+ and the contents of Sal B induced by SA before and after the application of extracellular calcium channel inhibitors (VP and LaCl3), intracellular calcium channel inhibitor (LiCl), as well as intracellular calmodulin antagonist (TFP). SA could induce the calcium burst, and the Ca2+ peak could last to 2-3 min in the guard cells of S. miltiorrhiza, which prompted the biosynthesis of Sal B after the Ca2+ burst. Both Vp or LaCl3, and LiCl or TFP could inhibit the burst of Ca2+ and the biosynthesis of Sal B. The above results demonstrated that Ca2+ from the extracellular and the intracellular calcium store regulate the biosynthesis of Sal B elicited by salicylic acid in S. miltiorrhiz young seedlings.

Topics: Benzofurans; Calcium; Plant Leaves; Salicylic Acid; Salvia miltiorrhiza; Seedlings; Signal Transduction

This study investigated phospholipids complex (PC) loaded pellets of poorly permeable Salvianolic acid B (SalB), in which PC was to improve the liposolubility and permeability of SalB. Transmission electron microscopy observation, differential scanning calorimetry measurement, infrared spectroscopy analysis, n-octanol/water partition coefficient study, and foam cell permeability research were employed to prove the complex formation. Pellets containing SalB phospholipids complex (SalB-PC) were prepared via extrusion/spheronization technique. The optimal pellets obtained with 30% SalB-PC, 15% Kollidon®CL-SF, 15% Flowlac®100, and 40% MCC exhibited a very homogeneous size distribution, the shortest disintegration time, highest crushing force, appreciable spherical shape, and a fast drug release behavior. Following hydration, the droplet size distribution of SalB-PC pellets was nearly same to its PC (85.4±16 and 73.5±12nm). In vivo performance showed SalB-PC pellets presented significantly larger AUC(0-)(t), which was 0.58 times more than that of physical mixtures (PMs) and 1.57 times more than that of SalB pellets. C(max) of SalB-PC pellets were also increased by 0.26-fold and 0.80-fold as that of PMs and SalB pellets, respectively. In conclusion, extrusion/spheronization could be a suitable technique to prepare PC loaded pellets, which could effectively preserve the properties of PC to improve the permeability and bioavailability of highly water-soluble drug.

Topics: Animals; Area Under Curve; Benzofurans; Biological Availability; Calorimetry, Differential Scanning; Cellulose; Excipients; Foam Cells; Lactose; Male; Microscopy, Electron, Transmission; Particle Size; Permeability; Phospholipids; Povidone; Rabbits; Solubility; Spectroscopy, Fourier Transform Infrared; Time Factors

The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as one of the causative factors of type 2 diabetes mellitus (T2DM). Salvia miltiorrhiza (Danshen), one of the most commonly used of traditional Chinese medicines, is often used in Compound Recipes for treating diabetes, however with unclear mechanisms. Since salvianolic acid B (SalB) is the most abundant bioactive ingredient of salvia miltiorrhiza water-extract. In this study, we tested whether SalB has any effect on the amyloidogenicity of hIAPP. Our results clearly suggest that SalB can significantly inhibit the formation of hIAPP amyloid and disaggregate hIAPP fibrils. Furthermore, photo-crosslinking based oligomerization studies suggest SalB significantly suppresses the toxic oligomerization of hIAPP monomers. Cytotoxicity protection effects on pancreatic INS-1 cells by SalB were also observed using MTT-based assays, potentially due to the inhibition on the membrane disruption effects and attenuated mitochondria impairment induced by hIAPP. These results provide evidence that SalB may further be studied on the possible pharmacological treatment for T2DM.

Topics: Amino Acid Sequence; Amyloid; Benzofurans; Cell Line; Cell Survival; Diabetes Mellitus, Type 2; Drugs, Chinese Herbal; Erythrocytes; Hemolysis; Humans; Insulin-Secreting Cells; Islet Amyloid Polypeptide; Mitochondria; Molecular Sequence Data; Protein Structure, Secondary; Salvia miltiorrhiza

The clinical use of arsenic trioxide (ATO), a potent anti-neoplastic agent, is often limited because of its severe cardiotoxicity. Salviae miltiorrhiza is widely used for the treatment of cardiovascular diseases. One of the most abundant ingredients of S. miltiorrhiza is salvianolic acid B (Sal B). The present study was designed to evaluate whether Sal B protects against ATO-induced cardiac cell injury in vitro. With MTT cell viability assay, LDH release, ROS generation, caspase-3 activity assay and Hoechst 33342/PI staining, we found that Sal B pretreatment provided significantly protection against ATO-induced cell death. The effect was correlated with the activation of the PI3K/Akt signal pathway. Conversely, blocking Akt activation with the PI3K inhibitor LY294002 effectively suppressed the protective effect of Sal B against ATO-induced cell apoptosis. In addition, the PI3K inhibitor partially blocked the effects of Sal B on the upregulation of Bcl-2 and Bcl-xl protein expression, and downregulation of Bax protein expression. Collectively, the results showed that Sal B decreased the apoptosis and necrosis of H9c2 cardiomyocytes caused by ATO treatment, and PI3K played a crucial role in enhancing cell survival during this process. These observations indicate that Sal B has the potential to exert cardioprotective effects against ATO toxicity.

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; bcl-2-Associated X Protein; bcl-X Protein; Benzofurans; Blotting, Western; Cardiovascular Diseases; Caspase 3; Cell Line; Cell Survival; L-Lactate Dehydrogenase; Myocytes, Cardiac; Oxides; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

When herbal products are used in combination therapy with drugs, alterations in pharmacokinetics, pharmacodynamics, and toxicity can result. Many active components of herbal products are organic anions, and human organic anion transporter 1 (hOAT1, SLC22A6), hOAT3 (SLC22A8), and hOAT4 (SLC22A11) have been identified as potential sites of drug-drug interactions. Therefore, we assessed the effects of lithospermic acid (LSA), rosmarinic acid (RMA), salvianolic acid A (SAA), salvianolic acid B (SAB), and tanshinol (TSL), components of the herbal medicine Danshen, on the function of these transporters. Kinetic analysis demonstrated a competitive mechanism of inhibition for all five. K(i) values (µM) were estimated as 20.8 ± 2.1 (LSA), 0.35 ± 0.06 (RMA), 5.6 ± 0.3 (SAA), 22.2 ± 1.9 (SAB), and 40.4 ± 12.9 (TSL) on hOAT1 and as 0.59 ± 0.26 (LSA), 0.55 ± 0.25 (RMA), 0.16 ± 0.03 (SAA), 19.8 ± 8.4 (SAB), and 8.6 ± 3.3 (TSL) on hOAT3. No significant inhibition of hOAT4 activity by TSL was observed. Using published human pharmacokinetic values, unbound C(max)/K(i) ratios were calculated as an indicator of in vivo drug-drug interaction potential. Analysis indicated a strong interaction potential for RMA and TSL on both hOAT1 and hOAT3 and for LSA on hOAT3. Thus, herb-drug interactions may occur in vivo in situations of co-administration of Danshen and clinical therapeutics known to be hOAT1/hOAT3 substrates.

Topics: Animals; Benzofurans; Caffeic Acids; CHO Cells; Cinnamates; Cricetulus; Depsides; Drugs, Chinese Herbal; HEK293 Cells; Herb-Drug Interactions; Humans; Lactates; Organic Anion Transport Protein 1; Organic Anion Transporters, Sodium-Independent; Phenanthrolines; Rosmarinic Acid; Salvia miltiorrhiza

The aim of this study was to develop novel multiple agents loaded poly (D,L-lactide-co-glycolide acid) (PLGA) nanoparticles (NPs) and evaluate their potential for brain delivery via inner ear administration. PLGA NPs loaded with salvianolic acid B (Sal B), tanshinone IIA (TS IIA) and panax notoginsenoside (PNS) were prepared by double emulsion/solvent evaporation method. It was observed that optimized NPs displayed satisfactory encapsulation efficiency and desired sustained-release characteristics. NPs following intratympanic administration (IT) in guinea pigs greatly improved drug distribution within the inner ear, cerebrospinal fluid (CSF) and brain tissues compared with intravenous administration (IV). Pharmacodynamic studies demonstrated that NPs following IT markedly inhibited oxidizing reactions and protected the brain from cerebral ischemia reperfusion (I/R) injury by upregulating superoxide dismutase (SOD) activity both in serum and brain tissues, simultaneously significantly reducing the levels of malondialdehyde (MDA) and nitric oxide synthase (NOS). Moreover intratympanic delivery did not cause injury of cochlear function by preliminary study on the toxicity. These findings suggested that PLGA NPs-based delivery system via inner ear administration was a promising candidate to brain delivery for the treatment of brain diseases.

Topics: Abietanes; Animals; Benzofurans; Brain; Carotid Arteries; Drug Administration Routes; Drug Carriers; Ear, Inner; Ginsenosides; Guinea Pigs; Lactic Acid; Male; Nanoparticles; Neuroprotective Agents; Panax notoginseng; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Reperfusion Injury

The application of near infrared (NIR) spectroscopy for on-line quantitative monitoring of alcohol precipitation of the Danhong injection was investigated. For the NIR measurements, two fiber optic probes designed to transmit NIR radiation through a 2mm path length flow cell were applied to collect spectra in real-time. Particle swarm optimization- (PSO-) based least square support vector machines (LS-SVM) and partial least squares (PLS) models were developed for quantitative analysis of the critical intermediate quality attributes: the soluble solid content (SSC) and concentrations of danshensu (DSS), protocatechuic aldehyde (PA), hydroxysafflor yellow A (HSYA) and salvianolic acid B (SAB). The optimal models were then used for on-line quantitative monitoring of alcohol precipitation. The results showed that the PSO-based LS-SVM with a radial basis function (RBF) kernel was slightly better than the conventional PLS method, even though both methods exhibited satisfactory fitting results and predictive abilities. In this study, successful models were built and applied on-line; these models proffer real-time data and instant feedback about alcohol precipitation.

Topics: Benzaldehydes; Benzofurans; Catechols; Chalcone; Chemical Precipitation; Drugs, Chinese Herbal; Ethanol; Lactates; Least-Squares Analysis; Microfluidic Analytical Techniques; Online Systems; Quinones; Spectroscopy, Near-Infrared; Support Vector Machine

The aim of the present study was to examine the effect and possible mechanism of salvianolic acid B (SalB) on pulmonary microcirculation disturbance induced by lipopolysaccharide (LPS) in rat. Male Sprague-Dawley rats were subjected to thoracotomy under continuous anesthesia and mechanical ventilation. Albumin leakage from pulmonary capillary and the numbers of leukocytes adherent to the pulmonary capillary wall were determined for 60 min by an upright microscope upon LPS (2 mg · kg(-1) · h(-1)) infusion with or without administration of SalB (5 mg · kg(-1) · h(-1)). Pulmonary tissue wet-to-dry weight ratio, tumor necrosis factor α, and interleukin 8 in plasma and bronchoalveolar lavage fluid were measured. In addition, the expressions of E-selectin, intercellular adhesion molecule 1, and myeloperoxidase in pulmonary tissue were assessed by immunohistochemistry. The expressions of aquaporin 1 (AQP-1), AQP-5, metalloproteinase 2 (MMP-2), and MMP-9 were assessed by Western blot assay. Pretreatment with SalB significantly attenuated LPS-induced pulmonary microcirculatory disturbance, including the increase in leukocyte adhesion and albumin leakage. In addition, LPS increased pulmonary tissue wet-to-dry weight ratio and tumor necrosis factor α and interleukin 8 levels in plasma and bronchoalveolar lavage fluid enhanced the expression of E-selectin, intercellular adhesion molecule 1, myeloperoxidase, MMP-2, and MMP-9, whereas it decreased the expression of AQP-1 and AQP-5 in pulmonary tissue, all of which were attenuated by SalB pretreatment. Salvianolic acid B pretreatment improves pulmonary microcirculation disturbance and lung injury on LPS exposure. More studies are required to evaluate the potential of SalB as an option for protecting lung from endotoxemia.

Topics: Acute Lung Injury; Animals; Benzofurans; Bronchoalveolar Lavage Fluid; Capillaries; Capillary Permeability; Cell Adhesion; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; E-Selectin; Intercellular Adhesion Molecule-1; Interleukin-8; Leukocytes; Lipopolysaccharides; Lung; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Microcirculation; Peroxidase; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

To establish a LC-MS/MS method for determining the concentration of tanshinone IIA, salvianolic acid B and paeoniflorin of refined coronary cataplasm in rabbit plasma, in order to determine the concentration of the three main ingredients in blood after transdermal administration and calculate their pharmacokinetic parameters.. Rabbits were given refined coronary cataplasm on the basis of 15 g x kg(-1) by transdermal administration to detect the plasma concentration of the three main ingredients using LC-MS/MS. Winnonlin software was used to calculate their major pharmacokinetic parameters.. Tanshinone IIA, salvianolic acid B and paeoniflorin showed good linearity (r>0.999) at 1-100, 50-1 000, 10-1 000 microg x L(-1) respectively in plasma, with average recovery rate of 96.57%, 91.90%, 95.93%, respectively. The RSD within day were less than 15%. After transdermal administration of refined coronary cataplasm in rabbits, the main pharmacokinetic parameters of tanshinone IIA, salvianolic acid B or paeoniflorin were as follows: Cmax (20.85 +/- 12.68), (636.25 +/- 386.91), (787.80 +/- 395.64) microg x L(-1); Tmax (0.49 +/- 0.28), (0.44 +/- 0.27), (0.46 +/- 0.30) h.. The LC-MS/MS method is highly selective and sensitive to determine the concentration of samples in rabbit plasma. The pharmacokinetic characteristics of tanshinone IIA, salvianolic acid B and paeoniflorin are suitable to assess the percutaneous absorption of refined coronary cataplasm.

Topics: Abietanes; Animals; Benzoates; Benzofurans; Bridged-Ring Compounds; Chromatography, Liquid; Coronary Disease; Drugs, Chinese Herbal; Glucosides; Humans; Male; Monoterpenes; Rabbits; Tandem Mass Spectrometry

To investigate the effects of Salvianolic-acid B on p38MAPK signaling pathway and its transcriptional factor activated by Transforming growth factor b1 in rat hepatic stellate cells.. Hepatic stellate cells were isolated from normal rat by in situ perfusion and Nycodenz density-gradient centrifugation method.TGFb1 (10 ng/ml), PD98059(50 mumol/L), SB203580(10 mumol/L) and SA-B (10-6 mol/L) were directly added to the medium of the isolated HSCs. Groups: (1)The detection of total p38, MKK3/6, MEF2A and MEF2C induced by TGFb1 in HSC: include control group, SA-B group, SA-B+TGFb1 group and TGFb1 group. (2)The detection of the phosphorylation of p38, MKK3/6 and a-SMA induced by TGFb1 in HSC: include control group, SA-B group, SA-B+TGFb1 group, TGFb1 group, PD98059 group, PD98059+SA-B group, PD98059+TGFb1 group and SA-B+PD98059+TGFb1 group. (3)The effects of SA-B on activity of MEF2 reporter and collagen a 1(I) reporter induced by TGFb1 in HSC: include mt group, wt group, TGFb1 group, SA-B+TGFb1 group, SA-B group, SB203580+TGFb1 group and SB203580 group. Total and phosphorylated p38 and MKK3/6, MEF2A, MEF2C and a-SMA were assayed by Western blot. HSCs were transfected with either MEF2 or collagen a1(I) luciferase reporter gene by Lipofectamine 2000 transfection method, Cellular extracts were assayed for both MEF2 and collagen a1(I) luciferase activities. Comparisons between groups were performed with Student-Newman-Keuls test.. The relative expression level of the phosphorylation of p38 of SA-B group is 0.33+/-0.05,obviously lower than control group(q=7.08, P less than 0.01); SA-B+TGFb1 group is 0.46+/-0.04, obviously lower than TGF b1 group(q=10.45, P less than 0.01); The relative expression level of the phosphorylation of MKK3/6 of SA-B group is 0.11+/-0.07, obviously lower than control group(q=3.944, P less than 0.05); SA-B+TGF b1 group is 0.28+/-0.07, obviously lower than TGFb1 group (q=7.91, P less than 0.01); The relative luciferase activity of MEF2 reporter of SA-B+TGFb1 group and SB203580+TGF b1 group is 2.93+/-0.09 and 2.50+/-0.05 respectively, both obviously lower than TGFb1 group(q=35.35 and 37.2, P less than 0.01); The relative expression level of MEF2C and MEF2A of SA-B group is 15.82+/-0.97 and 13.00+/-0.40 respectively, obviously lower than control group(q is 5.18 and 13.32, both P less than 0.01); SA-B+TGF b1 group is 13.40+/-0.72 and 20.47+/-0.83 respectively, obviously lower than TGFb1 group(q is 43.93 and 12.52,both P less than 0.01); The relative expression level of a-SMA of SA-B+TGFb1 group is 8.76+/-0.44, obviously lower than TGFb1 group(q=20.35, P less than 0.01); SA-B+SB203580+TGFb1 group is only 3.57+/-0.49, obviously lower than TGFb1 group(q=39.78, P less than 0.01); The relative luciferase activity of collagen a1(I) reporter of SA-B+TGF b1 group and SB203580+TGFb1 group is 1.61+/-0.05 and 1.42+/-0.07 respectively, obviously lower than TGFb1 group(q=26.4 and 27.62, both P less than 0.01).. SA-B could inhibit activation of HSC induced by TGFb1 through inhibiting p38MAPK signaling pathway in hepatic stellate cells.

Topics: Animals; Benzofurans; Cells, Cultured; Hepatic Stellate Cells; Male; MAP Kinase Signaling System; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta1

This article studied the possible effect of rifampicin (RIF), an inhibitor of organic anion transporting polypeptide (Oatp), on the pharmacokinetics of salvianolic acid B (SAB) in rats. Rifampicin was administered intravenously 15 min prior to SAB (5 mg/kg) in rats at doses of 0, 5.0, 10.0 and 20.0 mg/kg, respectively. The concentrations of SAB in plasma and bile were determined using a Shimadzu HPLC system coupled to a LC-MS-2010EV mass spectrometer. Compared with the control group, the AUC(0-t) and C(max) values of SAB were increased significantly, while the CL(total) and CL(bile) were decreased significantly. These results suggested that pretreatment with rifampicin prior to SAB administration could decrease significantly the total and bile elimination of SAB and alter its pharmacokinetic profiles. The influence of rifampicin on the pharmacokinetics of SAB may be attributed to the inhibition of Oatp-mediated influx.

Topics: Animals; Area Under Curve; Benzofurans; Bile; Biological Transport; Chromatography, High Pressure Liquid; Chromatography, Liquid; Enzyme Inhibitors; Herb-Drug Interactions; Male; Mass Spectrometry; Organic Anion Transporters; Plant Extracts; Rats; Rats, Sprague-Dawley; Rifampin; Salvia miltiorrhiza

This study investigated the effects of Danshen and its active ingredients on the protein expression and enzymatic activity of CYP1A2 in primary rat hepatocytes. The ethanolic extract of Danshen roots (containing mainly tanshinones) inhibited CYP1A2-catalyzed phenacetin O-deethylation (IC(50)=24.6 μg/ml) in primary rat hepatocytes while the water extract containing mainly salvianolic acid B and danshenshu had no effect. Individual tanshinones such as cryptotanshinone, dihydrotanshinone, tanshinone IIA inhibited the CYP1A2-mediated metabolism with IC(50) values at 12.9, 17.4 and 31.9 μM, respectively. After 4-day treatment of the rat hepatocytes, the ethanolic extract of Danshen and tanshinone I increased rat CYP1A2 activity by 6.8- and 5.2-fold, respectively, with a concomitant up-regulation of CYP1A2 protein level by 13.5- and 6.5-fold, respectively. CYP1A2 induction correlated with the up-regulation of mRNA level of aryl hydrocarbon receptor (AhR), which suggested a positive feedback mechanism of tanshinone I-mediated CYP1A2 induction. A formulated Danshen pill (containing mainly danshensu and salvianolic acid B and the tanshinones) up-regulated CYP1A2 protein expression and enzyme activity, but danshensu and salvianolic acid B, when used individually, did not affect CYP1A2 activity. This study was the first report on the Janus action of the tanshinones on rat CYP1A2 activity.

Topics: Abietanes; Animals; Benzofurans; Cell Survival; Cytochrome P-450 CYP1A2; Cytochromes; Drugs, Chinese Herbal; Enzyme Activation; Enzyme Induction; Ethanol; Hepatocytes; Male; Phenacetin; Phenanthrenes; Phenanthrolines; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Receptors, Aryl Hydrocarbon; RNA, Messenger; Salvia miltiorrhiza; Tacrine

To investigate protective effects of Salvianolic acid B (Sal B) on the intermittent high glucose (IHG)-induced oxidative stress, mitochondrial pathway activation and Schwann cell (SC) apoptosis in vitro.. SCs were primarily cultured and exposed to the different conditions. Apoptosis was confirmed by the Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and concentration of 8-hydroxy-2-deoxy Guanosine (8-OHdG) was detected by Elisa. Intracellular ROS generation and mitochondrial transmembrane potential (ΔΨm) were detected by flow cytometry analysis. Quantitative real-time reverse transcriptase PCR was performed to analyze the expression levels of Bax and BcL-2. Western blot was performed to analyze the expression levels of some important transcription factors and proteins.. Treatment with Sal B inhibited the IHG-induced oxidative stress by reducing ROS production and 8-OHdG levels, mitochondrial depolarization and apoptosis in SCs in a dose-dependent manner. Furthermore, treatment with Sal B down-regulated the IHG-induced release of cytochrome c, AIF nuclear translocation and Bax expression, but mitigated the IHG-mediated down-regulation of BcL-2 expression in SCs. In addition, treatment with Sal B attenuated the IHG-induced activation of caspase-9 and caspase-3 and minimized the cleavage of PARP in SCs.. Our results indicated that IHG induced SC apoptosis in both caspase-dependent and caspase-independent pathways by activating the mitochondrial pathway. Sal B inhibited the IHG-induced oxidative stress, activation of the mitochondrial pathway and apoptosis in SCs.

Topics: Animals; Animals, Newborn; Apoptosis; Benzofurans; Cells, Cultured; Drugs, Chinese Herbal; Glucose; Oxidative Stress; Rats; Rats, Sprague-Dawley; Schwann Cells

Oral cancer typically develops from hyperplasia through dysplasia to carcinoma with a multistep process of carcinogenesis involving genetic alterations resulting in aberrant cellular appearance, deregulated cell growth, and carcinoma. The metabolic transformation during the process of oral carcinogenesis and its implications for cancer therapy have not been extensively investigated. Here, we report a metabonomic study on a classical model of 7,12-dimethylbenz(a)anthracene (DMBA)-induced oral carcinogenesis in hamsters to delineate characteristic metabolic transformation during the carcinogenesis using gas chromatography time-of-flight mass spectrometry (GC-TOF MS). Salvianolic acid B (Sal-B), isolated from Salvia miltiorrhiza Bge, and Breviscapine, a flavonoid isolated from Herba Erigerontis, were used to treat the hamsters exposed to DMBA to investigate the molecular mechanism of the inhibitory effect of the two agents on oral carcinogenesis. The dynamic changes of serum metabolic profiles indicated that both Sal-B and Breviscapine were able to attenuate DMBA-induced metabolic perturbation, which is consistent with the histopathological findings that Sal-B and Breviscapine significantly decreased the squamous cell carcinoma (SCC) incidence in the two treatment groups. Significant alterations of key metabolic pathways, including elevated glutaminolysis and glycolysis, and decreased cholesterol and myo-inositol metabolism, were observed in the DMBA-induced model group, which were attenuated or normalized by Sal-B or Breviscapine treatment. Elevated inflammation and tumor angiogenesis at gene and metabolite expression levels were also observed in DMBA-induced oral dysplasia and SCC but were attenuated or normalized by Sal-B and Breviscapine along with significantly decreased incidences of SCC formation.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benzofurans; Carcinogens; Cricetinae; Drug Interactions; Flavonoids; Gas Chromatography-Mass Spectrometry; Histocytochemistry; Male; Mesocricetus; Metabolic Networks and Pathways; Metabolome; Metabolomics; Mouth Neoplasms; Neovascularization, Pathologic

Diabetic peripheral neuropathy (DPN) is one of the most common and debilitating microvascular complications of diabetes, and there is no effective therapy for the prevention or treatment of DPN. Oxidative stress triggers several pathways of injury and may be the unifying factor of hyperglycemia. The aim of this study was to investigate protective effect of Salvianolic acid B (Sal B) on the high glucose (HG)-induced oxidative stress-induced mitochondrial pathway activation and Schwann cells (SCs) apoptosis in vitro. We found that Sal B inhibited the HG-induced oxidative stress by reducing ROS and 8-hydroxy-2-deoxy Guanosine (8-OHdG) production, and mitochondrial depolarization and apoptosis in SCs in a dose-dependent manner. Furthermore, Sal B down-regulated the HG-mediated Bax expression and AIF nuclear translocation and the release of cytochrome c, but up-regulated the HG-induced BcL-2 expression in SCs. In addition, Sal B attenuated the HG-induced activation of caspase 3 and 9 and minimized the cleavage of PARP in SCs. Our results indicated that Sal B antagonized the HG-induced oxidative stress, activation of the mitochondrial pathway and apoptosis in SCs.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Animals, Newborn; Apoptosis; Base Sequence; Benzofurans; Cells, Cultured; Deoxyguanosine; DNA Primers; Glucose; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Schwann Cells

Lithospermic acid B (LAB), an active component of danshen, is known to inhibit the proliferation of vascular smooth muscle cells (VSMCs) and has pharmacological activity scavenging free radicals in VSMCs. However, the precise mechanism through which LAB exerts its antiproliferative effect is unclear. Therefore, we investigated how LAB regulates cellular proliferation in primary cultured rat VSMCs. Using fluorescein isothiocyanate (FITC)-conjugated LAB to track its cellular localization, we show that LAB localizes to the nucleus, specifically to the nucleolus, where it binds to histone H3, leading to the inhibition of the platelet-derived growth factor (PDGF)- induced phosphorylation of histone H3. LAB also only moves into the nucleus during the normal expression of nonmuscle myosin heavy chain (NMHC-IIA), which is associated with LAB in VSMCs. Notably, LAB suppressed the PDGF-induced phosphorylation of Akt and the expression of cyclin D2 in the presence of NMHC-IIA expression. Knockdown of NMHC-IIA expression impeded the function of LAB, which was then unable to inhibit the PDGF-induced proliferation of VSMCs. We conclude that LAB modulates the PDGF-induced proliferation of VSMCs by interacting with NMHC-IIA, which allows LAB to localize in the nucleus and to suppress the PDGF-induced proliferation of VSMCs.

Topics: Animals; Aorta, Thoracic; Benzofurans; Cell Nucleolus; Cell Nucleus; Cell Proliferation; Cells, Cultured; Cyclin D2; Cytoplasm; Depsides; Growth Inhibitors; Histones; Molecular Motor Proteins; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Myosin Heavy Chains; Phosphorylation; Platelet-Derived Growth Factor; Protein Binding; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; RNA, Small Interfering

We studied the effect of ultrasonication extraction technology combined with ammonium sulfate/ethanol aqueous two-phase system (ATPS) for the separation of lithospermic acid B (LAB) from Salvia miltiorrhiza Bunge. According to the literature and preliminary studies, ammonium sulfate concentration, ethanol concentration, pH, ultrasonication power, ultrasonication time and the ratio of solvent-to-solid were investigated using a single factor design to identify the factors affecting separation. Taking into consideration a simultaneous increase in LAB recovery (R (%)) and partition coefficient (K), the best performance of the ATPS was obtained at 25°C and pH 2 using ammonium sulfate 22% (w/w) and ethanol 30% (w/w). To keep the solvent-to-solid ratio at 10, response surface methodology was used to find the optimal ultrasonication power and ultrasonication time. Quadratic models were predicted for LAB yield in the upper phase. Optimal conditions of 572.1 W ultrasonication power and 42.2 min produced a maximum yield of LAB of 42.16 mg g(-1) sample. There was no obvious degradation of LAB with ultrasound under the applied conditions, and the experimental yield of LAB was 42.49 mg g(-1) sample and the purity was 55.28% (w/w), which was much higher than that obtained using conventional extraction. The present study demonstrated that ultrasound coupled with aqueous two-phase systems is very efficient tool for the extraction and purification of LAB from Salvia miltiorrhiza Bunge.

Topics: Ammonium Sulfate; Benzofurans; Chromatography, High Pressure Liquid; Depsides; Ethanol; Molecular Structure; Salvia miltiorrhiza; Sonication; Water

Drying is the most common and fundamental procedure in the post-harvest processing which contributes to the quality and valuation of medicinal plants. However, attention to and research work on this aspect is relatively poor. In this paper, we reveal dynamic variations of concentrations of five major bioactive components, namely salvianolic acid B (SaB), dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA, in roots of Salvia miltiorrhiza (Dashen) during the drying process at different oven temperatures. A minor amount of SaB was found in fresh materials while an noticeable increase in SaB was detected in drying at 50~160 °C. The maximal value occurred after 40 min of drying at 130 °C and its variation showed a reverse V-shaped curve. Production of SaB exhibited a significant positive correlation with drying temperatures and a significant negative correlation with sample moistures. The amounts of tanshinones were nearly doubled in the early stage of drying and their variations showed similar changing trends with drying temperatures and sample moistures. The results supported our speculation that postharvest fresh plant materials, especially roots, were still physiologically active organs and would exhibit a series of anti-dehydration mechanisms including production of related secondary metabolites at the early stage of dehydration. Hence, the proper design of drying processes could contribute to promoting rather than reducing the quality of Danshen and other similar medicinal plants.

Topics: Abietanes; Benzofurans; Chromatography, High Pressure Liquid; Desiccation; Drugs, Chinese Herbal; Medicine, Chinese Traditional; Phenanthrenes; Plant Roots; Salvia miltiorrhiza

The clinical application of salvianolic acid B (Sal B), a potential therapeutic agent for cardiovascular diseases isolated from Salvia miltiorrhiza, is greatly restricted by its short half-life and low bioavailability. To improve therapeutic effects and prolong the systemic circulation time of Sal B, liposomes, composed of soybean phosphatidylcholine and cholesterol were prepared by reverse-phase evaporation method. In addition, polyethylene glycol 2000-disteroylphosphoethanolamine (PEG-DSPE 2000) was included to give steric barrier to liposomes. A central composite design was employed to optimize liposomal formulation with high encapsulation efficiency and small particle size. Physicochemical characteristics such as particle size, zeta potential, encapsulation efficiency and in vitro release were investigated. In vivo pharmacokinetic properties of Sal B in beagle dogs and the effect of PEG on the blood circulation time of Sal B-loaded liposomes were also evaluated. An optimized formulation with encapsulation efficiency of 73.68% and mean particle size of 136.6nm were developed. Encapsulation of Sal B into conventional and PEGylated liposomes could prolong the half-life of Sal B by 5.8- and 17.5-fold and enhance the AUC(0-t) of Sal B by 6.7- and 13.3-fold compared with free Sal B, respectively. Therefore, the use of PEGylated liposomes could prolong the circulation time in blood and longevity effect of liposomes on Sal B was increased by PEG.

Topics: Animals; Benzofurans; Biological Availability; Capsules; Cardiovascular Agents; Chemistry, Pharmaceutical; Cholesterol; Dogs; Glycine max; Half-Life; Liposomes; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Polyethylene Glycols; Salvia miltiorrhiza

Salvia miltiorrhiza (Danshen), a traditional Chinese herbal medicine, is commonly used for the prevention and treatment of cardiovascular disorders including atherosclerosis. However, the mechanisms responsible for the vasoprotective effects of Danshen remain largely unknown. Salvianolic acid B (Sal B) represents one of the most bioactive compounds that can be extracted from the water-soluble fraction of Danshen. We investigated the effects of Danshen and Sal B on the inflammatory response in murine macrophages. Danshen and Sal B both induced the expression of heme oxygenase-1 (HO-1) and inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Inhibition of HO activity using Sn-protoporphyrin-IX (SnPP) abolished the inhibitory effect of Sal B on NO production and iNOS expression. Sal B increased macrophage arginase activity in a dose-dependent manner and diminished LPS-inducible tumor necrosis factor-α production. These effects were also reversed by SnPP. These data suggest that HO-1 expression plays an intermediary role in the anti-inflammatory effects of Sal B. In contrast to the observations in macrophages, Sal B dose-dependently inhibited arginase activity in murine liver, kidney, and vascular tissue. Furthermore, Sal B increased NO production in isolated mouse aortas through the inhibition of arginase activity and reduction of reactive oxygen species production. We conclude that Sal B improves vascular function by inhibiting inflammatory responses and promoting endothelium-dependent vasodilation. Taken together, we suggest that Sal B may represent a potent candidate therapeutic for the treatment of cardiovascular diseases associated with endothelial dysfunction.

Topics: Animals; Arginase; Benzofurans; Blotting, Western; Drugs, Chinese Herbal; Electrophoretic Mobility Shift Assay; Fibrinolytic Agents; Heme Oxygenase-1; Humans; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Nitric Oxide Synthase Type II; Phenanthrolines; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Salvia miltiorrhiza; Tumor Necrosis Factor-alpha; Vascular Diseases

Glucocorticoid (GC) induced osteoporosis (GIO) is caused by the long-term use of GC for treatment of autoimmune and inflammatory diseases. The GC related disruption of bone marrow microcirculation and increased adipogenesis contribute to GIO development. However, neither currently available anti-osteoporosis agent is completely addressed to microcirculation and bone marrow adipogenesis. Salvianolic acid B (Sal B) is a polyphenolic compound from a Chinese herbal medicine, Salvia miltiorrhiza Bunge. The aim of this study was to determine the effects of Sal B on osteoblast bone formation, angiogenesis and adipogenesis-associated GIO by performing marrow adipogenesis and microcirculation dilation and bone histomorphometry analyses. (1) In vivo study: Bone loss in GC treated rats was confirmed by significantly decreased BMD, bone strength, cancellous bone mass and architecture, osteoblast distribution, bone formation, marrow microvessel density and diameter along with down-regulation of marrow BMPs expression and increased adipogenesis. Daily treatment with Sal B (40 mg/kg/d) for 12 weeks in GC male rats prevented GC-induced cancellous bone loss and increased adipogenesis while increasing cancellous bone formation rate with improved local microcirculation by capillary dilation. Treatment with Sal B at a higher dose (80 mg/kg/d) not only prevented GC-induced osteopenia, but also increased cancellous bone mass and thickness, associated with increase of marrow BMPs expression, inhibited adipogenesis and further increased microvessel diameters. (2) In vitro study: In concentration from 10(-6) mol/L to 10(-7) mol/L, Sal B stimulated bone marrow stromal cell (MSC) differentiation to osteoblast and increased osteoblast activities, decreased GC associated adipogenic differentiation by down-regulation of PPARγ mRNA expression, increased Runx2 mRNA expression without osteoblast inducement, and, furthermore, Sal B decreased Dickkopf-1 and increased β-catenin mRNA expression with or without adipocyte inducement in MSC. We conclude that Sal B prevented bone loss in GC-treated rats through stimulation of osteogenesis, bone marrow angiogenesis and inhibition of adipogenesis.

Topics: Adipocytes; Adipogenesis; Animals; Benzofurans; beta Catenin; Bone and Bones; Bone Marrow Cells; Bone Morphogenetic Proteins; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Gene Expression; Intercellular Signaling Peptides and Proteins; Male; Neovascularization, Physiologic; Osteoblasts; Osteogenesis; Osteoporosis; PPAR gamma; Prednisone; Rats; Rats, Sprague-Dawley; Stromal Cells

To investigate the effects of salvianolic acid B (SA-B) on extracellular signal-regulated kinase (ERK) signal transduction pathway activated by transforming growth factor-β1 (TGF-β1) in rat hepatic stellate cells (HSCs).. HSCs were isolated from male Sprague-Dawley rats by in situ perfusion and Nycodenz density-gradient centrifugation method. TGF-β1 and SA-B were directly added to the serum-free medium of HSCs. Total and phosphorylated ERK, MEK, Raf and α-smooth muscle actin (α-SMA) and type I collagen were assayed by Western blotting.. Phosphorylation of MEK in HSCs with or without TGF-β1 was inhibited by SA-B; however, phosphorylation of Raf in HSCs with or without TGF-β1 was not inhibited by SA-B. Expression of α-SMA in HSCs with TGF-β1 was inhibited by SA-B. Combination of SA-B and the inhibitors of ERK (PD98059) can effectively inhibit the expression of α-SMA. SA-B also inhibited synthesization of type I collagen in HSCs with or without TGF-β1.. The action point of SA-B inhibiting ERK signaling induced by TGF-β1 in HSCs is the inhibition of the phosphorylation of MEK. SA-B reduces the increase of expression of α-SMA and protein synthesization of type I collagen induced by TGF-β1 by means of inhibiting ERK signaling in activated HSCs of rats.

Topics: Actins; Animals; Benzofurans; Collagen Type I; Extracellular Signal-Regulated MAP Kinases; Hepatic Stellate Cells; Male; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta1

Small molecules have been shown to modulate the neurogenesis processes. In search for new therapeutic drugs, the herbs used in traditional medicines for neurogenesis are promising candidates.. We selected a total of 45 natural compounds from Traditional Chinese herbal medicines which are extensively used in China to treat stroke clinically, and tested their proliferation-inducing activities on neural stem/progenitor cells (NSPCs). The screening results showed that salvianolic acid B (Sal B) displayed marked effects on the induction of proliferation of NSPCs. We further demonstrated that Sal B promoted NSPCs proliferation in dose- and time-dependent manners. To explore the molecular mechanism, PI3K/Akt, MEK/ERK and Notch signaling pathways were investigated. Cell proliferation assay demonstrated that Ly294002 (PI3K/Akt inhibitor), but neither U0126 (ERK inhibitor) nor DAPT (Notch inhibitor) inhibited the Sal B-induced proliferation of cells. Western Blotting results showed that stimulation of NSPCs with Sal B enhanced the phosphorylation of Akt, and Ly294002 abolished this effect, confirming the role of Akt in Sal B mediated proliferation of NSPCs. Rats exposed to transient cerebral ischemia were treated for 4 weeks with Sal B from the 7th day after stroke. BrdU incorporation assay results showed that exposure Sal B could maintain the proliferation of NSPCs after cerebral ischemia. Morris water maze test showed that delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats.. Sal B could maintain the NSPCs self-renew and promote proliferation, which was mediated by PI3K/Akt signal pathway. And delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats. These findings suggested that Sal B may act as a potential drug in treatment of brain injury or neurodegenerative diseases.

Topics: Adult Stem Cells; Animals; Benzofurans; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Drugs, Chinese Herbal; Gene Expression Regulation; Learning; Male; MAP Kinase Signaling System; Memory; Neural Stem Cells; Neurogenesis; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Receptors, Notch

To study the protective effect and mechanism of salvianolic acid B (Sal B) on glutamate-induced excito-toxicity.. Glutamate-induced PC12 cell injury model was established to detect the cell survival rate by MTT, the leakage rate of lactic dehydrogenases using LDH, and the cell apoptosis by using AO/EB double staining for fluorescence microscope and PI single staining flow cytometry which was also used to detect the content of intracellular reactive oxygen species. The expression of Caspase-3 protein was also detected by the Western blotting method.. Sal B is proved to inhibit glutamate-induced PC12 cells from injury and prevent them from releasing LDH within the range from 50 micromol x L(-1) to 200 micromol x L(-1). Meanwhile, Sal B has an effect on significantly reducing the expression of inhibit glutamate-induced active Caspase-3 protein, inhibiting accumulated glutamate-induced ROS and decreasing PC12 cell apoptosis rate within the range from 50 micromol x L(-1) to 200 micromol x L(-1).. The study proves that Sal B prevented against glutamate-induced cell injury via inhibiting ROS formation and Caspase-3 pathway-dependent apoptosis in PC12 cells.

Topics: Action Potentials; Animals; Apoptosis; Benzofurans; Caspase 3; Cell Proliferation; Drugs, Chinese Herbal; Excitatory Amino Acid Antagonists; Glutamic Acid; Lactate Dehydrogenases; PC12 Cells; Pheochromocytoma; Rats; Reactive Oxygen Species

To study the protective effect and mechanism of salvianolic acid B on isolated heart ischemia/reperfusion injury in rats.. Forty-eight SD rats were divided into 6 groups randomly(n = 8): the control group, the positive administration group (verapamil 150 microg x L(-1)), and high, middle and low-dose salvianolic acid B groups (10, 5, 2.5 mg x L(-1)). The myocardial ischemia/reperfusion injury model was established using the Langendorff method, re-perusing isolated working hearts for 30 min after ischemia for 25 min. A water-bag catheter was inserted in rat left atrium for recording the effect of salvianolic acid B on hemodynamics indexes-AST, LDH, SOD and MDA.. Various group with different doses showed that salvianolic acid B decreased AST, release of LDH and formation of MDA and increased SOD activity.. Salvianolic acid B showed a protective effect on myocardial ischemia/reperfusion injury. Its mechanism may be related with improvement of cardiac contractility, cleaning of oxygen free radicals and reduction of lipid peroxidation.

Topics: Animals; Benzofurans; Drugs, Chinese Herbal; Hemodynamics; In Vitro Techniques; L-Lactate Dehydrogenase; Male; Malondialdehyde; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Time Factors

The main purpose of this study was to illustrate the effect of borneol on pharmacokinetics of salvianolic acid B (SalB) in rats after oral administration of SalB with different doses of borneol. The concentrations of SalB in rat plasma were determined by an established and validated LC-MS/MS method. Our data showed that when 20, 40, and 80 mg kg⁻¹ of borneol were orally administrated with SalB at 50 mg kg⁻¹, C (max) of SalB was increased by 18.4%, 55.8%, and 103.2% compared with that of SalB alone. And AUC(0-t) of SalB in plasma was increased by 14.4%, 48.5%, and 123.3%, respectively. The results indicated that borneol is able to enhance the intestinal absorption and relative bioavailability of SalB, with a positive dose-dependent relationship. The described herb-drug interactions might prove the scientific rationality of the compatible ratio of traditional Chinese medicines.

Topics: Administration, Oral; Animals; Benzofurans; Camphanes; Dose-Response Relationship, Drug; Male; Medicine, Chinese Traditional; Rats

CD36, a class B scavenger receptor, has been implicated in the pathogenesis of a host of vascular inflammatory diseases. Through a high-throughput screening (HTS) assay for CD36 antagonist, we previously identified salvianolic acid B (SAB), a hydrophilic component derived from the herb Danshen, as a potential candidate. Danshen, the dried roots of Salvia miltiorrhiza, has been widely used in China for the prevention and treatment of atherosclerosis-related disorders. Previous studies showed that SAB acted as an anti-oxidant by preventing lipid peroxidation and oxidized LDL (oxLDL) formation. The present study was to investigate the specificity and efficacy of SAB in the inhibition of CD36-mediated lipid uptake. SAB reduced modified LDL (mLDL) uptake in a dose-dependent manner in phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 and RAW 264.7 cells. In the CD36 silenced THP-1 cells, SAB had no effect in reducing mLDL uptake, whereas its overexpression in CHO cells reinstates the effect, indicating a specific involvement of SAB in antagonizing the CD36's function. Surface plasmon resonance (SPR) analysis revealed a direct binding of SAB to CD36 with a high affinity (K(D) = 3.74 μM), confirming physical interactions of SAB with the receptor. Additionally, SAB reduced oxLDL-induced CD36 gene expression in the cultured cell lines and primary macrophages. In ApoE KO mice fed a high fat diet, SAB reduced CD36 gene expression and lipid uptake in macrophages, showing its ability to antagonize CD36 pathways in vivo. These results demonstrate that SAB is an effective CD36 antagonist and suggest SAB as a potential anti-atherosclerotic agent.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Benzofurans; Biological Transport; CD36 Antigens; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Dose-Response Relationship, Drug; Humans; Hyperlipidemias; Hypolipidemic Agents; Lipoproteins, LDL; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, LDL; RNA Interference; Surface Plasmon Resonance; Tetradecanoylphorbol Acetate; Time Factors; Transfection

To observe the proliferation state of transplanted cells in acute myocardial infarction (AMI) rats, and the endothelial progenitor cells (EPCs) preconditioned by salvianolic acid B in different ratios with the bone mesenchymal stem cells (BMSCs).. The cultivation and purification of EPCs were performed by density-gradient centrifugation and plastic adherence method. Two types of cells were identified by immunocytochemical method (CD34, CD133, and CD44). The rat model of AMI was prepared by ligation of left anterior descending artery. The EPCs were pre-treated with the optimal concentration of salvianolic acid B (8 microg/ mL). They were mixed with BMSCs in different proportions (EPCs/BMSCs in the ratio of 1:1, 2:1, 4:1, and 8:1, respectively). BMSCs and EPCs were injected into the myocardial infarction area. The infarcted area was determined by the N-BT staining and hematoxylin-eosin staining. The expression of Ki-67 was detected by immunohistochemical assay.. Compared with the model group (19.60% +/- 3.23%), the myocardial infarction area of each implanted group obviously decreased (P < 0.05). Of them, the decrease was most obvious in the 4:1 group (11.37% +/- 2.18%) and the 8:1 group (9.23% +/- 2.35%, P < 0.05). Compared with the model group (cell/high magnification, 5.17 +/- 2.31), the Ki-67 positive cell number of each implanted groups significantly increased (P < 0.05). Of them, the Ki-67 positive cell number was obviously higher in the 8:1 group (15.00 +/- 3.16, P < 0.05).. EPCs pretreated by salvianolic acid B combined with BMSCs could reduce the myocardial infarcted area, improve the proliferation of BMSCs in the peripheral infarction and local ischemia. Besides, along with the increase of the implant proportion of EPCs, the infarct area was gradually reduced, and the proliferative expression was gradually enhanced.

Topics: Animals; Benzofurans; Bone Marrow Cells; Cell Proliferation; Endothelial Cells; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Myocardial Infarction; Rats; Rats, Wistar; Transplantation Conditioning

Salvianolic acid B (SalB), one of the most abundant and bioactive compounds extracted from Salvia miltiorrhiza (Danshen), shows neuroprotective and anti-inflammatory activities in vivo and in vitro. This research was intended to investigate the antidepressant effect of SalB by forced swimming test (FST) and tail suspension test (TST).. SalB was extracted from S. miltiorrhiza roots and followed by HPLC analysis. Thirty five male C57BL/6 mice were divided into five groups: three SalB groups of different doses, one imipramine group, and one control group. The SalB groups received intraperitoneally (i.p.) 5mg/kg SalB, 10mg/kg SalB, and 20mg/kg SalB respectively. At the same time, the imipramine group received 20mg/kg imipramine, and the control group saline only. The behavioral tests including FST, TST and locomotor activity test were done after administration of drugs for consecutively three times, at 24, 1, and 0.5h before the tests.. SalB, from S. miltiorrhiza with purity of 95%, significantly reduced the immobility time in both the FST and TST tests (doses at 5, 10, 20mg/kg, i.p.), without changing locomotion in spontaneous motor activity.. This data suggests that besides neuroprotective and anti-inflammatory activities, SalB has promising therapeutic potential in treatment of depressive disorders.

Topics: Animals; Antidepressive Agents; Benzofurans; Depression; Hindlimb Suspension; Male; Mice; Mice, Inbred C57BL; Plant Extracts; Plant Roots; Swimming; Treatment Outcome

Salvianolic acid B (Sal B) is the most abundant phenolic compound in Salvia miltiorrhiza, which has been widely used for the treatment of cardiovascular diseases in Oriental medicine. To elucidate structure of the converted compounds of Sal B by decoction in aqueous solution, Sal B (200 mg) was decocted in an aqueous solution (200 mL) at pH 4.9 and the decocted solution was purified by Sephadex LH-20 column chromatography and preparative HPLC. The 13 converted compounds were isolated and the chemical structures were determined by NMR and MS. In addition to the 4 compounds previously reported as conversion products of Sal B by decoction, 9 compounds were first reported with the complete structure of compounds isolated from decocted Sal B solution and three of the compounds were determined to be novel compounds. In addition, a conversion mechanism of compounds converted by decoction was proposed on the basis of kinetics studies, which reasonably supported the conversion mechanism of Sal B. The 13 compounds seemed to be produced by the hydrolysis of an ester bond, decarboxylation, retro oxa-Michael reaction, hydration, and radical reaction during decoction in aqueous solution.

Topics: Benzofurans; Drugs, Chinese Herbal; Molecular Structure; Salvia miltiorrhiza

To establish the liver-depression and spleen-deficiency syndrome model in rats to screen the optimal extraction method of small compound Yueanjian on the basis of pharmacodynamic and chemical indicators.. The PMS liver-depression and spleen-deficiency syndrome model were established by the chronic restraint stress method and treatment with Yueanjian extracted by three methods: water-extraction, steam-distillation and alcohol-extraction. Behavioral performances and the contents of estradiol and progestin in serum were determined before and after the administration of the three extracts. The contents of salvianolic acid B in these three extracts were detected by HPLC. The optimal extraction method of Yueanjian was selected according to pharmacodynamic results.. The contents of estradiol and progestin in groups treated with steam distillations and alcoholic extraction were higher than the model group. In the open field test, the group treated with steam distillations showed much higher scores than the model group. HPLC showed that the content of salvianolic acid B extracted by steam-distillation was higher than the other two extracts.. On the basis of pharmacodynamic and chemical results, the steam-distillation was proved to be best extraction method of Yueanjian.

Topics: Animals; Benzofurans; Chromatography, High Pressure Liquid; Distillation; Drugs, Chinese Herbal; Estradiol; Female; Premenstrual Syndrome; Progestins; Rats; Rats, Wistar

To investigate the influence and mechanism of salvianolic acid B (SalB) on apoptosis inhibition in rat bone marrow-derived mesenchymal stem cells (BMSCs) induced by hypoxia and serum deprivation (hypoxia/SD).. SalB concentration of 0.1, 1, 10 or 100 mg/L (drug groups) were investigated for their ability to inhibit apoptosis in rat BMSCs. BMSCs in both the apoptosis model and drug groups were cultured under hypoxic conditions for 6 h, after which cell apoptosis and change in mitochondrial membrane potential (MMP) were detected using flow cytometry. Activation of caspase-3 was detected using western blot analysis.. Hypoxia/SD induced apoptosis in rat BMSCs. The early apoptosis rate was lower in the drug groups compared to the apoptosis model group (P < 0.05). SalB was found to inhibit the reduction in MMP and decrease the activation of caspase-3.. 0.1, 1 and 10 mg/L of SalB inhibits activation of caspase-3 and early apoptosis of rat BMSCs induced by hypoxia/SD and could therefore enhance the survival rate of grafted stem cells.

Topics: Animals; Apoptosis; Benzofurans; Cell Hypoxia; Cell Proliferation; Cells, Cultured; Culture Media, Serum-Free; Drugs, Chinese Herbal; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza

Resting cells of Fusarium oxysporum f. sp. Cucumerinum (F. oxsporum) were used for the biotransformation of salvianolic acid B (Sal B). Three transformed products, isolithospermic acid, prolithospermic acid and danshensu, were identified on the basis of chemical and spectroscopic data. The stability of the two ester bonds of Sal B was studied and two degradation routes were found. In the biotransformation system, Sal B was transformed into isolithospermic acid first which was then converted into prolithospermic acid. In alkaline solutions, Sal B was transformed into lithospermic acid first which was then converted into prolithospermic acid. This is the first reports of the NMR spectra of isolithospermic acid and this result may indicate the metabolic pathways of Sal B in vivo.

Topics: Benzofurans; Depsides; Fusarium; Magnetic Resonance Spectroscopy

This study provides a desirable candidate gene resource (SmAOC) to increase the content of valuable natural products via appropriate JA pathway genetic engineering. Jasmonates (JAs) are important signal molecules in plants. They regulate transcripts of defense and secondary biosynthetic metabolite genes in response to environmental stresses. Currently, JAs are widely used as elicitors to improve the content of useful secondary metabolism in plants. Synthesis of the naturally occurring enantiomer of various jasmonates is catalyzed by allene oxide cyclase (AOC, EC 5.3.99.6). Here, we cloned and characterized the AOC gene (SmAOC) from Salvia miltiorrhiza. As expected, SmAOC expression was induced by abiotic stimuli such as methyl jasmonate (MeJA), ultraviolet radiation (UV) and low temperature (4 °C) in S. miltiorrhiza plantlets. To demonstrate whether the engineered internal JAs pool by overexpressing AOC gene could promote secondary metabolism production, the SmAOC was incorporated into S. miltiorrhiza hairy roots. The results revealed that SmAOC overexpression significant enhanced the yields of tanshinone IIA, rosmarinic acid (RA) and lithospermic acid B (LAB) in S. miltiorrhiza hairy roots. In addition, expression levels for key genes involved in the biosynthetic pathway of diterpenes and phenolic acids were also altered. These suggest that genetic manipulation of AOC would be helpful for improving the production of valuable secondary metabolites by regulating the biosynthesis of JAs.

Topics: Abietanes; Acetates; Benzofurans; Cinnamates; Cloning, Molecular; Cold Temperature; Cyclopentanes; Depsides; Diterpenes; Escherichia coli; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Genetic Engineering; Genetic Vectors; Hydroxybenzoates; Intramolecular Oxidoreductases; Oxylipins; Plant Roots; Rosmarinic Acid; Salvia miltiorrhiza; Transgenes; Ultraviolet Rays

The contraction of hepatic stellate cells (HSCs) has a critical role in the regulation of intrahepatic vascular resistance and portal hypertension. Previous studies have confirmed that salvianolic acid B (Sal B) is effective against liver fibrosis. In the present study, we evaluated the effect of Sal B on portal hypertension and on HSCs contractility. Liver cirrhosis was induced in rats by peritoneal injection of dimethylnitrosamine and the portal pressure was measured. HSCs contraction was evaluated by collagen gel contraction assay. Glycerol-urea gel electrophoresis was performed to determine the phosphorylation of myosin light chain 2 (MLC2). F-actin stress fiber polymerization was detected by fluorescein isothiocyanate-labeled phalloidin. Intracellular Ca(2+) and RhoA signaling activation were also measured. Sal B effectively reduced the portal pressure in DMN-induced cirrhotic rats. It decreased the contraction by endothelin-1 (ET-1)-activated HSCs by ∼66.5% and caused the disassembly of actin stress fibers and MLC2 dephosphorylation. Although Sal B reduced ET-1-induced intracellular Ca(2+) increase, blocking Ca(2+) increase completely by BAPTA-AM, a Ca(2+) chelator, barely affected the magnitude of contraction. Sal B decreased ET-1-induced RhoA and Rho-associated coiled coil-forming protein kinase (ROCK) II activation by 66.84% and by 76.79%, respectively, and inhibited Thr(696) phosphorylation of MYPT1 by 80.09%. In vivo, Sal B lowers the portal pressure in rats with DMN-induced cirrhosis. In vitro, Sal B attenuates ET-1-induced HSCs contraction by inhibiting the activation of RhoA and ROCK II and the downstream MYPT1 phosphorylation at Thr(696). We consider Sal B a potential candidate for the pharmacological treatment of portal hypertension.

Topics: Actins; Animals; Benzofurans; Cardiac Myosins; Cells, Cultured; Dimethylnitrosamine; Endothelin-1; Hepatic Stellate Cells; Histocytochemistry; Liver; Liver Cirrhosis, Experimental; Male; Myosin Light Chains; Phosphorylation; Portal Pressure; Protein Phosphatase 1; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Stress Fibers

Hepatic fibrosis, a precursor of liver cirrhosis, is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. Salvianolic acid B (SA-B) is one of water soluble compounds derived from Salvia miltiorrhiza Bunge (Danshen in Chinese) widely used for chronic liver diseases. In this study we investigated the protective effects of SA-B on CCl(4)-induced hepatic fibrosis.. Hepatic fibrosis in rats was induced by carbon tetrachloride (CCl(4)). Rats were divided into four groups, including normal controls (N group), model (M group), low SA-B of 10mg/kg body weight (L group), or high SA-B of 20mg/kg body weight (H group). After 6 weeks, macroscopic features of the liver and weight ratio of liver to body were measured. Liver fibrosis of the rats was evaluated by HE and Massion staining. Activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were checked with automated biochemistry analyzer. Serum levels of hyaluronic acid (HA), type IV collagen (IV-C), Laminin (LN) and procollagen III peptide (PIIIP) were detected by radioimmunoassay (RIA). The expression of NF-κB and IκBα was detected by western blotting.. SA-B was shown to reduce CCl(4)-induced hepatic fibrosis in rats. The serum levels of ALT, AST, and TBIL were significantly lower in the SA-B treatment groups than in the M group. Compared the M group, the serum levels of HA, LN, IV-C and PIIIP were decreased markedly after treatment with SA-B, especially in the H group. Treatment with SA-B at 10-20mg/kg (L and N groups, respectively) dose-dependently decreased the expression of NF-κB in the nucleolus and increased the expression levels of NF-κB and IκBα protein in the cytoplasm compared to that of the M group.. This study reveals that SA-B could prevent the progression of liver angiogenesis and alleviate liver fibrosis possibly by regulating the expression of NF-κB and IκBα.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Benzofurans; Bilirubin; Carbon Tetrachloride; Collagen Type IV; Hyaluronic Acid; I-kappa B Proteins; Laminin; Liver Cirrhosis, Experimental; Male; NF-kappa B; NF-KappaB Inhibitor alpha; Peptide Fragments; Procollagen; Protective Agents; Rats; Rats, Sprague-Dawley; Salvia

Salviae miltiorrhizae, a traditional Chinese medicine, is widely used in the treatment of cardiovascular and cerebrovascular diseases. Salvianolic acid B is identified as one of the most important water-soluble active ingredients in Salviae miltiorrhizae and associated with the activation of Ca(2+) channel of cytomembrane. But the further mechanism of action was not very clearly. In our study, we investigated the vasodilation activity of salvianolic acid B using the isolated thoracic aortic rings from Japanese white rabbit. Salvianolic acid B significantly released the contraction of the isolated thoracic aortic rings induced by phenylephrine and CaCl(2) while had no effects on the aortic rings with KCl stimulated. Different with Di-ao-xin-xue-kang capsule, salvianolic acid B caused an increase of Ca(2+) in cytoplasm from not only activation of Ca(2+) channel in cytomembrane but also release of endogenous Ca(2+). Then, a series of endogenous Ca(2+) inhibitors were pretreated to explore the mechanism of salvianolic acid B, and the results provided further evidences that salvianolic acid B causes intracellular calcium release in ryanodine receptors-dependent manners. Moreover, combining l-arginine (l-Arg) with salvianolic acid B promoted the vasodilation activity suggesting a relationship with nitric oxide (NO). To further investigated its mechanism, both guanylate cyclase (GC) inhibitor and NO Synthase inhibitor were used and demonstrated to block vasodilation activity of the aortic rings. Our findings reveal a NO-sGC-cGMP signals dependence mechanism of salvianolic acid B on its vasodilation activity which provide an evidence for its subsequent application in clinic.

Topics: Animals; Aorta, Thoracic; Benzofurans; Calcium; Calcium Channel Blockers; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Nitric Oxide; Nitric Oxide Synthase; Rabbits; Receptors, Cytoplasmic and Nuclear; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Soluble Guanylyl Cyclase; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Our previous study has demonstrated the therapeutic potential of bone marrow derived-neural stem cells (BM-NSCs) in CNS disorders; however, the beneficial effects are modest due to the poor survival and low neural differentiation frequency. Here, we demonstrate that salvianolic acid B (Sal B), a potent aqueous of a well known Chinese medicine herb, Salvia miltiorrhiza, possesses the ability to promote BM-NSCs proliferation in a dose dependent manner as verified by growth curve and Bromodeoxyuridine (BrdU) incorporation assays; While in differentiation medium, Sal B promoted nestin(+) BM-NSCs differentiated into greater numbers of NF-M(+) neurons and NG2(+) oligodendrocyte precursors, but fewer GFAP(+) astrocytes as verified by triple immunostaining and quantitative analysis; upon exposure to H(2)O(2), Sal B facilitated the cells survival, reduced LDH leakage, and inhibited apoptosis, displaying a dose-dependent neuroprotective effect on BM-NSCs. Sal B induced brain-derived neurotrophic factor (BDNF) production by BM-NSCs, which may be beneficial for the cells survival and differentiation in unfavourable environment. The collective evidence indicates that Sal B may be a potential drug to upgrade the therapeutic efficiency of BM-NSCs in CNS diseases.

Topics: Animals; Antigens; Apoptosis; Benzofurans; Biomarkers; Bone Marrow Cells; Brain-Derived Neurotrophic Factor; Cell Proliferation; Cell Survival; Cytoprotection; Dose-Response Relationship, Drug; Glial Fibrillary Acidic Protein; Hydrogen Peroxide; Immunohistochemistry; L-Lactate Dehydrogenase; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neural Stem Cells; Neurofilament Proteins; Neurogenesis; Neuroprotective Agents; Oxidants; Phenotype; Proteoglycans; Spheroids, Cellular; Time Factors

This study aimed to investigate the effects of different light quality on the growth, accumulation of active ingredients and enzymes activities of Salvia miltiorrhiza.. The seedlings of S. miltiorrhiza were treated by different light quality, and relative parameters were measured. The data was statistically processed.. Plant height was significantly decreased with supplemental blue light (WB), and the root length, root diameter, root fresh weight and root dry weight were significantly increased with supplemental red light (WR). Salvianolic acid B concentration in S. miltiorrhiza was highly increased by supplemental blue and red light, but tanshinone IIA concentration was not significantly affected by supplemental blue and red light. Enzymes activities of SOD, POD, PAL, TAT and PPO in S. miltiorrhiza were significant increased by supplemental blue light, and enzymes activities of POD, TAT and PPO were significant increased by supplemental red light.. The root growth of S. miltiorrhiza was greatly promoted by supplemental red light (WR). Salvianolic acid B concentration in S. miltiorrhiza was highly increased by supplemental blue and red light. Enzymes activities of TAT and PPO in S. miltiorrhiza were significant increased by supplemental blue light and red light.

Topics: Benzofurans; Light; Salvia miltiorrhiza

Hydrogen peroxide (H2O2), one of reactive oxygen species, is widely generated in many biological systems, and it mediates various physiological and biochemical process in plants. To investigate the role of H2O2 as a signaling molecule in the process of salicylic acid (SA)-induced Salvianolic acid B (Sal B) accumulation, we separately inspected the cultured cells of Salvia miltiorrhiza with SA, H2O2, catalase (CAT), 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (DMTU) and Imidazole (IMD) to investigate the influence on the activity of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) and the accumulation of Sal B. Treatment of S. miltiorrhiza cells with SA resulted in an increase of H2O2, the increase of PAL and TAT and accumulation of Sal B. Exogenous application of 10-30 mmol/L H2O2 was found to effectively increase PAL and TAT activity as well as the Sal B content. CAT, a H2O2 scavenger, eliminated the Sal B-accumulating effects of exogenous H2O2 and SA. These indicated that H2O2 may serve as an upstream signaling molecule in the SA-induced accumulation of Sal B signal transduction pathway. Disposed by DMTU, a chemical trap for H2O2, as observed to be effective in inhibiting SA-induced accumulation of Sal B. IMD strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H2O2 formation in plant cells. IMD treatment strongly inhibited the accumulation of Sal B in cultured cells of S. miltiorrhiza, but the effects of IMD, can be partially reversed by the exogenous SA. The accumulation of Sal B was blocked once the generation of H2O2 by NADPH oxidase was inhibited, and H2O2 served as signaling molecule mediated the SA-induced Sal B accumulation.

Topics: Benzofurans; Cell Culture Techniques; Cells, Cultured; Hydrogen Peroxide; Salicylic Acid; Salvia miltiorrhiza; Signal Transduction

Atherosclerosis and restenosis are inflammatory responses involving free radicals and lipid peroxidation and may be prevented/cured by antioxidant-mediated lipid peroxidation inhibition. Salvianolic acid (Sal B), a water-soluble antioxidant obtained from a Chinese medicinal herb, is believed to have multiple preventive and therapeutic effects against human vascular diseases. In this study the in vitro and in vivo inhibitory effects of Sal B on oxidative stress were determined.. In human aortic endothelial cells (HAECs), Sal B reduced oxidative stress, inhibited low-density lipoprotein (LDL) oxidation and reduced oxidised LDL-induced cytotoxicity. Sal B inhibited Cu(2+) -induced LDL oxidation in vitro (with a potency 16.3 times that of probucol) and attenuated HAEC-mediated LDL oxidation as well as reactive oxygen species (ROS) production. In cholesterol-fed New Zealand White rabbits (with probucol as positive control), Sal B intake reduced Cu(2+) -induced LDL oxidation, lipid deposition in the thoracic aorta, intimal thickness of the aortic arch and thoracic aorta and neointimal formation in the abdominal aorta.. The data obtained in this study suggest that Sal B protects HAECs from oxidative injury-mediated cell death via inhibition of ROS production. The antioxidant activity of Sal B may help explain its efficacy in the treatment of vascular diseases.

Topics: Animals; Antioxidants; Aorta; Benzofurans; Cholesterol, Dietary; Copper; Drugs, Chinese Herbal; Endothelial Cells; Humans; Hypercholesterolemia; Hyperplasia; Lipid Metabolism; Lipid Peroxidation; Lipoproteins, LDL; Oxidative Stress; Phytotherapy; Rabbits; Reactive Oxygen Species; Salvia miltiorrhiza; Tunica Intima; Vascular Diseases

(2E)-2-{6-[(E)-2-carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid, a novel compound designated SMND-309, is a new metabolite of salvianolic acid B. The present study was carried out to investigate the effects of SMND-309 on experimental liver fibrosis in rats induced by subcutaneous injection of carbon tetrachloride and explore its possible mechanisms on the basis of biochemical, histopathologic and immunohistochemical studies. The results showed that intragastrical treatment with SMND-309 ameliorated liver function and decreased the elevation of serum hyaluronic acid, laminin, procollagen type III levels and hydroxyproline content in liver tissue. It also decreased the elevation in the malondialdehyde level and restored the decrease in superoxide dismutase and glutathione peroxidase activities. Upon histopathologic examination, the SMND-309-treated rats reduced the liver damage and the liver fibrosis grade. Moreover, the results of immunohistochemical examination showed that SMND-309 powerfully down-regulated the expression of connective tissue growth factor (CTGF) rather than transforming growth factor-beta1 (TGF-β1) in serum and liver. Meanwhile, SMND-309 exhibits significantly higher potency compared with salvianolic acid B (Sal B) at the same dose. The antifibrotic mechanisms of SMND-309 might be associated with its ability to suppress the expression of CTGF as well as scavenge lipid peroxidation products and increase endogenous antioxidant enzyme activity.

Topics: Animals; Benzofurans; Caffeic Acids; Collagen Type III; Connective Tissue Growth Factor; Disease Models, Animal; Glutathione Peroxidase; Hyaluronic Acid; Hydroxyproline; Immunohistochemistry; Laminin; Liver; Liver Cirrhosis; Male; Malondialdehyde; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Transforming Growth Factor beta1

To investigate the protective or lethal role of autophagy and the effects of Salvianolic acid B (Sal B) on autophagy in starving myocytes.. Cardiac myocytes were incubated under starvation conditions (GD) for 0, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sal B (50 μmol/L) in the presence or absence of chloroquine (3 μmol/L) under GD 3 h, the amount of LC3-II, the abundance of LC3-positive fluorescent dots in cells, cell viability and cellular ATP levels were determined using immunoblotting, immunofluorescence microscopy, MTT assay and luminometer, respectively. Moreover, electron microscopy (EM) and immunofluorescent duel labeling of LC3 and Caspase-8 were used to examine the characteristics of autophagy and apoptosis.. Immunoblot analysis showed that the amount of LC3-II in starving cells increased in a time-dependent manner accompanied by increased LC3-positive fluorescence and decreased cell viability and ATP content. Sal B (50 μmol/L) inhibited the increase in LC3-II, reduced the abundance of LC3 immunofluorescence and intensity of Caspase-8 fluorescence, and enhanced cellular viability and ATP levels in myocytes under GD 3 h, regardless of whether chloroquine was present.. Autophagy induced by starvation for 3 h led to cell injury. Sal B protected starving cells by blocking the early stage of autophagic flux and inhibiting apoptosis that occurred during autophagy.

Topics: Adenosine Triphosphate; Animals; Autophagy; Benzofurans; Cell Survival; Cells, Cultured; Microtubule-Associated Proteins; Myocytes, Cardiac; Rats

Tanshinone IIA (T), salvianolic acid B (S) and ginsenoside Rb1 (G) are the three major active ingredients of Compound Danshen Formula (CDF) for its protective effects on myocardial ischemia (MI). In this study, we aimed to investigate therapeutic and synergistic effects of TSG (combination of T, S and G) on MI rats with metabolomic strategy.. MI model were induced in Sprague-Dawley rats by left anterior descending coronary artery ligation. MI rats were respectively administrated T, S, G, TSG and CDF. Plasma was analyzed by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Partial least squares discriminate analysis (PLS-DA) models were built to evaluate the therapeutic and synergistic effects of TSG at whole level. 22 MI biomarkers in rat plasma were also investigated to explain that.. TSG brings nearly equal therapeutic effects on MI as CDF and it plays more stable regulated action on those 22 identified metabolites than single compound.. Overall, there were few methods for the study of synergistic effects of Chinese medicine. Our results suggested that metabolomics offers a new idea for Chinese medicine research.

Topics: Abietanes; Animals; Benzofurans; Chromatography, Liquid; Ginsenosides; Male; Mass Spectrometry; Metabolomics; Rats; Rats, Sprague-Dawley

Salvianolic acid B (SalB), a bioactive compound isolated from the Chinese medicinal herb Danshen, has been shown to exert various anti-oxidative and anti-inflammatory activities in in vitro and in vivo studies. Here, we investigated the protective effects of SalB on traumatic brain injury (TBI) in mice. When administered within 2 h after TBI onset, SalB (25 mg/kg) reduced brain edema, lesion volume and motor functional deficits, and improved spatial learning and memory abilities. Moreover, SalB treatment inhibited the neutrophil infiltration and microglial activation at 48 h after TBI. Enzyme-linked immunosorbent assay (ELISA) for brain tissue homogenates was performed at 24 h after TBI to evaluate the expression of inflammation-related cytokines. The results showed that SalB suppressed the expression of pro-inflammatory cytokines TNF-α and IL-1β, whereas enhanced the expression of anti-inflammatory cytokines IL-10 and TGF-β1. All of these findings extended the protective role of SalB in the model of TBI and suggested that these protective effects might be associated with its anti-inflammatory activities. Thus SalB may have therapeutic potential for patients with TBI and perhaps other forms of acute brain injury.

Topics: Animals; Benzofurans; Brain Edema; Brain Injuries; Inflammation; Interleukin-10; Interleukin-1beta; Learning; Male; Memory; Mice; Mice, Inbred C57BL; Molecular Structure; Space Perception; Tumor Necrosis Factor-alpha

The interaction between salvianolic acid B (Sal B) and human hemoglobin (HHb) under physiological conditions was investigated by UV-vis absorption, fluorescence, synchronous fluorescence and circular dichroism spectroscopic techniques. The experimental results indicate that the quenching mechanism of fluorescence of HHb by Sal B is a static quenching procedure, the binding reaction is spontaneous, and the hydrophobic interactions play a major role in binding of Sal B to HHb. Based on Förster's theory of non-radiative energy transfer, the binding distance between Sal B and the inner tryptophan residues of HHb was determined to be 2.64 nm. The synchronous fluorescence experiment revealed that Sal B can not lead to the microenvironmental changes around the Tyr and Trp residues of HHb, and the binding site of Sal B on HHb is located at α(1)β(2) interface of HHb. Furthermore, the CD spectroscopy indicated the secondary structure of HHb is not changed in the presence of Sal B.

Topics: Benzofurans; Circular Dichroism; Drugs, Chinese Herbal; Hemoglobins; Humans; Molecular Structure; Spectrum Analysis; Tyrosine

Salvia miltiorrhiza (Danshen) has been widely used for the treatment of cardiac and cerebrovascular disease throughout history. The objective of this study is to further elucidate the mechanisms underlying Danshen's cardiac protective effects to support its clinical evidence.. AND RESULTS: Salvianolic acid B (Sal B) and Tanshinone IIA (Tan IIA) are two of the major components in Danshen. We observed that Sal B and Tan IIA have cardioprotective effects in an in vivo myocardial infarction model of C57 mice, have vasodilator action in a ex vivo micro-artery system through the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway and are involved in the regulation of the L-arginine/eNOS/NO pathways in human umbilical vein endothelial cells (HUVECs). Both Sal B and Tan IIA inhibited cardiac hypertrophy and infarction sizes and improved cardiac function at 4 weeks after induction of infarction. Furthermore, an eNOS inhibitor (L-NAME) obliterated the observed effects. Sal B and Tan IIA mediated vasodilatation in mice coronaries ex vivo, the effect of which was decreased with either L-NAME or PI3K inhibitor (LY294002). In addition, Sal B and Tan IIA-induced vasodilatation was observed ex vivo in the microvessels of eNOS-/- mice. Sal B and Tan IIA also stimulated eNOS phosphorylation in a concentration- and time-dependent manner in the HUVEC culture, which was diminished by LY294002. In addition, Sal B and Tan IIA were found to stimulate the phosphorylation of AMPK (Thr(172)) and Akt (Ser(473)), while compound C significantly decreased the phosphorylation of Akt (Ser(473)) mediated by both. Finally, Sal B and Tan IIA were found to increase NO production, induce [(3)H]-L-arginine uptake and increase the CAT-1 and CAT-2B mRNA levels in HUVEC culture.. These findings suggest that both Sal B and Tan IIA have cardioprotective function in certain levels through multiple targets related with NO production, such as eNOS phosphorylation, L-arginine uptake and CAT expression, which may have major clinical implications.

Topics: Abietanes; Amino Acid Transport Systems, Basic; AMP-Activated Protein Kinases; Analysis of Variance; Animals; Arginine; Benzofurans; Cardiovascular Agents; Cationic Amino Acid Transporter 1; Cells, Cultured; Chromones; Disease Models, Animal; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Endothelial Cells; Enzyme Inhibitors; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Myocardial Infarction; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA, Messenger; Signal Transduction; Time Factors; Vasodilation

Lithospermic acid B (LAB) has been reported to protect OLETF rats, an established type 2 diabetic animal model, from the development of diabetes-related vascular complications. We investigated whether magnesium lithospermate B (LAB) has a protective role under cytokine-induced apoptosis in INS-1 cells in vitro and whether it slows the development of diabetes in OLETF rats in vivo. Pretreatment with 50 μM LAB significantly reduced the 1000 U/mL INF-γ and 100 U/mL IL-1β-induced INS-1 cell death. LAB significantly alleviated cytokine-induced phosphorylations of p38 and JNK in accordance with a decrease in cleaved caspase-3 activity in beta-cells. LAB also protected against the cytokine-induced caspase-3 apoptotic pathway via significant activation of Nrf2-HO (heme-oxygenase)-1 and Sirt1 expression. OLETF rats treated with 40 mg/kg/day LAB showed a significant improvement in glucose tolerance compared to untreated OLETF control rats in vivo. Our results suggest that the cytoprotective effects of LAB on pancreatic β-cells are related with both alleviating apoptotic pathways and activating anti-apoptotic pathways of Nrf2-HO-1 and Sirt1.

Topics: Animals; Apoptosis; Benzofurans; Cells, Cultured; Cytokines; Depsides; Diabetes Mellitus, Type 2; Heme Oxygenase (Decyclizing); Insulin-Secreting Cells; Male; NF-E2-Related Factor 2; Protective Agents; Random Allocation; Rats; Rats, Inbred OLETF; Rats, Long-Evans; Signal Transduction; Sirtuin 1

Platelet-derived growth factor (PDGF) induces cell proliferation together with oxidative stress. The present study investigated the effects of salvianolic acid A (Sal A) and B (Sal B) on the PDGF-induced signaling cascades in hepatic stellate cells (HSCs). HSC-T6, a rat hepatic stellate cell line, was stimulated with PDGF (10 ng/mL). The inhibitory effects of Sal A and B on oxidative stress-related signaling pathways were assessed in vitro. The protein levels were measured by Western blotting. FACS analysis was applied to detect the thioredoxin (Trx) level. Sal A and B showed different inhibitory abilities on the PDGF-related pathway. Sal A inhibited 70-kDa ribosomal S6 kinase (p70(s6k)) and associated proteins. Sal B attenuated PDGF-induced c-jun-N-terminal kinase (JNK), p38, and PKC- δ phosphorylations. Both Sal A and B diminished the activation of PKD, Trx, heme-oxygenase (HO)-1, and Nrf2. Taken together, our results showed that Sal A and B attenuated PDGF-induced ROS formation in HSCs, possibly through different signaling pathways.

Topics: Animals; Benzofurans; Caffeic Acids; Cell Line; Cell Proliferation; Hepatic Stellate Cells; Lactates; Liver; Liver Cirrhosis; Oxidative Stress; Phosphorylation; Plant Roots; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Salvia miltiorrhiza; Signal Transduction

Salvianolic acid B (SB) is a natural compound with protective effect against ischemia-reperfusion heart injury. However, the signal network of SB including both direct target proteins and downstream signal-related proteins has not been clarified. In the present study, epidermal growth factor receptor (EGFR) was predicted to be the most possible direct protein target of SB by INVDOCK, a ligand-protein inverse-docking algorithm. Possible signal-related proteins of SB in H9C2 cells, including both under normal condition and under ischemia-reperfusion injury, were searched using 2-DE analysis. Totally, 14 signal-related proteins were found. Finally, signal network from EGFR to the signal-related proteins was established using bioinformatic analysis. Interestingly, 9 of the 14 signal-related proteins could be included in a network together with EGFR through direct interaction or only one intermediate partner. The signal cascade from EGFR to heat shock protein 27 (HSP27) and mitofilin (IMMT, inner membrane mitochondrial protein) might be the most important cascade. The signal network was certified by measuring the binding affinity of SB to EGFR in vitro, the effect of SB on internalization and phosphorylation of EGFR, the effect of SB on viability and proliferation of H9C2 cells, and the expression of inner membrane mitochondrial protein in the presence of EGFR inhibitor AG 1478.

Topics: Animals; Benzofurans; Blotting, Western; Cell Line; Computational Biology; Epidermal Growth Factor; ErbB Receptors; HSP27 Heat-Shock Proteins; Mitochondrial Proteins; Muscle Proteins; Protein Binding; Proteomics; Rats; Reperfusion Injury; Signal Transduction

Salvianolic acid B (SB) is an active component isolated from Danshen, a traditional Chinese medicine widely used for the treatment of cardiovascular disorders. Previous study suggested that SB might inhibit adhesion as well as aggregation of platelets by a mechanism involving the integrin α2β1. But, the signal cascades in platelets after SB binding are still not clear.. In the present study, a differential proteomic analysis (two-dimensional electrophoresis) was conducted to check the protein expression profiles of rat platelets with or without treatment of SB. Proteins altered in level after SB exposure were identified by MALDI-TOF MS/MS. Treatment of SB caused regulation of 20 proteins such as heat shock-related 70 kDa protein 2 (hsp70), LIM domain protein CLP-36, copine I, peroxiredoxin-2, coronin-1 B and cytoplasmic dynein intermediate chain 2C. The regulation of SB on protein levels was confirmed by Western blotting. The signal cascades network induced by SB after its binding with integrin α2β1 was predicted. To certify the predicted network, binding affinity of SB to integrin α2β1 was checked in vitro and ex vivo in platelets. Furthermore, the effects of SB on protein levels of hsp70, coronin-1B and intracellular levels of Ca²+ and reactive oxygen species (ROS) were checked with or without pre-treatment of platelets using antibody against integrin α2β1. Electron microscopy study confirmed that SB affected cytoskeleton structure of platelets.. Integrin α2β1 might be one of the direct target proteins of SB in platelets. The signal cascades network of SB after binding with integrin α2β1 might include regulation of intracellular Ca²+ level, cytoskeleton-related proteins such as coronin-1B and cytoskeleton structure of platelets.

Topics: Animals; Benzofurans; Blood Platelets; Integrin alpha2beta1; Male; Metabolic Networks and Pathways; Models, Biological; Platelet Adhesiveness; Platelet Aggregation; Proteins; Proteome; Proteomics; Rats; Rats, Sprague-Dawley

The plant density-dependent variations in the root yield and content, and the yield of biomarkers in Australian grown Salvia miltiorrhiza Bunge, a commonly used Chinese medicinal herb for the treatment of cardiovascular diseases, were investigated in a field trial involving six different plant densities. The key biomarker compounds cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B were quantified by a validated RP-HPLC method, and the root yields were determined per plant pair or unit area. There were significant variations (p<0.05) in the root yields and contents and the yields of the biomarkers between the different plant densities. Positive linear correlations were observed between the contents of the three tanshinones, whereas negative linear correlations were revealed between the contents of the tanshinones and salvianolic acid B. The highest root yield per plant pair was achieved when the plants were grown at 45×30 cm or 45×40 cm, whereas the highest root production par unit area was obtained for a plant density of 30×30 cm. The highest contents of the three tanshinones and the most abundant production of these tanshinones per unit area were achieved when the plants were grown at 30×30 cm. However, the highest content of salvianolic acid B was found for a density of 45×40 cm, while its highest yield per unit area was obtained for densities of 30×40 cm or 45×30 cm. The findings suggest that the plant density distinctly affects the root yield and content and the yield of tanshinones and salvianolic acid B in Australian grown S. miltiorrhiza, which may be used as a guide for developing optimal agricultural procedures for cultivating this herb.

Topics: Abietanes; Australia; Benzofurans; Phenanthrenes; Plant Roots; Salvia miltiorrhiza

To evaluate the pharmacokinetics of phenolic compounds after oral administration of Danshen extract in rat brain.. Blood and brain microdialysis probes were inserted into jugular vein and cerebral cortex of rat under anesthesia and perfused with ringer's solution at the rate of 2.0 and 0.8 μL/min, respectively. Blank microdialysates were collected after 2h post-implantation equilibrium time. Danshen extract (danshensu 40 mg/kg BW, protocatechuic aldehyde 149 mg/kg BW, and salvianolic acid B 50mg/kg BW) was administrated intragastrically, and then blood and brain microdialysates were collected at 15 and 30 min time intervals for 4h, respectively. The concentrations of phenolic compounds were determined by high-performance liquid chromatography coupled with chemiluminescence detection. Pharmacokinetic parameters were estimated using non-compartmental methods.. Danshensu and protocatechuic acid could be detected in both blood and brain microdialysates, while protocatechuic aldehyde and salvianolic acid B were not detected. Brain-to-blood (AUC(brain)/AUC(blood)) distribution ratio were 0.25±0.04 and 0.09±0.02 for danshensu and protocatechuic acid, respectively.. Danshensu can readily permeate the blood brain barrier after oral administration of Danshen extract, and protocatechuic acid is a potential oxidative metabolite of protocatechuic aldehyde.

Topics: Animals; Benzaldehydes; Benzofurans; Blood-Brain Barrier; Catechols; Drugs, Chinese Herbal; Hydroxybenzoates; Lactates; Male; Microdialysis; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza

Salvianolic acid B (Sal B) is the most abundant bioactive molecule from Radix Salviae Miltiorrhizae, and has recently been used for treating renal fibrosis in traditional Chinese medicine. Here we investigated the ability reversal of Sal B to reverse the transdifferentiation of human kidney proximal tubular epithelial cells that was induced by transforming growth factor-beta 1 (TGF-β1). The effects of Sal B on HK-2 cell morphology were observed by phase contrast microscopy, while alpha smooth muscle actin and E-cadherin were studied by immunocytochemistry and real-time reverse transcription polymerase chain reaction, respectively. Exposure of HK-2 cells to TGF-β1 for 72 h induced a complete conversion of the epithelial cells to myofibroblasts. When HK-2 cells were co-incubated with Sal B and TGF-β1 for a further 72 h, the morphology of myofibroblasts returned to that of proximal tubular epithelial cells, whereas the myofibroblast phenotype was maintained after exposure of cells to TGF-β1 for 144 h. Sal B reduced alpha smooth muscle actin levels and increased E-cadherin levels compared with their epithelial-to-mesenchymal transition controls. The reversal effect of Sal B was dose-dependent. That Sal B reverses the epithelial-to-mesenchymal transition in vitro suggests that it could possibly facilitate the repair of tubular epithelial structures and the regression of renal fibrosis in injured kidneys.

Topics: Benzofurans; Cell Culture Techniques; Cell Line; Drugs, Chinese Herbal; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibrosis; Humans; Kidney; Mesoderm; Microscopy, Phase-Contrast; Transforming Growth Factor beta

Salviae miltiorrhizae is one of the most commonly used herbal plants in the treatment of numerous ailments including cardiovascular diseases for hundreds of years. According to the theory of traditional Chinese herbal medicine, S. miltiorrhizae is always used in combination with borneol to obtain better pharmacological effects. The purpose of this study was to investigate the effects of borneol on the pharmacokinetic and bioavailability of S. miltiorrhizae. The pharmacokinetics studying on rosmarinic acid, salvianolic acid A and salvianolic acid B which are the main active compounds of S. miltiorrhizae in rat plasma, was achieved using a optimal high-performance liquid chromatographic technique coupled with liquid-liquid extraction method. After administration of either single salvianolic acids or salvianolic acids in combination with borneol, plasma concentrations of rosmarinic acid, salvianolic acid A and salvianolic acid B of male Sprague-Dawley rats were determined at different time points (5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 300, and 360 min). In comparison with salvianolic acid extract alone, there were statistically significant differences in pharmacokinetic parameters of rosmarinic acid, salvianolic acid B and salvianolic acid A, and the bioavailability of the three salvianolic acids increased by different degrees when the salvianolic acid extract and borneol were administered together. These results indicated that borneol could enhance the intestinal absorption, decrease the distribution and inhibit the metabolism of salvianolic acids.

Topics: Animals; Benzofurans; Biological Availability; Caffeic Acids; Camphanes; Cinnamates; Depsides; Drugs, Chinese Herbal; Lactates; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Rosmarinic Acid; Salvia miltiorrhiza; Sensitivity and Specificity

In order to provide a scientific water management for the standardized cultivation, the effects of soil water content on the seedlings growth and active ingredients of S. miltiorrhiza were studied.. A water stress experiment with pot culture was applied to study the effects of different soil water content on the seedlings growth, biomass and the accumulation of tanshinone, salvianolic acid B and mineral nutrition of S. miltiorrhiza.. Soil water content had serious influence on the growth, yield, outer appearance and inner quality of S. miltiorrhiza when the soil was under severe drought or with too much water. But the shoot and root biomass of S. miltiorrhiza was increased significantly under mild drought. As well as the content and cumulant of dihydrotanshinone I , cryptotanshinone, tanshinone I , tanshinone IIA and salvianolic acid B in root of mild drought were increased. It also enhanced the P, K, Ca, Mg, Mn, Zn, Cu and Fe contents of S. miltiorrhiza under mild drought.. The overall results from the experiment suggest that the appropriate soil water content is 55% to 60% in seedling stage of S. miltiorrhiza. And it will be strongly recommended that the ridge culture and suitable soil moisture management must be carry out in production of S. miltiorrhiza in order to improve the yield and quality of medicinal materials.

Topics: Abietanes; Benzofurans; Biomass; Minerals; Plant Roots; Salvia miltiorrhiza; Seedlings; Soil; Stress, Physiological; Water

To study the chemical changes of salvianolic acid B and lithospermic acid of Salvia miltiorrhiza under the conditions of high temperature and high pressure and explore the reaction mechanism.. S. miltiorrhiza extracts, salvianolic acid B and lithospermic acid were put in the reactor under the conditions of high temperature and high pressure (120 degrees C, 0.2 MPa), and the chemical changes and stability was studied.. Salvianolic acid A was the primary product in salvianolic acid B and lithospermic acid's conversion process, and lithospermic acid was an intermediate in the conversion process of salvianolic acid B. Compared with salvianolic acid B, lithospermic acid could convert into more salvianolic acid A and fewer other products in the same conditions. Salvianolic acid A was not stable under the conditions of high temperature and high pressure, and could sequentially convert into other small molecules.. Referring to the chemical conversion of salvianolic acid B and lithospermic acid, a method of large-scale preparation of salvianolic acid A can be developed.

Topics: Benzofurans; Caffeic Acids; Depsides; Hot Temperature; Lactates; Pressure; Salvia miltiorrhiza

To observe the effect of salvianolic acid B (SAB), an extract from Radix Salviae miltiorrhizae, on expression of leucocyte differentiation antigen 14 (CD14) in the liver tissue of experimental rats with carbon tetrachloride (CCl4)-induced liver fibrosis.. Thirty SD rats were randomly divided into three groups, the model group, the treated group, and the control group. The pathological fibrosis changes in liver of rats were observed. Meantime, their liver function was detected by automatic biochemical analyzer. Serum content of endotoxin was assayed by matrix staining, and plasma content of tumor necrosis factor-alpha (TNF-alpha) was detected by radioimmunoassay. mRNA and protein expressions of CD14 in the liver tissue were measured using reverse transcriptional-polymerase chain reaction and immunohistochemistry respectively.. All the laboratory parameters, including liver function, degree of liver fibrosis, serum endotoxin levels, plasma TNF-alpha contents, and CD14 mRNA and protein expressions in the model group were higher than those in the control group (all P<0.01). All the aforesaid indices were lowered more in the treated group than in the model group (all P<0.01).. SAB could antagonize the CCl4, induced liver fibrosis in rats. Its mechanism of action was possibly correlated with its effects on down-regulating hepatic CD14 expression and blocking the endotoxin signal transduction pathway.

Topics: Animals; Benzofurans; Lipopolysaccharide Receptors; Liver; Liver Cirrhosis, Experimental; Male; Rats; Rats, Sprague-Dawley

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is known to be effective in the prevention of atherosclerosis. Here, we tested the hypothesis that the anti-atherosclerotic effect of Sal B might be mediated by suppressing maturation of human monocyte-derived dendritic cells (h-monDC).. h-monDC were derived by incubating purified human monocytes with GM-CSF and IL-4. h-monDC were pre-incubated with or without Sal B and stimulated by oxidized low-density lipoprotein (ox-LDL) in the presence or absence of PPARγ siRNA. Expression of h-monDC membrane molecules (CD40, CD86, CD1a, HLA-DR) were analysed by FACS, cytokines were measured by elisa and the TLR4-associated signalling pathway was determined by Western blotting.. Ox-LDL promoted h-monDC maturation, stimulated CD40, CD86, CD1a, HLA-DR expression and IL-12, IL-10, TNF-α production; and up-regulated TLR4 signalling. These effects were inhibited by Sal B. Sal B also triggered PPARγ activation and promoted PPARγ nuclear translocation, attenuated ox-LDL-induced up-regulation of TLR4 and myeloid differentiation primary-response protein 88 and inhibited the downstream p38-MAPK signalling cascade. Knocking down PPARγ with the corresponding siRNA blocked these effects of Sal B.. Our data suggested that Sal B effectively suppressed maturation of h-monDC induced by ox-LDL through PPARγ activation.

Topics: Antigens, CD; Base Sequence; Benzofurans; Blotting, Western; Cytokines; Dendritic Cells; DNA Primers; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Lipoproteins, LDL; Monocytes; Myeloid Differentiation Factor 88; PPAR gamma; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Toll-Like Receptor 4

Adsorption on polyamide resin was investigated as a means of separating lithospermic acid B (LAB) from a crude extract of the roots of the traditional Chinese medicine Salvia miltiorrhiza Bunge ("Danshen"). Variables affecting adsorption capacity (solution pH, contact time on resin, initial LAB concentration) were studied. Adsorption was strongly dependent upon the initial concentration of LAB and pH. In all conditions, the polyamide resin gave optimal adsorption of LAB at an initial concentration of 2.66 mg/mL and pH <3.0. The adsorption isotherm correlated well with the Langmuir-type adsorption isotherm. Maximal adsorption capacity was calculated to be 380 mg/g at pH 2.0 and 25°C. LAB purity of 85.30% could be obtained by polyamide resin adsorption followed by elution with 70% ethanol solution, and the recovery was 87.1%. After preparative HPLC, the maximum HPLC purity obtained was 99.28% with a recovery of 75.2%. This method provides an efficient and low-cost method for LAB purification for industrial applications.

Topics: Adsorption; Benzofurans; Chromatography, High Pressure Liquid; Depsides; Drugs, Chinese Herbal; Ethanol; Hydrogen-Ion Concentration; Kinetics; Nylons; Plant Roots; Reproducibility of Results; Salvia miltiorrhiza

Our previous studies showed that Salvianolic acid B (Sal B) inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters and such anti-cancer effects might be related to the inhibition of angiogenesis. This study was aimed to further investigate the anti-proliferative effect of Sal B on the most common type of oral cancer, oral squamous cell carcinoma (OSCC) and the possible mechanisms of action with respect to angiogenesis inhibition.. Two well-characterized oral squamous cell carcinoma cell lines, CAL27 and SCC4, and premalignant leukoplakia cells were treated with different concentrations of Sal B. Cytotoxicity was assessed by MTT assay. cDNA microarray was utilized to evaluate the expression of 96 genes known to be involved in modulating the biological processes of angiogenesis. Real-time reverse transcription-polymerase chain reaction analysis was conducted to confirm the cDNA microarray data.. Sal B induced growth inhibition in OSCC cell lines but had limited effects on premalignant cells. A total of 17 genes showed a greater than 3-fold change when comparing Sal B treated OSCC cells to the control. Among these genes, HIF-1α, TNFα and MMP9 are specifically inhibited, expression of THBS2 was up-regulated.. Sal B has inhibitory effect on OSCC cell growth. The antitumor effect can be attributed to anti-angiogenic potential induced by a decreased expression of some key regulator genes of angiogenesis. Sal B may be a promising modality for treating oral squamous cell carcinoma.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents, Phytogenic; Benzofurans; Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leukoplakia; Matrix Metalloproteinase 9; Mouth Neoplasms; Oligonucleotide Array Sequence Analysis; Phytotherapy; Reverse Transcriptase Polymerase Chain Reaction; Salvia miltiorrhiza; Thrombospondins; Tumor Necrosis Factor-alpha; Up-Regulation

NAD(P)H: quinone oxidoreductase1 (NQO1) is an important detoxification enzyme that can protect mammalian cells against toxic quinones and reduce the risk of tumorigenesis. In this study, it was found that salvianolic acid B (SaB), lithospermic acid (LA), and rosmarinic acid (RA), three main hydrophilic constituents in Danshen, conjugated with glutathione (GSH) easily in vitro but exhibited no NQO1-inducing activities in Hepa 1c1c7 cells, which might attribute to their poor absorptions. After a simple methylation strategy that aimed at improving the liposolubility, both the NQO1-inducing activities and the absorptions in cells of the phenolic acids improved obviously, without losing the GSH-conjugating abilities. The concentration to double the specific activity of NQ01 values of methylated products of lithospermic acid and rosmarinic acid were 17.86 ± 2.34 μg/mL and 11.97 ± 0.60 μg/mL, respectively. The findings indicated that methylation is an effective strategy to improve the NQO1-inducing activities of phenolic acids in Danshen.

Topics: Animals; Benzofurans; Cell Line, Tumor; Cinnamates; Depsides; Glutathione; Hydroxybenzoates; Methylation; Mice; NAD(P)H Dehydrogenase (Quinone); Rosmarinic Acid; Salvia miltiorrhiza

The studies on the interaction between HSA and drugs have been an interesting research field in life science, chemistry and clinical medicine. There are also many metal ions present in blood plasma, thus the research about the effect of metal ions on the interaction between drugs and plasma proteins is crucial. In this study, the interaction of Salvianolic acid B (Sal B) with human serum albumin (HSA) was investigated by the steady-state, synchronous fluorescence and circular dichroism (CD) spectroscopies. The results showed that Sal B had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. Binding parameters calculated showed that Sal B was bound to HSA with the binding affinities of 10(5) L mol(-1). The thermodynamic parameters studies revealed that the binding was characterized by positive enthalpy and positive entropy changes, and hydrophobic interactions were the predominant intermolecular forces to stabilize the complex. The specific binding distance r (2.93 nm) between donor (HSA) and acceptor (Sal B) was obtained according to Förster non-radiative resonance energy transfer theory. The synchronous fluorescence experiment revealed that Sal B cannot lead to the microenvironmental changes around the Tyr and Trp residues of HSA, and the binding site of Sal B on HSA is located in hydrophobic cavity of subdomain IIA. The CD spectroscopy indicated the secondary structure of HSA is not changed in the presence of Sal B. Furthermore, The effect of metal ions (e.g. Zn(2+), Cu(2+), Co(2+), Ni(2+), Fe(3+)) on the binding constant of Sal B-HSA complex was also discussed.

Topics: Benzofurans; Binding Sites; Circular Dichroism; Drug Interactions; Humans; Ions; Metals; Models, Biological; Models, Molecular; Protein Binding; Protein Conformation; Serum Albumin; Spectrometry, Fluorescence; Thermodynamics

Radix Salvia miltiorrhiza (RSM), a traditional Chinese medicinal herb, has been alleged to possess therapeutic effects against senile dementia, also known as Alzheimer's disease (AD). However, the effects of the major components in RSM on cytotoxicity induced by amyloid-β peptide (Aβ) and on acetylcholinesterase activity have not been studied in depth to date. In this report, the effects of RSM aqueous/ethanol extracts, total polyphenols, total tanshinones and 3 phenolic compounds against toxicity mediated by Aβ(25-35) were tested with PC-12 cells. The results showed that Aβ(25-35)-induced cytotoxicity was revised by RSM aqueous/ethanol extracts and total polyphenols and that danshensu and salvianolic acid B could protect PC-12 cells by blocking Aβ(25-35)-induced Ca(2+)-intake, lactate dehydrogenase release, cell viability decrease and apoptosis. In addition, the activities of RSM extracts and relevant constituents in their inhibition of acetylcholinesterase were investigated using rat brain homogenates as an enzyme resource. Galanthamine hydrobromide, an accepted acetylcholinesterase inhibitor, was employed as a positive control agent. Our preliminary studies demonstrated that RSM ethanol extract, total tanshinones, tanshinone I and dihydrotanshinone I had remarkable inhibition effects on acetylcholinesterase in vitro. These findings suggest that both tanshinones and polyphenols in RSM are the active constituents responsible for the beneficial effects of this herb in AD treatment.

Topics: Abietanes; Amyloid beta-Peptides; Animals; Antioxidants; Apoptosis; Benzofurans; Calcium; Cell Survival; Cholinesterase Inhibitors; Hydroxybenzoates; L-Lactate Dehydrogenase; Male; Neuroprotective Agents; PC12 Cells; Phosphatidylinositols; Plant Extracts; Polyphenols; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Tetrazolium Salts; Thiazoles

Study the yield and quality of Salvia miltforthiza in the poplars and apple trees intercropping system.. Assay the yield, heavy metals, organochlorine pesticides and active components of Salvia miltforthiza in different intercropping systems are different.. The contents of Cd exceeds the allowed figure seriously though other heavy metals such as Pb, As, Hg, Cu fell in the standard range. The contents of organochlorine pesticides of different Salvia miltforthiza fell in the standard range. The active components of biennial Salvia miltforthiza intercropping with apple trees, including Tanshinone II A and Salvianolic acid B complied with the quality standards of Pharmacopoeia.. Soil fertility, heavy metals of intercropping system and planting years should be considered in order to ensure the quality and stability of Chinese medicine.

Topics: Abietanes; Agriculture; Benzofurans; Biomass; China; Malus; Metals, Heavy; Pesticide Residues; Plants, Medicinal; Quality Control; Salvia; Soil; Trees

Alzheimer's disease (AD) is a neurodegenerative disorder associated with progressive cognitive and memory loss and neuronal cell death. Current therapeutic strategies for AD are very limited; thus, traditional herbal medicines or their active constituents receive much attention. The aim of this study was to investigate the cognitive enhancing effects of salvianolic acid B (SalB) isolated from Salvia miltiorrhiza and its ameliorating effects on various drug-induced amnesic models using the passive avoidance, Y-maze, and Morris water maze tasks. Drug-induced amnesia was induced by administering scopolamine, diazepam, muscimol, or amyloid-β (Aβ)(25-35) peptide. SalB (10 mg/kg, p.o.) was found to significantly reverse the cognitive impairments induced by scopolamine (1 mg/kg, i.p.) or Aβ(25-35) (10 nmol/5 μl, i.c.v.) injection. This ameliorating effect of SalB was antagonized by the GABA(A) receptor agonists, muscimol or diazepam, respectively. In addition, SalB alone was capable of improving cognitive performances. Furthermore, SalB (100 μM) was found to inhibit GABA-induced outward Cl(-) currents in single hippocampal CA1 neuron. These results suggest that the observed ameliorations of cholinergic dysfunction- or Aβ(25-35)-induced memory impairment by SalB were mediated, in part, via the GABAergic neurotransmitter system after a single administration.

Topics: Amyloid beta-Peptides; Animals; Animals, Newborn; Benzofurans; Cholinergic Antagonists; Cholinesterase Inhibitors; Cognition Disorders; Disease Models, Animal; Dose-Response Relationship, Drug; Female; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Injections, Intraventricular; Male; Maze Learning; Membrane Potentials; Mice; Neurons; Patch-Clamp Techniques; Peptide Fragments; Pregnancy; Rats; Tacrine

Targeting cellular function as a system rather than on the level of the single target significantly increases therapeutic potency. In the present study, we detect the target pathway of salvianolic acid B (SalB) in vivo. Acute myocardial infarction (AMI) was induced in rats followed by the treatment with 10 mg/kg SalB. Hemodynamic detection and pathological stain, 2-dimensional electrophoresis, MALDI-TOF MS/MS, Western blot, pathway identification, apoptosis assay and transmission electron microscope were used to elucidate the effects and mechanism of SalB on cardioprotection. Higher SalB concentration was found in ischemic area compared to no-ischemic area of heart, correlating with improved heart function and histological structure. Thirty-three proteins regulated by SalB in AMI rats were identified by biochemical analysis and were classified as the components of metabolism and apoptosis networks. SalB protected cardiomyocytes from apoptosis, inhibited poly (ADP-ribose) polymerase-1 pathway, and improved the integrity of mitochondrial and nucleus of heart tissue during AMI. Furthermore, the protective effects of SalB against apoptosis were verified in H9c2 cells. Our results provide evidence that SalB regulates multi-targets involved in the apoptosis pathway during AMI and therefore may be a candidate for novel therapeutics of heart diseases.

Topics: Animals; Apoptosis; Benzofurans; Blotting, Western; Cell Line; Cell Nucleus; Electrophoresis, Gel, Two-Dimensional; Hemodynamics; In Situ Nick-End Labeling; Male; Microscopy, Electron, Transmission; Mitochondria; Myocardial Infarction; Myocardium; Myocytes, Cardiac; NF-kappa B; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Rats; Rats, Wistar; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry

Cardiac fibroblasts play the key role in cardiac function and matrix metalloproteinases-9 (MMP-9) is a well known contributor to the development of myocardial remodeling. However, the direct regulation of MMP-9 on the function of cardiac fibroblasts and the underlying mechanism are far from elucidation. In the present research, recombinant protein encoding catalytic domain of MMP-9 (MMP-9 CD) was constructed and the function of neonatal cardiac fibroblasts was investigated by cell proliferation assay, migration assay, picrosirius red assay, multiplex cytokine assay and fibroblast phenotype detection. 200 nM MMP-9 CD stimulated cardiac fibroblasts migration (169.4±22.5% versus 100±0%, p<0.01), increased collagen synthesis (1.5±0.2 fold, p<0.05), up-regulated the secretion of ICAM (574.0±40.1 versus 268.5±8.6pg/ml, p<0.01), TNF-α (192.6±11.0 versus 14.4±1.8pg/ml, p<0.001), IL-6 (1500.9±70.2 versus 323.4±40.6pg/ml, p<0.001) and sVCAM-1 (30.3±4.3 versus 7.0±0.1 pg/ml, p<0.05) and down-regulated VEGF (436.5±148.9 versus 1034.3±28.1 pg/ml, p<0.05) significantly with modest effects on proliferation. Accompanying with these regulations, transition of fibroblasts to myofibroblast was confirmed by immunofluorescent stain of α-smooth muscle actin (α-SMA) with MMP-9 CD treatment. Furthermore, salvianolic acid B (SalB) inhibited the effects of MMP-9 CD significantly. In conclusion, our results provide evidence for a direct influence of MMP-9 on cardiac fibroblast migration, collagen and cytokine secretion, which can be attenuated by SalB.

Topics: Animals; Benzofurans; Cell Movement; Collagen; Cytokines; Enzyme Induction; Fibroblasts; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Medicine, East Asian Traditional; Myocardium; Phytotherapy; Plant Extracts; Protein Structure, Tertiary; Rats; Rats, Wistar; Recombinant Proteins; Salvia miltiorrhiza; Up-Regulation

Enzymes involved in drug and xenobiotic metabolism have been considered to exist in two groups: phase I and phase II enzymes. Cytochrome P450 isoenzymes (CYPs) are the most important phase I enzymes in the metabolism of xenobiotics. The products of phase I metabolism are then acted upon by phase II enzymes, including glutathione S-transferases (GSTs). Herbs that inhibit CYPs such as CYP3A4 or that induce GSTs may have the potential to protect against chemical carcinogenesis since the mutagenic effects of carcinogens are often mediated through an excess of CYP-generated reactive intermediates. This study was designed to investigate the effects of salvianolic acid B (Sal B), a pure compound extracted from Radix Salviae Miltiorrhizae, a Chinese herb, on cell proliferation and CYP1A2 and CYP3A4 mRNA expression in the presence or absence of rifampicin, a potent inducer of CYPs and GST protein expression in HepG2 cells.. HepG2 cells were incubated with different concentrations of Sal B. Cell proliferation was determined by SYTOX-Green nucleic acid staining. CYP3A4 and CYP1A2 mRNA expression was assayed by real-time PCR. GST protein expression was analyzed by Western blotting.. Low concentrations of Sal B (0-20 μmol/L) had no significant effects on cell proliferation, while higher concentrations (100-250 μmol/L) significantly inhibited proliferation in a concentration-dependent manner. Ten μmol/L Sal B, but not 1 μmol/L, down-regulated CYP3A4 and CYP1A2 mRNA expression after 24 hours of incubation, whereas both 1 and 10 μmol/L Sal B down-regulated CYP3A4 mRNA expression after 96 hours of incubation; moreover, 1 and 10 μmol/L Sal B inhibited CYP3A4 mRNA expression induced by rifampicin. Both 1 μmol/L and 10 μmol/L Sal B increased GST expression.. Sal B inhibits CYP3A4 and CYP1A2 mRNA expression and induces GST expression in HepG2 cells.

Topics: Benzofurans; Blotting, Western; Cell Proliferation; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP3A; Dose-Response Relationship, Drug; Enzyme Induction; Enzyme Repression; Gene Expression Regulation, Neoplastic; Glutathione Transferase; Hep G2 Cells; Humans; Liver Neoplasms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Rifampin; RNA, Messenger; Time Factors

Dysfunction of the endothelium contributes to pathological conditions of the arterial wall including atherosclerosis as a result of immunological and/or inflammatory responses. Salvianolic acid B (Sal B), a pure and active compound extracted from the Chinese herb Salvia miltiorrhizae (SM) was characterized for its anti-inflammatory and anti-oxidant properties on vascular system.. Sal B pretreatment significantly inhibited the IFN-γ-induced phosphorylations of JAK2 (Tyr 1007/1008) and STAT1 (Tyr701 and Ser727). Consistently, IFN-γ-induced STAT1 downstream targets CXC chemokines' IP-10, Mig, and I-TAC were suppressed by Sal B pretreatment. Sal B inhibited promoter activities of IP-10 and the secretion of IP-10 protein. The monocyte adhesion to IFN-γ-treated ECs was observed to be reduced after Sal B pretreatment. ECs treated with Sal B alone also increased the expression of PIAS1 and SOCS1 which may also contribute to its inhibitory effect on JAK-STAT1 signaling pathways.. The anti-inflammatory properties of Sal B on IFN-γ-induced JAK-STAT1 activation were demonstrated in the present study which provides a molecular basis for possible therapeutic usage on vascular disorders.

Topics: Animals; Benzofurans; Cattle; Drug Interactions; Endothelial Cells; Humans; Interferon-gamma; Janus Kinase 1; Jurkat Cells; Phosphorylation; STAT1 Transcription Factor; Transfection

Salvianolic acid B (SalB) represents the most characteristic constituent of Salvia miltiorrhiza Bge. with a strong free radicals scavenger activity. This property may be useful in the treatment of some severe chronic diseases, where there is an imbalance of reactive oxygen species formation and where intracellular reactive oxygen and nitrogen species level can cause severe cell damage and even cell death. In particular, SalB can protect against the oxidative stress as well as the antioxidant superoxide dismutase and reduced activity of glutathione, important determinants of neuropathological and behavioural consequences in neuropathic pain. This is a chronic disease defined by the WHO as an untreatable illness because therapeutics are unsatisfactory in many cases and there is an urgent need to discover and develop novel active drugs. In the present work, SalB has been extracted and purified with an efficient and rapid method from the roots and rhizome of S. miltiorrhiza Bge. It was firstly submitted to pharmacological studies using the paw-pressure test, in an animal model of neuropathic pain where a peripheral mono neuropathy was produced by a chronic constriction injury of the sciatic nerve. SalB was effective against mechanical hyperalgesia when administered intraperitoneally at the dose of 100mg/kg, 15 min after administration. Due to the poor chemical stability and bioavailability of SalB, liposomes were developed as drug carriers for parental administration. SalB-loaded liposomes were characterised in terms of particle size, polydispersity index, encapsulation efficacy and morphology. According to the in vivo studies, encapsulation, especially into PEGylated liposomes, increased and prolonged the antihyperalgesic activity 30 min after i.p. administration and the effect was still significant at 45 min. Thus, PEGylated formulation ameliorated the performance of drug delaying, increasing and prolonging in time its antihyperalgesic effect.

Topics: Analgesics; Animals; Benzofurans; Hyperalgesia; Ligation; Male; Microscopy, Electron, Transmission; Neuralgia; Pain Measurement; Pain Threshold; Particle Size; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Sciatica; Static Electricity; Surface Properties; Unilamellar Liposomes

Salvianolic acid A, salvianolic acid B, danshensu, protocatechuic aldehyde, rosmarinic acid and lithospermic acid are the six major active constituents in Danshen injection. In this study, a rapid, sensitive and specific liquid chromatographic-electrospray ionization-mass spectrometry method for the simultaneous quantitative determination of these compounds in rat plasma was developed. After a single step of liquid-liquid extraction with ethyl acetate, they were eluted by a Hypersil C18 column (5 µm, i.d. 4.6 × 200 mm) within 4 min with a mobile phase consisting of acetonitrile and 0.1% formic acid water solution (35:65, v/v). The assay was linear in the concentration range of 0.05-10 µg mL(-1). Absolute recoveries were above 60%. The precisions and accuracies determined within three consecutive days were within acceptable limits. The method was successfully applied to a pharmacokinetic study in rats after an intravenous administration of Danshen injection.

Topics: Animals; Benzaldehydes; Benzofurans; Caffeic Acids; Catechols; Chromatography, High Pressure Liquid; Chromatography, Liquid; Cinnamates; Depsides; Drug Stability; Drugs, Chinese Herbal; Injections, Intravenous; Lactates; Liquid-Liquid Extraction; Male; Mass Spectrometry; Plant Preparations; Rats; Reference Standards; Rosmarinic Acid; Salvia miltiorrhiza; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization

To investigate the effect of salvianolic acid B (SAB) on the proliferation of human embryonic lung fibroblast MRC-5, and the secretion of procollagen I and endogenous transforming growth factor-beta1, (TGF-beta1).. The MRC-5 cells were randomly divided into four groups as follows: the control group: cells cultured with DMEM but with no TGF-beta1, or SAB; the TGF-beta1, group: cell cultured with 10 ng/mL TGF-beta1; the SAB1 group: cell cultured with medium with 10 ng/mL TGF-beta1 and 1 pmol/L SAB; the SAB2 group: cell cultured with medium with 10 ng/mL TGF-beta1, and 10 pmol/L SAB. The proliferation of cells was assayed by MTT incorporation. The concentration of amino-terminal propeptide of type I procollagen (PINP), a marker of collagen synthesis, was measured by radioimmunoassay. The endogenous TGF-beta1, levels were measured using ELISA.. The optical density, procollagen I contents, and endogenous TGF-beta1, levels significantly increased when compared with those of the control group (P<0.05). Compared with the TGF-beta1, group, the optical density was obviously lowered, the procollagen I contents and endogenous TGF-beta1, levels significantly decreased in the SAB1 group and the SAB2 group, and better in the SAB2 group, showing statistical difference (P<0.05).. SAB could inhibit the proliferation of MRC-5 cells induced by TGF-beta1 and attenuate the roles of secreting collagen and endogenous TGF-beta1. It had the potential of postponing or delaying the progressive developing of pulmonary fibrosis.

Topics: Benzofurans; Cell Proliferation; Cells, Cultured; Collagen Type I; Fibroblasts; Humans; Lung; Transforming Growth Factor beta1

To investigate the role of the TLR4-NFκB-TNFα inflammation pathway on: lipopolysaccharide (LPS)-induced neonatal rat cardiomyocyte injury and the possible protective effects of salvianolic acid B (Sal B).. Wistar rat (1-2 days old) cardiomyocytes were isolated and cultured. Sal B 10(-5)mol/L, 10(-6)mol/L and 10(-7)mol/L were pre-treated for 6 h in the culture medium. LPS (1 μg/mL) was added to mol/the culture medium and kept for 6 h to induce inflammation injury. The concentration of lactate dehydrogenase (LDH) in the supernatant was detected by spectrophotometry. The concentrations of tumor necrosis factor α (TNFα) and heat shock protein 70 (HSP70) in the supernatant were detected by enzyme linked immunosorbent assay. The protein expressions of toll, such as receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were detected by immunohistochemistry. The mRNA expressions of TLR4 and NFκB were detected by real-realtime reverse transcription polymerase chain reaction (RT-PCR).. (1) The concentrations of LDH and: TNFα in the LPS control group were significantly higher than those in the control group (561.41±67.39 U/L and 77.94±15.08 pg/mL, versus 292.13±26.02 U/L and 25.39±16.53 pg/mL, respectively, P<0.01, P<0.05). Compared with the LPS control group, the concentrations of LDH and TNFα were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (451.76±83.96 U/L and 34.00±10.38 pg/mL, respectively, P<0.05). (2) The TLR4 and NFκB protein expression area in the LPS control group were significantly higher than those in the control group (1712.41±410.12 μm(2) and 2378.15±175.29 μm(2), versus 418.62±24.42 μm(2) and 1721.74±202.87 μm(2), respectively, P<0.01). The TLR4 and NFκB protein expression internal optical density (IOD) values in the LPS control group were also significantly higher than those in the control group (3.06±0.33 and 7.20±1.04, versus 0.91±0.21 and 4.24±0.48, respectively, P<0.05 and P<0.01). Compared with the LPS control group, the TLR4 and NFκB protein expression areas were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (1251.54±133.82 μm(2) and 1996.37±256.67 μm(2), respectively, P<0.05), the TLR4 and NFκB protein expression IOD values were also significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.92±0.28 and 5.17±0.77, respectively, treated P<0.05). (3) The TLR4 and NFκB mRNA expressions (2(-ΔΔ)CT value) in the LPS control group were significantly higher than those in the control group (3.16±0.38 and 5.03±0.43 versus 1.04±0.19 and 1.08±0.21, respectively, P<0.01). Compared with the LPS control group, the TLR4 and NFκB mRNA expressions (2(-ΔΔ) -CT value) were significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.34±0.22 and 1.74±0.26, respectively, treated P<0.05). The concentration of HSP70 did not show any 0.05).. The TLR4-NFκB-TNFα pathway was quickly activated: and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. The protective effects of Sal B may be through inhibiting the TLR4-NFκB-TNFα pathway and are dose-dependent.

Topics: Animals; Animals, Newborn; Benzofurans; Gene Expression Regulation; HSP70 Heat-Shock Proteins; L-Lactate Dehydrogenase; Lipopolysaccharides; Myocytes, Cardiac; NF-kappa B; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; Subcellular Fractions; Toll-Like Receptor 4; Transcription, Genetic; Tumor Necrosis Factor-alpha

Danshen (Salvia miltiorrhiza Bunge) hairy roots were obtained by infecting Danshen leaves with Agrobacterium rhizogenes 9402. Besides rosmarinic acid (RA) and salvianolic acid B (SAB), the hairy root could also produce salvianolic acid K (SAK), salvianolic acid L, ethyl salvianolic acid B (ESAB), methyl salvianolic acid B (MSAB), and a compound with a molecular weight of 538 (compound 538) identified by using LC-MS. Effects of methyl jasmonate (MeJA) and yeast elicitor (YE) on the accumulation of these compounds had been investigated. MeJA increased the accumulation of SAB, RA, SAK, and compound 538 from 4.21%, 2.48%, 0.29%, and 0.01% of dry weight to 7.11%, 3.38%, 0.68%, and 0.04%, respectively. YE stimulated the biosynthesis of RA from 2.83% to 5.71%, but depressed the synthesis of SAB, SAK and compound 538. It was indicated in all the results that these Danshen hairy roots could be used as alternative resources to produce salvianolic acids. Analysis of the content variation of these compounds after elicitation suggested that SAK and compound 538 might be the intermediates in the biosynthesis from RA to SAB in Danshen hairy roots.

Topics: Acetates; Alkenes; Benzofurans; Cinnamates; Cyclopentanes; Depsides; Oxylipins; Phenylpropionates; Plant Growth Regulators; Plant Roots; Plants, Medicinal; Polyphenols; Rosmarinic Acid; Salvia miltiorrhiza; Yeasts

To observe the effects of the separate and joint use of salvianolic acid B (SalB) and tetrahydropalmatine (THP) on the L-type calcium channel of rat ventricular myocytes.. Single isolated ventricular myocytes of rats were obtained using acute enzymolysis separation. The current of the L-type calcium channel was recorded using whole-cell patch clamp technique. Changes of the current peak value of the calcium channel (the vertical distance between the peak value point after activation of the calcium electric current and the electric current track after complete inactivation) were observed before and after medication.. The inhibition rate of using SalB (at the dose of 1, 10, and 100 micromol/L) alone on the current peak value of the calcium channel was respectively (25.3 +/- 16.4)% (n=4), (44.6 +/- 24.0)% (n=6), and (86.0 +/- 20.4)% (n =4). That of using THP (at the dose of 10, 30, and 100 micromol/L) alone on the current peak value of the calcium channel was respectively (22.2 +/- 6.4)% (n=5), (27.4 +/- 1.6)% (n= 3), and (51.0 +/- 23.0)% (n=9). The inhibition potency of joint use of SalB (1 micromol/L) and THP (10 micromol/L) on the current peak value of the calcium channel was stronger than using SalB (1 micromol/L) alone or THP (10 micromol/L) alone, showing statistical difference ( P< 0.05). Atropine hydrochloric acid (14 mmol/L) could reverse the inhibition of THP on the L-type calcium channel, while strengthening the inhibition of SalB.. Both SalB and THP showed inhibition on the L-type calcium channel of rat ventricular myocytes. They could generate synergistic effects. Besides, their action mechanisms for regulating the L-type calcium channel were different.

Topics: Animals; Benzofurans; Berberine Alkaloids; Calcium Channels, L-Type; Heart Ventricles; Male; Myocytes, Cardiac; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley

To establish the quality standard for Danmo capsule.. TLC was used for the qualitative identification of Salviae Miltiorrhizae Radix Et Rhizoma and Lonicerae Japonicae Flos. HPLC was used to determine the content of Salvianolic acid B.. TLC spots were clear and well-separated without negative interference. The linear range of Salvianolic acid B was 0.120042 - 2.40084 microg (r = 0.9999) with an average recovery of 103.63%, RSD was 0.6% (n = 6).. The method is simple, accurate and reliable. It can be used for the quality control of Danmo capsule.

Topics: Benzofurans; Capsules; Chlorogenic Acid; Chromatography, High Pressure Liquid; Drug Combinations; Drugs, Chinese Herbal; Ethanol; Flowers; Lonicera; Plants, Medicinal; Quality Control; Reproducibility of Results; Rhizome; Salvia miltiorrhiza

We explored the effects of combination of acupuncture and Chinese medicinal herbs in treating model rats with polycystic ovarian syndrome (PCOS) and to explore whether acupuncture has positive effects on the absorption of salvianolic acid B in the extracts of a Chinese medicine formula when treating the model rats. 60 female Sprague-Dawley (SD) rats were randomly divided into Groups A, B, C, D, E and F, with ten rats in each group. Except Group F, all of the other rats were induced to PCOS with oral administration of letrozole. The rats in Group F served as normal controls. Group A was treated with acupuncture. Group B was treated with oral administration of the extracts of the Chinese medicine formula. Group C was treated with a combination of oral administration of the extracts of Chinese medicine and acupuncture. Group D received western medicine as positive controls. After treatment, the serum levels of follicle stimulating hormone (FSH), luteinizing hormone(LH) and testosterone (T) in each group were detected with the Enzyme-Linked ImmunoSorbent Assay (ELISA) and the serum concentration of salvianolic acid B were determined using High Performance Liquid Chromatography (HPLC). The serum levels of T and the ratio of LH/FSH in Group A, B. C, D, and F were significantly lower than those of Group E, indicating the model rats with PCOS were successfully established. Compared with Groups A, B, D and E, the serum levels of T and the ratio of LH/FSH in Group C were significantly lower respectively, indicating combination of acupuncture and Chinese medicinal herbs can significantly enhance curative effects in treating model rats with PCOS. The concentration of serum salvianolic acid Group C was significantly higher than Group B, indicating that acupuncture might improve the absorption of salvianolic acid B from the extracts of Salvia miltiorrhiza Bunge in the Chinese medicine formula. Combination of acupuncture and Chinese medicinal herbs significantly enhance curative effects in treating model rats with PCOS and acupuncture has positive effects in improving the absorption of salvianolic acid B in the extracts of the Chinese medicine formula when treating the model rats with PCOS.

Topics: Acupuncture Therapy; Administration, Oral; Animals; Benzofurans; Chromatography, High Pressure Liquid; Disease Models, Animal; Drugs, Chinese Herbal; Enzyme-Linked Immunosorbent Assay; Female; Follicle Stimulating Hormone; Letrozole; Luteinizing Hormone; Nitriles; Polycystic Ovary Syndrome; Rats; Rats, Sprague-Dawley; Testosterone; Treatment Outcome; Triazoles

A simple and sensitive capillary electrophoresis method with field-enhanced stacking concentration for the analysis of protocatechuic aldehyde, protocatechuic acid, danshensu, rosmarinic acid and salvianolic acid B in Salvia miltiorrhiza var. miltiorrhiza f. alba was developed. The separation was achieved with a fused-silica capillary (75 microm x 50.2 cm, effective length was 40 cm) and a running buffer 15 mmol x L(-1) borax (pH 10.0) containing 20% CH3 OH. The UV detection wavelength was 210 nm. The applied voltage was 28 kV, and the cartridge temperature was 25 degrees C. Water plug was introduced from the anode by 0.5 psi x 4 s before injection. Sample was injected by electrokinetic injection - 8 kV x 3 s. The linear range of protocatechuic aldehyde is 3.0-60.00 mg x L(-1) (R2 = 0.999 8); that of protocatechuic acid, danshensu, rosmarinic acid and salvianolic acid B are 1.0-20.00 mg x L(-1) (R2 are 0.999 1, 0.999 4, 0.998 9 and 0.999 8, respectively), and the limits of detection of five analyts are 0.55, 0.40, 0.25, 0.32, 0.38 microg x L(-1), respectively, Stacking factor is higher and precision is satisfactory. The recoveries ranges were from 97.3% to 99.8%. The proposed method was used to determine the protocatechuic aldehyde, protocatechuic acid, danshensu, rosmarinic acid and salvianolic acid B in S. miltiorrhiza var. miltiorrhiza f. alba. The proposed method is simple, rapid, accurate and high sensitivity, and can be used to control of the quality of S. miltiorrhiza var. miltiorrhiza f. alba.

Topics: Benzaldehydes; Benzofurans; Catechols; Cinnamates; Depsides; Electrophoresis, Capillary; Hydroxybenzoates; Lactates; Plant Extracts; Quality Control; Rosmarinic Acid; Salvia miltiorrhiza; Sensitivity and Specificity; Solubility; Water

Enhanced oxidative stress is associated with hepatic fibrosis. Salvianolic acids A (Sal A) and B (Sal B) have been reported to be strong polyphenolic antioxidants and free radical scavengers. The present study is to investigate if Sal A and B could attenuate oxidative stress and liver fibrosis in rats. A cell line of rat hepatic stellate cells (HSCs) was stimulated with platelet-derived growth factor (PDGF, 10 ng/ml). The inhibitory effects of Sal A and B on intracellular hydrogen peroxide levels were measured with dichlorofluorescein diacetate (DCF-DA) dye assay. alpha-Smooth muscle actin (alpha-SMA), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits were measured by Western blotting. Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA, 200 mg/kg) twice per week for 6 weeks. Sal A (10 mg/kg), Sal B (50 mg/kg) or S-adenosylmethionine (SAMe, 10 mg/kg), was given by gavage twice per day consecutively for 4 weeks starting 2 weeks after TAA injection. In vitro, PDGF increased the accumulation of hydrogen peroxide in HSCs, which was attenuated by Sal A (10 muM) and Sal B (200 muM). Sal A and B attenuated the PDGF-stimulated expressions of alpha-SMA and NADPH oxidase subunits gp91(phox) and p47(phox) in membrane fractions. In vivo studies showed that the hepatic levels of collagen, malondialdehyde, TNF-alpha, IL-6, and IL-1beta, fibrosis scores and protein expressions of alpha-SMA, heme-oxygenase-1, iNOS, and gp91(phox), and serum levels of ALT, AST, IL-6, and IL-1beta were increased in TAA-intoxicated rats, all of which were attenuated by 4-week treatment of Sal A or Sal B. Our results showed that Sal A and B attenuated PDGF-induced ROS formation in HSCs, possibly through inhibition of NADPH oxidase. Sal A and B treatments were also effective against hepatic fibrosis in TAA-intoxicated rats.

Topics: Actins; Animals; Benzofurans; Blotting, Western; Caffeic Acids; Hydrogen Peroxide; Lactates; Liver Cirrhosis; Male; Oxidative Stress; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley

(2E)-2-{6-[(E)-2-carboxyvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid, a novel compound designated SMND-309, is a new derivate of salvianolic acid B. The present study was designed to investigate the cardioprotective potential of SMND-309 and to elucidate the possible mechanisms on the basis of biochemical, histopathological and immunohistochemical studies in a rat model of acute myocardial infarction induced by permanent ligation of the left coronary artery. The results showed that treatment with SMND-309 via tail vein at doses of 10 and 20 mg/kg significantly prevented the elevation in ST segment level and the increase in serum creatine kinase-MB, lactate dehydrogenase, alanine aminotransferase and cardiac troponin T content. Meanwhile, SMND-309 significantly increased the activities of superoxide dismutase, catalase and glutathione peroxidase, decreased the content of malondialdehyde in myocardium, and reduced the myocardium necrosis scores and the number of apoptosis cardiocytes in accordance with the up-regulated expression of anti-apoptotic protein, Bcl-2 and the down-regulated expression of proapoptotic protein, Bax. Moreover, SMND-309 exhibits significantly higher potency compared to salvianolic acid B at the same mg/kg but not the same mol/kg. These findings indicate that SMND-309 has a protective potential against myocardial infarction injury and the protective effects may be due to its scavenging lipid peroxidation products, increasing endogenous antioxidant defence enzymes and attenuating cardiocyte apoptosis.

Topics: Animals; Antioxidants; Apoptosis; Benzofurans; Caffeic Acids; Cardiotonic Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Lipid Peroxidation; Male; Myocardial Infarction; Rats; Rats, Sprague-Dawley

The present study aimed to investigate the effects of salvianolic acid B (SalB) isolated from Radix Salvia miltiorrhizae on the oral pharmacokinetics of metoprolol (MET) and metoprolol acid (META) in rats. The pharmacokinetic parameters of MET and META were measured after oral (15 mg/kg) administration of MET in rats in the presence and absence of SalB. Compared to the control given MET alone, with the concurrent administration of SalB (50 mg/kg), the AUC and C(max) of MET increased by 51.7% and 27.7%, and the AUC and C(max) of META decreased by 26.5% and 19.6%, respectively. With the presence of SalB, the metabolic ratio was markedly decreased by 50.8%. However, no significant changes were observed in the pharmacokinetic parameters of SalB when it was combined with MET.

Topics: Animals; Area Under Curve; Benzofurans; Female; Male; Metoprolol; Rats; Rats, Wistar; Salvia miltiorrhiza

Salvianolic acid B is one of the effective components from the Chinese traditional drug Salvia miltiorrhiza (Danshen), which is widely used as a usual clinic drug for atherosclerosis-related disorder patients in China. But the targeting protein of salvianolic acid B is still not known. The possible targeting proteins of salvianolic acid B were explored by high throughput screening in this paper. Attached to the magnetic nanoparticles, salvianolic acid B was used for screening the high-affinity protein from the displaying cDNA peptide library phage. After biopanning, the selected protein or peptide sequences were used to explore the whole proteins containing the selected sequences in the National Center for Biotechnology Information website using blast. One of the selected phages was carried out by affinity analysis with salvianolic acid B using capillary electrophoresis (CE). The CE results indicated that the protein or peptide on the surface of the selected phages could bind the drug salvianolic acid B. The results are helpful to preliminarily explain the pharmacology of salvianolic acid B.

Topics: Bacteriophage T7; Benzofurans; Drugs, Chinese Herbal; Humans; Kinetics; Magnetics; Nanoparticles; Nanotechnology; Peptide Library; Phenanthrolines; Protein Binding; Proteins; Salvia miltiorrhiza

This study examined whether Salvianolic acid B (Sal B), a major active component of Chinese herb Radix Salviae Miltiorrhizae, may exert an anti-inflammatory effect in microglia and may be neuroprotective by regulating microglial activation. Our results showed that Sal B significantly reduced the production of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and reactive oxygen species (ROS) induced by lipopolysaccharide (LPS) treatment in rat primary microglia in a dose-dependent manner. Sal B had no effects on ATP-dependent IL-1beta release and interferon (IFN)-gamma-induced NO production. Sal B also suppressed LPS-induced inducible nitric oxide synthase (iNOS), TNF-alpha, and IL-1beta mRNA expression, which was accompanied by inhibiting transcription factor NF-kappaB activation. Sal B could protect neurons through inhibition of microglial activation in a microglia-neuron coculture system. In conclusion, these data demonstrate that anti-inflammatory activity of Sal B in microglia contributes to its neuroprotective effect and suggest that it may be useful for preventing microglia-mediated neuroinflammation.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Benzofurans; Cell Survival; Coculture Techniques; Interleukin-1beta; Lipopolysaccharides; Microglia; Neurons; Neuroprotective Agents; NF-kappa B; Nitric Oxide; Rats; Rats, Wistar; Reactive Oxygen Species; RNA, Messenger; Tumor Necrosis Factor-alpha

To investigate the mechanism of salvianolic acid B (Sal B) action against liver fibrosis through preventing lipid peroxidation and regulating MMP-2 activity in liver.. The liver fibrotic model was induced through intraperitoneally injection of DMN at a dose of 10 microg x kg(-1) for every other day and lasting for 4 weeks. Sal B was administered (10 mg x kg(-1)), and perindopril (5 mg x kg(-1)) was used as positive control. Hepatic inflammation and collagen were observed with HE and sirius red staining. The liver function including serum ALT, AST activity, Alb and total bilirubin (T. Bil) level were determined. The hepatic lipid peroxidation including SOD and GST activities and GSH content were measured. Hepatic hydroxyproline (Hyp) content was detected with Jamall's method. The activity of metalloproteinase was assayed by gelatin zymography. The expressions of alpha-SMA, Col I in liver tissue were analyzed by Western blot.. The model rats had higher serum T. Bil content, ALT and AST activities but lower Alb content than the normal rats, also had remarkable inflammatory necrosis and collagen deposition in liver, with much higher Hyp content, protein expression of alpha-SMA and collagen I and MMP-2 activity in liver, but had a decreased GSH content, SOD and GST activities. Both Sal B and perindopril attenuated hepatic injury and collagen deposition in model rats, decreased serum ALT activity and hepatic Hyp content, down-regulated alpha-SMA and collagen I protein expressions and metalloproteinase-2 activity than those in the model group, but increased SOD activity and GSH content, and Sal B decreased serum T. Bil content and increased GST activity. Sal B had a much better comprehensive actions than perindopril.. Sal B has a good preventive action against liver fibrosis, the action mechanism is related to the prevention from lipid peroxidation and down-regulation of metalloproteinase-2 activity in fibrotic liver.

Topics: Animals; Benzofurans; Lipid Peroxidation; Liver; Liver Cirrhosis; Male; Matrix Metalloproteinase 2; Rats; Rats, Sprague-Dawley

To investigate the effect of salvianolic-acid B (SA-B) on epithelia-mesenchymal transition in human renal proximal tubular cells (HK2), induced by transforming growth factor beta1 (TGF-beta1).. Epithelia-mesenchymal transition (EMT) was induced with TGF-beta1 in HK2 cultured in vitro. Different concentrations (2, 5, 10, 20 microg x L(-1)) and stimulant periods (12, 24, 48 h) were tried to find the perfect condition for EMT. At the same time bone morphogenetic protein-7 (BMP-7, positive control) and the SA-B intervention were given to observe their effect on EMT. Western blot and immunofluorescent microscopy were used to analyze the expression of E-cadherin and alpha-smooth muscle actin (alpha-SMA) in HK2.. BMP-7 significantly inhibited the down-regulation of E-cadherin and the up-regulation of alpha-SMA induced by TGF-beta1 (P < 0.05), and SA-B significantly inhibited the up-regulation of alpha-SMA expression induced by TGF-beta1 (P < 0.05), but not the down-regulation of E-cadherin induced by TGF-beta1.. SA-B and BMP-7 can inhibit TGF-beta1-induced EMT in HK2. Their common role is to inhibit the up-regulation of alpha-SMA, and the effect of SA-B on the regulation of E-cadherin needs further study to be confirmed.

Topics: Benzofurans; Bone Morphogenetic Protein 7; Cadherins; Cell Differentiation; Cell Line; Epithelial Cells; Humans; Mesoderm; Transforming Growth Factor beta1

To investigate the pharmacokinetic interactions induced by content variation of the main water-soluble components of Danshen injection in rats.. Intravenous Danshen injection (control) or Danshen injection with danshensu (DSS), protocatechuic aldehyde (PAL), salvianolic acid A (Sal A) or salvianolic acid B (Sal B) were administered to female Sprague Dawley rats. Plasma concentrations of DSS, Sal A, PAL and its oxidative metabolite protocatechuic acid (PA) were analyzed simultaneously with LC-MS/MS; concentrations of Sal B were determined by the LC-MS method. Non-compartmental pharmacokinetic parameters were calculated and compared for identifying the pharmacokinetic interactions among these components.. Compared with the control group, the DSS, Sal A, and Sal B groups had significant increases in AUC(0-infinity) in response to elevated concentrations of PAL (by 78.1%, 51.0%, and 82.9%, respectively), while the clearances (CL) were markedly reduced (by 42.5%, 32.9%, and 46.8%, respectively). Similarly, Sal A increased the AUC(0-infinity) of DSS and Sal B (26.7% and 82.4%, respectively) and substantially decreased their clearances (21.4% and 45.6%, respectively). In addition, the pharmacokinetics of DSS and Sal B were significantly affected by the content variation of the other major components; the AUC(0-infinity) increased by 45.1% and 52.1%, respectively, the CL dropped by 29.6% and 27.1%, respectively, and the T(1/2) was decreased by 22.0% and 19.6%, respectively.. Complex, extensive pharmacokinetic interactions were observed among the major water-soluble constituents in the Danshen injection. The content variation of PAL had the most significant effect on the pharmacokinetic behaviors of other major constituents. Furthermore, the pharmacokinetics of DSS and Sal B were the most susceptible to the content change of other components.

Topics: Animals; Benzaldehydes; Benzofurans; Caffeic Acids; Catechols; Drugs, Chinese Herbal; Female; Lactates; Phenanthrolines; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza

Salvianolic Acid B (Sal B) is a water-soluble component from Danshen (a traditional Chinese herb widely used for chronic renal diseases) with anti-oxidative and cell protective properties. Sal B also has potential protective effects on renal diseases. Tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT), which plays an important role in the pathogenesis of renal interstitial fibrosis (RIF) and is mainly regulated by TGF-beta1/Smads pathway. The aims of the study are to investigate the effect of Sal B on tubular EMT in vivo and in vitro, and to elucidate its underlying mechanism against EMT related to TGF-beta1/Smads pathway.. For in vivo experiments, RIF was induced in rats by oral administration of HgCl2 and prophylaxised with Sal B and vitamin E. The protein expression of E-cadherin was down-regulated, while the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3 and the activity of matrix metalloproteinase-2 (MMP-2) were up-regulated in kidneys of model rats when compared with those of normal rats. In contrast, Sal B and vitamin E significantly attenuated the expression of alpha-SMA, TGF-beta1, TbetaR-I, p-Smad2/3, and MMP-2 activity, but increased E-cadherin expression. For in vitro experiments, HK-2 cells were incubated with TGF-beta1 to induce EMT, and the cells were co-cultured with 1 and 10 microM Sal B or SB-431542 (a specific inhibitor of TbetaR-I kinase). TGF-beta1 induced a typical EMT in HK-2 cells, while it was blocked by Sal B and SB-431542, as evidenced by blocking morphologic transformation, restoring E-cadherin and CK-18 expression, inhibiting alpha-SMA expression and F-actin reorganization, and down-regulating MMP-2/9 activities in TGF-beta1 mediated HK-2 cells. Furthermore, Sal B and SB-431542 profoundly down-regulated the expressions of TbetaR-I and p-Smad2/3 but prevented the decreased expression of Smad7 in TGF-beta1 stimulated HK-2 cells.. Sal B can prevent tubular EMT in the fibrotic kidney induced by HgCl2 as well as HK-2 cells triggered by TGF-beta1, the mechanism of Sal B is closely related to the regulation of TGF-beta1/Smads pathway, manifested as the inhibition of TGF-beta1 expression, suppression of TbetaR-I expression and function, down-regulation of Smad2/3 phosphorylation, and restoration of the down-regulation of Smad7, as well as inhibition of MMP-2 activity.

Topics: Actins; Animals; Benzofurans; Cell Differentiation; Cell Line; Drugs, Chinese Herbal; Epithelial Cells; Fibrosis; Humans; Kidney; Mesoderm; Phosphorylation; Protein Serine-Threonine Kinases; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta1; Up-Regulation

Salvianolic acid B (Sal B) is a water-soluble compound found in the traditional Chinese medicine, Radix Salviae miltiorrhizae, and has been widely used to treat a variety of diseases in Asian cultures. Sal B was shown to inhibit apoptosis in many cell types, but its effect on hepatocyte apoptosis is unknown. In this study, we attempt to show that Sal B attenuates hepatocyte apoptosis and hepatic injury induced by lipopolysaccharide and D-galactosamine in mice. Sal B also inhibits apoptosis that is induced by the death receptor in the HL-7702 hepatocyte cell line. Apoptosis in vitro is determined by flow cytometry, DNA electrophoresis and high content screening assay. The antiapoptotic effect is generated by reducing the expression of tumor necrosis factor alpha receptor type 1, balancing the expression of Bcl-2 family members, decreasing the release of cytochrome C from the mitochondria into the cytosol and inhibiting activated Caspase-3. These findings suggest that Sal B can effectively inhibit hepatocyte apoptosis as well as the underlying mechanisms related to regulating mediators in death receptor and mitochondrial pathways.

Topics: Animals; Apoptosis; Benzofurans; Cell Line; DNA; DNA Fragmentation; Electrophoresis, Agar Gel; Flow Cytometry; Galactosamine; Hepatocytes; Humans; Inflammation; Lipopolysaccharides; Liver Diseases; Liver Function Tests; Male; Mice; Mitochondria; Receptors, Death Domain; Signal Transduction; Tumor Necrosis Factor-alpha

Head and neck squamous cell carcinoma (HNSCC) development is closely associated with inflammation. Cyclooxygenase-2 (COX-2) is an important mediator of inflammation. Therefore, celecoxib, a selective inhibitor of COX-2, was hailed as a promising chemopreventive agent for HNSCC. Dose-dependent cardiac toxicity limits long-term use of celecoxib, but it seems likely that this may be diminished by lowering its dose. We found that salvianolic acid B (Sal-B), isolated from Salvia miltiorrhiza Bge, can effectively suppress COX-2 expression and induce apoptosis in a variety of cancer cell lines. In this study, we report that combination of Sal-B with low-dose celecoxib results in a more pronounced anticancer effect in HNSCC than either agent alone. The combination effects were assessed in four HNSCC cell lines (JHU-06, JHU-011, JHU-013, and JHU-022) by evaluating cell viability, proliferation, and tumor xenograft growth. Cell viability and proliferation were significantly inhibited by both the combined and single-agent treatments. However, the combination treatment significantly enhanced anticancer efficacy in JHU-013 and JHU-022 cell lines compared with the single treatment regimens. A half-dose of daily Sal-B (40 mg/kg/d) and celecoxib (2.5 mg/kg/d) significantly inhibited JHU-013 xenograft growth relative to mice treated with a full dose of Sal-B or celecoxib alone. The combination was associated with profound inhibition of COX-2 and enhanced induction of apoptosis. Taken together, these results strongly suggest that combination of Sal-B, a multifunctional anticancer agent, with low-dose celecoxib holds potential as a new preventive strategy in targeting inflammatory-associated tumor development.

Topics: Animals; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzofurans; Carcinoma, Squamous Cell; Celecoxib; Cell Division; Cell Line, Tumor; Cyclooxygenase 2 Inhibitors; Dinoprostone; Drug Synergism; Head and Neck Neoplasms; Humans; In Situ Nick-End Labeling; Male; Mice; Mice, Nude; Pyrazoles; Salvia miltiorrhiza; Sulfonamides; Xenograft Model Antitumor Assays

The study was conducted to investigate the effect of Salvianolic acid B (Sal B) on TNF-alpha-stimulated adhesion molecule expression i.e. vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin in human aortic endothelial cells (HAECs) under laminar shear stress (LSS) condition. Exposure of HAECs to LSS (12 dynes/cm(2) for 6 h decreased the TNF-alpha-induced protein expression of adhesion molecules i.e. VCAM-1, ICAM-1 and E-selectin. Pre-treatment of HAECs with Sal B (10 microg/ml) then exposed to LSS (12 dynes/cm(2)) for 6 h significantly inhibited VCAM-1, ICAM-1 and E-selectin expression stimulated by TNF-alpha. Moreover, combined Sal B and LSS treatment inhibited the adhesiveness of monocytic U937 cells to TNF-alpha-stimulated HAECs. We further examined the molecular mechanisms and found that the combination of Sal B and LSS treatment dramatically inhibited TNF-alpha-induced NF-kappaB activation evidenced by IkappaBalpha degradation and p65 nuclear translocation in HAECs. This study provides the first biomechanopharmacological evidence that Sal B has a combination effect with LSS to reduce the expression of three adhesion molecules, leading to reduced monocyte adhesion to HAECs, at least in part, by inhibiting the NF-kappaB signaling pathway. Data from this study thus support the potential clinical application of Sal B in vascular inflammatory diseases.

Topics: Anti-Inflammatory Agents; Benzofurans; Cell Adhesion; E-Selectin; Endothelium, Vascular; Humans; Intercellular Adhesion Molecule-1; NF-kappa B; Signal Transduction; Stress, Mechanical; Tumor Necrosis Factor-alpha; U937 Cells; Vascular Cell Adhesion Molecule-1

The present work investigated the effects of salicylic acid (SA) on the accumulation of phenolic compounds and the activities of PAL, TAT, SOD, CAT and POD enzymes in the Salvia miltiorrhiza cell culture. When SA is applied to the cell culture, phenolic compounds will increase and PAL, TAT, SOD, CAT, and POD enzymes will become more active. The accumulations of phenolic compounds and the PAL activity were stimulated 8h after the treatment with SA. The TAT activity was stimulated after 48 h. The resulting antioxidative enzymes' activities were greatly improved. SA elicitation on the phenolic acid accumulation was depended upon the application dosage and the time-duration. The suitable SA concentration for eliciting phenolic compound accumulations was 6.25-22.5mg/L. The elicitation effect of SA on phenolic compound accumulations correlated with the PAL activity, but not with the TAT activity. This indicates that PAL may be the key enzyme for the biosynthesis of salvianolic acid B and caffeic acid. The raised PAL activity leads to the improvement of the quantity of phenolic compounds. This could be of particular significance by using plant cell culture systems for biotechnological production of plant secondary metabolites such as salvianolic acid B and caffeic acid.

Topics: Benzofurans; Biomass; Caffeic Acids; Oxidoreductases; Phenylalanine Ammonia-Lyase; Plant Proteins; Salicylic Acid; Salvia miltiorrhiza; Tyrosine Transaminase

Salvianolic-acid B (SA-B) is an effective component of Radix Salviae miltiorrhizae for anti-hepatic fibrotic herbs. MAPK signaling pathway has been implicated in hepatic stellate cells (HSC) stimulated by TGF-(1. We have investigated the effect of SA-B on MAPK pathway in rat HSC.. To observe the pharmacological effect of SA-B on HSC, SA-B was added into the medium of primary HSC. TGF-(1 was added during last 2h, and PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor) were added just 30 min before adding TGF-(1. MEF2 and Col. I were measured by luciferase reporter gene assay and Western blot. (-SMA, MEF2, Raf, ERK, p-ERK, MEK, p-MEK, p38, p-p38, MKK3 and p-MKK3/6 were assayed by Western blot. Activity of MMP-2 and MMP-9 was analyzed by zymography. Each experiment was repeated for three times.. The expression of (-SMA and Col. I in HSC was inhibited by SA-B. There was no effect of SA-B on the activity of MMP-2 or MMP-9 in the media of cultured HSC. Phosphorylation of ERK1/2 in HSC stimulated with or without TGF-(1 was inhibited by SA-B. Specifically, phosphorylation of MEK (upstream kinase of ERK pathway) was inhibited by SA-B. SA-B also inhibited phosphorylation of MKK3/6 (upstream kinases of p38 pathway) and inhibited the synthesis of MEF2.. SA-B performs anti-hepatic fibrosis through inhibiting ERK and p38 MAPK pathway in HSC. SA-B inhibits ERK pathway via inhibiting phosphorylation of MEK and inhibits p38 MAPK pathway via blocking phosphorylation of MKK3/6 and inhibiting expression of MEF2 in HSC with or without TGF-(1 stimulation.

Topics: Animals; Benzofurans; Cell Line; Drugs, Chinese Herbal; Extracellular Signal-Regulated MAP Kinases; Hepatic Stellate Cells; Liver Cirrhosis; MAP Kinase Kinase 3; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Matrix Metalloproteinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Rats; Signal Transduction; Transforming Growth Factor beta1

Leaf of Salvia miltiorrhiza Bunge, the waste part during the root harvest, is rich in health-promoting phenolics and is a novel resource of natural antioxidants. The acetone and methanol extracts of leaves (AL and ML, respectively) of S. miltiorrhiza were evaluated by various in vitro antioxidant assays. The total phenolic contents of AL and ML were 39.0+/-1.13 and 54.3 +/-1.1mg gallic acid equivalents/g extract tested, respectively. EC(50) of ML was 7.0+/-0.28 microg/mL in DPPH radical scavenging assay and 246.5 +/-10.35 microg/mL in superoxide radical quenching assay. It was also found that ML has prominent effects on the inhibition of linoleic acid oxidation (93.2%), which was equivalent to the positive control, butylated hydroxytoluene (BHT, p>0.05), and was significantly higher than α-tocopherol (VE, p<0.05). The reducing power of leaf extracts was as strong as roots (p>0.05). HPLC and correlation analysis show that salvianolic acid B and rosmarinic acid constitute the most abundant phenolic compounds. They are the major contributors to antioxidant activities. The results suggested that S. miltiorrhiza leaves could be considered as a new potential source of natural phenolic antioxidants for food, pharmaceutical, cosmetics or nutraceutical industries.

Topics: Acetone; Antioxidants; Benzofurans; beta Carotene; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Ferric Compounds; Indicators and Reagents; Methanol; Phenols; Plant Extracts; Plant Leaves; Plant Roots; Reducing Agents; Rosmarinic Acid; Salvia miltiorrhiza; Solvents; Spectrophotometry, Ultraviolet

Salvianolic acid B is isolated from Salvia miltiorrhiza, the root of which is widely used as a traditional Chinese medicine to treat stroke. However, little is known about how salvianolic acid B influences growth characteristics of neural stem cells (NSCs). The purpose of the present study was to evaluate the effects of salvianolic acid B on proliferation, neurite outgrowth and differentiation of NSCs derived from the cerebral cortex of embryonic mice using MTT, flow cytometry, immunofluorescence and RT-PCR. It was found that 20 microg mL(-1) and 40 microg mL(-1) salvianolic acid B had similar effects on proliferation of NSCs, and a suitable concentration of salvianolic acid B increased the number of NSCs and their derivative neurospheres. The growth-promoting activity of salvianolic acid B was dependent on and associated with an accumulation in the G2/S-phase cell population. Salvianolic acid B also promoted the neurite outgrowth of NSCs and their differentiation into neurons. The mRNA for tau, GFAP and nestin were present in differentiating neurospheres induced by salvianolic acid B. However, high-level expression of tau mRNA and low-level expression of GFAP mRNA was detected in differentiated cells, in contrast to the control conditions. This collective evidence indicates that exogenous salvianolic acid B is capable of promoting proliferation of neurospheres and differentiation towards the neuronal lineage in vitro and may act in the proliferation of NSCs and may promote NSC differentiation into neuronal cells.

Topics: Animals; Benzofurans; Cell Cycle; Cell Differentiation; Cell Proliferation; Cerebral Cortex; Female; Flow Cytometry; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Mice; Neurites; Pregnancy; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stem Cells; tau Proteins

To improve the survival and/or differentiation of grafted BMSCs (bone marrow stem cells) represents one of the challenges for the promising cell-based therapy. Considerable reports have implicated Sal B (salvianolic acid B), a potent aqueous extract of Salvia miltiorrhiza, in enhancing the survival of cells under various conditions. In this study, we investigated the effect of Sal B on H₂O₂-induced apoptosis in rat BMSCs, focusing on the survival signalling pathways. Results indicated that the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] inhibitor (PD98059) and 10 μM Sal B remarkably prevented BMSCs from H₂O₂-induced apoptosis through attenuating caspase-3 activation, which is accompanied by the significant up-regulation of Bcl-2. In addition, the ROS (reactive oxygen species) accumulation was also reduced after Sal B treatment. Furthermore, Sal B inhibited the ERK1/2 phosphorylations stimulated by H₂O₂. Taken together, our results showed that H₂O₂-induced apoptosis in BMSCs via the ROS/MEK/ERK1/2 pathway and Sal B may exert its cytoprotection through mediating the pathway.

Topics: Animals; Apoptosis; Benzofurans; Bone Marrow Cells; Cytoprotection; Extracellular Signal-Regulated MAP Kinases; Flavonoids; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Oxidative Stress; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Stem Cells

The rhodamine B (RhB) covalently grafted SBA-15-structured mesoporous silica nanoparticles (MSNs-RhB) of high surface area (750 m(2) g(-1)), large pore volume (0.7 cm(3) g(-1)), uniform particle size (about 400 nm) and positively charged surface (29.6 +/- 5.0 mV), has been developed as a drug delivery system (SAB@MSNs-RhB) for anti-ROS (reactive oxygen species)/hepatic fibrosis by loading a negatively charged drug salvianolic acid B (SAB). The dosage formulation SAB@MSNs-RhB effectively protected the loaded drug SAB from decomposition. The multi-release experimental results showed that SAB@MSNs-RhB exhibited an outstanding SAB sustained-release property, and relatively high SAB release rates and concentrations in a long term after the consumption of previously released SAB as compared to SAB loaded MSNs (SAB@MSNs) of negatively charged surface (-31.1 +/- 2.6 mV). The influences of the drug concentration, incubation time, drug formula and drug carrier on the ROS level, proliferative activity and cytotoxicity of LX-2 cells were evaluated. The results showed that the inhibiting effect of SAB@MSNs-RhB on the ROS level and proliferative activity of LX-2 cells was more remarkable than free SAB in a long term (72 h), and became more intensive with the increase of the sample concentration and the incubation time. SAB@MSNs-RhB enhanced the cellular drug uptake, the drug bioaccessability and efficacy for anti-ROS/hepatic fibrosis via the nanoparticles-mediated endocytosis and the sustained release of the drug. There was no visible cytotoxicity of free SAB, MSNs-RhB and SAB@MSNs-RhB against LX-2 cells in a broad concentration range (0.5-100 microm) and incubation time periods up to 72 h. The blood compatibility of the carrier MSNs-RhB was evaluated by investigating the hemolysis and coagulation behaviors in a broad concentration range (50-500 microg mL(-1)) under in vitro conditions. The results suggested that MSNs-RhB possessed good blood compatibility.

Topics: Benzofurans; Biocompatible Materials; Cell Line; Cell Proliferation; Drug Carriers; Drug Delivery Systems; Drugs, Chinese Herbal; Fluorescent Dyes; Hemolysis; Humans; Liver Cirrhosis; Materials Testing; Molecular Structure; Nanoparticles; Particle Size; Porosity; Reactive Oxygen Species; Rhodamines

Parkinson's disease (PD) is associated with mitochondrial dysfunction, oxidative stress, and activation of the apoptotic cascade. In the study, we investigated the effects of salvianolic acid B (Sal B) on 1-methyl-4-phenylpyridinium (MPP(+))-treated SH-SY5Y cells, a classic in vitro model for PD. We found Sal B inhibited the loss of cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The underlying mechanisms of Sal B action were further studied. Treatment of SH-SY5Y cells with MPP(+) caused a loss of cell viability and mitochondrial membrane potential, condensation of nuclei, elevation in the level of reactive oxygen species (which was associated with cytochrome c release), an increase in the Bax/Bcl-2 mRNA ratio, and activation of caspase-3. Sal B ameliorated the MPP(+)-altered phenotypes. These results indicate that the Sal B protected SH-SY5Y cells against MPP(+)-induced apoptosis by relieving oxidative stress and modulating the apoptotic process. Our findings suggest that salvianolic acid B may be a promising agent to prevent PD.

Topics: 1-Methyl-4-phenylpyridinium; Antiparkinson Agents; Apoptosis; bcl-2-Associated X Protein; Benzofurans; Biological Assay; Caspase 3; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Humans; Membrane Potential, Mitochondrial; Molecular Structure; Neuroblastoma; Parkinson Disease; Proto-Oncogene Proteins c-bcl-6; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction

Infarct-induced left ventricular (LV) remodeling is a deleterious consequence after acute myocardial infarction (MI) which may further advance to congestive heart failure. Therefore, new therapeutic strategies to attenuate the effects of LV remodeling are urgently needed. Salvianolic acid B (SalB) from Salviae mitiorrhizae, which has been widely used in China for the treatment of cardiovascular diseases, is a potential candidate for therapeutic intervention of LV remodeling targeting matrix metalloproteinase-9 (MMP-9).. Molecular modeling and LIGPLOT analysis revealed in silico docking of SalB at the catalytic site of MMP-9. Following this lead, we expressed truncated MMP-9 which contains only the catalytic domain, and used this active protein for in-gel gelatin zymography, enzymatic analysis, and SalB binding by Biacore. Data generated from these assays indicated that SalB functioned as a competitive inhibitor of MMP-9. In our rat model for cardiac remodeling, western blot, echocardiography, hemodynamic measurement and histopathological detection were used to detect the effects and mechanism of SalB on cardio-protection. Our results showed that in MI rat, SalB selectively inhibited MMP-9 activities without affecting MMP-9 expression while no effect of SalB was seen on MMP-2. Moreover, SalB treatment in MI rat could efficiently increase left ventricle wall thickness, improve heart contractility, and decrease heart fibrosis.. As a competitive inhibitor of MMP-9, SalB presents significant effects on preventing LV structural damage and preserving cardiac function. Further studies to develop SalB and its analogues for their potential for cardioprotection in clinic are warranted.

Topics: Animals; Benzofurans; Binding Sites; Computer Simulation; Down-Regulation; Drug Design; Drugs, Chinese Herbal; Fibrosis; Heart Ventricles; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Molecular Conformation; Myocardial Contraction; Myocardial Infarction; Protease Inhibitors; Random Allocation; Rats; Rats, Wistar; Recombinant Fusion Proteins; Ventricular Remodeling

To study the relaxant effects of glycyrrhizinate and salvianolic acid B on rat portal vein in vitro.. Healthy female Wistar rats were canalized from hepatic artery, portal vein and hepatic vein in vitro. Remained blood in liver was eliminated with heparinized Krebs-Henseleit solution through hepatic artery, then the liver was isolated under infusing manner. Being constricted with phenylephrine and relaxed with acetylcholine, and infused with glycyrrhizinate or salvianolic acid B, the portal pressures of infused rat livers were consistently monitored by BL-420S physiological experiment system. The median effective concentration (EC50) of the two agents were analyzed with non-linear various slope regression using Prism-4 software.. EC50 of glycyrrhizinate in relaxing the phenylephrine-contracted portal was 1.5556 x 10(-9) mol/L, suggesting one of the mechanism of action of diammonium glycyrhizinate for the treatment of portal hypertension was direct relaxation. Salvianolic acid B showed constrictive action on the phenylephrine-retracted portal vein, the EC50 was 1.4639 x 10(-9) mol/L, indicating that its indirect control action was took part in the portal hypertension therapy synergistically.. Under the mode with both controlled-velocity and monitored pressure, glycyrrhizinate showed relaxation and salvianolic acid B showed constriction on portal pressure in vitro.

Topics: Animals; Benzofurans; Blood Pressure; Female; Glycyrrhizic Acid; Hypertension, Portal; Phenylephrine; Portal Vein; Rats; Rats, Wistar; Vasoconstrictor Agents; Vasodilator Agents; Vasomotor System

To investigate the effect of total salvianolic acid (TSA) on ischemia-reperfusion (I/R)-induced rat mesenteric microcirculatory dysfunctions.. Male Wistar rats were randomly distributed into 5 groups (n = 6 each): Sham group and I/R group (infused with saline), TSA group, TSA + I/R group and I/R + TSA group (infused with TSA, 5 mg/kg per hour). Mesenteric I/R were conducted by a ligation of the mesenteric artery and vein (10 min) and subsequent release of the occlusion. TSA was continuously infused either starting from 10 min before the ischemia or 10 min after reperfusion. Changes in mesenteric microcirculatory variables, including diameter of venule, velocity of red blood cells in venule, leukocyte adhesion, free radicals released from venule, albumin leakage and mast cell degranulation, were observed through an inverted intravital microscope. Meanwhile, the expression of adhesion molecules CD11b/CD18 on neutrophils was evaluated by flow cytometry. Ultrastructural evidence of mesenteric venules damage was assessed after microcirculation observation.. I/R led to multiple responses in mesenteric post-capillary venules, including a significant increase in the adhesion of leukocytes, production of oxygen radicals in the venular wall, albumin efflux and enhanced mast cell degranulation in vivo. All the I/R-induced manifestations were significantly reduced by pre- or post-treatment with TSA, with the exception that the I/R-induced increase in mast cell degranulation was inhibited only by pre-treatment with TSA. Moreover, pre- or post-treatment with TSA significantly attenuated the expression of CD11b/CD18 on neutrophils, reducing the increase in the number of caveolae in the endothelial cells of mesentery post-capillary venules induced by I/R.. The results demonstrated that TSA protects from and ameliorates the microcirculation disturbance induced by I/R, which was associated with TSA inhibiting the production of oxygen-free radicals in the venular wall and the expression of CD11b/CD18 on neutrophils.

Topics: Animals; Benzofurans; Blood Flow Velocity; Caffeic Acids; CD11b Antigen; CD18 Antigens; Cell Degranulation; Cinnamates; Lactates; Leukocytes; Male; Mast Cells; Mesentery; Microcirculation; Neutrophils; Phenylpropionates; Plant Extracts; Random Allocation; Rats; Rats, Wistar; Reperfusion Injury; Venules

An improved everted gut sac method was applied to the study of prescription compatibility effect on the major components in Danshen extracts. With the separation and detection by HPLC-ECD, 5 major peaks could be detected in intestinal absorbed solution after prescription administration. Following the identification by HPLC-MS/MS, peak 2, 3, 4, and 5 were rosmaric acid, lithospermic acid, salvianolic acid B, and salvianolic acid A, respectively, which also confirmed with reference standards of those components. Through paralleling substance identification, peak 2, 3, 4, and 5 could be found as the major components in Danshen extracts, except Salvianolic acid E which is undetectable in intestinal solution. The contents of peak 2, 3, and 4 did not show difference before and after compatible prescription administrated, where the peak 5 had a significant increase in the same process. Those results revealed that peak 5, salvianolic acid A, might lead to an increasing pharmacological effect after prescription compatibility.

Topics: Animals; Benzofurans; Caffeic Acids; Chromatography, High Pressure Liquid; Depsides; Drugs, Chinese Herbal; In Vitro Techniques; Intestinal Absorption; Lactates; Male; Plants, Medicinal; Rats; Rats, Wistar; Salvia miltiorrhiza; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

The aim of the study is to investigate the effect of salvianolic acid B (SalB) on blood-brain barrier (BBB) in rats after cerebral ischemia-reperfusion, and to illustrate its possible mechanisms. Cerebral ischemia-reperfusion was induced by middle cerebral artery occlusion in rats. The break-down of BBB was indicated by extravasations of immunoglobulin (IgG) monitored with immunohistochemistry. The expression of MMP-9 and NOS2 in the brain was determined by immunohistochemistry, and the expression of p-p38 and p-ERK1/2 was detected by Western blotting. It was shown that on day 2 after ischemia-reperfusion the IgG accumulated around the vascular boundary zone, suggesting the break-down of BBB, and the expression of MMP-9 and NOS2 up-regulated at the same time. The result of Western blotting suggested that the expression of p-p38 and p-ERK1/2 increased. On day 7 after ischemia-reperfusion the. expression of MMP-9 and NOS2 was about the same level as day 2, the expression of p-p38 was higher than that on day 2 and the expression of p-ERK1/2 was slightly lower than that on day 2. SalB (1 and 10 mg x kg(-1)) significantly alleviated the extravasations of immunoglobulin induced by cerebral ischemia-reperfusion (P < 0.05). On day 2 and day 7 SalB attenuated the expression of MMP-9 and NOS2 (P < 0.05). SalB (10 mg x kg(-1)) reduced the expression of p-p38 and p-ERK1/2 apparently on day 2 and 7 after ischemia-reperfusion (P < 0.05). SalB (1 mg x kg(-1)) inhibited the expression of p-p38 on day 7 after ischemia-reperfusion (P < 0.05). The results indicate that SalB protects blood-brain barrier in rats after cerebral ischemia-reperfusion by inhibiting the MAPK pathway.

Topics: Animals; Benzofurans; Blood-Brain Barrier; Brain Ischemia; Drugs, Chinese Herbal; Infarction, Middle Cerebral Artery; Male; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plants, Medicinal; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Salvia miltiorrhiza

This study aimed to investigate the effects of concentration, intestinal section and borneol on the intestinal absorption of salvianolic acids. The experiment not only studied the intestinal absorption properties of three concentrations of rosmarinic acid, salvianolic acid B and salvianolic acid A at duodenum, jejunum and ileum, but also of salvianolic acids compatible with borneol at different concentrations using single-pass intestinal perfusion model in rat with phenol red as the marker. The results showed that salvianolic acids was stable under weak-acid condition and affected by metabolism enzyme; The Peff and Ka significantly different among three concentrations of rosmarinic acid and salvianolic acid B, whose intestinal absorption were saturated in high concentration, suggesting that the transport mechanisms of rosmarinic acid and salvianolic acid B were similar to active transport or facilitated diffusion; However, there was inconspicuousness in the Peff and Ka of salvianolic acid A at different concentrations, whose absorption was not saturated in high concentration, indicating that the transport mechanisms of salvianolic acid A was passive diffusion; The Peff and Ka in the ileum obviously higher than those in the duodenum and jejunum, namely the ileum was the best absorption section; When concentration of borneol increased, the enhancing effect of intestinal absorption of salvianolic acids increased, but significantly decreased when borneol increased to some degree. The enhancing effect of medium borneol concentration was the optimum. This implied that borneol can enhance the intestinal absorption of salvianolic acids, and the capacity of enhancing effect was influenced by the concentration of borneol.

Topics: Animals; Benzofurans; Caffeic Acids; Camphanes; Cinnamates; Depsides; Dose-Response Relationship, Drug; Duodenum; Ileum; Intestinal Absorption; Jejunum; Lactates; Male; Perfusion; Plants, Medicinal; Rats; Rats, Sprague-Dawley; Rosmarinic Acid; Salvia miltiorrhiza

This paper is aimed to report the development of a method for the determination of the binding rate of plasma protein with salvianolic acid B. In vitro, equilibrium dialysis method was used to imitate the binding process between salvianolic acid B and plasma protein, in vivo, ultrafiltration method was used and the binding rate with HPLC was determined. Plasma samples were treated with methanol to precipitate the protein, and the buffer solution was directly determined after filtering. The calibration curve of the buffer solution was linear in the range of 0.5-20 microg mL(-1). The calibration curve of the plasma was linear in the range of 2-200 microg mL(-1). The extract recovery was 68.6%-81.9%. RSDs of intra- and inter-day precisions were all less than 8.5%. The binding rates of plasma protein with salvianolic acid B in vitro was 75.2% and in vivo was 92.1%. This paper shows the high binding power of salvianolic acid B to plasma protein with high sensitivity, good reproduction, simple management and fulfilling the requirement.

Topics: Animals; Benzofurans; Blood Proteins; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Male; Protein Binding; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Salvia miltiorrhiza; Sensitivity and Specificity; Ultrafiltration

To optimize the dynamic extraction process of salvianolic acids.. Salvianolic acids was selected as index. The effects of extraction temperature, granularity, solvent multiple, circle value and extraction time were studied on the process of dynamical extraction. The orthogonal experiment was employed to investigate the influence of different parameters.. The optimal extracting method of salvianolic acids was as follows: temperature was 80 degrees C, granularity was 4 mm, circle value was 30 L x h(-1), solvent multiple was 10 times and extraction time was 150 min.. By comparison to static extraction, dynamic extraction can improve the extraction efficiency, reduce the solvent and energy consumption, as well as lower the burden of post-processing.

Topics: Benzofurans; Chemical Fractionation; Drugs, Chinese Herbal; Temperature

In order to clarify the chemical composition of Tongmai Keli, a HPLC fingerprint was established, and the 22 peaks were characterized by LC-DAD-MS. The herbal sources of these peaks were assigned. The results implied that genistin 8-C-glucoside, puerarin, daidzein 8-C-apiosyl (1, 6)-O-glucoside, daidzin, and salvianolic acid B were the main constituents of in Tongmai Keli. Ten batches of Tongmai Keli produced by different pharmaceutical companies were analyzed. Although different batches contained similar compounds, the contents of major compounds were significantly different. The method established in this study could be used for the quality control of Tongmai Keli.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Drug Combinations; Drugs, Chinese Herbal; Isoflavones; Ligusticum; Plants, Medicinal; Pueraria; Quality Control; Salvia miltiorrhiza; Tandem Mass Spectrometry

To investigate the effect and mechanism of salvianolic acid B (SA-B) on renal interstitial fibrosis in rats induced by unilateral ureteral obstruction (UUO).. 18 male SD rats were randomly divided into 3 groups, 6 in each group. After the models were established, the rats were treated with SA-B for 2 weeks. Then their renal pathology were examined by hight microscope and electron microscopy Protein expression levels of alpha-smooth muscle actin (alpha-SMA) and E-cadherin (E-cad) in the obstructed kidney were analyzed by Western blot and Biochemistry assay.. Pathological examination of the kidney in model group showed tubules lumen widened and many inflammatory cells infiltrated, a part of renal tubule expanded and part of them atrophied. The tubular epithelial cells were karyorrhexis or karyolysis, some tubulars were atrophy. The protein expression of alpha-SMA were significantly up-regulated (P < 0.01) and E-cad were significantly down-regulated (P < 0.01) in the model group. After intervention with SA-B, the renal pathological status in the treatment group was significantly improved, the expression of alpha-SMA were significantly down-regulated (P < 0.05), but E-cad only a little up-regulated (P > 0.05).. SA-B could antagonize renal interstitial fibrosis mainly by maintaining epithelial phenotype, inhibiting the protein of alpha-SMA which is the principal effect cells that are responsible for the progressive kidney fibrosis.

Topics: Actins; Animals; Benzofurans; Blotting, Western; Cadherins; Disease Models, Animal; Fibrosis; Immunohistochemistry; Kidney; Kidney Diseases; Kidney Tubules; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Ureteral Obstruction

Cerebral ischemia-reperfusion injury is the main reason for the loss of neurons in the ischemic cerebrovascular disease. Therefore, to deeply understand its pathogenesis and find a new target is the key issue to be solved. This research aimed to investigate the neuroprotective effects of salvianolic acid B (SalB) against oxygen-glucose deprivation/reperfusion (OGD/RP) damage in primary rat cortical neurons.. The primary cultures of neonatal Wister rats were randomly divided into the control group, the OGD/RP group and the SalB-treatment group (10 mg/L). The cell model was established by depriving of oxygen and glucose for 3 hours and reperfusion for 3 hours and 24 hours, respectively. The neuron viability was determined by MTT assay. The level of cellular reactive oxygen species (ROS) was detected by fluorescent labeling method and spin trapping technique respectively. The activities of neuronal Mn-superoxide dismutase (Mn-SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) were assayed by chromatometry. The mitochondria membrane potential (ΔΨ(m)) was quantitatively analyzed by flow cytometry. The release rate of cytochrome c was detected by Western blotting. The neuronal ultrastructure was observed by transmission electron microscopy. Statistical significance was evaluated by analysis of variance (ANOVA) followed by Student-Newman-Keuls test.. OGD/RP increased the level of cellular ROS, but decreased the cell viability and the activities of Mn-SOD, CAT and GSH-PX; SalB treatment significantly reduced the level of ROS (P < 0.05); and enhanced the cell viability (P < 0.05) and the activities of these antioxidases (P < 0.05). Additionally, OGD/RP induced the fluorescence value of ΔΨ(m) to diminish and the release rate of cytochrome c to rise notably; SalB markedly elevated the level of ΔΨ(m) (P < 0.01) and depressed the release rate of cytochrome c (P < 0.05); it also ameliorated the neuronal morphological injury.. The neuroprotection of SalB may be attributed to the elimination of ROS and the inhibition of apoptosis.

Topics: Animals; Apoptosis; Benzofurans; Catalase; Cells, Cultured; Cerebral Cortex; Cytochromes c; Glutathione Peroxidase; Hypoxia-Ischemia, Brain; Membrane Potential, Mitochondrial; Neuroprotective Agents; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase

To investigate the effects of salvianolic acid B (Sal-B) on pancreatic damage in experimental chronic pancreatitis.. Chronic pancreatitis was induced by infusion of trinitrobenzene sulfonic acid into the pancreatic duct in male Sprague-Dawley rats. From the beginning of 5 weeks, the rats in group 2 were treated with Sal-B by gavage for 8 weeks. Salvianolic acid B was given at a daily dose of 10 mg/kg body weight. At the end of 12 weeks, the levels of serum biochemical indexes were measured on an automatic biochemical analyzer; serum hyaluronic acid and laminin levels were determined by radioimmunoassay; pancreatic tissue malondialdehyde (MDA) was analyzed, and the degree of pancreatic damage was determined.. The level of serum biochemical indexes were similar in all groups (P > 0.05 for all). Salvianolic acid B treatment did not obviously reduce hyaluronic acid and laminin concentration in blood (P > 0.05). Salvianolic acid B treatment decreased MDA concentration in pancreatic tissue (P < 0.01). Salvianolic acid B clearly improved pancreatic histological findings and prevented the activation of pancreatic stellate cells.. Sal-B treatment decreased MDA concentration in pancreatic tissue, attenuated morphological pancreatic damage, and prevented the activation of pancreatic stellate cells in experimental chronic pancreatitis.

Topics: Animals; Benzofurans; Biomarkers; Body Weight; Disease Models, Animal; Hyaluronic Acid; Laminin; Male; Malondialdehyde; Organ Size; Pancreas; Pancreatitis, Chronic; Protective Agents; Rats; Rats, Sprague-Dawley; Trinitrobenzenesulfonic Acid

The purposes of the present study were to both evaluate the protective effects of Salvianolic acid B (Sal B) and to determine the possible molecular mechanisms by which Sal B protects endothelial cells from damage caused by oxidative stress.. Pretreatment with Sal B markedly attenuated H(2)O(2)-induced viability loss, lactate dehydrogenase leakage and apoptosis in human umbilical vein endothelial cells (HUVECs). The mechanism of Sal B protection was studied using two-dimensional gel electrophoresis coupled with hybrid quadrupole time-of-flight mass spectrometry. Database searching implicated that glucose-regulated protein 78 (GRP78), a central regulator for endoplasmic reticulum (ER) stress, was up-regulated in Sal B-exposed HUVECs. GRP78 expression regulation was confirmed by western blot and RT-PCR (reverse transcription-polymerase chain reaction) analyses. Additionally, GRP94, which shares significant sequence homology with GRP78, was also up-regulated in Sal B-treated cells. Sal B caused pancreatic ER kinase (PKR)-like ER kinase (PERK) activation followed by the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2 alpha) and the expression of activating transcription factor 4 (ATF4). Knockdown of endogenous ATF4 expression partially repressed Sal B-induced GRP78 induction. Further investigation showed that ATF6 was also activated by Sal B. Knockdown of GRP78 by siRNA significantly reduced the protective effects of Sal B.. The results suggest that Sal B induces the expression of GRP78 by activating ATF6 and the PERK-eIF2 alpha-ATF4 pathway. Furthermore, up-regulation of GRP78 by Sal B may play an important role in protecting human endothelial cells from oxidative stress-induced cellular damage.

Topics: Activating Transcription Factor 4; Activating Transcription Factor 6; Apoptosis; Benzofurans; Cells, Cultured; Drugs, Chinese Herbal; eIF-2 Kinase; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endothelium, Vascular; Heat-Shock Proteins; Humans; Hydrogen Peroxide; Membrane Glycoproteins; Molecular Chaperones; Oxidative Stress; RNA, Small Interfering; Signal Transduction; Transcription Factors; Umbilical Veins

Overexpression of cyclooxygenase-2 (COX-2) in oral mucosa has been associated with increased risk of head and neck squamous cell carcinoma (HNSCC). Celecoxib is a nonsteroidal anti-inflammatory drug, which inhibits COX-2 but not COX-1. This selective COX-2 inhibitor holds promise as a cancer preventive agent. Concerns about cardiotoxicity of celecoxib, limits its use in long-term chemoprevention and therapy. Salvianolic acid B (Sal-B) is a leading bioactive component of Salvia miltiorrhiza Bge, which is used for treating neoplastic and chronic inflammatory diseases in China. The purpose of this study was to investigate the mechanisms by which Sal-B inhibits HNSCC growth. Sal-B was isolated from S. miltiorrhiza Bge by solvent extraction followed by 2 chromatographic steps. Pharmacological activity of Sal-B was assessed in HNSCC and other cell lines by estimating COX-2 expression, cell viability and caspase-dependent apoptosis. Sal-B inhibited growth of HNSCC JHU-022 and JHU-013 cells with IC(50) of 18 and 50 microM, respectively. Nude mice with HNSCC solid tumor xenografts were treated with Sal-B (80 mg/kg/day) or celecoxib (5 mg/kg/day) for 25 days to investigate in vivo effects of the COX-2 inhibitors. Tumor volumes in Sal-B treated group were significantly lower than those in celecoxib treated or untreated control groups (p < 0.05). Sal-B inhibited COX-2 expression in cultured HNSCC cells and in HNSCC cells isolated from tumor xenografts. Sal-B also caused dose-dependent inhibition of prostaglandin E(2) synthesis, either with or without lipopolysaccharide stimulation. Taken together, Sal-B shows promise as a COX-2 targeted anticancer agent for HNSCC prevention and treatment.

Topics: Animals; Apoptosis; Benzofurans; Blotting, Western; Carcinoma, Squamous Cell; Caspases; Cells, Cultured; Colony-Forming Units Assay; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Drugs, Chinese Herbal; Female; Flow Cytometry; Head and Neck Neoplasms; Humans; In Vitro Techniques; Keratinocytes; Magnetic Resonance Imaging; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mouth Mucosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Salvia miltiorrhiza; Survival Rate; Transplantation, Heterologous

SMND-309, a novel compound (2E)-2-[6-[(E)-2-carboxyvinyl]-2,3-dihydroxyphenyl]-3-(3,4-dihydroxyphenyl) acrylic acid, is a new derivate of salvianolic acid B. The present study elucidates the effects of SMND-309 on the cultured rat cortical neuron damage induced by oxygen-glucose deprivation. The results show that SMND-309 treatment obviously attenuates apoptosis and ameliorates mitochondrial energy metabolism in rat cortical neurons by increasing cell survival rate, mitochondrial antioxidant enzyme activities, mitochondrial respiratory enzymes activities, mitochondrial respiratory control ratio and the adenosine triphosphate content, and by decreasing mitochondrial malondialdehyde content, lactate dehydrogenase release, intracellular Ca(2+) level and caspase-3 activity in a concentration-dependent manner. Moreover, SMND-309 exhibits significantly higher potency as compared to salvianolic acid B. These findings indicate that SMND-309 has a protective potential against cerebral ischaemic injury and its protective effects may be due to the suppression of intracellular Ca(2+) elevation and caspase-3 activity, and improvement of mitochondrial energy metabolism and antioxidant property.

Topics: Animals; Apoptosis; Benzofurans; Caffeic Acids; Calcium; Caspase 3; Cell Culture Techniques; Cell Survival; Cells, Cultured; Cerebral Cortex; Energy Metabolism; Lipid Peroxidation; Mitochondria; Molecular Structure; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley

Ischemia-reperfusion injury (IRI) is a serious complication of liver surgery, especially for extended hepatectomy and liver transplantation. The aim of this study was to evaluate the protective effect of combined ischemic preconditioning (IPC) and salvianolic acid-B (Sal-B) pretreatment against IRI-induced hepatocellular injury.. Sixty male Wistar rats weighing around 200 g were randomized into five groups (n=12): sham group: only anesthesia and laparotomy; IR group: 90 min sustained ischemia by blocking the left ortal vessels; IPC group: 10 min ischemia and 10 min reperfusion prior to the sustained ischemia; Sal-B group: 10 mg/kg injection of Sal-B intravenously 10 min prior to the sustained ischemia; IPC+Sal-B group: same IPC procedure as in IPC group, but proceeded by intravenous administration of Sal-B 10 min prior to sustained ischemia. After 5 h of reperfusion, serum levels of ALT and AST were measured; the amount of malondialdehyde (MDA) and adenine nucleotides in liver tissue was determined; the expression of Bcl-2 and caspase-3 was detected by immunofluorescent and western blotting techniques; the severity of apoptosis and pathological alterations was evaluated by TUNEL and H&E staining, respectively.. The serum aminotransferases, hepatic MDA concentration, and apoptotic index in groups IPC, Sal-B, and IPC+Sal-B were significantly lower than those in the IR group (P<0.001), while the IPC+Sal-B group had the lowest values among these groups (P<0.05). Compared with the IR group, groups IPC and Sal-B not only had statistically higher ATP levels and energy charge (EC) values (P<0.01), but also had upregulated Bcl-2 expression and downregulated cleaved caspase-3 expression in liver tissue. All these effects were further augmented in the IPC+Sal-B group. Liver histopathological findings were consistent with these results.. Based on these results, the combined IPC and Sal-B pretreatment had a synergistically protective effect on liver tissue against IRI, which might be due to decreased post-ischemic oxidative stress, improved energy metabolism, and reduced hepatocellular apoptosis.

Topics: Adenine Nucleotides; Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Benzofurans; Blotting, Western; Caspase 3; Combined Modality Therapy; Cytoprotection; Disease Models, Animal; Energy Metabolism; Fluorescent Antibody Technique; In Situ Nick-End Labeling; Ischemic Preconditioning; Liver; Male; Malondialdehyde; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reperfusion Injury; Time Factors

We have used microarray technology to detect the effect of Guanxin No.2 decoction on gene expression in different areas of the myocardial infarcted heart of rats.. Male Sprague-Dawley rats (180-200 g) were randomly divided into three groups: sham-operated; coronary artery ligation; and coronary artery ligation plus administration of Guanxin No.2 decoction (10.0 g raw materials/kg per day by gavage). The experiment was carried out on day seven after ligation.. We found that the gene expression using microarray technology showed many differences in the border infarcted left ventricular area compared with the remote noninfarcted left ventricular area after administration of Guanxin No.2 decoction.. Guanxin No.2 decoction has a long history in treating ischaemic cardiomyopathy in China, but the molecular mechanism has been unclear. In this study we found that some important genes may have contributed to the cardioprotective effect of Guanxin No.2 decoction.

Topics: Animals; Benzaldehydes; Benzoates; Benzofurans; Bridged-Ring Compounds; Cardiotonic Agents; Carthamus tinctorius; Catechols; Chalcone; Coumaric Acids; Dalbergia; Drugs, Chinese Herbal; Gene Expression; Gene Expression Profiling; Glucosides; Heart Ventricles; Hydroxybenzoates; Lactates; Ligusticum; Male; Monoterpenes; Myocardial Infarction; Oligonucleotide Array Sequence Analysis; Paeonia; Plant Extracts; Quinones; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Salvia miltiorrhiza

LAB (lithospermic acid B) is a dimer of RA (rosmarinic acid) and has been suggested to be derived from RA, but the detailed biosynthesis process has not yet been identified. The accumulation of RA has been intensively investigated in the plant species of Boraginaceae and Lamiaceae. In the present study, we report that silver ions (Ag+; 15 microM), an abiotic elicitor, did not stimulate RA accumulation but dramatically enhanced LAB from approx. 5.4% to 18.8% of dry weight in Salvia miltiorrhiza hairy root cultures, and the rise in LAB was found to be coincident with the decline of RA content at each time point after treatment. Meanwhile, a profiling analysis of genes and metabolites (intermediates) involved in the RA synthesis pathway was performed; the result indicated that several gene transcripts and metabolite accumulations show temporal changes in abundance consistent with LAB production. Thus a potential (putative) biosynthetic route from RA to LAB was presumed, which was suggested to be significantly activated by Ag+ in S. miltiorrhiza hairy root cultures. Further intermediate monitoring and compound feeding experiments were performed to rank the strength of this hypothesis. Our study, for the first time, provides evidence that RA is a precursor leading to LAB synthesis.

Topics: Benzofurans; Cells, Cultured; Cinnamates; Depsides; Gene Expression Regulation, Plant; Molecular Structure; Plant Proteins; Plant Roots; Rosmarinic Acid; Salvia miltiorrhiza; Silver; Stress, Physiological

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which as a group can degrade essentially all extracellular matrix components. The proteolytic property of the MMPs is important during wound healing to remove debris and facilitate cell migration. Targeting towards the decreased MMPs activities is a new treatment strategy for healing chronic wounds. Salvia miltiorrhiza is a popular Chinese herb that could promote chronic ulcers healing for topical use. Salvianolic acid B (Sal B) is the most abundant bioactive component in Salvia miltiorrhiza. The research was designed to explore the inhibitory effects of Sal B on MMP-1, MMP-2 and MMP-9 activities.. Pure human interstitial collagenase (MMP-1) or gelatinase A (MMP-2) was activated by p-aminophenylmercuric acetate (APMA), and was incubated with Sal B for 1 h. The activities were observed by quenched fluorescent substrate. Gelatinase B (MMP-9) is rich in polymorphonuclear neutrophils (PMN), so the rat PMN was used as a source of MMP-9 for MMPs activity assays. In vitro MMP-9 from rats' PMN lysate was incubated with Sal B for 1 h, and its activity was tested by gelatin zymography.. Sal B dose-dependently inhibited the human MMP-1 and MMP-2 activities in the range of 0.002 4 to 0.3 g/L, with 50% inhibiting concentration (IC(50)) of (0.090<0.015) g/L and (0.080<0.005) g/L respectively. In the range of 0.003 to 0.3 g/L, Sal B could inhibit the MMP-9 activity (P<0.01).. The broad-spectrum inhibitory effects of Sal B on MMPs may reveal one of the mechanisms for the effects of Salvia miltiorrhiza on chronic wounds.

Topics: Animals; Benzofurans; Female; Humans; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Rats; Rats, Wistar

To observe the effects of Salviae miltiorrhizae and its component, salvianolic acid B (SA-B), on the microcirculation of liver in mice with portal hypertension induced by endothelin-1 (ET-1).. Eighty-four Kunming mice were randomly divided into 7 groups: untreated group, endothelin A receptor (ETAR) blocker group, Astragali mongolici group, Astragalus polysaccharides (APS) group, Corydalis Yanhusuo group, Salviae miltiorrhizae group and SA-B group. There were 12 mice in each group. Mice were pretreated with a corresponding equivalent volume of drug or distilled water for 3 days, and then the portal hypertension in mice was induced by continuous injection of ET-1 into coccygeal vein using a micro-injection pump. Six mice in each group were used to observe the average liver blood flow volume by laser-Doppler flow instrument before and after injection of ET-1, and the other six rats were used to observe the hepatic microcirculation velocity in vivo by an inverted microscope.. The average blood flow of liver in mice decreased in each group after ET-1 injection. But the changes of average blood flow in the SA-B group and the ETAR blocker group were less than that in the untreated group (P<0.01). The changes of average blood volume in the Astragali mongolici group and the APS group were similar to that in the untreated group, but more than that in the SA-B group after injection of ET-1. The change of average blood flow in the SA-B group showed no significant difference when compared with the ETAR blocker group. The microcirculatory flow velocity in liver also decreased in each group after ET-1 injection. But the changes of microcirculatory flow velocity in the SA-B group and the ETAR blocker group were less than that in the untreated group (P<0.05, P<0.01). There were no significant differences in the changes of microcirculatory flow velocity among the Salviae miltiorrhizae group, the SA-B group and the ETAR blocker group.. Salviae miltiorrhizae and SA-B can decrease the average blood flow and microcirculatory flow velocity in liver in mice with portal hypertension, which may be one of the mechanisms of Salviae miltiorrhizae and SA-B in decreasing portal hypertension.

Topics: Animals; Benzofurans; Hypertension, Portal; Liver; Male; Mice; Mice, Inbred Strains; Microcirculation; Salvia miltiorrhiza

Danshen (Salvia miltiorrhiza Bunge, Lamiaceae) is a commonly used and highly valued Chinese medicinal herb grown widely in China. In the present work, we studied cultivar variations of Australian-grown Danshen in order to select optimal cultivars for local herbal production. Root yields of seven cultivars, V1-V7, were monitored in a one-year field trial, and bioactive markers, including cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B, were quantitatively determined using a validated RP-HPLC method. Significant variations were found in root yields, root production efficiencies, and contents of the bioactive marker compounds. Linear correlations were observed among the contents of three tanshinones but not among those of tanshinones and salvianolic acid B. Among the cultivars, V6 was the best cultivar for production of tanshinones, and V4 and V5 were best for production of salvianolic acid B. The findings indicate that it is possible to achieve optimal root yields, and high contents of tanshinones and salvianolic acid B by selecting specific Danshen cultivars.

Topics: Abietanes; Australia; Benzofurans; Chromatography, High Pressure Liquid; Medicine, Chinese Traditional; Phenanthrenes; Plant Roots; Salvia miltiorrhiza

Water-soluble salvianolic acid A (Sal A) and salvianolic acid B (Sal B) were successfully isolated and purified from the crude extract of Salvia miltiorrhiza by high-speed counter-current chromatography (HSCCC). The solvent system was n-hexane-ethyl acetate-methanol-water (3:6:6:10, v/v/v/v). 4.27 mg of Sal A and 32.09 mg of Sal B were obtained from 260 mg of the crude sample. The purities of Sal A and Sal B were 96.67% and 97.43%, respectively. Their structures were identified by (1)H NMR and (13)C NMR. Antioxidant activities of Sal A and Sal B were also evaluated and compared by the methods of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS(+)) radical cation decolourisation assay. Both Sal A and Sal B showed high radical scavenging activities with their EC(50) values being 1.43+/-0.09 and 1.81+/-0.01 microg/ml in DPPH radical method. The ABTS results showed that Sal A and Sal B exhibited high total antioxidant activities, their EC(50) values were 1.35+/-0.00 and 1.43+/-0.01 microg/ml, respectively.

Topics: Benzofurans; Caffeic Acids; Countercurrent Distribution; Free Radical Scavengers; Lactates; Salvia miltiorrhiza

A large-scale purification of salvianolic acid B from Salvia miltiorrhiza Bunge is presented. The method development began with selection of the solvent system, then optimization of the operating parameters and ended up with linear scale-up from an analytical to a preparative instrument. Three factors were used for method optimization and scale-up estimation: purity, process throughput and process efficiency. Preparation was achieved using a two-phase solvent system comprising hexane-ethyl acetate-methanol-acetic acid-water (1:5:1.5:0.00596:5, v/v). This preparation yielded 475 mg of salvianolic acid B with a purity of 96.1% from 1.5 g of crude extract. The process throughput of crude was 2.23 g/h while process efficiency per gram of target compound was 0.769 g/h. Two factors-process environmental risk factor and process evaluation factor were used for evaluation of the separation process.

Topics: Benzofurans; Countercurrent Distribution; Drugs, Chinese Herbal; Salvia miltiorrhiza

Salvianolic acid B was separated and purified from Salvia miltiorrhiza Bunge (danshen) by microbial transformation together with chromatography of microsphere resin. The aqueous extract of danshen was transformed by Fusarium graminearum in a bioreactor containing phosphate buffer (PBS), in which rosmarinic acid was transformed into danshensu and caffeic acid and the yield of salvianolic acid B was higher than 85%. After biotransformation, salvianolic acid B was purified by microsphere resin. A parallel test for making a comparison of microsphere resin chromatography between elution by methanol water solution and water was done. The purity of salvianolic acid B was up to 95% at the yield of 62% when impurities and salvianolic acid B were eluted by 45% and 55% methanol solution respectively. The purity of salvianolic acid B was up to 99% at the yield of 90% when distilled water was used to elute the impurities and salvianolic acid B. The total yield of salvianolic acid B was up to 75% at the purity over 99% while biotransformation combined with microsphere resin chromatography by water elution. Microbial biotransformation together with water elution of microsphere resin supplied an efficient method to eliminate the micromolecular impurities and a possible method to purify water-soluble compounds in traditional Chinese medicine.

Topics: Adsorption; Benzofurans; Biotransformation; Chromatography, Liquid; Drugs, Chinese Herbal; Fusarium; Phenanthrolines; Plant Roots; Resins, Synthetic; Salvia miltiorrhiza

To observe the contraction effect of endothelin-1 (ET-1) on human hepatic stellate cells (HSCs) and the inhibition of salianic-acid B (SA-B) on ET-1, to explore the acting link and the possible mechanism.. HSC were isolated from human normal liver tissue by enzyme digestion and Nycondenz density gradient centrifugation. The contraction of ET-1 on passage HSCs and the intervention of SA-B with three doses (low-, middle-, and high-) on the contraction were observed by collagen gel contraction. ET-1 and SA-B were directly added to the serum-free medium of HSCs, then calcium ion concentration was detected by laser scanning confocal microscope.. Collagen gel contraction experiments showed that ET-1 could induce the contraction of HSC directly (P < 0.01). Three doses of SA-B significantly inhibited the contraction effects of ET-1 on HSCs (all P < 0.01). After adding the ET-1, HSCs morphology changed obviously with the number of cells decreased. However, SA-B inhibited the changes. Laser scanning confocal microscope experiments revealed that ET-1 stimulated the transiently rapid increase of intracellular calcium ion concentration, and the effects was obviously inhibited when SA-B was added.. SA-B could inhibit the contraction of HSCs induced by ET-1, and its mechanism might be related to the lowing of free calcium ion concentration in HSCs. This anti-contraction effect of SA-B is perhaps one of the mechanisms of its anti-fibrosis and anti-portal hypertension effects.

Topics: Benzofurans; Cells, Cultured; Endothelin-1; Hepatic Stellate Cells; Humans; Isometric Contraction

Seasonal variations in contents of bioactive markers in Australian-grown Salvia miltiorrhiza roots were investigated in a two-year field trial. Cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B were quantitatively determined by reversed-phase (RP) HPLC. Similar accumulation patterns were observed for the three tanshinones throughout the trial period, although roots harvested in the first year was found to contain relatively higher contents of these compounds. In contrast, the content of salvianolic acid B was peaked at 250 days after planting in the first year, and subsequently maintained at a plateau level in the second-year period. Linear correlations between the contents of individual tanshinones were observed, but not between those of tanshinones and salvianolic acid B. The findings suggest that tanshinones and salvianolic acid B have different accumulation patterns in Australian-grown Salvia miltiorrhiza roots, which should be critically considered for optimum harvesting of the roots for pharmaceutical applications.

Topics: Abietanes; Australia; Benzofurans; Biomarkers; Chromatography, High Pressure Liquid; Phenanthrenes; Plant Roots; Salvia miltiorrhiza; Seasons

3,4-dihydroxy-phenyl lactic acid (DLA) and salvianolic acid B (SAB) are two major water-soluble components of Salvia miltiorrhiza (SM). Previous works have revealed the ability of DLA and SAB to scavenge oxygen free radicals, inhibiting the expression of adhesion molecules CD11b/CD18 in neutrophil. Cardiotonic pills (CP), which is a traditional Chinese medicine compound preparation containing DLA and SAB, was found to inhibit venular thrombosis induced by photochemical reaction (PR) in rat mesentery. The present study addressed the effect of DLA and SAB on PR-induced thrombosis in rat mesentery by utilizing a microcirculation dynamic viewing system. The result demonstrated that both DLA and SAB delayed thrombus-initiation time, while DLA also prolonged thrombus half-size time. The experiments explored the mechanism underlying that the dihydrorhodamine 123 (DHR) fluorescence in the mesenteric venular walls after PR challenge was diminished by pretreatment with either DLA or SAB, the expression of CD18 in neutrophils elicited by PR was depressed by administration of DLA, while mast cell degranulation in rat mesentery induced by PR was damped by SAB. The antioxidant potential of the two substances is likely to be responsible for their most beneficial effects on thrombosis, through either directly scavenging the peroxides produced and/or indirectly depressing the expression of adhesion molecules in neutrophil.

Topics: Animals; Antioxidants; Benzofurans; Camphanes; CD18 Antigens; Drugs, Chinese Herbal; Hematoporphyrin Photoradiation; Lactates; Male; Mast Cells; Mesenteric Vascular Occlusion; Mesenteric Veins; Neutrophils; Panax notoginseng; Rats; Salvia miltiorrhiza; Venous Thrombosis

Epithelial-mesenchymal transition (EMT) is an important mechanism in kidney fibrosis. While Salvianolic acid-B (Sal B) has been well appreciated to show a protective effect of tissue fibrosis, the objective of this study was to investigate the influence of Sal B on the transdifferentiation of renal tubular epithelial cells. Human kidney proximal tubular cell line (HK-2) was used as the proximal tubular cell model and EMT was induced with 5 ng/ml of human transforming growth factor (TGF)-beta1. The effects of the Sal B on cell morphology were observed by phase contrast microscopy, and the possible mechanisms were studied by immunocytochemistry and real-time reverse transcription-polymerase chain reaction. Our results revealed that Sal B could inhibit TGF-beta1-induced myofibroblast phenotype and restored the epithelial morphology in a dose-dependent manner. It was partially through modulating alpha-smooth muscle actin (SMA) increase and E-cadherin reduction. These observations strongly suggest that Sal B is a potent inhibitor of TGF-beta1-induced EMT and might be a promising agent for treating tubulointerstitial fibrosis.

Topics: Benzofurans; Cell Line; Cell Transdifferentiation; Epithelial Cells; Fibrosis; Humans; Immunohistochemistry; Kidney Tubules, Proximal; Mesoderm; Nephritis, Interstitial; Protective Agents; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta1

In this Letter, we report the natural products salvianolic acid A, salvianolic acid B, and caftaric acid as inhibitors of the protein-protein interactions mediated by the SH2 domains of the Src-family kinases Src and Lck, two established disease targets. Moreover, we propose a binding mode for the inhibitors based on molecular modeling, which will facilitate chemical optimization efforts of these important lead structures for drug discovery.

Topics: Benzofurans; Biological Products; Caffeic Acids; Drug Discovery; Humans; Lactates; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Models, Molecular; Phenols; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins pp60(c-src); src Homology Domains; src-Family Kinases

Acute myocardial infarction (AMI) remains the leading cause of mortality in the world. Early intervention using salvianolic acids (SA) can substantially improve clinical outcomes. However, in spite of the great achievements that have been made in elucidating the protective effects of SA on AMI, the effects of SA on the contractile performance of the left ventricle (LV) and the underlying mechanism are still not so clear. In the present study, AMI was introduced by ligation of the left anterior descending coronary artery near the main pulmonary artery. Administration of SA significantly decreased infarct size, improved LV function and appearance of the myocardium and decreased myocardial malondialdehyde levels compared with the AMI group. Furthermore, treatment with SA significantly downregulated the mRNA expression level and activity of matrix metalloproteinase-9 (MMP-9), but did not regulate the tissue inhibitor of metalloproteinase-1 (TIMP-1) expression level at the infarct area. Lisinopril (an angiotensin converting enzyme inhibitor), which holds potential effects on cardioprotection, was chosen as the positive control in this study. Lisinopril elevated LV function and appearance of the myocardium, decreased malondialdehyde levels without an influence on infarct size, and regulated the MMP-9 enzyme level but not the MMP-9 mRNA and TIMP-1 protein levels. These findings suggest that early SA treatment is effective to improve LV function; and SA may exert preventative effects against myocardial remodeling after infarction.

Topics: Animals; Benzofurans; Caffeic Acids; Cardiotonic Agents; Down-Regulation; Lactates; Lisinopril; Male; Malondialdehyde; Matrix Metalloproteinase 9; Myocardial Contraction; Myocardial Infarction; Plant Extracts; Rats; Rats, Wistar; RNA, Messenger; Salvia miltiorrhiza; Tissue Inhibitor of Metalloproteinase-1; Ventricular Remodeling

A sensitive and specific liquid chromatographic-electrospray ionization mass spectrometric method was developed for quantification of salvianolic acid B in rat plasma with resveratrol as the internal standard. The analytes were separated on a reversed-phase column with acetonitrile (40%) and water (60%) containing 0.75% formic acid as mobile phase at a flow rate of 1 mL/min. Liquid-liquid extraction was adopted for the sample preparation, and the analytes were determined using electrospray negative ionization mass spectrometry in the selective monitoring mode. The method was validated over the concentration range 0.1-40 microg/mL using 0.1 mL of plasma with coefficients of correlation >0.999. The intra- and inter-day precisions of analysis were <10%, and accuracy ranged from 94 to 101%. This method was successfully applied to a pharmacokinetics of salvianolic acid B in rats.

Topics: Animals; Area Under Curve; Benzofurans; Chromatography, Liquid; Drug Stability; Least-Squares Analysis; Male; Plant Roots; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Salvia miltiorrhiza; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Temperature

Salvianolic acid B (SalB) is an active component isolated from Chinese herbal medicine Salvia miltiorrhiza. The aim of this study was to investigate the extent of absolute oral bioavailability (F) of SalB in beagle dogs and the effect on blood viscosity after intravenous and oral administration of Salvianolic acids (SAs). A gradient elution HPLC method was developed and validated to determine the concentration of SalB and its three possible metabolites in plasma. After SAs (180 mg/kg, p.o.; 9 mg/kg, i.v.) were given, the AUCs of SalB were 1680 +/- 670 and 7840 +/- 1140 ng/mL.h, respectively. The F of SalB in dogs was calculated to be only 1.07 +/- 0.43%. The blood viscosity was remarkably decreased after a single intravenous injection of SAs (9 mg/kg). However, no significant change of blood viscosity was observed after a single oral administration of SAs (180 mg/kg). The results suggested that the F of SalB was extremely low and single oral administrated SAs had no effect on ameliorating blood viscosity in beagle dogs.

Topics: Administration, Oral; Animals; Area Under Curve; Benzofurans; Biological Availability; Biotransformation; Blood Viscosity; Chromatography, High Pressure Liquid; Dogs; Drugs, Chinese Herbal; Hemorheology; Injections, Intravenous; Male; Reproducibility of Results; Spectrophotometry, Ultraviolet

SMND-309, a novel compound named (2E)-2-{6-[(E)-2-carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid, is a new derivate of salvianolic acid B. The present study was conducted to investigate whether SMND-309 has a protective effect on brain injury after focal cerebral ischemia, and if it did so, to investigate its effects on brain mitochondria. Adult male SD rats were subjected to middle cerebral artery occlusion (MCAO) by bipolar electro-coagulation. Behavioral tests and brain patho-physiological tests were used to evaluate the damage to central nervous system. Origin targets including mitochondria production of reactive oxygen species, antioxidant potentia, membrane potential, energy metabolism, mitochondrial respiratory enzymes activities and mitochondria swelling degree were evaluated. The results showed that SMND-309 decreased neurological deficit scores, reduced the number of dead hippocampal neuronal cells in accordance with its depression on mitochondria swelling degree, reactive oxygen species production, improvements on mitochondria swelling, energy metabolism, membrane potential level and mitochondrial respiratory chain complex activities. All of these findings indicate that SMND-309 exerted potent neuroprotective effects in the model of permanent cerebral ischemia, contributed to its protections on brain mitochondrial structure and function.

Topics: Animals; Antioxidants; Benzofurans; Brain; Brain Ischemia; Caffeic Acids; Male; Mitochondria; Neuroprotective Agents; Phytotherapy; Plant Extracts; Plants, Medicinal; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza

The current study aims to investigate the effect of sodium caprate on the intestinal absorption and bioavailabilities of danshensu and salvianolic acid B, the major active components in Salvia miltiorrhiza Bge (Danshen). Biopharmaceutics and pharmacokinetics properties of the two compounds have been characterized by in vitro, in situ models as well as in vivo in rats. Based on the identified biopharmaceutics characteristics of the two compounds, effect of sodium carparate as absorption enhancer on the intestinal absorption and pharmacokinetics of danshensu and salvianolic acid B in pure compound form as well as extract form were investigated both in vitro and in vivo. Both danshensu and salvianolic acid B demonstrated very limited intestinal permeabilities, leading to oral bioavailabilities of only 11.09% and 3.90% in rats, respectively. Results from both in vitro and in vivo studies consistently indicated that sodium caprate could significantly enhance intestinal permeabilities as well as the in vivo bioavailabilities of both danshensu and salvianolic acid B. The current findings not only identified the usefulness of sodium caprate for the improved delivery of Danshen product but also demonstrated the importance of biopharmaceutics characterization in the dosage form development of traditional Chinese medicine.

Topics: Administration, Oral; Animals; Benzofurans; Biological Availability; Caco-2 Cells; Decanoic Acids; Excipients; Humans; Intestinal Absorption; Lactates; Male; Permeability; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza

An optimized microwave-assisted extraction method using water (MAE-W) as the extractant and an efficient HPLC analysis method were first developed for the fast extraction and simultaneous determination of D(+)-(3,4-dihydroxyphenyl) lactic acid (Dla), salvianolic acid B (SaB), and lithospermic acid (La) in radix Salviae Miltiorrhizae. The key parameters of MAE-W were optimized. It was found that the degradation of SaB was inhibited when using the optimized MAE-W and the stable content of Dla, La, and SaB in danshen was obtained. Furthermore, compared to the conventional extraction methods, the proposed MAE-W is a more rapid method with higher yield and lower solvent consumption with a reproducibility (RSD <6%). In addition, using water as extractant is safe and helpful for environment protection, which could be referred to as green extraction. The separation and quantitative determination of the three compounds was carried out by a developed reverse-phase high-performance liquid chromatographic (RP-HPLC) method with UV detection. Highly efficient separation was obtained using gradient solvent system. The optimized HPLC analysis method was validated to have specificity, linearity, precision, and accuracy. The results indicated that MAE-W followed by HPLC-UV determination is an appropriate alternative to previously proposed method for quality control of radix Salviae Miltiorrhizae.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Depsides; Lactates; Microwaves; Molecular Structure; Particle Size; Reproducibility of Results; Salvia miltiorrhiza; Temperature; Time Factors; Water

Salvia miltiorrhiza f. alba (Baihua Danshen) is a Chinese medicinal herb commonly used for treating cardiovascular disease. It has been grown in Australia, although the quality of its main medicinal part (dried root) has not been assessed. In this study, we investigated HPLC profiles and biomarker contents of Australian-grown S. miltiorrhiza f. alba roots. Patterns of HPLC profiles were established in MeOH, and aqueous extracts in terms of number of common characteristic peaks and their relative retention times. The contents of three tanshinone biomarkers (cryptotanshinone (3), tanshinone I (1), and tanshinone IIA (2)) were significantly higher (p<0.05) in the roots of one-year-old plants than those of two-year-old plants. In contrast, salvianolic acid B (4) content was significantly higher in the roots of two-year-old plants than in those of one-year-old plants. The findings suggest that the biomarker contents in Australian-grown S. miltiorrhiza f. alba roots vary with the growth periods of the plants, which may be important in determining the optimal harvest time for the plant roots with targeted levels of tanshinones and salvianolic acid B (4).

Topics: Abietanes; Australia; Benzofurans; Biomarkers; Chromatography, High Pressure Liquid; Phenanthrenes; Plant Roots; Plants, Medicinal; Salvia miltiorrhiza

Inefficient cardiomyocyte differentiation limits the therapeutic use of embryonic stem (ES) cell-derived cardiomyocytes. While large collections of proprietary chemicals had been screened to improve ES cell differentiation into cardiomyocytes, the natural product library remained unexplored. Using a mouse ES cell line transfected with a cardiomyocyte-specific alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein (EGFP) reporter, we screened 24 natural products with known cardioprotective actions. Salvianolic acid B (saB), while produced minimal effect on its own, concentration-dependently synergized with vitamin C in inducing cardiomyocyte differentiation, as demonstrated by an increase in EGFP(+) cells, beating area in embryoid bodies, and expression of cardiomyocyte maturity markers. This synergy is specific to cardiomyocyte differentiation, and is involved with collagen synthesis. The present study demonstrates the saB-vitamin C synergy in inducing ES cell differentiation into matured and functional cardiomyocytes, and this may lead to a practicable cocktail approach to generate ES cell-derived cardiomyocytes for cardiac stem cell therapy.

Topics: Animals; Ascorbic Acid; Benzofurans; Cardiotonic Agents; Cell Differentiation; Cell Line; Collagen; Drug Evaluation, Preclinical; Drug Synergism; Embryonic Stem Cells; Mice; Myocytes, Cardiac; Vitamins

To investigate the effects of Salvianolic acid B preconditioned endothelial progenitor cells (EPCs) on the Nkx2.5 and GATA-4 gene expressions at the early stage of cell differentiation of bone mesenchymal stem cells (BMSc) transplanted into infarcted myocardium, in order to find out the best synergism for co-transplantation of the two kinds of cells.. BMSc and EPCs of rats were isolated and cultured, and rats were modeled into acute myocardial infarction (AMI) by left coronary artery ligation. Then the EPCs preconditioned with different concentrations of Salvianolic acid B and BMSc or DMEM medium were implanted into heart ischemia area. Expressions of Nkx2.5 and GATA-4 mRNA expressions in myocardium were detected by Real-time RT-PCR 4 weeks later.. Compared with those in the non-implanted model rats' myocardium, the gene expression of Nkx2.5 and GATA-4 mRNA were significantly higher in all the transplantation receptive groups, comparisons between the implanted groups showed that the highest value of expressions (2. 654 +/- 0.606 of Nkx2.5 and 1.573 +/- 0.372 of GATA-4) displayed in the group contained more EPCs, for 8-fold to BMSc in volume.. BMSc can differentiate into cardiac muscle like cells, and condition of their differentiation is related with the degree of the internal environment improved.

Topics: Animals; Benzofurans; Cells, Cultured; Endothelial Cells; Gene Expression; Male; Mesenchymal Stem Cell Transplantation; Myocardial Infarction; Myocardium; Random Allocation; Rats; Rats, Wistar; Stem Cell Transplantation; Stem Cells

The removal of the colloidal impurities in the purification of salvianolic acid B (SB) was carried out on a macroporous adsorption resin column with a modified elution method on the principle of combining frontal chromatography with displacement chromatography. Two serially connected columns were packed with macroporous adsorption resin in a certain proportion. The latter column was eluted with 50% methanol, when the colloidal impurities reached saturation adsorption in the former column. Comparative study suggested that the SB product was superior to that from the routine method with the weighted average purity risen from 59.6% to 64.5%, and the recovery from 69.75% to 80.0%. The ethanol concentration was lowered in the eluent. The method is simple and can be used for the production of SB in large scale.

Topics: Adsorption; Benzofurans; Chromatography; Colloids; Drugs, Chinese Herbal; Phenanthrolines; Porosity; Resins, Synthetic; Salvia miltiorrhiza; Technology, Pharmaceutical

To establish capillary electrophoresis fingerprint (CEFP) of Compound Danshen Dropping Pill (CDDP) by capillary zone electrophoresis, the electrophoretic separation was performed by using a 75 cm x 75 microm (the effective length of 63 cm) uncoated fused silica capillary with 50 mmol/L sodium borate and 200 mmol/L boric acid (1:1, v/v) containing 1.1% triethylamine as the background electrolyte. The running voltage was 18 kV while the detection wavelength was set at 290 nm. The CEFPs were produced by the electropherograms from 10 batches of CDDP and the 8 co-possessing peaks were selected as the fingerprint peaks of CDDP's CEFP by choosing protocatechualdehyde peak as the referential peak. The quality of CDDP was evaluated by systematic quantified fingerprint method through assessing the CEFP. Ten batches of CDDP were classified by systematic quantified fingerprint method. Among the 10 batches of CDDP, the contents of 1 batch were obviously lower while the others were almost similar. The CEFPs of CDDP were established with good precision and reproducibility, which can be served as a novel reference to identify and control the quality of CDDP.

Topics: Benzaldehydes; Benzofurans; Catechols; Drug Compounding; Electrophoresis, Capillary; Quality Control; Salvia miltiorrhiza; Tablets

A sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was applied for the quantification of each component: tetrahydropalmatine (THP), dehydrocorydaline (DHC), salvianolic acid B (SAB), ginsenoside Rg1, Re, Rb1 and Rd in the Chinese herbal component SSTG (Shuangshentongguan) with different combinations. The pharmacokinetic data were analyzed with WinNonlin 5.2 software. The results showed that combination can increase the THP AUC value while the AUC values of SAB, ginsenoside Rg1, Re, Rb1 and Rd were reduced. These results showed significant differences. The AUC value of ginsenoside Rb1 was increased when combined with Danshen or Yanhusuo, but reduced when combined with Danshen and Yanhusuo. The DHC concentration in serum was too low to be determined.

Topics: Benzofurans; Drug Combinations; Drugs, Chinese Herbal; Ginsenosides; Phenanthrolines; Salvia miltiorrhiza

To investigate the effect of salvianolic acid B (SA-B) on renal interstitial fibrosis due to unilateral ureteral obstruction.. Thirty-six SD male rats were randomly divided into 3 groups, 12 in each group, the sham-operated group, the model group and the SA-B treated group. The rat model of renal interstitial fibrosis was successfully established by unilateral ureteral obstruction (UUO). Rats in the SA-B treated group was intragastrically administrated with SA-B (12.5 mg x kg(-1)) daily after modeling. Rats of each group were killed respectively at day 14 and day 21 after UUO. Pathological changes of renal tissue were observed by hematoxylin and eosin (HE) staining. The expression of alpha-smooth muscle actin (alpha-SMA) in kidney was determined with immunohistochemistry. And the expressions of cytokeratinl9 (ck19) mRNA in renal tissue were detected using reverse transcription polymerase chain reaction (RT-PCR).. Renal interstitial fibrosis was obviously ameliorate in SA-B treated group. The expression of alpha-SMA was significantly decreased in SA-B treated group as compared with that in model group at day 14. And the expression of ck19 was significantly lower than that determined in model group at day 21.. SA-B could ameliorate renal interstial fibrosis due to UUO, probable by inhibiting epithelial-to-myofibroblast transdifferentiation and the activation of myofibroblast.

Topics: Animals; Benzofurans; Disease Models, Animal; Drugs, Chinese Herbal; Fibroblasts; Fibrosis; Gene Expression; Humans; Keratin-19; Kidney Diseases; Male; Muscle, Smooth; Random Allocation; Rats; Rats, Sprague-Dawley

To establish a new method using AOTF-Near infrared spectroscopy (NIRS) for quick determination of tanshinone II(A) and salvianolic acid B in Fufang Danshen tablets.. HPLC was used as a reference method to determine the contents of tanshinone II(A) and salvianolic acid B in Fu Fang Dan Shen tablets. Multivariate calibration models based on PLS1 algorithm were developed to correlate the spectra and the corresponding values determined by the reference methods.. RMSECV (root-mean-square error of cross-validation) of the models for tanshinone II(A) and salvianolic acid B were 0.0103 and 0.1868 respectively. The correlation coefficients of the calibration models were 0.9873 and 0.9832 respectively. External validation with external validation samples proved that the relative coefficient of the predicted value and the truth value were R2 = 0.9743 and R2 = 0.9886 respectively, with measured recycle rates of 103.0% and 99.0%.. NIRS can be used in the determination of tanshinone II(A) and salvianolic acid B, which sets up the foundation of product-line control of Fuang Danshen tablets.

Topics: Abietanes; Benzofurans; Drugs, Chinese Herbal; Phenanthrenes; Reproducibility of Results; Spectroscopy, Near-Infrared; Tablets

An increased propensity towards cardiac arrhythmias and aggravated heart function is observed in myocardial infarction (MI), the development of which is associated with the calcium handling system in the myocardium. It was hypothesized that the abnormal changes in the MI model may be a consequence of the abnormal expression and function of the RyR2-FKBP12.6 channel complex and that these abnormalities may be related to an over-activated endothelin (ET) system. Salvianolic acid B is expected to suppress life-threatening arrhythmias and to restore the abnormality of the RyR2-FKBP12.6 complex in rats. MI was produced by ligating the coronary artery for 4 weeks. Salvianolic acid B (100 mg/kg/day, p.o. for 4 weeks) was administered to rats 0.5 h before surgery. Measurements of cardiac arrhythmias, cardiac function, calcium transient, cardiac calcium release channel handling proteins and the endothelin system were conducted. The aggravated arrhythmia and compromised cardiac function in MI rats was accompanied by elevated diastolic Ca(2+) levels in the cytosol and a significant down-regulation of expression of RyR2-FKBP12.6. These were closely linked with an over-activated ET pathway in the myocardium. After a 4-week treatment with salvianolic acid B, all abnormalities were reversed significantly. Salvianolic acid B was capable of normalizing FKBP12.6 expression levels and decreasing the propensity towards arrhythmias by attenuating the up-regulated ET pathway.

Topics: Animals; Arrhythmias, Cardiac; Benzofurans; Calcium; Disease Models, Animal; Drugs, Chinese Herbal; Gene Expression; Hemodynamics; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Receptors, Endothelin; Ryanodine Receptor Calcium Release Channel; Signal Transduction; Tacrolimus Binding Proteins

Metabolism and pharmacokinetic studies on rat were conducted for lithospermic acid B, one of the components from Radix Salviae Miltiorrhizae (danshen) that shows many bioactivities. Liquid chromatography-electrospray ionization mass spectrometry method was applied for the determination of lithospermic acid B and its metabolites in samples from in vitro and in vivo metabolism studies. Rat plasma samples collected after intravenous administration were analyzed for obtaining pharmacokinetic data of lithospermic acid B. Four O-methylated metabolites, namely one monomethyl-, two dimethyl- and one trimethyl-lithospermic acid B, were detected when lithospermic acid B was incubated in rat hepatic cytosol. These four metabolites were also detected in rat bile, plasma and feces samples after intravenous administration of lithospermic acid B. The in vitro and in vivo results indicate that the methylation is the main metabolic pathway of lithospermic acid B. The danshen component and its methylated metabolites were excreted to rat bile and feces.

Topics: Animals; Benzofurans; Bile; Chromatography, High Pressure Liquid; Depsides; Feces; Injections, Intravenous; Mass Spectrometry; Models, Animal; Plant Extracts; Rats; Salvia miltiorrhiza; Sensitivity and Specificity; Tandem Mass Spectrometry

Salvianolic acid B (Sal B) is one of the major water-soluble compounds isolated from Radix Salviae Miltiorrhizae (Danshen in Chinese) that has been reported to be beneficial to treatment of diabetic complications. However, the mechanisms involved in these effects are not discussed in relation to mesangial proliferation via modulation of NF-kappaB. To explain this, human mesangial cells were pretreated with or without Sal B (0.1, 1, 10 microM) for 24 h and stimulated with high glucose (30 mM). Then the effects of Sal B on mesangial cells proliferation, extracellular matrix production and the possible mechanisms were evaluated by methylthiazoletetrazolium assay, flow cytometry assay, enzyme-linked immunosorbent assay, gelatin zymography assay and western blot assay. These results indicated that Sal B could inhibit high glucose-induced mesangial cells proliferation and extracellular matrix production in a dose-dependent manner, partially through modulating the cell-cycle progress and MMP-2 and MMP-9 activities via suppressing NF-kappaB activation, suggesting that Sal B may be a promising agent for treating diabetic nephropathy.

Topics: Antioxidants; Benzofurans; Blotting, Western; Cell Cycle; Cell Nucleus; Cell Proliferation; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Fibronectins; Flow Cytometry; Glucose; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mesangial Cells; NF-kappa B; Signal Transduction; Tetrazolium Salts; Thiazoles

Salvianolic acid B (SAB) is a component of Danshen, a herb widely used in Chinese medicine, and was previously shown to exert a number of biological activities including inhibition of platelet function, but the exact mechanisms involved are unclear. SAB dose-dependently inhibited platelet deposition from flowing, anticoagulated whole blood to immobilized collagen at both venous and arterial shear rate, whereas platelet deposition to immobilized fibrinogen was not affected. The inhibitory effect of SAB on platelet adhesion to collagen was independent of alphaIIbbeta3, since SAB still inhibited platelet deposition in the presence of a alphaIIbbeta3-blocking peptide. SAB inhibited static platelet adhesion to a synthetic peptide specific for the collagen receptor alpha2beta1, whereas platelet adhesion to a glycoprotein VI-specific peptide was not affected. SAB inhibited binding of an antibody against alpha2beta1 to platelets as studied by flow cytometry, and inhibited the interaction of soluble alpha2beta1 to immobilized collagen in a solid phase binding assay. These combined results indicate that SAB inhibits platelet adhesion to immobilized collagen by interfering with the collagen receptor alpha2beta1.

Topics: Benzofurans; Cell Adhesion; Hemorheology; Integrin alpha2beta1; Plant Roots; Platelet Adhesiveness; Protein Binding; Receptors, Collagen; Salvia; Solubility; Stress, Mechanical

To study the adsorption of the macroporous resin for the five salvianolic acids (danshen su, rosmarinic acid, protocate chualdehyde, salvianolic acid B, salvianolic acid A, extracted from Salviae mitiorrhizae.. The five salvianolic acids were employed as an index, and the change of them in the static and dynamic absorbent was detected by HPLC, respectively.. HP20 resin was a suitable marcoporous resin to purify salvianolic acids. The dynamic adsorption capacity of rosmarinic acid, salvianolic acid B and salvianolic acid A was 30.506 mg x g(-1) (dry resin), 36.996 mg x g(-1), (dry resin), 43.85 mg x g(-1) (dry resin) respectively.. It is not suitable that danshensu and protocate chualdehyde are the evaluation indexes for using 8 macroporous resins to purify salvianolic acids.

Topics: Adsorption; Benzofurans; Caffeic Acids; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Lactates; Plant Extracts; Resins, Synthetic; Rosmarinic Acid; Salvia miltiorrhiza

With a simple and feasible method, a salvianolic acid B-phospholipid complex was prepared to increase the absorption of salvianolic acid B in the gastrointestinal (GI) tract. Because the solubility of the complex in water was very poor, the complex was encapsulated into nanoparticles to facilitate its administration. The physicochemical properties of the complex were investigated by differential scanning calorimetry (DSC), infrared scanning, ultraviolet scanning and X-ray diffraction (XRD), and the solubility of salvianolic acid B and the complex in water or n-octanol was measured. The pharmacokinetic characteristics and bioavailability were compared after oral administration of salvianolic acid B (500 mg/kg) and the complex nanoparticles (450 mg/kg equivalent to salvianolic acid B). The results proved the drastic decrease in the solubility of salvianolic acid B in water after successful formation of the drug-phospholipid complex. After oral administration the peak plasma concentration (Cmax) of salvianolic acid B given by the complex nanoparticles was 3.4 microg/ml which was much higher than that of salvianolic acid B sample (Cmax = 0.9 microg/ml), with Tmax of 75 min, 45 min and AUC of 664, 257 microg/ml min, respectively. The relative bioavailability (F) reached 286%.

Topics: 1-Octanol; Animals; Antioxidants; Area Under Curve; Benzofurans; Biological Availability; Calibration; Calorimetry, Differential Scanning; Chromatography, High Pressure Liquid; Drug Carriers; Drug Compounding; Male; Nanoparticles; Phospholipids; Rats; Rats, Sprague-Dawley; Solubility; Solvents; Spectrophotometry, Infrared; X-Ray Diffraction

To establish a new quality evaluation method for traditional Chinese medicine preparations, using one chemical reference substance to calcutate multi-components simultaneously.. Employed puerarin as the maker component, puerarin relative correction factors (RCF) of salvianolic acid B to puerarin and paidzein to puerarin were calcatated in the chromatographic conditions for determination of the three components in Guanmaikang capsules. The contents of Puerarin were determined by external standard method, and those of salvianolic acid B and paidzein were calculated by puerarin and their RCF. The accuracy of the new method was evaluated by comparing the calculated contents with the determined.. The analysis methods were established, and it has been no significant difference between the calculated contents and determined contents.. The method can control the components without providing salvianolic acid B and paidzein reference. It is to be a suitable quality evaluation pattern for TCM Preparation.

Topics: Benzofurans; Drugs, Chinese Herbal; Isoflavones; Medicine, Chinese Traditional; Reference Standards

To investigate the effects of salvianolic acid B (SA-B) on cardiovascular endothelial cell function and platelet activation during myocardial ischemia-reperfusion in rabbits.. A total of 24 New Zealand white rabbits were randomly divided into sham-operated group, ischemia-reperfusion group (untreated group) and SA-B group. The hearts of rabbits in untreated group and SA-B group underwent half an hour of left anterior descending coronary artery (LADCA) occlusion via ligation technology, which was followed by 4 hours of reperfusion to prepared ischemia-reperfusion injury model in vivo. For sham-operated group, the animals were not subjected to occlusion of LADCA. In SA-B treatment group the rabbits were intravenously administered SA-B immediately after LADCA occlusion, and the other two groups were given normal saline in the same way instead of SA-B. The jugular vein bloods of animals were collected before LADCA ligation, half an hour after ligation and after 1-, 4-hour reperfusion, respectively. The content of plasma nitric oxide (NO) was determined by nitrate reductase process. Radioimmunoassay was applied to detect the endothelin (ET) content in plasma and the count of alpha-granule membrane protein-140 (GMP-140) on platelet surface to identify the activation of the platelet.. No significant difference was observed before and after sham LADCA occlusion in sham-operated group in the contents of NO and ET in plasma (P>0.05), neither was the count of GMP-140 on platelet surface (P>0.05). The content of NO in plasma detected 0.5 h after LADCA occlusion was significantly decreased in untreated group compared with the sham-operated group at the corresponding time, and they were also much lower than that before LADCA occlusion in the sham-operated group (P<0.05). The plasma content of NO in untreated group showed a progressive decrease in response to the myocardial reperfusion. However, the content of ET in plasma and the count of GMP-140 on platelet surface were remarkably increased after myocardial ischemia as compared with those before LADCA ligation and those detected in sham-operated group (P<0.05). The content of ET and the count of GMP-140 in the untreated group were further increased corresponding to the aggressive reperfusion. The content of NO was significantly increased while the content of ET and the count of GMP-140 were both significantly decreased in SA-B group as compared with untreated group after 1- and 4-hour myocardial reperfusion, respectively (P<0.01).. The results show that endothelial dysfunction and platelet activation occur during ischemia-reperfusion in rabbit hearts in vivo and SA-B protects cardiovascular endothelium cells against ischemia-reperfusion injury and inhibits the activation of platelet during myocardial ischemia and reperfusion.

Topics: Animals; Benzofurans; Endothelial Cells; Endothelins; Endothelium, Vascular; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; P-Selectin; Platelet Activation; Rabbits

To investigate the feasibility of extracting the fat-soluble and the water-solubility substances from Salviae Miltiorrhizae with O/W Microemulsion.. With the yield of Tanshinone IIA and Salvianolic acid B as index, compere with efficiency of extracting Salviae Miltiorrhizae by suing the different media (water, alcohol and microemulsion), using different extraction methods (heat, microwave, ultrasonic), using different formulations of O/W microemulsion.. The extracting yield of Tanshinone IIA and Salvianolic acid B are more than 70% by using microemulsion at mean time. It is good of the abilities of extraction of liposolubility compounds such as the Tanshinone IIA by the microemulsion. The formulations of microemulsion have remarkable effect for extracting Salviae Miltiorrhizae.. It is feasibiale that extracted Salviae Miltiorrhizae by using O/W microemulsion as a solvents.

Topics: Abietanes; Benzofurans; Drugs, Chinese Herbal; Emulsions; Oils; Phenanthrenes; Reproducibility of Results; Salvia miltiorrhiza; Water

A sensitive liquid chromatography-mass spectrometric (LC-MS) method for the quantification of salvianolic acid B in rat plasma was developed and validated. The effect of different combination on the salvianolic acid B were studied to investigate SSTG'pharmacokinetics mechanism.. Different combination methods were used for oral administration. The compounds were extracted by solvent in rat plasma and determined using LC-MS-MS method. The results were analysis by DAS software.. Chromatographic separation was achieved on a reversed-phase SB-C18 column with the mobile phase of acetonitrile-methanolformic acid (0.1%) and step gradient elution resulted. The analytes were detected by using an electrospray negative ionization mass spectrometry in the MRM mode. The method showed excellent linearity over the concentration range 0.5-1000 microg x L(-1) (R2=0.999). The low limit of quantification was 0.5 microg x L(-1). The extract recoveries of analytes were from 30.88% to 38.18%. The precisions, the accuracy and the stability all meet the requirements for all analytes. The HPLC-MS-MS technique provided an excellent method for the quantification of SAB in rat plasma.. The method is simple, sensitive and rapid. The plasma concentration of Rg1 and Re were too low to determine. Ginseng & Yanhusuo and Ginseng & Danshen combination can decrease the AUC value and increase the Vd and CL.

Topics: Animals; Benzofurans; Chromatography, Liquid; Drugs, Chinese Herbal; Male; Rats; Rats, Wistar; Reproducibility of Results; Tandem Mass Spectrometry

Salvia miltiorrhiza is a Chinese medicine widely used for treatment of various cardiovascular diseases. However, little is known about the role of dihydroxylphenyl lactic acid (DLA) and salvianolic acid B (SAB), the main ingredients of S. miltiorrhiza, in the microcirculation. This study aimed to investigate the effect of DLA and SAB on LPS-elicited microcirculatory disturbance, focusing especially on leukocyte adhesion and its potential mechanism. Mesenteric venular diameter, velocity of red blood cells in venules, shear rate of the venular wall, numbers of leukocytes adherent to and emigrated across the venular wall, and mast cell degranulation were determined by an inverted microscope in rats after LPS infusion with or without DLA or SAB. Expression of CD11b and CD18 and production of superoxide anion (*O2-) and hydrogen peroxide (H2O2) by neutrophils were evaluated in vitro by flow cytometry. LPS exposure induced a significant increase in the number of adherent and emigrated leukocytes and mast cell degranulation, and a prominent decrease in the velocity of red blood cells in venules and shear rate of the venular wall. Additionally, in vitro experiments revealed an apparent enhancement in expression of CD11b and CD18 and production of *O2- and H2O2 by rat neutrophils by LPS stimulation. Treatment with DLA or SAB significantly ameliorated LPS-induced microcirculatory disturbance in rat mesentery and inhibited both the expression of CD11b and CD18 and the production of *O2- and H2O2 by neutrophils caused by LPS.

Topics: Animals; Benzofurans; CD11b Antigen; CD18 Antigens; Cell Adhesion; Flow Cytometry; Hydrogen Peroxide; Lactic Acid; Leukocytes; Lipopolysaccharides; Male; Mast Cells; Mesentery; Microcirculation; Molecular Structure; Neutrophils; Peroxides; Rats; Rats, Sprague-Dawley; Venules

Danshensu (3-(3,4-dihydroxyphenyl) lactic acid) and salvianolic acid B, two natural phenolic acids of caffeic acid derivatives isolated from Salvia miltiorrhiza root of the most widely used traditional Chinese medicine for the treatment of various cardiovascular diseases, have been reported to have potential protective effects from oxidative injury. To better understand their biological functions, the in vitro radical scavenging and antioxidant activities of danshensu and salvianolic acid B were evaluated along with vitamin C. Both danshensu and salvianolic acid B exhibited higher scavenging activities against free hydroxyl radicals (HO()), superoxide anion radicals (O(2)(-)), 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals and 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals than vitamin C. In contrary, danshensu and salvianolic acid B showed weaker iron chelating and hydrogen peroxide (H(2)O(2)) scavenging activities than vitamin C. As expressed as vitamin C equivalent capacity (VCEAC), the relative VCEAC values (mg/100ml) were in the order of salvianolic acid B (18.59) > danshensu (12.89) > vitamin C (10.00) by ABTS radical assay. The protective efficiencies against hydrogen peroxide induced human vein vascular endothelial cell damage were correlated with their antioxidant activities. Analysis of structure-activity relationship of these two compounds showed that the condensation and conjugation of danshensu and caffeic acid appears important for antioxidant activity. These results indicated that danshensu and salvianolic acid B are efficient radical scavengers and antioxidants, and salvianolic acid B is superior to danshensu. Their radical scavenging and antioxidant properties might have potential applications in food and healthcare industry.

Topics: Antioxidants; Ascorbic Acid; Benzofurans; Benzothiazoles; Biphenyl Compounds; Cell Survival; Chelating Agents; Endothelial Cells; Ferrous Compounds; Free Radical Scavengers; Humans; Hydrogen Peroxide; Hydroxyl Radical; Lactates; Oxidants; Picrates; Plant Roots; Salvia; Sulfonic Acids; Tetrazolium Salts; Thiazoles

Oxidative stress caused by dopamine may play an important role in the pathogenesis of Parkinson's disease. Salvianolic acid B is an antioxidant derived from the Chinese herb, Salvia miltiorrhiza. In this study, we investigated the neuroprotective effect of salvianolic acid B against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with salvianolic acid B significantly reduced 6-hydroxydopamine-induced generation of reactive oxygen species, and prevented 6-hydroxydopamine-induced increases in intracellular calcium. Our data demonstrated that 6-hydroxydopamine-induced apoptosis was reversed by salvianolic acid B treatment. Salvianolic acid B reduced the 6-hydroxydopamine-induced increase of caspase-3 activity, and reduced cytochrome C translocation into the cytosol from mitochondria. The 6-hydroxydopamine-induced decrease in the Bcl-x/Bax ratio was prevented by salvianolic acid B. Additionally, salvianolic acid B decreased the activation of extracellular signal-regulated kinase and induced the activation of 6-hydroxydopamine-suppressed protein kinase C. These results indicate that the protective function of salvianolic acid B is dependent upon its antioxidative potential. Our results strongly suggest that salvianolic acid B may be effective in treating neurodegenerative diseases associated with oxidative stress.

Topics: Adrenergic Agents; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Benzofurans; Calcium; Caspase 3; Cell Line, Tumor; Cytochromes c; Drugs, Chinese Herbal; Humans; Neuroblastoma; Neuroprotective Agents; Oxidative Stress; Oxidopamine; Protein Kinase C; Reactive Oxygen Species; Salvia miltiorrhiza; Sympatholytics

In this study, we have investigated the actions of cryptotanshinone, an active, lipophilic component of the medicinal herb danshen (Salvia miltiorrhiza), on rat isolated coronary artery rings precontracted with 1 microM 5-hydroxytryptamine (5-HT) and its action compared to the ethanol-extractable fraction of the herb. Extraction of the ethanol-soluble fraction from danshen provided a yield of 1%. The amount of cryptotanshinone determined in this ethanol extract was 3.682%, and it was 6 times more potent than the extract in relaxing 5-HT-precontracted coronary artery rings; IC(50) values were 2.65+/-0.15 microg/ml and 15.82+/-1.07 microg/ml, respectively. Involvement of endothelium-dependant mechanisms in their dilator effects were investigated by pretreatment of the artery rings with a cyclooxygenase inhibitor flurbiprofen (10 microM), a nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), a muscarinic receptor antagonist atropine (100 nM), and by mechanical removal of the endothelium; none of these procedures produced a significant change on their dilator actions. Involvement of endothelium-independent mechanisms was investigated in endothelium-denuded artery rings pretreated with a beta-adrenoceptor antagonist propranolol (100 nM), an adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536, 100 microM), a guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), and a potassium channel inhibitor tetraethylammonium (TEA, 100 mM); these also produced no change on their dilator actions. The possible involvement of Ca(2+) channels was investigated in artery rings incubated with Ca(2+)-free buffer and primed with 1 microM 5-HT for 5 min prior to adding CaCl(2) to elicit contraction. The danshen ethanol extract (100 microg/ml) abolished the CaCl(2)-induced vasoconstriction, whereas, cryptotanshinone (30 microg/ml) produced 59% inhibition. These findings suggest their vasorelaxant effects are independent of pathways mediated by the endothelium, muscarinic receptors, beta-adrenoceptors, adenylyl cyclase, and guanylyl cyclase, whereas, inhibition of Ca(2+) influx in the vascular smooth muscle cells is important for their vasodilator actions. The high vasodilator potency and the quantity of salvianolic acid B contained in danshen ethanolic extract suggest it is an important constituent in this medicinal herb.

Topics: Adenylyl Cyclases; Animals; Benzofurans; Calcium Signaling; Coronary Vessels; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Endothelium, Vascular; Ethanol; Guanylate Cyclase; In Vitro Techniques; Male; Nitric Oxide Synthase; Phenanthrenes; Potassium; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Receptors, Muscarinic; Salvia miltiorrhiza; Serotonin; Signal Transduction; Solvents; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Lithospermic acid B (LAB), an active component isolated from Salvia miltiorrhizae, has been reported to have renoprotective effects in type 1 diabetic animal models. In the present study we investigated the effects of LAB on the prevention of diabetic nephropathy in type 2 diabetic Otsuka Long-Evans-Tokushima Fatty (OLETF) rats. LAB (20 mg/kg) was given orally once daily to 10-week-old male OLETF rats for 28 weeks. Treatment of OLETF rats with LAB had little effects on body weight and blood glucose levels. Treatment with LAB resulted in significant reduction in blood pressure. LAB markedly attenuated albuminuria and significantly lowered levels of lipid peroxidation, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-beta (TGF-beta1) expression in renal tissues of OLETF rats. In addition, LAB inhibited the progression of glomerular hypertrophy, mesangial expansion, and expansion of the extracellular matrix in the renal cortex. Collectively, these results suggest that LAB has beneficial effects on the diabetic nephropathy in OLETF rats by decreasing blood pressure, oxidative stress, and MCP-1 expression. Our results suggest that LAB might be a new therapeutic agent for the prevention of nephropathy in type 2 diabetes.

Topics: Administration, Oral; Aldehyde Reductase; Animals; Benzofurans; Blood Glucose; Blood Pressure; Chemokine CCL2; Depsides; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Gene Expression Regulation; Male; Oxidative Stress; Rats; Rats, Inbred OLETF; Rats, Long-Evans; Salvia miltiorrhiza

The traditional Chinese medicine, Salvia miltiorrhiza (Danshen), promotes blood circulation and relieves blood stasis, also demonstrating good antioxidant activity. In the present study, therefore, the antioxidant activities of medicinal plant materials prepared using nanotechnology or traditional grinding methods were compared using three biological assays. It was found that the nanotechnology preparation had stronger antioxidant bioactivities. Complementary quantitative analysis of four active constituents, salvianolic acid B, cryptotanshinone, tanshinone I and tanshinone IIA, by HPLC revealed only marked differences for salvianolic acid B. The results indicate that the polar active constituent in the nanotechnology samples was released faster compared to the traditionally powdered samples.

Topics: Antioxidants; Benzofurans; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Free Radical Scavengers; Iron Chelating Agents; Nanotechnology; Particle Size; Phenanthrolines; Reducing Agents; Salvia miltiorrhiza

Stem cells hold great promise for brain and spinal cord injuries (SCI), but cell survival following transplantation to adult central nervous system has been poor. Salvianolic acid B (Sal B) has been shown to improve functional recovery in brain-injured rats. The present study was designed to determine whether Sal B could improve transplanted mesenchymal stem cell (MSC) survival in SCI rats.. SCI rats were treated with Sal B. The Basso-Beatie-Bresnahan (BBB) scale was used to test the functional recovery. Sal B was used to protect MSC from being damaged by TNF-alpha in vitro. Bromodeoxyuridine-labeled MSC were transplanted into SCI rats with Sal B intraperitoneal injection, simultaneously. MSC were examined, and the functional recovery of the SCI rats was tested.. Sal B treatment significantly reduced the lesion area from 0.26+/-0.05 mm2 to 0.15+/-0.03 mm2 (P<0.01) and remarkably raised the BBB scores on d 28, post-injury, from 7.3+/-0.9 to 10.5+/-1.3 (P<0.05), compared with the phosphate-buffered saline (PBS) control group. MSC were protected from the damage of TNF-alpha by Sal B. The number of surviving MSC in the MSC plus Sal B groups were 1143.3+/-195.6 and 764.0+/-81.3 on d 7 and 28, post-transplantation, more than those in the MSC group, which was 569.3+/-72.3 and 237.0+/-61.3, respectively (P<0.05). Rats with MSC transplanted and Sal B injected obtained higher BBB scores than those with MSC transplanted alone (P<0.05) and PBS (P<0.01).. Sal B provides neuroprotection to SCI and promotes the survival of MSC in vitro and after cell transplantation to the injured spinal cord in vivo.

Topics: Animals; Antioxidants; Behavior, Animal; Benzofurans; Cell Survival; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Tumor Necrosis Factor-alpha

Although doxorubicin is an effective antitumor agent, the serious cardiotoxicity mediated by the production of reactive oxygen species has remained a considerable clinical problem. In China, salvianolic acids has been widely used for cardioprotection. To test whether salvianolic acids holds the potential to be protective against cardiotoxicity of doxorubicin, we created an acute cardiac injury mice model. Therapeutic treatment with salvianolic acids (40 mg/kg for 3 connective days) significantly reduced doxorubicin-induced (15 mg/kg) toxicity, including elevation of body weight and heart weight/tibia length ratio, decrease of creatine kinase, improvement of electrocardiography and heart vacuolation. Furthermore, the antioxidative effects of salvianolic acids were verified by oxygen radicals absorbance capacities assay in vitro and malondialdehyde detection in vivo, suggesting one possible mechanism of salvianolic acids on cardioprotection through blocking oxidative stress.

Topics: Animals; Antibiotics, Antineoplastic; Benzofurans; Body Weight; Caffeic Acids; Cardiotonic Agents; Creatine Kinase; Doxorubicin; Electrocardiography; Heart Diseases; Lactates; Male; Malondialdehyde; Mice; Myocardium; Oxidative Stress; Reactive Oxygen Species; Tissue Fixation

Apoptosis induced by high shear stress has been reported for the dysfunction of various vascular endothelial cells. We investigated the protective effects of tetramethylpyrazine (TMP) and salvianolic acid B (SAB) from Chinese medicine on the shear-induced early and late stages of apoptosis in cultured rat cerebral microvascular endothelial cells (rCMECs) under pathological high shear stress. Near-confluent cultures of rCMECs were pretreated with TMP or SAB and their combinational dosages, and exposed to high shear stress generated by a rheometer. Apoptotic death rate of rCMECs was assessed by immunofluorescence microscopy of Annexin V-FITC and propidium iodide (PI). We found that early and late stage apoptosis occurred at 3.0 Pa for a short duration of 450 sec but did not occur at 1.5 Pa. SAB inhibited the cells from apoptosis at concentrations from 10 microM to 20 microM in a dose-dependent manner, while effect of TMP at 0.37 mM and 0.73 mM did not significantly differ. Moreover, the combined use of TMP and SAB had synergistic anti-apoptotic effects (P<0.01). The results indicate that the anti-apoptotic effect of TMP and SAB on rheologically induced endothelial injury is likely involved in their efficacy.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Benzofurans; Cells, Cultured; Cerebrovascular Circulation; Drugs, Chinese Herbal; Endothelium, Vascular; Hemorheology; Male; Microcirculation; Pyrazines; Rats; Rats, Sprague-Dawley; Stress, Mechanical

Three major active components of the traditional Chinese medicinal herb Salvia miltiorrhiza Bunge, 3,4-dihydroxyphenyllactic acid, salvianolic acid B, and protocatechualdehyde, are separated and purified from a crude water extract in one step by isocratic hydrogen bond adsorption chromatography on cross-linked 12% agarose (Superose 12 HR 10/30). Separation is achieved by stepwise elution with mobile phases composed of mixtures of ethanol and acetic acid: 0-50 mL, 5% ethanol, 5% acetic acid; 50-100 mL, 20% ethanol, 20% acetic acid; and 100-200 mL, 30% ethanol, 30% acetic acid. The 3,4-dihydroxyphenyllactic acid is obtained with a purity of 97.3% and with a recovery of 88.1%. The corresponding figures for protocatechualdehyde are a purity of 99.4% with a recovery of 90.7%, and for salvianolic acid B a purity of 90.4% with a recovery of 50.3%, respectively. At a sample load of 40 mg crude extract dissolved in 0.5 mL mobile phase (corresponding to a load of 1.6 mg/mL gel), a 3,4-dihydroxyphenyllactic acid purity of approximately 94% with a recovery of 80.2% is obtained.

Topics: Adsorption; Benzaldehydes; Benzofurans; Catechols; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Hydrogen Bonding; Lactates; Salvia miltiorrhiza; Sepharose

The purpose of the present study was to examine the effects of Panax quinquefolium protopanaxadiol saponins (PQDS) extracts on the plasma protein binding and pharmacokinetic of salvianolic acids extracts extracted from the traditional Chinese medical Salvia miltiorrhiza,. Salvianolic acids are used to treat myocardial ischemia, and PQDS has similar functions. It is expected to achieve a better therapeutic efficacy if the two extracts are developed as a compound prescription for injection.. An established high-performance liquid chromatographic technique coupled with microdialysis was used. Male Sprague-Dawley rats were given salvianolic acids extracts and a compound of the two extracts via femoral vein.. It was found that there were significant differences in the percentage protein binding as well as the pharmacokinetic parameters. The rat plasma protein binding of the four salvianolic acids increased by different degrees at three dose levels (25, 50, 100mg/kg of salvianolic acid B) when the two extracts were administered together. Also, their elimination half-life was prolonged, and their plasma concentrations remained stable longer after administration of a dose of 50mg/kg (salvianolic acid B).. The results indicated that the PQDS extracts could delay the excretion of salvianolic acids as well as maintain the blood concentration higher than salvianolic acids extracts administered alone.

Topics: Animals; Benzofurans; Blood Proteins; Chromatography, High Pressure Liquid; Injections, Intravenous; Male; Microdialysis; Panax; Protein Binding; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sapogenins; Saponins

To study the cardioprotective effect of salvianolic acid B (Sal B) on cardiac dysfunction. We hypothesized that hyperleptinemia may correlate with abnormal expression of sarco/endoplasmic reticulum ATPase 2a (SERCA2a), phospholamban (PLB) and endothelin-reactive oxygen species (ET-ROS) pathways in rats with large myocardial infarction (MI).. Large MI was produced by coronary artery ligation for 4 weeks in rats. The rats were divided into four groups: sham, MI, MI+l-Sal B (50 mg/(kg d)), p.o. for 4 weeks), and MI+h-Sal B (100 mg/(kg d)), p.o. for 4 weeks).. In MI rats, hemodynamic and echocardiographic abnormalities, cardiac remodeling, and histological changes with features of cardiac failure were associated with hyperleptinemia accompanied by oxidative stress and upregulated OB-Rb, ET pathway mRNA expression and downregulated SERCA2a and PLB mRNA and protein expressions in the myocardium.. The studies demonstrated that an activated leptin pathway correlated with abnormal expression of SERCA2a, PLB and an activated ET-ROS system in the affected myocardium. Sal B exerts beneficial actions on cardiac function in rats with large MI, mainly suppressing upregulation of leptin and ET pathways and oxidative stress, and recovering the normal expressions of SERCA2a and PLB in myocardium.

Topics: Animals; Benzofurans; Calcium-Binding Proteins; Cardiotonic Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelins; Gene Expression Regulation; Leptin; Male; Myocardial Infarction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sarcoplasmic Reticulum Calcium-Transporting ATPases

The aim of this study was to compare the cardioprotective effects of salvianolic acid B (Sal B) and the angiotension-converting enzyme inhibitor, benazepril, in rats with chronic myocardial infarction (MI) that resulted from a coronary artery ligation for 4 weeks. The rats were divided into four groups: those undergoing a sham operation; a MI group; a MI+SalB group (100 mg/kg by a gavage, once a day for 4 weeks); a MI+benazepril group (10 mg/kg by a gavage, once a day for 4 weeks). The following parameters were measured: echocardiographic, hemodynamic and hemorheological changes, angiogenesis, infarct size and cardiac remodeling and the messenger ribonucleic acid (mRNA) of vascular endothelium growth factor (VEGF). Rats treated with SalB or benazepril manifested the following: (1) marked improvements in echocardiographic, hemodynamic and hemorheological parameters; (2) significant reduction of infarct size; (3) significantly attenuated heart, kidney and lung hypertrophies, left ventricular (LV) dilatation and fibrosis. The unique effects of SalB were angiogenesis and augmented VEGF expression in the border and remote noninfarcted left ventricular area. These results suggest that both SalB and benazepril exerted beneficial cardioprotective effects in our experimental system, but that the modality of Sal B was different from that of benazepril. The additional beneficial effects of Sal B relative to benazpril, augmenting VEGF expression and promoting angiogenesis, may result in improved myocardial microcirculation.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Antioxidants; Benzazepines; Benzofurans; Blood Viscosity; Cardiomegaly; Chronic Disease; Collagen; Electrocardiography; Hemodynamics; Immunohistochemistry; Male; Myocardial Infarction; Myocardium; Neovascularization, Pathologic; Protective Agents; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A; Ventricular Remodeling

Salviae miltiorrhizae (SM), a clinical, commonly used herb, can activate blood circulation and resolve stasis. We have investigated the effects of salvianolic acid B (Sal B), a pure compound extracted from the dried SM roots, on fibrinolytic (tissue-type plasminogen activator and plasminogen activator inhibitor, t-PA and PAI) and anticoagulant (thrombomodulin,TM) properties of cultured human umbilical vein endothelial cells (HUVECs). When HUVECs were treated with Sal B, a dose- (0.0125-0.5 mg/ml) and a time-dependent decrease in PAI activity were observed. PAI type 1 (PAI-1) antigen and PAI-1 mRNA expression significantly decreased compared to control values in the conditioned media of HUVECs pretreated with Sal B for 12 h. Moreover, TM activity reached a maximum stimulation of 1.25-fold over control levels in the pretreatment of Sal B for 12 h and t-PA and TM specific mRNA expression also increased (1.7- and 1.8-fold, respectively). In conclusion, Sal B increased the fibrinolytic and anticoagulant potential of cultured HUVECs by up-regulating the expression of t-PA and TM and by down-regulating the expression of PAI-1. These data suggest that Sal B is clinically effective because of its ability to change the gene expression profile of endothelial cells thereby preventing vascular events.

Topics: Benzofurans; Cell Survival; Cells, Cultured; Drugs, Chinese Herbal; Endothelial Cells; Fibrinolytic Agents; Hemostasis; Humans; Plasminogen Activator Inhibitor 1; Plasminogen Inactivators; RNA, Messenger; Thrombomodulin; Tissue Plasminogen Activator; Umbilical Veins

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo-E-deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP-2 and MMP-9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo-E-deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP-2 and MMP-9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP-2 and barely detectable levels for MMP-9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS-induced cell migration through the inactivation of MMP-2 and MMP-9 protein synthesis as well as the downregulation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These results demonstrate that Sal B has anti-migration properties on smooth muscle cells and may explain its anti-atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.

Topics: Animals; Aorta; Apolipoproteins E; Benzofurans; Cell Movement; Cells, Cultured; Cholesterol; Cyclooxygenase 2; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Myocytes, Smooth Muscle; Phosphorylation; Signal Transduction

The hydrolytic kinetics of lithospermic acid B (LAB) extracted from the roots of Salvia miltiorrhiza (Chinese herb: danshen) was investigated by using reversed-phase high-performance liquid chromatography (HPLC) with UV-vis detection. The influences of initial drug concentration, pH and temperature on hydrolysis of LAB were studied in aqueous solutions. The results showed that initial concentration of LAB has no effect on the degradation rate at pH 2.0. The hydrolysis followed pseudo-first-order kinetics at 90 degrees C. The log k(obs)-pH profile indicated that the optimal stability range was at pH 2.0-5.0. The rate constant of overall hydrolysis as a function of temperature under the given conditions obeyed the Arrhenius equation. Analysis of the acid-induced degraded solution of LAB by liquid chromatography-mass spectrometry (LC-MS) revealed at least four degradation products [M-H](-) ion at m/z 197, 137, 537 and 537, respectively. Three of these degradation products, i.e. danshensu (DSU), protocatechuic aldehyde (PRO), and lithospermic acid, were further identified by comparing the retention times with standard samples. According to the structure of LAB and its hydrolysis behavior in solution, the other product was proposed to be the isomer of lithospermic acid.

Topics: Benzaldehydes; Benzofurans; Catechols; Chromatography, High Pressure Liquid; Depsides; Drug Stability; Drugs, Chinese Herbal; Half-Life; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lactates; Mass Spectrometry; Models, Chemical; Molecular Structure; Plant Roots; Reference Standards; Salvia miltiorrhiza; Solutions; Spectrophotometry, Ultraviolet; Temperature

The degradation of lithospermic acid B (LAB) was investigated as a function of buffer concentration, pH and temperature. Stability tests were performed using a stability-indicating high-performance liquid chromatography (HPLC) with UV-vis detection. The degradation followed pseudo-first-order kinetics under all experimental conditions. The maximum stability of LAB was observed at pH 2.0. The logk(pH)-pH profile described by specific acid-base catalysis and water molecules agreed with the experimental results. The overall degradation rate constant as a function of the temperature under the given conditions obeyed the Arrhenius equation. The chemical fate of LAB in mild acidic solution was investigated, and nine degradation products were detected and tentatively identified by LC-MS analysis. The primary degradation pathway involving the cleavage of ester bond and ring-opened of benzofuran in the LAB are proposed.

Topics: Benzofurans; Buffers; Chromatography, Liquid; Depsides; Drug Stability; Hydrogen-Ion Concentration; Kinetics; Mass Spectrometry; Models, Molecular; Solutions; Temperature; Water

Salviae miltiorrhiza, a traditional Chinese medical herb known as "Danshen," has been widely used in clinics to improve blood circulation, relieve blood stasis, and treat coronary heart disease. Depside salts from S. miltiorrhiza are a novel drug in which magnesium lithospermate B and its analogs are the active components. The pharmacokinetics, tissue distribution, metabolism, and excretion of three of the major components, lithospermic acid B, rosmarinic acid (RA), and lithospermic acid (LA), were studied by liquid chromatography-tandem mass spectrometry following intravenous administration in Sprague-Dawley rats. The elimination half-lives for LSB, RA, and LA were 1.04, 0.75, and 2.0 h, respectively, when 60 mg/kg S. miltiorrhiza depside salts were administrated. The areas under the curve for LSB, RA, and LA were 51.6, 6.6, and 25.2 mg . h/l, respectively, and the values decreased in the individual tissues in the following order: kidney > lung > liver > heart > spleen > brain for LSB; kidney > lung > heart > liver > spleen > brain for RA; and heart > lung > kidney > liver > spleen > brain for LA. After intravenous administration of 60 mg/kg S. miltiorrhiza depside salts, 86% of the LSB was excreted in the bile within 6 h. The main metabolites M1 and M2 were found in the serum. Overall, the results show that depside salts from S. miltiorrhiza are rapidly and widely distributed to tissues after intravenous administration in rats but that they are also rapidly cleared and excreted.

Topics: Animals; Benzofurans; Cinnamates; Depsides; Male; Rats; Rats, Sprague-Dawley; Rosmarinic Acid; Salvia miltiorrhiza; Tissue Distribution

Three kinds of polyphenols of Salvia miltiorrhiza Bunge, 3,4-dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde, were separated and purified in one step with solvent system n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1.5:8) by high-speed counter-current chromatography. Acetic acid was successfully used to increase the partition of high polar target compounds in organic phase to modify partition coefficient value. 3,4-Dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde were purified from 100mg water extracted crude sample of Salvia miltiorrhiza Bunge at purity of 97.6%, 94.2% and 98.2% and at yield of 98.6%, 73.6% and 90.2%. High-speed counter-current chromatography together with organic/aqueous solvent system supplied an efficient method to purify water-soluble compounds directly from crude samples of traditional Chinese medicines.

Topics: Benzaldehydes; Benzofurans; Catechols; Chromatography, High Pressure Liquid; Countercurrent Distribution; Lactates; Salvia miltiorrhiza

A number of studies indicate that reactive oxygen species (ROS) are involved in neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The neuroprotective effects of salvianolic acid B (SalB) from Radix Salviae miltiorrhizae (RSM) against hydrogen peroxide (H2O2)-induced rat pheochromocytoma line PC12 injury were evaluated in the present study. Vitamin E, a potent antioxidant, was employed as a positive control agent. Following exposure of cells to H2O2 (150 microM), a marked decrease in cell survival and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as increased levels of malondialdehyde (MDA) production and lactate dehydrogenase (LDH) release were observed. In parallel, H2O2 caused significant elevation in intracellular Ca2+ level and caspase-3 activity, and induced apoptotic death as determined by flow cytometric assay. However, pretreatment of the cells with SalB (0.1-10 microM) prior to H2O2 exposure blocked these H2O2-induced cellular events noticeably. Moreover, SalB exhibited significantly higher potency as compared to Vitamin E. The present findings indicated that SalB exerts neuroprotective effects against H2O2 toxicity, which might be of importance and contribute to its clinical efficacy for the treatment of neurodegenerative diseases.

Topics: Animals; Apoptosis; Benzofurans; Calcium; Caspase 3; Hydrogen Peroxide; Lipid Peroxidation; Molecular Structure; Oxidoreductases; PC12 Cells; Rats; Salvia miltiorrhiza

To investigate the effects of salvianolic acid B (SA-B) on portal hypertension induced by endothelin-1 in rats.. Twenty-eight Sprague-Dawley rats were randomly divided into four groups: ET-1 group, ET-1+SA-B group, ET-1+ET(A)R blocker (BQ-123) group and ET-1+ET(B)R blocker (BQ-788) group. The rats of ET-1+SA-B group underwent intragastrical administration of salvianolic acid B for five days before ET-1 injection, while in three other groups' drinking water was given. In BQ-123 group or BQ-788 group, an intravenous injection of BQ-123 or BQ-788 via femoral vein was administered 30 minutes prior to ET-1 injection. Then changes of portal pressure, cervical artery pressure and heart rate were monitored continuously.. After ET-1 injection, the portal pressure of all rats in the ET-1 group increased significantly, while slightly in groups that pretreated with SA-B, BQ-123 or BQ-788.. SA-B can attenuate the elevated portal pressure induced by ET-1 with effect similar to ETR blocker.

Topics: Animals; Antihypertensive Agents; Benzofurans; Drugs, Chinese Herbal; Endothelin Receptor Antagonists; Endothelin-1; Hypertension, Portal; Injections, Intravenous; Male; Oligopeptides; Peptides, Cyclic; Piperidines; Portal Pressure; Random Allocation; Rats; Rats, Sprague-Dawley

A sensitive solid-phase extraction/high-performance liquid chromatographic method with ultraviolet detection was established for the analysis of salvianolic acid B in rabbit plasma. The analyte was separated on a reversed-phase column with trifluoroacetic acid-methanol-acetonitrile (70:10:20, v/v/v) as mobile phase at a flow rate of 1 mL/min, and ultraviolet detection at 315 nm. The calibration curve for salvianolic acid B was linear over the range 35-1400 microg/L with coefficients of correlation >0.999. The inter-day and intra-day precisions of analysis were <15%, and assay accuracy ranged from 95.3 to 109.1%. This method is suitable for determining salvianolic acid B in plasma and thus investigating the pharmacokinetics of salvianolic acid B.

Topics: Animals; Benzofurans; Chromatography, High Pressure Liquid; Female; Male; Rabbits; Sensitivity and Specificity; Solid Phase Extraction

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the simultaneous determination of lithospermic acid B and its three main O-methylated metabolites in rat serum with silibinin as the internal standard. The calibration curves for LSB, and the three metabolites were linear over the ranges of 16-4096, and 8-2048 ng/ml, respectively, with coefficients of correlation >0.998. For LSB, the intra-assay coefficient of variance (CV) was less than 9.3% and the inter-assay CV was less than 8.9%. The inter-assay mean accuracy was between 92.8% and 104.7%. For the three metabolites, the intra-assay CV was less than 8.7% and the inter-assay CV was less than 9.9%. The inter-assay mean accuracy was between 92.5% and 107.9%. This quantitation method was successfully applied to a pharmacokinetic study of LSB in rats. Also, a total recovery of 5.2% was found in bile after oral administration.

Topics: Absorption; Administration, Oral; Animals; Autoanalysis; Benzofurans; Bile Acids and Salts; Biological Availability; Chromatography, Liquid; Depsides; Drug Stability; Formates; Injections, Intravenous; Methylation; Molecular Structure; Molecular Weight; Rats; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Silybin; Silymarin; Tandem Mass Spectrometry; Temperature; Time Factors; Water

Salvia Miltiorrhiza Bunge (SMB), a traditional Chinese medicinal herb, has been alleged to support bone healing. However, the effects of the isolated major components of SMB on osseous cells and their corresponding effective doses are still unclear. In the present study, the effects of three components of SMB, including tanshinone IIA (Ts), salvianolic acid B (salB) and protocatechuic aldehyde (Pca), on mesenchymal bone marrow cells with the potential for osteoblastic differentiation were investigated. Various concentrations of Ts, salB and Pca were added to a rat bone marrow cell culture. The total metabolic activity and differentiation of bone marrow cells were evaluated by a metabolic assay and alkaline phosphatase (ALP) activity, respectively. The morphology and number of cells was observed by phase contrast microscopy and fluorescent microscopy after propidium iodide staining, respectively. Ts suppressed the growth and differentiation of bone precursor cells. SalB exhibited a biphasic effect: the high concentration of 160 microg/mL significantly depressed the population of bone marrow cells, however, lower concentrations (3-80 microg/mL) enhanced the total metabolic activity and their ALP expression. Pca suppressed the bone marrow cell population in a dose-dependent manner. Therefore, SalB has the potential to ameliorate bone healing by stimulating both the total metabolic activity and ALP activity of osteoblastic cells. Aqueous extracts, which preferably contain salB over Pca and are free of Ts therefore are recommended for bone formation.

Topics: Abietanes; Alkaline Phosphatase; Animals; Benzaldehydes; Benzofurans; Bone Marrow Cells; Catechols; Cell Differentiation; Cells, Cultured; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Male; Mesenchymal Stem Cells; Osteoblasts; Osteogenesis; Phenanthrenes; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza

The pharmacokinetics of the main components of protocatechualdehyde, salvianolic acid B, tanshinone II(A), cryptotanshinone, and the hydrophilic or lipophilic extracts of Salvia Miltiorrhiza Bge., in rat plasma were studied after oral administration separately to explore the interactions between them. Some components in the hydrophilic extract depress the absorption of the protocatechualdehyde, on the contrary, enhance the absorption of the salvianolic acid B and depress its elimination rate. The concomitant components in the lipophilic extract might enhance the absorption of cryptotanshinone and its distribution from the centre compartment to the peripheral compartment, and the metabolism to tanshinone II(A). The 'concomitant components' in the extract of Chinese material medica had significant effect on the pharmacokinetics of its 'marker components'. It can not only be rival, synergic, but also have the effects on metabolism. Therefore the traditional Chinese medicine was a complicated system, It should be taken a scientific and dialectic view in the research and development processes.

Topics: Abietanes; Animals; Area Under Curve; Benzaldehydes; Benzofurans; Catechols; Drug Interactions; Female; Male; Metabolic Clearance Rate; Molecular Structure; Phenanthrenes; Plant Extracts; Plants, Medicinal; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza

A sensitive and selective high-performance liquid chromatography method was developed and validated to determine the prototype of salvianolic acid B and the metabolites of phenolic acids (protocatechuic acid, vanillic acid and ferulic acid) in rat tissues after oral administration of total phenolic acids and salvianolic acid B extracted from the roots of Salvia miltiorrhiza, respectively. The tissue samples were treated with a simple liquid-liquid extraction prior to HPLC. Analysis of the extract was performed on a reverse-phase C(18) column with a mobile phase consisting of acetonitrile and 0.05% trifluoracetic acid. The calibration curves for the four phenolic acids were linear in the given concentration ranges. The intra-day and inter-day relative standard deviations in the measurement of quality control samples were less than 10% and the accuracies were in the range of 88-115%. The average recoveries of all the tissues ranged from 78.0 to 111.8%. This method was successfully applied to evaluate the distribution of the four phenolic acids in rat tissues after oral administration of total phenolic acids of Salvia miltiorrhiza or salvianolic acid B and the possible metabolic pathway was illustrated.

Topics: Administration, Oral; Animals; Benzofurans; Chemical Fractionation; Chromatography, High Pressure Liquid; Coumaric Acids; Drugs, Chinese Herbal; Hydrophobic and Hydrophilic Interactions; Hydroxybenzoates; Male; Metabolic Networks and Pathways; Plant Roots; Rats; Rats, Sprague-Dawley; Reference Standards; Salvia miltiorrhiza; Specimen Handling; Spectrophotometry, Ultraviolet; Tissue Distribution

Dihydroxy stilbene derivatives were designed based on lithospermic acid B and were prepared from 4-(chloromethyl)benzoic acid. The inhibitory activities of the novel compounds against protein tyrosine phosphatase 1B (PTP1B) were evaluated. 3,4-Dihydroxy stilbene carbonyl compounds (7, 11b, 27b) inhibited PTP1B with IC50 values comparable to molybdate, while the conjugation-extended compound (15b) showed inhibition 3-fold better than preclinical RK682. The introduction of electron withdrawing groups or amides into the second phenyl ring, or extension of the conjugation into the stilbene molecule may increase stability of the generated radicals.

Topics: Antioxidants; Benzofurans; Depsides; Drug Design; Models, Molecular; Molecular Structure; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Stilbenes; Structure-Activity Relationship

Exposure of endothelial cells to tumour necrosis factor-alpha (TNF-alpha) results in increased endothelial permeability, accompanied by a loss of cell-cell adherence junctions. The importance of tyrosine phosphatase and kinase activity in oxidant-mediated loss of cell junction structures has been demonstrated. The purpose of this study was to determine whether tyrosine phosphorylation contributes to TNF-alpha-mediated disorganization of endothelial cell junctions and how an extract of Salvia miltiorrhiza (ESM) and its active ingredients, Danshensu (DSS) and salvianolic acid B (Sal B), exert their protective effect in maintaining cell integrity. Immunoblotting results indicated that TNF-alpha exposure resulted in tyrosine phosphorylation of junctional proteins such as vascular endothelial cadherin and beta-catenin, which was attenuated by ESM and its active ingredients DSS and Sal B. In addition, immunoprecipitation showed ESM and its active ingredients prevented beta-catenin disassociation from the cytoskeleton in TNF-alpha-treated human umbilical vein endothelial cells. The results suggest that TNF-alpha produced biological effects at least partly by junctional protein phosphotyrosine modifications by increasing the total cellular phosphorylation level. It could be concluded that ESM and its active ingredients were effective at eliminating the factors leading to the rise in cellular phosphorylation, thus helping to maintain the integrity of endothelial junction structure.

Topics: Adherens Junctions; Analysis of Variance; Benzaldehydes; Benzofurans; beta Catenin; Cadherins; Catechols; Cell Membrane Permeability; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelial Cells; Enzyme Activation; Fluorescent Antibody Technique; Humans; Lactates; Phosphorylation; Phosphotyrosine; Plant Extracts; Salvia miltiorrhiza; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Endothelial Growth Factor A

Danshen root (Salvia miltiorrhiza Radix et Rhizoma) is a representative Chinese herb containing multiple components contributing to its polyvalent bioactivities. Advanced analysis approaches are needed to obtain a comprehensive picture of the targeting constituents in complete matrix. In this study, a chromatographic fingerprinting method to monitor simultaneously the hydrophilic and lipophilic constituents was developed for the quality evaluation of Danshen root and its preparations. Ten hydrophilic and nine lipophilic components were identified using HPLC-diode array detection-electrospray-MS (HPLC-DAD-ESI/MS) by comparing with the available references and reported data. Using the established method, 13 Danshen root samples collected from different sources, and 21 batches of Danshen preparations including tablets, injections, capsules, and dropping pills produced by different manufacturers were analyzed and their chromatographic fingerprints (CFP) were constructed. The results showed that the products of Danshen roots such as the tablets and capsules contained both the hydrophilic and lipophilic components, while the injections and dropping pills contained mainly the hydrophilic components. Principal components analysis (PCA) was applied for the statistical analysis of the fingerprinting data of crude herb and its preparations. The established CFPs demonstrate the representative chemical profiling of the existing components and can be applied to the authentication and quality assessment of Danshen roots and other Danshen containing formulated preparations.

Topics: Abietanes; Benzofurans; Caffeic Acids; Chromatography, High Pressure Liquid; Cinnamates; Diterpenes; Drugs, Chinese Herbal; Lactates; Molecular Structure; Phenanthrenes; Phenylpropionates; Plant Extracts; Plant Roots; Reference Standards; Salvia miltiorrhiza; Spectrometry, Mass, Electrospray Ionization

A sensitive and specific HPLC-UV method was developed for the simultaneous determination of major active components of danshen in rat plasma. Both water-soluble and lipid-soluble compounds were included, i.e. danshensu, salvianolic acid B and tanshinone IIA. Protocatechuic aldehyde and diazepam were used as internal standards. The chromatographic separation was achieved on a reversed-phase C(18) column by gradient elution using acetonitrile and 0.025% (v/v) phosphoric acid solution as mobile phase, at a flow rate of 1.0 mL/min. Salvianolic acid B, danshensu and internal standards were detected at 281 nm, while the detection of tanshinone IIA was carried out at 272 nm. All calibration curves showed good linearity (r(2) > 0.999) within test ranges. The limit of detection and the limit of quantification for danshensu, salvianolic acid B and tanshinone IIA in plasma were 0.065, 0.043, 0.022, 0.131, 0.085 and 0.044 microg/mL, respectively. This is the first report on the determination and pharmacokinetic study of danshensu, salvianolic acid B and tanshinone IIA in rat plasma and the results indicated that this method was reliable for the determination of the major active components of danshen in rat plasma.

Topics: Abietanes; Animals; Benzofurans; Calibration; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Hydrophobic and Hydrophilic Interactions; Lipids; Molecular Structure; Phenanthrenes; Phenanthrolines; Rats; Rats, Sprague-Dawley; Reference Standards; Reproducibility of Results; Salvia miltiorrhiza; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Water

This study was conducted to compare the in vivo metabolites of salvianolic acid B (Sal B) between normal rats and antibiotic-treated rats and to clarify the role of intestinal bacteria on the absorption, metabolism and excretion of Sal B. A valid method using LC-MS(n) analysis was established for identification of rat biliary and fecal metabolites. And isolation of normal rat urinary metabolites by repeated column chromatography was applied in this study. Four biliary metabolites and five fecal metabolites in normal rats were identified on the basis of their MS(n) fragmentation patterns. Meanwhile, two normal rat urinary metabolites were firstly identified on the basis of their NMR and MS data. In contrast, no metabolites were detected in antibiotic-treated rat urine and bile, while the prototype of Sal B was found in antibiotic-treated rat feces. The differences of in vivo metabolites between normal rats and antibiotic-treated rats were proposed for the first time. Furthermore, it was indicated that the intestinal bacteria showed an important role on the absorption, metabolism and excretion of Sal B. This investigation provided scientific evidence to infer the active principles responsible for the pharmacological effects of Sal B.

Topics: Animals; Bacteria; Benzofurans; Chromatography, High Pressure Liquid; Male; Mass Spectrometry; Molecular Structure; Neomycin; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Spectrometry, Mass, Electrospray Ionization; Streptomycin

This study is to observe the effect of salvianolic acid B (Sal B) on neural cells damage and neurogenesis in sub-granular zone (SGZ) and sub-ventricular zone (SVZ) after brain ischemia-reperfusion (I/R) in rats. A modified middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia-reperfusion was used. The rats were divided into four groups: sham control group, ischemia-reperfusion group, Sal B 1 and 10 mg x kg(-1) groups. Sal B was consecutively administrated once a day by ip injection after MCAO. The neurogenesis in SGZ and SVZ was investigated by BrdU method 7 days after MCAO. The Nissl staining for neurons in the hippocampal CA1 and cerebral cortex was performed 14 days after MCAO. A beam-walking test was used to monitor the motor function recovery. We found that brain ischemia resulted in an increase of BrdU positive cells both in ipsilateral SGZ and SVZ at 7th day after MCAO. Sal B (10 mg x kg(-1)) significantly increased further the number of BrdU positive cells both in SGZ and SVZ (P < 0.01). Ipsilateral hippocampal neuron damage occurred and CA1 almost lost 14 days after MCAO. Sal B (10 mg x kg(-1)) obviously attenuated the neuron damage and increased the number of neuron both in ipsilateral CA1 and cerebral cortex (P < 0.01). We also observed an obvious improvement of motor function recovery when Sal B (10 mg x kg(-1)) administrated. From the results above we concluded that Sal B stimulated neurogenesis process both in SGZ and SVZ after brain ischemia, and also alleviated neural cells loss and improved motor function recovery after brain ischemia in rats.

Topics: Animals; Benzofurans; Cell Count; Cerebral Cortex; Cerebral Ventricles; Dentate Gyrus; Hippocampus; Infarction, Middle Cerebral Artery; Male; Motor Activity; Neurogenesis; Neurons; Plants, Medicinal; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Salvia miltiorrhiza

pH-Zone-refining countercurrent chromatography was successfully applied to the separation of salvianolic acid B from the Chinese medicinal plant, Salvia miltiorrhiza Bunge, using a multilayer coil planet centrifuge. A 2.0 g quantity of sample was separated using the following two-phase solvent system: methyl tert-butyl ether (MtBE)-water, 10 mM TFA in organic stationary phase and 10 mM ammonia in aqueous mobile phase. The obtained fractions were analyzed by HPLC and ESI-MS. The separation yielded 572 mg of the main component of salvianolic acid B with a purity of 94.1%.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Countercurrent Distribution; Hydrogen-Ion Concentration; Molecular Structure; Plants, Medicinal; Salvia miltiorrhiza

To observe the effect of salvianolic acid B (SalB) on high energy phosphate and activity of ATPase of cerebral ischemia in mice, and to study the role of SalB on hydrocephalus further.. NIH mice were divided into four groups randomly: Sham-operated group, cerebral ischemia group, SalB-treated group and Nimodipine (Nim)-collated group. In Sal B-treated group, mice were injected with SalB (22.5 mg x kg(-1)) in vena caudalis at 30 min before the experiment. In Nim-collated group, Nim (0.03 mg x kg(-1)) was injected into tail vein at the same time, while the mice in Sham-operated group and cerebral ischemia group were injected the same volume normal saline. The acute cerebral ischemia model was established by ligating bilateral common carotid arteries for 30 min in mice, then the mice were killed and the content of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), phosphocreatine (PCr) were observed, and the cerebral energy charge (EC) was computed. At the same time, activity of Na(+) -K(+) -ATPase and Ca2(+) -ATPase, content of water in brain tissue were measured.. Compared with cerebral ischemia group, EC and content of ATP, ADP, PCr in SalB-treated group heightened evidently (P < 0.01). Moreover, activity of Na(+)-K+ ATPase and Ca2+ ATPase in SalB-treated group had a remarkable increase (P < 0.01). But the content of water in brain tissue decreased markedly (P < 0.05).. The mechanism that SalB can relieve content of water in brain tissue of cerebral ischemia in mice, may be associated with improving the content of high-energy phosphoric acid compounds and enhancing the activity of ATPase.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Benzofurans; Brain; Brain Ischemia; Calcium-Transporting ATPases; Energy Metabolism; Male; Mice; Phosphocreatine; Plants, Medicinal; Random Allocation; Salvia miltiorrhiza; Sodium-Potassium-Exchanging ATPase; Water

Salvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H(2)O(2)) is implicated in the pathogenesis of cerebrovascular disorders.. By examining the effect of Sal B on H(2)O(2)-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H(2)O(2)-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H(2)O(2) induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor (LY294002) blocked ERK activation caused by H(2)O(2 )and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H(2)O(2)-induced apoptosis, suggesting that Sal B prevents H(2)O(2)-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway.. Our findings provide the first evidence that H(2)O(2) induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H(2)O(2)-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway.

Topics: Animals; Apoptosis; Benzofurans; Cells, Cultured; Endothelium, Vascular; Hydrogen Peroxide; In Situ Nick-End Labeling; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction

The application of accelerated solvent extraction (ASE) technique for the Analysis of active components in traditional Chinese medicinal herbs was introduced by using two kinds of herbs as examples. The conditions including extraction temperature, static extraction time, the ratio of material to solvent and solvent of ASE for extraction of salvianolic acid B in Salvia miltiorrhiza were optimized by orthogonal experiments, and the optimal conditions were obtained. Different extraction methods (ASE, steam distillation, ultrasonic wave and Soxhlet extraction) were used to extract volatile oil in Aucklandia lappa Decne. Results of the comparative experiments indicated that ASE was the most effective method in this case. All the results from these studies demonstrate that ASE is indeed a powerful tool in the preparation of herbal extracts for downstream chromatographic analysis.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Gas Chromatography-Mass Spectrometry; Oils, Volatile; Salvia miltiorrhiza; Solvents

To study the optimum conditions for the isolation and purification of water-soluble substance from Salvia miltiorrhiza Bge.. The optimum macroporous resin was selected and the separation and purification process was evaluated by measuring the content of Salvianolic acid B in the fractions by HPLC.. The XDA-5 macroporous resin was the most effective compared with other macroporous resins. The optimum conditions were screened, which were 18 mg/ml corresponding to Salvianolic acid B for concentration of extract, pH was 4, and the volume of 70% (V/V) ethanol as eluant was 3 BV. By this method, the elution efficiency of Salvianolic acid B exceeded 90%.. The method is more effective for large-scale isolation and purification of water-soluble substance from Salvia miltiorrhiza Bge.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Ethanol; Hydrogen-Ion Concentration; Plant Roots; Plants, Medicinal; Resins, Synthetic; Rhizome; Salvia; Technology, Pharmaceutical; Water

To provide some evidence for breeding excellent lines of Salvia miltiorrhiza.. Contents of tanshinone II(A), cryptotanshinone, salvialic acid A and salvianolic acid B in root of S. miltiorrhiza from different populations were determined by HPLC. Total tanshinone and total salvianolic acids were determined by spectrophotometry.. The biomass of individual plant from Zhongjiang of Sichuan province, content of tanshinone II(A), from Shanxi province, cryptotanshinone and total tanshinone from Zhongjiang of Sichuan province, salvialic acid A of High-Stem cultivation from Hebei province, salvianolic acid B and total salvianolic acids of tetroploid were higher than the other samples, respectively. With the value of biomass of individual plant multiplying contents of every active components as indicative constituents, the multiplying value from Zhongjiang of Sichuan province was higher than the other samples.. With biomasses and effective components as indicative constituents, it suggested that S. miltiorrhiza from Zhongjiang of Sichuan province was a better derivative material.

Topics: Abietanes; Benzofurans; Biomass; Caffeic Acids; China; Chromatography, High Pressure Liquid; Ecosystem; Lactates; Phenanthrenes; Plant Roots; Plants, Medicinal; Salvia miltiorrhiza; Spectrophotometry, Ultraviolet

Mice pathological model of acute cerebral ischemia was established. In order to observe the effect of salvianolic acid B (Sal B) on brain energy metabolism and hydrocephalus in the brain of mice at different ischemic times, the energy charge (EC), content of phosphocreatine (PCr), level of lactic acid (Lac), activity of Na+ -K+ -ATPase, brain index and water content of brain were measured at 6, 12, 18, 24, and 30 min, separately after ligating bilateral common carotid arteries in mice. NIH mice were randomly divided into sham-operated group (sham), cerebral ischemia group (ischemia), Sal B-treated group (Sal B) and nimodipine-collated group (Nim). At 6 min after cerebral ischemia, EC, content of PCr and activity of Na +-K -ATPase began to decrease, while level of Lac, brain index and water content of brain increased gradually. However, Sal B (22.5 mg x kg(-1) improved pathophysiological changes at different ischemic times. Especially at 30 min after cerebral ischemia in Sal B group, EC (P < 0.01), content of PCr (P < 0.01 and activity of Na+ -K+ -ATPase ( < 0.05) increased significantly. Meanwhile, level of Lac (P < 0.01, brain index (P < 0.01) and water content of brain (P < 0.05) were lower obviously than those of cerebral ischemia group. Sal B could alleviate hydrocephalus by the improvement of energy metabolism in mice with acute cerebral ischemia, that provides scientific evidence that Sal B can be used for the clinical application of ischemic diseases.

Topics: Animals; Benzofurans; Brain; Brain Ischemia; Drugs, Chinese Herbal; Energy Metabolism; Hydrocephalus; Lactic Acid; Male; Mice; Phosphocreatine; Plants, Medicinal; Random Allocation; Salvia miltiorrhiza; Sodium-Potassium-Exchanging ATPase; Time Factors; Water

Salvia miltiorrhiza (SM) has been used clinically in Asian countries to improve the microcirculation in the human body. Salvianolic acid B (Sal B), a pure compound extracted from SM, has been reported to be effective against fibrosis and ischemia-reperfusion injury, possibly through its anti-lipid peroxidation action. But the effect of Sal B on oral premalignant lesion and oral carcinogenesis remains unexplored. It is our interest to investigate the chemopreventive effect of Sal B on 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters with respect to angiogenesis. Seventy male Syrian golden hamsters were randomly divided into five groups, with two of 20 and three of 10. DMBA solution (0.5% in acetone) was applied topically to the left cheek pouch of male Syrian golden hamsters in Groups A and B, while animals in Group C were painted with acetone, three times a week for 6 weeks. For the next 18 weeks, animals in Groups B and D received Sal B daily (10 mg/kg body wt/day) by gavage, animals in Groups A and C received same volume of saline. Animals in Group E received no treatment and served as blank control. At the end of the experiment, animals were killed and tissue samples were collected for histopathological and immunohistochemical examinations. The results showed that Sal B significantly decreased the squamous cell carcinoma (SCC) incidence from 64.7 (11/17) to 16.7% (3/18) (P=0.004); angiogenesis was inhibited in dysplasia and SCC (P<0.01), with a simultaneous decrease in the immunostaining of hypoxia-inducible factor 1alpha and vascular endothelium growth factor protein (P<0.05). The results suggested that Sal B had inhibitory effect against the malignant transformation of oral precancerous lesion and such inhibition may be related to the inhibition of angiogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Administration, Oral; Animals; Benzofurans; Carcinogens; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Chemoprevention; Cricetinae; Male; Mesocricetus; Mouth Neoplasms; Neovascularization, Pathologic; Precancerous Conditions; Random Allocation

The production of reactive oxygen species (ROS) is believed to be involved in liver injury and hepatic fibrosis. Activation of hepatic stellate cells (HSCs) is a key feature of liver fibrosis. Salvia miltiorrhiza is a traditional Chinese herb used in the treatment of cardiovascular and liver diseases to resolve stasis. The effects of salvianolic acid B (Sal B), a major component of Salvia miltiorrhiza, on oxidative damage include free radical DPPH scavenging, malondialdehyde (MDA) formation and ROS generation in primary rat hepatocytes and HSCs, and on alpha-SMA, and collagen expression in transforming growth factor-beta1 (TGF-beta1)-stimulated HSCs were examined. Results indicated that Sal B scavenged DPPH potently with an IC50 2.2+/-0.2 microg/ml (3.06+/-0.3 microM), inhibited lipid peroxidation and eliminated ROS accumulation in a concentration-dependent manner on primary rat hepatocytes and HSCs. Sal B also reduced alpha-SMA and collagen synthesis and deposition in HSCs, and had no direct cytotoxicity on both hepatocytes and HSCs. Our results suggest that Sal B ameliorated oxidative damage and eliminated ROS accumulation in hepatocytes, and attenuated HSC activation, potentially conferring hepatoprotective and anti-fibrogenic effects.

Topics: Actins; Animals; Benzofurans; Cells, Cultured; Free Radical Scavengers; Hepatocytes; In Vitro Techniques; Liver; Male; Malondialdehyde; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Transforming Growth Factor beta

Inflammation plays an essential role in atherosclerosis and post-angioplasty restenosis and the synthesis and release of inflammatory cytokines from vascular smooth muscle cells is an important contributor to these pathologies. It is assumed that drugs that prevent the overproduction of inflammatory cytokines may inhibit cardiovascular disorders. In the present study, the effects of a water-soluble antioxidant, salvianolic acid B (Sal B), derived from a Chinese herb, on the expression of cyclooxygenase (COX) in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMCs) and in the aortas of cholesterol-fed apoE deficient mice were investigated. In unstimulated HASMCs, COX-2 mRNA and protein were almost undetectable, but were strongly upregulated in response to LPS. In contrast, HASMCs with or without LPS treatment showed constitutive expression of COX-1 mRNA and protein. The activation of COX-2 protein synthesis in LPS-stimulated HASMCs was shown to involve the activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathway. Incubation of HASMCs with Sal B before LPS stimulation resulted in pronounced downregulation of COX-2 expression. Sal B treatment suppressed ERK1/2 and JNK phosphorylation and attenuated the increase in prostaglandin E(2) production and NADPH oxidase activity in LPS-treated HASMCs. When apoE-deficient mice were fed a 0.15% cholesterol diet with or without supplementation with 0.3% Sal B for 12 weeks, the intima/media area ratio in the thoracic aortas was significantly reduced in the Sal B group (0.010 +/- 0.009%) compared to the apoE-deficient group (0.114 +/- 0.043%) and there was a significant reduction in COX-2 protein expression in the thickened intima. These results demonstrate that Sal B has anti-inflammatory properties and may explain its anti-atherosclerotic properties. This new mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.

Topics: Animals; Aorta; Apolipoproteins E; Benzofurans; Cells, Cultured; Cyclooxygenase 2; Dinoprostone; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Humans; Intercellular Adhesion Molecule-1; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Myocytes, Smooth Muscle; NADPH Oxidases; Phosphorylation; Protein Transport; RNA, Messenger

To study on the release of compound Danshen sustained-release tablet and the evaluate method of Chinese material medica compound sustained-release preparation.. Rotating basket method and HPLC were employed.. Through the determination of 6 time-point samples, the water-soluble compositions of compound Danshen sustained-release tablet had a Well-balanced release behavior with a zero-grade release model or Higuchi release model.. Compound Danshen sustained-release tablet had a zero-grade release model. The method was rapid and stable and could be applied to evaluate the water-soluble composition release of compound Danshen sustained-release Tablet.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Delayed-Action Preparations; Drug Combinations; Drug Stability; Drugs, Chinese Herbal; Ginsenosides; Panax; Plants, Medicinal; Salvia miltiorrhiza; Solubility; Tablets

Establishment of hydrophilic organic/salt-containing aqueous two-phase system and purification of salvianolic acid B from crude extract of S. miltiorrhiza by counter-current chromatography with said system were studied. Ethanol and n-propanol were selected to constitute biphasic systems with ammonia sulphate, sodium chloride and phosphate separately, and related system characteristics including phase diagrams, phase ratio, separation time were tested. The partition coefficient of crude salvianolic acid B was also tested in above systems and further finely adjusted by altering the constitution of phosphate in n-propanol/phosphate system. Salvianolic acid B was purified to 95.5% purity by counter-current chromatography in 36% (w/w) n-propanol/8% (w/w) phosphate system with the ratio between dipotassium hydrogen phosphate and sodium dihydrogen phosphate of 94:6. One hundred and eight milligrams of salvianolic acid B was purified from 285 mg crude extract with the recovery of 89%.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Countercurrent Distribution; Plant Extracts; Salts; Salvia miltiorrhiza

The present study was to investigate the protective effects of salvianolic acids (SA) on deformability of red blood cells (RBCs) and its mechanism during the development of acute lung injury (ALI) induced by oleic acid (OA) in rabbits. 32 rabbits were randomized into four groups, normal control group, OA-treated group (0.15 ml/kg), SA-treated group and OA+SA treated group. The blood samples were collected at 0, 10, 30, 60, 90, 120 and 180 min after OA injection. The RBC deformation index, Orientation index and small deformation index were measured by ektacytometry. The concentration of malondialdehyde (MDA) in RBCs was detected by the assay kit. Meanwhile, the pulmonary pathological examination and the blood gas analysis were also performed. The results showed that the deformation index, orientation index and small deformation index decreased during the early phase of ALI, while the concentration of MDA in RBCs increased during the course. Pre-treatment with SA increased the deformability and orientability of RBC significantly and decreased the concentration of MDA in RBCs compared with OA group. Meanwhile, the hypoxia and pulmonary pathological damage were much improved. These results suggest that there were erythrocyte deformability changes in the early phase of ALI. SA has the protective effects on erythrocyte deformability during the development of ALI induced by OA, which might be due to its antioxidant effect. These results are valid in rabbits and in a model of ARDS, it would be interesting to see the effects of SA in patients.

Topics: Acute Disease; Animals; Benzofurans; Blood Gas Analysis; Caffeic Acids; Disease Models, Animal; Erythrocyte Deformability; Lactates; Lung Injury; Male; Oleic Acid; Rabbits; Respiratory Distress Syndrome; Time Factors

Activated matrix metalloproteinases (MMPs) in patients with acute coronary syndromes may contribute to plaque destabilization. Tumor necrosis factor-alpha (TNF-alpha) enhances NAD (P) H oxidase-dependent reactive oxygen species (ROS) formation and ROS induce MMP-2. In the present study, the effects of a potent water-soluble antioxidant, salvianolic acid B (SalB), derived from a Chinese herb, Salvia miltiorrhiza, on the expression of MMP-2 by TNF-alpha-treated human aortic smooth muscle cells (HASMCs) were investigated. In this study, salvianolic acid B scavenged H2O2 in a dose-dependent manner in test tube. We found that SalB, as well as NADPH oxidase inhibitors, DPI or apocynin, and antioxidant NAC, inhibited TNF-alpha-induced MMP-2 mRNA, protein expression, and gelatinolytic activity in HASMCs in a concentration-dependent manner. We also observed a dose-dependent decrease in ROS production and NADPH oxidase activity induced by TNF-alpha in the presence of SalB. SalB also significantly inhibited angiotensin II or H2O2-induced MMP-2 mRNA and protein expression and gelatinolytic activity in HASMCs. Our data point out that the importance of NADPH oxidase-dependent ROS generation in the control of SalB inhibition of TNF-alpha-induced MMP-2 expression and activity.

Topics: Acetophenones; Acetylcysteine; Angiotensin II; Aorta; Benzofurans; Cells, Cultured; Humans; Hydrogen Peroxide; Matrix Metalloproteinase 2; Muscle, Smooth, Vascular; NADPH Oxidases; Onium Compounds; Reactive Oxygen Species; Salvia miltiorrhiza; Tumor Necrosis Factor-alpha; Up-Regulation

T o determine the quality standard of Yixin Tablet.. The nature differentiation of Radix Ginseng, Radix Astragali, Rhizoma Chuanxiong, Fructus Schisandrae was determined by TLC and content of salvianolic acid B in Yixin Tablet was determined by HPLC.. The nature differentiation separate degree is kind and special attribute was strong. The compounds were base-isolated on the column of C18 which gradient eluted with acetonitrile and H2O (0.05% H3PO4) and detective wavelength was 280 nm. The linearity was obtained over the range of 0.12504-2.50080 microg (r = 0.9999) and the average recovery rate was 99.35%.. The method is simple, convenient and accurate. The sensitivity is high. It can be used in the quality control of Yixin Tablet.

Topics: Astragalus propinquus; Benzofurans; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Drug Combinations; Drug Stability; Drugs, Chinese Herbal; Panax; Plants, Medicinal; Quality Control; Reproducibility of Results

To explore the anti-hepatic fibrosis mechanisms of salvianolic acid B (SA-B).. Hepatic stellate cells (HSCs) isolated from rats were primarily cultured in uncoated plastic culture dish for 7 days, then were incubated with 10(-6) mmol/L SA-B and stimulated with 10 ng/ml transforming growth factor-beta1 (TGF-beta1) or platelet-derived growth factor-BB (PDGF-BB). Expressions of extracellular-regulated kinase (ERK) and its phosphorylation in HSC, and expressions of TGF beta1, receptor I (TbetaR I) and II (TbetaR II) and PDGF receptor beta (PDGFR-beta) on the surface of HSC induced by TGF-beta1 or PDGF-BB were detected with Western blot assay.. SA-B inhibited the phosphorylation of ERK1/2 in HSC primary normally cultivated for 9 days stimulated or un-stimulated by TGF-beta1, but could not affect the expressions of TbetaR I and TbetaR II on the HSC surface; it down-regulated the expression of PDGFR-beta, but had no obvious effect on the phosphorylation of ERK1/2 in those HSC stimulated or un-stimulated by PDGF-BB.. SA-B inhibits the ERK signal transduction induced by TGF-beta1 in HSC, which is independent of the expressions of TbetaR on HSC surface and also free from the ERK signal transduction stimulated by PDGF-BB. And its inhibition on PDGF-BB signal transduction in HSC is by way of restraining the expression of PDGFR in HSC.

Topics: Animals; Becaplermin; Benzofurans; Cells, Cultured; Hepatocytes; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta

Several lines of evidence support that beta-amyloid (Abeta)-induced neurotoxicity is mediated through the generation of reactive oxygen species (ROS) and elevation of intracellular calcium. Salvianolic acid B (Sal B), the major and most active anti-oxidant from Salvia miltiorrhiza, protects diverse kinds of cells from damage caused by a variety of toxic stimuli. In the present study, we investigated the effects of Sal B against beta-amyloid peptide 25-35 (Abeta(25-35))-induced neurotoxicity, focused mainly on the neurotoxic effects of Abeta(25-35) and the neuroprotective effects of Sal B on the expression of brain-pancreas relative protein (BPRP), which is a new protein and mainly expressed in brain and pancreas. Following exposure of PC12 cells to 20 microM Abeta(25-35), a marked reduction in the expression of BPRP was observed, accompanied with decreased cell viability and increased cell apoptosis, as well as increased ROS production and calcium influx. Treatment of the PC12 cells with Sal B significantly reversed the expression of BPRP and cell viability while it decreased ROS production and intracellular calcium. These data indicate that Abeta(25-35) decreases the expression of BPRP via enhanced formation of intracellular ROS and increased intracellular calcium, and that Sal B, as an anti-oxidant, protects against Abeta(25-35)-induced reduction in expression of BPRP through its effects on suppressing the production of ROS, calcium flux, and apoptosis. However, the role(s) of BPRP in AD and the definite mechanisms by which Sal B protects against Abeta(25-35)-induced reduction in the expression of BPRP require further study.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Benzofurans; Calcium; Nerve Tissue Proteins; PC12 Cells; Peptide Fragments; Rats; Reactive Oxygen Species

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. The aim of this study was to apply an automated blood sampling system coupled to a simple liquid chromatographic system to determine the bioavailability of salvianolic acid B in stress-free rats. The plasma sample (25 microl) was vortex-mixed with 50 microl of internal standard solution (chloramphenicol 10 microg/ml in acetonitrile) to achieve protein precipitation. Salvianolic acid B in the rat plasma was separated using a reversed-phase C18 column (250 mm x 4.6 mm, 5 microm) with a mobile phase of acetonitrile-methanol-20mM NaH(2)PO(4) (adjusted to pH 3.5 with H(3)PO(4)) (20:10:70 v/v/v) containing 0.1mM 1-octanesulfonic acid, and the flow-rate of 1 ml/min. The UV detection wavelength was 286 nm. The concentration-response relationship from the present method indicated linearity over a concentration range of 0.5-200 microg/ml. Intra- and inter-assay precision and accuracy of salvianolic acid B fell well within the predefined limits of acceptability (<15%). The plasma sample of salvianolic acid B was further identified by LC-MS/MS in the negative ion mode using mass transition m/z 358.2 to the product ion m/z 196.9. After salvianolic acid B (100mg/kg, i.v.; 500 mg/kg, p.o.) was given in conscious and freely moving rats, the AUC were 5030+/-565 and 582+/-222 min microg/ml for intravenous (100 mg/kg) and oral (500 mg/kg) doses, respectively. The oral bioavailability of salvianolic acid B in freely moving rats was calculated to be 2.3%.

Topics: Animals; Automation; Benzofurans; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Chromatography, Liquid; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Ions; Mass Spectrometry; Models, Chemical; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Ultraviolet Rays

To investigate and compare the antioxidant activities of salvianolic acid B (SalB) and Ginkgo biloba extract (EGb 761) in aqueous solution, rat microsomes and the cellular system.. Superoxide anion (O2-.) was generated using xanthine/xanthine oxidase system and phenazine methosulate/NADH system, and the effects of SalB and EGb 761 on the generation of O2-.were achieved by spectrophotometric measurement of the product formed on reduction of nitro blue tetrazolium. Two different methods were used to assess the scavenging effects of the extracts on hydroxyl radical (. OH): HPLC method was used for quantitation of . OH by oxy-radical trapping of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form DMPO-OH adducts in Fe2+-EDTA-H2O2 system. To confirm the HPLC data, .OH was also measured by spectrophotometry using a commercial detection kit. The anti-lipid peroxidation effects of the extracts in microsomes of rat brain, liver and kidney induced by ascorbate-NADPH were determined by thiobarbituric acid (TBA) method. The protective effects of the extracts on peroxide hydrogen (H2O2)-induced oxidative damage in SH-SY5Y cells were investigated by assessing cell viability assay, the level of lipid peroxidation, and the lactate dehydrogenase(LDH) release.. Both SalB and EGb 761 were able to scavenge O2-. and . OH, inhibit lipid peroxidation of microsomes, and protect SH-SY5Y cells against H2O2-induced oxidative damage. However, the concentration of SalB was far lower than that of EGb 761 when a similar effect was obtained.. The antioxidant efficiency of SalB was greater than that of EGb 761. These results suggest that SalB, like EGb 761, has promising potential in treating oxidative damage-derived neurodegenerative disorders.

Topics: Animals; Antioxidants; Benzofurans; Brain; Cell Line, Tumor; Drugs, Chinese Herbal; Ginkgo biloba; Humans; Male; Neuroblastoma; Plant Extracts; Plants, Medicinal; Rats; Rats, Wistar; Salvia miltiorrhiza

In this study, we examined the effects of Salvia miltiorrhiza (Danshen) crude extract, some of its lipid-soluble components (tanshinone I, tanshinone II(A), cryptotanshinone, dihydroisotanshinone I) and the water-soluble compounds (danshensu and salvianolic acid B) on the K(+) channels such as the iberiotoxin-sensitive Ca(2+)-activated K(+) (BK(Ca)) channels and the glibenclamide-sensitive ATP-dependent K(+) (IK(ATP)) channels of the porcine left anterior descending coronary artery smooth muscle cells. Cumulative application of salvianolic acid B (30-300 microM) caused a l-NNA (100 microM)-insensitive, potentiation of the outward BK(Ca) current amplitude with no apparent effect on the IK(ATP) channels opening. Salvianolic acid B (300 microM) caused an ODQ (10 microM, a guanylate cyclase inhibitor)-sensitive enhancement of the outward BK(Ca) current amplitude. In contrast, none of the other isolated chemical constituents of S. miltiorrhiza modified the openings of the two types of K(+) channels studied. In conclusion, our results suggest that salvianolic acid B, a major hydrophilic constituent found in Radix S. miltiorrhiza, activated the opening of the BK(Ca) channels of the porcine coronary artery smooth muscle cells through the activation of guanylate cyclase without the involvement of the nitric oxide synthase activation.

Topics: Animals; Benzofurans; Coronary Vessels; Cromakalim; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Enzyme Activation; Enzyme Inhibitors; Glyburide; Guanylate Cyclase; In Vitro Techniques; Ion Channel Gating; Membrane Potentials; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Oxadiazoles; Patch-Clamp Techniques; Peptides; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Calcium-Activated; Quinoxalines; Receptors, Drug; Salvia miltiorrhiza; Swine

Salvianolic acid B and lithospermic acid B are the major components of Salvia miltiorrhiza, which is one of the most popular herbal traditional medicines in Asian countries. Salvianolic acid B and lithospermic acid B are reported to have identical structures except for the configurational assignments of two stereocenters. Through chemical correlation between a degradation product of salvianolic acid B and synthetic material, the absolute configuration of salvianolic acid B has been corrected to establish that salvianolic acid B and lithospermic acid B are in fact the same compound.

Topics: Benzofurans; Depsides; Molecular Structure; Plants, Medicinal; Salvia miltiorrhiza

In a previous short-term study, salvianolic acid B was reported to have a protective effect on cerebral ischemia. Here, we investigated whether salvianolic acid B improves the recovery of motor function after cerebral ischemia in a 14-day investigation. Cerebral ischemia was induced by middle cerebral artery occlusion in rats. Motor function was evaluated with beam-walking performance. Neural cell injury in both the sensorimotor cortex and CA1 of the hippocampus ipsilateral to ischemia was studied by Nissl stain with methylene blue. The integrity of cerebral microvessels was monitored by immunoglobulin extravasations. Neurogenesis in the subgranular zone in the dentate gyrus of the hippocampus was detected with 5'-bromo-2'-deoxyuridine incorporation. Animals receiving salvianolic acid B at a dose of 10 mg/kg had a more rapid recovery of beam-walking performance than vehicle-treated ischemia animals, and the improvement became significant at 10 and 14 days after ischemia (P<0.05). Treatment with salvianolic acid B at a dose of 10 mg/kg also significantly prevented neural cell loss in CA1 of the hippocampus. Neural cells in the sensorimotor cortex were also preserved in animals that received salvianolic acid B at a high dose of 10 mg/kg. Salvianolic acid B (10 mg/kg) also improved the integrity of microvessels after ischemia. We observed a slight increase in 5'-bromo-2'-deoxyuridine-positive cells in the subgranular zone of the hippocampus in the animals treated with salvianolic acid B at a dose of 10 mg/kg. These data indicate that salvianolic acid B could improve the recovery of motor function after cerebral ischemia in rats.

Topics: Animals; Benzofurans; Cell Survival; Hippocampus; Infarction, Middle Cerebral Artery; Male; Microcirculation; Motor Activity; Motor Skills; Nerve Regeneration; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Somatosensory Cortex

To compare the effects of salvianolic acid B (Sal B) and Ginkgo biloba extract EGb 761 on beta-amyloid peptide (beta-AP) fibril formation and cytotoxicity to PC12 cells.. The inhibitory effects of Sal B and EGb 761 on beta-AP1-40 fibril formation were determined by using fluorescence analysis with Thioflavin T (ThT) and electron microscopic image. beta-AP25-35 was aged by incubating at 37 degrees C for 7 d, then the protein was incubated with PC12 cells. The protective effects of Sal B and EGb 761 against cytotoxicity induced by aged beta-AP25-35 in PC12 cells were evaluated by MTT reduction assay and flow cytometric analysis. beta-AP25-35-induced accumulation of intracellular reactive oxygen species (ROS) was determined by fluorescence analysis.. Both Sal B and EGb 761 inhibited the formation of amyloid fibrils, protected PC12 cells from beta-AP25-35-induced cytotoxicity, and decreased ROS accumulation caused by beta-AP25-35. The effective doses of Sal B were far lower than those of EGb 761.. Sal B was much more efficient than EGb 761 in inhibiting beta-AP aggregation and in protecting PC12 cells from beta-AP-induced cytotoxicity.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Benzofurans; Cell Survival; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Flow Cytometry; Ginkgo biloba; Intracellular Fluid; Microscopy, Electron; Neuroprotective Agents; PC12 Cells; Peptide Fragments; Plant Extracts; Plants, Medicinal; Rats; Reactive Oxygen Species; Salvia miltiorrhiza

The technics of optimal extraction for compound shenzhutiaozhi capsule is investigated in this study.. Orthogonal test were employed for selecting the optimum of extraction technics and the contents of oleanolic acid and salvianolic acid B were detected by TLC-scanning and HPLC, respectively.. Five volume of 70% alcohol, extracting 3 times with half an hour for each time, and seven volume of water, extracting 3 times with half an hour for each time were considered the optimum extraction technics of Frugtus Ligustri lucid, Salvie miltiorrhizae as well as Reizaoma Atractylodis Macrocfphalae, respectively.. The considerable extraction rate of active components in the drugs is achieved by applying the selected technics.

Topics: Benzofurans; Capsules; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Drugs, Chinese Herbal; Ethanol; Fruit; Ligustrum; Methanol; Oleanolic Acid; Plants, Medicinal; Rhizome; Salvia miltiorrhiza; Technology, Pharmaceutical; Water

To develop a method for the determination of salvianolic acid B and paeoniflorin in Shuangdan granules.. The chromatographic separation was performed on a Zorbax SB C18 column with a linear gradient elution of phosphoric acid-water (0.05:100) and phosphoric acid-acetonitrile-methanol (0.05:4:96) was used. The flow rate was 1 mL x min(-1) and column temperature was set at 25 degrees C. The UV detection wavelength was set at 230 nm.. The linear ranger was of 0.710-22.7 microg for salvianolic acid B and 0.103-3.30 microg for paeoniflorin. Three batches of Shuangdan granules were analyzed, and the content of salvianolic acid B ranged from 6.46-11.6 mg x g(-1) and paeoniflorin ranged from 1.44-1.78 mg x g(-1).. The method was accurate, reproducible, reliable, and could be used for quantitative control of Shuangdan granules.

Topics: Benzoates; Benzofurans; Bridged-Ring Compounds; Chromatography, High Pressure Liquid; Drug Combinations; Drugs, Chinese Herbal; Glucosides; Monoterpenes; Paeonia; Plants, Medicinal; Quality Control; Reproducibility of Results; Salvia miltiorrhiza

In this study, we have investigated the mechanism of action through which salvianolic acid, a constituent of the medicinal herb danshen (Salvia miltiorrhiza), causes relaxation of isolated coronary artery rings precontracted with 1 microM 5-hydroxytryptamine (5-HT). The vasorelaxant effects of salvianolic acid B were also compared with the aqueous extract of danshen. Extraction of the water-soluble fraction from danshen provided a yield of 17.5%. The amount of salvianolic acid B determined in this aqueous extract was 3.9%, and the extract was 6.3 times less potent than salvianolic acid B in relaxing 5-HT-precontracted coronary artery rings; IC(50) values were 930.3+/-133.5 microg/ml and 147.9+/-17.4 microg/ml, respectively. Removal of the endothelium did not significantly affect their vasodilator potencies; IC(50) values were 842.1+/-123.8 mictog/ml and 160.3+/-25.9 microg/ml. On the other hand, a potassium channel inhibitor tetraethylammonium (TEA, 10 mM) shifted their concentration-response curves by 1.7 and 2.9 folds. The possible involvement of Ca(2+) channels was investigated in artery rings incubated with Ca(2+)-free buffer and primed with 1 microM 5-HT or 60 mM KCl for 5 min prior to adding CaCl(2) to elicit contraction. In 5-HT-primed preparations, 2 mg/ml danshen aqueous extract and 0.72 mg/ml salvianolic acid B abolished the CaCl(2)-induced vasoconstriction, whereas, in KCl-primed preparations, 10 mg/ml danshen aqueous extract and 1.44 mg/ml salvianolic acid B produced 90% inhibition. These findings suggest the vasorelaxant effects of danshen aqueous extract and salvianolic acid B were produced by inhibition of Ca(2+) influx in the vascular smooth muscle cells. The opening of K(+) channels had a minor contribution to their effects, but endothelium-dependent mechanisms were not involved. The high vasodilator potency and the quantity of salvianolic acid B contained in danshen aqueous extract suggest it is an important vasodilator constituent in this medicinal herb.

Topics: Animals; Benzofurans; Calcium Channel Blockers; Calcium Channels; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Coronary Vessels; Endothelium, Vascular; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nifedipine; Plant Extracts; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Salvia; Serotonin; Tetraethylammonium

Rapid nondestructive determination of salvianolic acid B and tanshinone IIA in Radix Salviae Miltiorrhizae with near-infrared reflectance spectroscopy.. A quantitive model was built up with near-infrared diffuse reflectance spectroscopy.. The RMSEP in quantitative calibration model for salvianolic acid B and tanshinone IIA were 0.259 and 0.0232 respectively.. NIR technique can dispose the samples without complicated pretreatment. You can achieve the results rapidly and correctly. It owns many remarkable advantages that cannot be displayed by traditional analysis methods. It is qualified to rapidly analyze traditional Chinese medicine whose components are complex. NIR can control the quality in production process of traditional Chinese medicine.

Topics: Abietanes; Benzofurans; Electronic Data Processing; Least-Squares Analysis; Models, Theoretical; Phenanthrenes; Plant Roots; Plants, Medicinal; Quality Control; Reproducibility of Results; Salvia miltiorrhiza; Spectroscopy, Near-Infrared

A precise and reproducible HPLC method has been established and validated for determination of salvianolic acid B (SalB) in rat plasma after oral administration of Radix Salviae Miltiorrhizae extract. Liquid-liquid extraction was adopted for the sample preparation. Separation was accomplished on a C(18) column with a linear gradient elution consisting of acetonitrile and aqueous phosphoric acid. Ultraviolet detection was at 280 nm. The method was validated over the concentration range 10.8-259.4 microg/mL using 1 mL of plasma. The assay was linear over this concentration range with a coefficient of variation less than 7%. The extraction recovery of SalB was within the range 71-83% with RSD 11%. The mean recovery of the internal standard was 84% (n = 6) with RSD of 5.6%. This method is suitable to determine SalB in plasma and to investigate the pharmacokinetics of SalB.

Topics: Acetonitriles; Administration, Oral; Animals; Benzofurans; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Male; Phosphoric Acids; Plant Extracts; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Salvia miltiorrhiza; Sensitivity and Specificity

Herbal medicine has become an increasing popular therapeutic alternative among patients suffering from various inflammatory disorders. The Salvia miltiorrhizae water-soluble extract (SME) have been shown to possess antioxidant and anti-inflammatory properties in vitro. However, the mechanism of action and impact of SME on LPS-induced gene expression is still unknown. We report that SME significantly abrogated LPS-induced IkappaB phosphorylation/degradation, NF-kappaB transcriptional activity and ICAM-1 gene expression in rat IEC-18 cells. Chromatin immunoprecipitation assay demonstrated that LPS-induced RelA recruitment to the ICAM-1 gene promoter was inhibited by SME. Moreover, in vitro kinase assay showed that SME directly inhibits LPS induced IkappaB kinase (IKK) activity in IEC-18 cells. To investigate the physiological relevance of SME inhibitory activity on NF-kappaB signalling, we used small intestinal explants and primary intestinal epithelial cells derived from a transgenic mouse expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of NF-kappaB cis-elements (cis-NF-kappaB(EGFP)). SME significantly blocked LPS-induced EGFP expression and IkappaBalpha phosphorylation in intestinal explants and primary IECs, respectively. However, salvianolic acid B, an activate component of SME did not inhibit NF-kappaB transcriptional activity and IkappaB phosphorylation/degradation in IEC-18 cells. These results indicate that SME blocks LPS-induced NF-kappaB signalling pathway by targeting the IKK complex in intestinal epithelial cells. Modulation of bacterial product-mediated NF-kappaB signalling by natural plant extracts may represent an attractive strategy towards the prevention and treatment of intestinal inflammation.

Topics: Animals; Benzofurans; Cells, Cultured; Chromatin Immunoprecipitation; Drugs, Chinese Herbal; Epithelial Cells; Gene Expression Regulation; I-kappa B Proteins; Intercellular Adhesion Molecule-1; Intestinal Mucosa; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Transgenic; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Salvia miltiorrhiza; Signal Transduction; Solubility; Translocation, Genetic; Water

To study the therapeutic mechanism of salvianolic acid B (SA-B) in treating hepatic fibrosis.. Twenty-four Wistar rats were randomly divided into 3 groups: normal control group, untreated group and SA-B-treated group. Rats in the untreated and SA-B-treated groups were injected intraperitoneally with 0.5% dimethylnitrosamine (DMN) for 4 weeks, daily for 3 days each week at a dose of 10 microg/kg, to induce hepatic fibrosis. Then, rats in the SA-B-treated group were given SA-B orally for another 4 weeks. Hydroxyproline (Hyp) contents in liver tissue of the rats in 3 groups were determined with HCl hydrolysis, and collagen type I, transforming growth factor-beta 1 (TGF-beta1), transforming growth factor-beta receptor type I (TbetaRI) and transforming growth factor-beta receptor type II (TbetaRII) were detected by Western blotting.. The Hyp content and the expressions of collagen type I, TGF-beta1, TbetaRI and TbetaRII in the liver tissue of rats in the untreated group increased significantly as compared with those in the normal control group, while those in SA-B-treated group decreased significantly as compared with those in the untreated group.. The therapeutic mechanism of SA-B in treating hepatic fibrosis may be related to inhibiting the expressions of TGF-beta1 and its receptors.

Topics: Animals; Benzofurans; Collagen Type I; Dimethylnitrosamine; Hydroxyproline; Liver; Liver Cirrhosis, Experimental; Male; Protein Serine-Threonine Kinases; Random Allocation; Rats; Rats, Wistar; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1

Salvia miltiorrhiza Bunge, a traditional Chinese herbal medicine, is often used for prevention and treatment of cardiovascular disorders such as atherosclerosis. To understand its mechanism of pharmacological action, its effects on endothelial monolayer permeability are studied. The present study demonstrated that extract of S. miltiorrhiza (ESM) and its major ingredients, Danshensu (DSS) and salvianolic acid B (Sal B), inhibited tumor necrosis factor (TNF-alpha) induced endothelial permeability, whereas the other major ingredient, protocatechualdehyde, was ineffective. ESM, DSS and Sal B also repressed expression of vascular endothelial growth factor (VEGF) and extracellular signal-regulated kinase (ERK) activation in TNF-alpha induced HUVEC cells. Furthermore, it was found that ESM attenuated the disorganization of vascular endothelial (VE)-cadherin induced by TNF-alpha. The effect of ESM on TNF-alpha induced endothelial permeability and redistribution of VE-cadherin is likely due to a reduction of VEGF protein expression as a result of modulation of the ERK signaling pathway. Endothelial cell hyperpermeability is implicated in inflammation and subsequent ischemic reperfusion injury and atherosclerosis. Data from this study suggest that one of the mechanisms S. miltiorrhiza exerts its pharmacological effect is through its modulation of endothelial cell permeability.

Topics: Benzaldehydes; Benzofurans; Cadherins; Capillary Permeability; Catechols; Cells, Cultured; Endothelial Cells; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Humans; Lactates; Plant Extracts; Salvia miltiorrhiza; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Plasminogen activator inhibitor type 1 (PAI-1), which plays a role in the development of atherosclerosis, is produced by endothelial cells following stimulation with various inflammatory cytokines such as tumor necrosis factor (TNF-alpha). In the present study, we investigated the effects of a potent water-soluble antioxidant, salvianolic acid B (SalB; derived from the Chinese herb, Salvia miltiorrhiza), on the expression of PAI-1 in TNF-alpha-treated human umbilical vein endothelial cells (HUVECs). We found that SalB inhibited TNF-alpha-induced PAI-1 mRNA production and protein secretion in HUVECs. Treatment with SalB (0.05 and 0.15 microM) notably attenuated TNF-alpha induced expression of PAI-1 to 90.5% and 74.6%, respectively, after 12 h, and to 75.1% and 64.2%, respectively, after 18 h. We also observed a dose-dependent decrease in PAI-1 protein production in the presence of SalB. We then used pathway inhibitors to investigate which step of the TNF-alpha induced signaling pathway was targeted by SalB. We found that the c-Jun N-terminal kinase (JNK) inhibitor, SP600125, increased the inhibitory effects of SalB on TNF-alpha-induced PAI-1 secretion, whereas the nuclear factor-kappaB (NF-kappaB) inhibitor, emodin, and the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, did not. A gel shift assay further showed that SalB inhibited the TNF-alpha-activated NF-kappaB and AP-1 DNA binding activities in a dose-dependent manner. Collectively, these results indicate that the NF-kappaB and ERK-AP-1 pathways are possible targets of SalB in the regulation of TNF-alpha-stimulated PAI-1 production in HUVECs.

Topics: Benzofurans; Cells, Cultured; Drugs, Chinese Herbal; Endothelial Cells; Endothelium, Vascular; Humans; NF-kappa B; Plasminogen Activator Inhibitor 1; RNA, Messenger; Signal Transduction; Tissue Plasminogen Activator; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Umbilical Veins

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been developed for continuous monitoring of protein-unbound salvianolic acid B in rat blood and bile. Microdialysis probes were inserted into the jugular vein/right atrium and bile duct of Sprague-Dawley rats, and a dose of 100 mg/kg salvianolic acid B was then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Salvianolic acid B was eluted using a microbore reversed-phase ODS 5 microm (150 mm x 1 mm I.D.) column. Isocratic elution of salvianolic acid B was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of acetonitrile-methanol-20 mM monosodium phosphoric acid (pH 3.5) (10:30:60, v/v/v) containing 0.1 mM 1-octanesulfonic acid with 0.05 ml/min. The wavelength of the UV detector was set at 290 nm. Salvianolic acid B in both blood and bile dialysates was adequately determined using the liquid chromatographic conditions described, although the blank bile pattern was more complex. The retention times of salvianolic acid B in rat blood and bile dialysates were found to be 7.2 min. Peak-areas of salvianolic acid B were linear (r2 > 0.995) over a concentration range of 0.1-50 microg/ml. In vivo recoveries of microdialysis probes of salvianolic acid B in rat blood and bile averaged 22 +/- 2% and 41 +/- 1%, respectively. This study indicates that salvianolic acid B undergoes hepatobiliary excretion.

Topics: Animals; Benzofurans; Bile; Chromatography, High Pressure Liquid; Male; Microdialysis; Rats; Rats, Sprague-Dawley; Reference Standards

To purify salvianolic acids by macroreticular resin,then mensurate the contents of salvianolic acids and analyse the chromatogram with HPLC.. Make salvianolic acids with macroreticular resin; mensurate the content of Salvianolic acids with UV spestrophotometry: the control compound is protocaechuic aldehyde, and the wavelength is 281 nm. Analysis the chromatogram with HPLC, and compare the chromatogram in different technics: zorbax ODS column (4.6 mm x 250 mm, 5 microm), mobilephase: 1% aceticacid-water and methanol in different proportions, the wavelength is 281 nm.. The contents of salvianolic acids is 53.8%; HPLC chromatogram indicate that the method is reasonable to make salvianolic acids.. Determination of contents and HPLC chromatogram can control the quality of Salvianolic acids more accurately.

Topics: Benzofurans; Caffeic Acids; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Ion Exchange Resins; Lactates; Salvia miltiorrhiza

The absorption and pharmacokinetics of an active component of Salvia miltiorrhiza, lithospermic acid B (LSB), was investigated after intravenous and oral administration of doses of 10 or 50 mg LSB/kg to rats. Concentrations of LSB were determined by a validated liquid chromatography/mass spectrometry (LC/MS/MS) assay method. After intravenous administration of 50 mg/kg, dose-normalized (10 mg/kg) area under the curve (AUC) (993 microg.min/ml) was significantly greater than that at 10 mg/kg (702 microg.min/ml). The slower clearance Cl-at 50 mg/kg could be due to saturable metabolism of LSB in rats, and this could be supported by significantly slower Cl(NR) and significantly greater 24-h urinary excretion of LSB at 50 mg/kg than at 10 mg/kg. Following oral administration of LSB, the extent of LSB recovered from the entire gastrointestinal tract at 24 h ranged from 41.2% to 23.3%. Although LSB was not detected (limit of quantitation 10 ng/ml) in plasma after oral dose of 10 mg/kg, the absolute oral bioavailability at 50 mg/kg was 5%. Since LSB was shown to have low permeability through the Caco-2 cell monolayers, the low bioavailability of LSB could be due to poor absorption and metabolism.

Topics: Administration, Oral; Animals; Benzofurans; Biological Availability; Caco-2 Cells; Cell Membrane Permeability; Depsides; Humans; Hydrogen-Ion Concentration; Injections, Intravenous; Male; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza

Salvianolic acid B (SA-B), one of water soluble compounds derived from Radix salviae miltiorrhizae, had good action against liver fibrosis of patients with chronic hepatitis. Hepatic stellate cells (HSCs) is the cellular resource for liver fibrogenesis, while transforming growth factor-beta1 (TGF-beta1) is most potent fibrogenic factor. In this study we investigated the mechanism of SA-B action against liver fibrosis relating to the interference with TGF-beta1 signaling at HSC.. Hepatic stellate cells (HSCs) were isolated, cultured, and incubated with SA-B. The TGF-beta1 content in the supernatant of subcultured HSCs was assayed with ELISA. Type I collagen and Smad3 protein in TGF-beta1-stimulated primarily cultured HSCs for 4 days were detected by Western blot.. TGF-beta1 secreted in activated HSCs was more than in primary HSCs, and SA-B significantly decreased TGF-beta1 secretion in activated HSCs. TGF-beta1 increased the expression of type I collagen and Smad3 protein in d4 primary HSCs, while SA-B inhibited their expression.. SA-B inhibits TGF-beta1 secretion in activated HSCs and counteracts the expression of TGF-beta1 stimulated type I collagen and Smad3. These actions are associated with the effect of SA-B on liver fibrosis.

Topics: Animals; Benzofurans; Blotting, Western; Cells, Cultured; Collagen Type I; DNA-Binding Proteins; Enzyme-Linked Immunosorbent Assay; Hepatocytes; Male; Models, Animal; Rats; Rats, Wistar; Reference Values; Sensitivity and Specificity; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

Two phenylpropanoid sucrose esters were isolated from dry rhizomes of Canna edulis Ker Gawl., along with a known phenylpropanoid sucrose ester and four known phenylpropanoids. On the basis of analysis of spectroscopic data and chemical evidence, these two phenylpropanoid sucrose esters were shown to be 3-O-p-coumaroyl-6-O-feruloyl-beta-D-fructofuranosyl 6-O-acetyl-alpha-D-glucopyranoside and 3,6-di-O-p-coumaroyl-beta-D-fructofuranosyl 6-O-acetyl-alpha-D-glucopyranoside.

Topics: Benzofurans; Caffeic Acids; Chromatography, High Pressure Liquid; Cinnamates; Depsides; Esters; Magnetic Resonance Spectroscopy; Marantaceae; Molecular Structure; Optical Rotation; Phenylpropionates; Rosmarinic Acid; Sucrose

The present study was designed to examine whether lithospermic acid B (LSB) isolated from Salvia miltiorrhiza has an ameliorative effect on renal functional parameters in association with the expression of aquaporin 2 (AQP 2) and Na,K-ATPase in the ischemia-reperfusion induced acute renal failure (ARF) rats. LSB showed strong antioxidant activity against production of reactive oxygen species (ROS), ROS-induced hemolysis, and production of lipid peroxide in a dose-dependent manner. Polyuria caused by down-regulation of renal AQP 2 in the ischemia-reperfusion induced ARF rats was partially restored by administration of LSB (40 mg/kg, i.p.), restoring expression of AQP 2, in renal inner and outer medulla. The expression of Na,K-ATPase alpha1 subunit in outer medulla of the ARF rats was also restored in the ARF rats by administration of LSB, while beta1 subunit level was not altered. The renal functional parameters including creatinine clearance, urinary sodium excretion, urinary osmolality, and solute-free reabsorption were also partially restored in ischemia-ARF rats by administration of LSB. Histological study also showed that renal damages in the ARF rats were abrogated by administration of LSB. Taken together, these data indicate that LSB ameliorates renal defects in rats with ischemia-reperfusion induced ARF, most likely via scavenging of ROS.

Topics: Acute Kidney Injury; Animals; Aquaporin 2; Aquaporins; Benzofurans; Blotting, Western; Depsides; Enzyme Inhibitors; Erythrocytes; Free Radical Scavengers; Hemolysis; Hydroxyl Radical; In Vitro Techniques; Kidney; Kidney Function Tests; Lipid Peroxidation; Male; Oxidants; Plant Roots; Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Salvia miltiorrhiza; Sodium-Potassium-Exchanging ATPase; Superoxides

Tetramethylpyrazine (TMP) and salvianolic acid B (SAB) are effective ingredients of Rhizoma Ligustici chuanxiong Hort. and Radix Salviae miltiorrhizae Bge., accordingly. The inhibitive effects of TMP, SAB and their combination on shear-induced platelet aggregation (SIPA) were investigated in the present study. SD rats were used as blood donors to collect anticoagulated blood, the concentration of platelet-rich-plasma (PRP) was adjusted to 5 x 10(5) microl. HAAKE rheometer RS 600 with sensor C60/0.5 degrees was used as shear generator. Different doses of TMP and SAB and their combinations were added to the PRP. After constant shear of 15 Pa at 37 degrees C for 360 seconds, PRP was transferred to a platelet aggregometer and SIPA was determined by turbidity. SIPA was inhibited by TMP and SAB in a dose-dependent manner. SIPA was decreased from 48.6 +/- 4.6% of the control to 12.5 +/- 2.1% in the presence of TMP (1.46 mM) and SAB (10 microM) (P < 0.0001). In conclusion, TMP and SAB have additive effects on inhibiting platelet aggregation induced by high shear stress.

Topics: Animals; Benzofurans; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Synergism; Drugs, Chinese Herbal; Hemorheology; Nephelometry and Turbidimetry; Platelet Aggregation; Platelet Aggregation Inhibitors; Pyrazines; Rats; Stress, Mechanical

To investigate the inhibiting effect of salianic-acid B (SA-B) on mitogen-activated protein kinase (MAPK) Signaling in activated rat hepatic stellate cells (HSCs).. HSCs were isolated from normal rat by in situ perfusion and Nycodenz density-gradient centrifugation method. HSCs were primarily cultured on uncoated plastic for 7 days. Then cells were stimulated with 10ng/ml transforming growth factor-beta1 (TGF-beta1) after incubated with 10-6 M/L SA-B. The effects of SA-B on Extracellular-regulated kinase (ERK) expression and its phosphorylation. Transforming growth factor beta1 receptor I (TbetaR I) and transforming growth factor beta1 receptor II (TbetaR II) on HSCs, type I collagen expression in HSC Induced by TGF-beta1 were detected with western blot assay. Quantity of Type I collagen in the medium of HSCs was detected by ELISA. Matrix metalloproteinase 2, 9, 13 (MMP-2, MMP-9 and MMP-13) in the medium of HSCs was tested by Zymography.. The phosphorylation of ERK1/2 in HSCs with or without TGF-beta1 was inhibited by SA-B. The expression of TbetaR I and TbetaR II on HSCs can not be affected by SA-B. The synthesization of Type I collagen in HSCs was decreased by SA-B; The synthesization and secretion of type I collagen in HSCs with TGF-beta1 were reduced by SA-B too. SA-B had no effect on the activity of MMP-2 and MMP-13, but induced the activity of MMP-13.. SA-B inhibits ERK signaling induced by TGF-b1 in HSC. This inhibition has no association with the expression of TbetaR I and TbetaR II on HSCs. SA-B reduces the synthesization and secretion of Type I collagen in HSC by means of inhibiting TGF-beta1 signaling, which might be not related to the degrading activities of MMPs.

Topics: Animals; Benzofurans; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Liver; Male; Mitogen-Activated Protein Kinase Kinases; Rats; Rats, Sprague-Dawley; Signal Transduction; Transforming Growth Factor beta

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/ MS) method for the determination of lithospermic acid B (LSB) in rat serum was developed. LSB and internal standard, 7-hydroxy-3-phenyl-chromen-4-one (HPC) were extracted from rat serum with methyl-tert-butyl ether at acidic pH and analyzed on a Luna C8 column with the mobile phase of acetonitrile-ammonium formate (10 mM, pH 6.5) (50:50, v/v). The analytes were detected using a negative electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r2= 0.997) over the concentration range of 10.0-500 ng/mL. The coefficient of variation and relative error for intra- and interassay at three QC levels were 1.1 approximately 6.2% and -10.3 approximately -2.7%, respectively. The recovery of LSB from serum sample ranged from 73.2 to 79.5%, with that of HPC (internal standard) being 75.1 %. The lower limit of quantification for LSB was 10 ng/mL using 50 microL of serum sample.

Topics: Animals; Benzofurans; Chromatography, Liquid; Depsides; Male; Mass Spectrometry; Plant Roots; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza; Spectrometry, Mass, Electrospray Ionization

To screen angiotensin converting enzyme inhibitor (ACEI) from traditional Chinese medicine, Salvia miltiorrhiza.. Hydrophilic and lipophilic fractions of S. miltiorrhiza were isolated, and their effective components were screened by a fluorimetric assay for inhibition of angiotensin converting enzyme (ACE).. Water-soluble fractions, total salvianolic acids and salvianolic acid B markedly decreased ACE activity of rabbit lung tissue. Their IC50 value were (2.45 +/- 0.07), (0.24 +/- 0.02), (0.02 +/- 0.01) g x L(-1) respectively. Lipophilic components or phenanthraquinones including tanshinone I and II showed no changes on the activity of ACE.. S. miltiorrhiza inhibits angiotensin converting enzyme and its active components are in aqueous extract, in which the main were salvianolic acids including salvianolic acids B.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Benzofurans; Drugs, Chinese Herbal; In Vitro Techniques; Lung; Peptidyl-Dipeptidase A; Plant Roots; Plants, Medicinal; Rabbits; Rats; Salvia miltiorrhiza

Salvia miltiorrhiza (SM) has been used clinically in Asian countries to improve the microcirculation in the human body. Although a pure compound extracted from SM, salvianolic acid B (Sal B), has been reported to be effective against fibrosis and ischemia-reperfusion injury, possibly through its anti-lipid peroxidation action, the effect of SM on angiogenesis remains unclear. It is our interest to investigate the role of SM on the regulation of the angiogenic process. By using the SVR endothelial cell line as an in vitro system, the effects of Sal B on the gene expression of vascular endothelial growth factor (VEGF) and its receptors VEGF-R1, VEGF-R2 were evaluated by morphology, differentiation assay, reverse-transcribed polymerase chain reaction (RT-PCR) and Western blot analysis. The results showed that both the crude extract of SM and the pure compound Sal B had enhancing effects on cell growth and differentiation. The gene expression of matrix metalloproteinase-2 (MMP-2) was up-regulated after Sal B treatment for 2 h, while VEGF and VEGF-R2 gene expression were up-regulated 40 min after Sal B treatment. We conclude that the crude extract of SM and Sal B enhance angiogenic processes on SVR cells through up-regulation of VEGF and VEGF receptors genes.

Topics: Animals; Benzofurans; Blotting, Western; Cell Line; Drugs, Chinese Herbal; Endothelial Growth Factors; Endothelium, Vascular; Gene Expression Regulation; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Mice; Neovascularization, Physiologic; Phytotherapy; Receptors, Vascular Endothelial Growth Factor; Salvia miltiorrhiza; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To investigate the effects of salvianolic acids on human umbilical vein endothelial cells (HUVEC) against damage induced by cholestane-3beta-5alpha-6beta-triol (chol-triol).. The viability of HUVEC was measured by MTT method. The apoptosis of HUVEC induced by chol-triol was detected by flow cytometry and TUNEL assay. The production of malondialdehyd (MDA) in HUVEC was tested by thiobarbaturic acid (TBA) assay.. The viability of HUVEC treated with chol-triol 100 micro mol/L decreased by 39.8% while salvianolic acids 100 micro g/ml increased by 27.9%. The apoptotic rate of HUVEC measured by PI staining increased from 6% - 8% to 17% - 20% after chol-triol treatment for 12 h. Salvianolic acids 100 micro g/ml reduced the apoptotic rate to 10% - 14% after treatment HUVEC for 1 h prior to chol-triol treatment. In another experiment, chol-triol increased the number of TUNEL-positive cells 5 times, but salvianolic acids 10 micro g/ml and 100 micro g/ml reduced the number of TUNEL-positive cells by 36.9% and 61.2%, respectively. The production of MDA in HUVEC increased by 120.7% after chol-triol treatment for 12 h. Salvianolic acids 10 micro g/ml and 100 micro g/ml also decreased the concentration of MDA by 28.7% and 39.8%, respectively.. Salvianolic acids has protective effect on endothelial cells against damage induced by chol-triol.

Topics: Apoptosis; Benzofurans; Caffeic Acids; Cell Survival; Cells, Cultured; Cholestanols; Cinnamates; Depsides; Endothelium, Vascular; Humans; Lactates; Malondialdehyde; Rosmarinic Acid

The roots of Salviae miltiorrhiza (RSM) have been traditionally used for treatment of hypertensive disease in China, Korea, and Japan. Bioassay guided fractionation and purification as assayed by angiotensin converting enzyme (ACE) inhibitory assay resulted in the isolation of lithospermic acid B (LSB) as an active principle. The ACE plasma activities were significantly inhibited by the addition of LSB in a dose-dependent manner of which IC50 value was 86 microg/ml (120 microM). Moreover, angiotensin I-induced contraction was markedly attenuated by prior exposure of endothelium-intact aortic rings to LSB. These results suggest that RSM-induced antihypertensive effect may be, at least in part, due to ACE inhibitory effect of LSB.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Aorta; Benzofurans; Depsides; Dose-Response Relationship, Drug; Humans; Hypertension; Inhibitory Concentration 50; Male; Muscle Contraction; Phytotherapy; Plant Extracts; Plant Roots; Rats; Rats, Sprague-Dawley; Salvia miltiorrhiza

Insufficient angiogenesis and microcirculatory intravascular clotting have been implicated in the pathophysiology of skin flap failure. Salvianolic acid B (Sal B), isolated from Salvia miltiorrhiza, has been reported to enhance angiogenesis in vitro. This study was aimed to determine the efficacy of Sal B on ischemia-reperfusion injury of the skin flap in Sprague-Dawley rats. Sal B was administered intraperitoneally 2 h before operation, and on the 2nd and 4th days after surgical elevation of an extended epigastric adipocutaneous flap (5 x 7 cm) in ketamine-anesthetized rats. Flap ischemia was achieved by ligating the right superficial epigastric artery and vein and clamping the left superficial epigastric artery and vein for 3 h and then released. Percentage of flap necrosis area (FNA) and plasma levels of aspartate aminotransferase, alanine aminotransferase, creatinine, and malondialdehyde were measured at 7 days after the operation. Animals were divided into six groups, including: vehicle, Sal B low dose (5 mg/kg), Sal B high dose (50 mg/kg) and each with [mesh(+)] or without mesh [mesh(-)] placement. In the three groups with mesh(+), FNA in control flaps was 53.7 +/- 6.9%, whereas low-dose and high-dose Sal B significantly improved flap survival with FNA 27.4 +/- 3.8% and 25.3 +/- 4.3%, respectively (P < 0.05, one-way ANOVA). In the three groups with mesh(-), control flaps were 35.9 +/- 4.5%, whereas high-dose Sal B also significantly improved flap survival with FNA 17.9 +/- 4.7% (P < 0.05, one-way ANOVA). There were no differences in aspartate aminotransferase, alanine aminotransferase, creatinine, or malondialdehyde between groups. We conclude that Sal B attenuates ischemia-reperfusion injury of skin flap, and provides therapeutic potential in reconstructive plastic surgery.

Topics: Animals; Benzofurans; Cell Line; Disease Models, Animal; Drugs, Chinese Herbal; Gene Expression; In Vitro Techniques; Kidney; Liver; Male; Malondialdehyde; Matrix Metalloproteinase 2; Necrosis; Neovascularization, Physiologic; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Skin; Surgical Flaps; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

To investigate the mechanism of salvianolic acid-B (SA-B) action against liver fibrosis relating to mediating hepatic stellate cell (HSC) activation and transforming growth factor-beta1(TGF-beta1) intracellular signal transduction.. HSC was isolated from normal rat through in situ perfusion of liver with pronase E and density-gradient centrifugation with 11 % nycondenz, then cells were subcultured. Cell proliferation was observed by [3H]TdR uptake. Cellular collagen deposition was measured with Ponceau S stain and semi-quantified with image analytic system. Type I collagen secretion in the supernatant was detected with ELISA. The gene expression of type I pro-collagen was analyzed by RT-PCR. The supernatant was acidified and active TGF-beta1 contents were assayed with ELISA. Mitogen-activated protein kinase (MAPK) activity was analyzed with immunoprecipitation and Western blot.. SA-B 0.1, 1, 10, and 100 micromol/L suppressed HSC proliferation concentration-dependently as determined by [3H]TdR uptake by 94.1 %, 82.4 %, 62.7 %, and 4 % of the control respectively (P<0.05 or P<0.01). SA-B 1, 10, and 100 micromol/L inhibited soluble type I collagen secretion by 75.3 %, 69.8 %, and 63.5 % of the control and decreased the matrix collagen deposition to 86.2 %, 75.4 %, and 73.4 % (P<0.05 or P<0.01). SA-B 1 and 10 micromol/L decreased the cell active TGF-beta1 secretion by 63.3 % and 15.6 % of the control, down-regulated pro-collgen alpha1(I) mRNA expression to 77.0 % and 51.8 % respectively (P<0.05). SA-B 1 and 10 micromol/L also inhibited MAPK activity by 1 to 2 fold respectively.. SA-B inhibited HSC proliferation and collagen production as well as decreased the cells' TGF-beta1 autocrine and MAPK activity, which might contribute to the mechanism of SA-B action against hepatic fibrosis.

Topics: Animals; Benzofurans; Cells, Cultured; Collagen Type I; Hepatocytes; Male; Mitogen-Activated Protein Kinase Kinases; Rats; Rats, Wistar; Signal Transduction; Transforming Growth Factor beta

Cell suspension cultures of Lithospermum erythrorhizon produced a large amount of lithospermic acid B, a caffeic acid tetramer, as well as shikonin derivatives (each ca. 10% of dry wt.) when cultured in shikonin production medium M-9. Various culture factors for increasing the production of lithospermic acid B were investigated. Lithospermic acid B production was inhibited by 2, 4-D or NH4+, whereas it was stimulated by Cu2+. These regulatory patterns were similar to those for the production of shikonin derivatives in these cell cultures, suggestive of close relations and similar metabolic regulation between the production of these compounds. Cultivation under light illumination, however, showed that these metabolisms were independently regulated. In particular, blue light showed a stimulatory effect on lithospermic acid B production, while shikonin production was strongly inhibited, indicative of an effective condition for lithospermic acid B production.

Topics: Benzofurans; Cell Culture Techniques; Depsides; Lithospermum; Naphthoquinones; Plant Structures

This study was conducted to observe the effect of salvianolic acids (SA) on regional cerebral blood flow (rCBF) in rats and on platelet aggregation in vitro and in vivo. Cerebral ischemia was produced in rats by occluding of the right middle cerebral artery, together with the right common carotid artery. rCBF was monitored by H2 clearance method with a tissue blood-flow meter. Platelet aggregation induced by collagen, ADP, and AA was measured in vitro and in vivo by platelet aggregometer. Doses of SA at 6 and 10 mg/kg body wt. (i.v.) improved rCBF in rats after ischemia, but had no obvious effect on normal rCBF. In vitro, SA inhibited significantly the platelet aggregation induced by collagen, ADP, and AA with IC50 values of 0.197, 2.22 and 3.29 x 10(3) mg/l, respectively. In vivo, doses of SA at 6 and 10 mg/kg body wt. inhibited significantly the platelet aggregation induced by collagen, and SA at 10 mg/kg body wt. inhibited remarkably platelet aggregation induced by ADP. The results suggest that SA could improve rCBF in the ischemic hemisphere and inhibit platelet aggregation in rats.

Topics: Animals; Benzofurans; Caffeic Acids; Cerebrovascular Circulation; Drugs, Chinese Herbal; In Vitro Techniques; Lactates; Platelet Aggregation Inhibitors; Rats; Rats, Wistar

To investigate hepatic stellate cells (HSC) responses at different differentiation stages on transforming growth factor-beta 1, and to elucidate the mechanisms of salvianolic acid - B (SA-B), a water soluble compound from Salvia miltiorrhiza, against hepatic fibrosis, relating to interference with TGF-beta 1 stimulated HSC activation and intracellular signal transduction via Smads.. HSC was isolated from rat by in situ perfusion of liver and 8.2% nycondenz gradient centrifugation, and primarily cultured on uncoated plastic for 1 d, 4 d and 7 d respectively, which represented quiescent, intermediate and activated phenotypes. The cells were stimulated with 100 pmol/L TGF-beta 1, cell phenotypes were observed under inverted microscope, alpha-actin expression was checked by Western blot, and collagen secretion was measured with [(3)H] proline incorporation and collangenase digestion, then HSC at one definite differentiation stage that responded most sensitively to TGF-beta 1 was selected as the cell model for the following study. 0.1 micromol/L - 1 mmol/L SA-B was incubated with HSC and the cell proliferation was measured by intracellular [(3)H] thymidine pulse. SA-B was also incubated with TGF-beta 1 stimulated HSC, the collagen secretion was measured as above, alpha-actin and plasmin activator inhibitor-1 (PAI-1) were checked with Western blot, and alpha1 (I) procollagen mRNA levels were analyzed with Northern blot. The cytoplasmic and nuclear proteins were extracted, and cytoplasmic and nuclear Smad2, 3 expression and phosphorylation levels were measured with Western blot.. As culture duration prolonged, HSC phenotypes underwent activation gradually, accompanied by the increase of alpha-actin expression and collagen secretion. TGF-beta 1 increased the basal collagen levels at d1, d4 and d7 by 128.6%, 207.7% and 188.2% of the control respectively, while d4 HSC had the most sensitive response, and this intermediate HSC was used as cell model for the following study. Except 0.1 mmol/L-1 mmol/L SA-B caused parts of HSC death, 0.1 micromol/L-10 micromol/L SA-B had no influence on cell shape, but decreased HSC proliferation in a dose depend manner, by 76%, 60.1% and 47.8% of the control respectively. 1 micromol/L-10 micromol/L SA-B remarkably inhibited the collagen secretion of TGF-beta 1 stimulated HSC by 68.6% and 56.1% of the control, PAI-1 and alpha-actin expression, and down-regulated alpha 1 (I) pro-collagen gene expression. 0.1 micro mol/L approximately 10 micro mol/L SA-B decreased the cytoplasmic and nuclear Smad2, 3 protein expression, especially inhibited Smad2 phosphorylation and nuclear translocation.. SA-B obviously inhibits intermediate HSC proliferation, decreases TGF-beta 1 stimulated HSC activation and matrix protein and gene expression, and inhibited stimulated HSC Smad2, 3 protein expression, phosphorylation and nuclear translocation. The inhibition of TGF-beta 1 signaling in HSC and its biological responses is the important mechanism of SA-B against hepatic fibrosis.

Topics: Actins; Animals; Benzofurans; Blotting, Northern; Blotting, Western; Cell Size; Cells, Cultured; Collagen; Collagen Type I; DNA-Binding Proteins; Drugs, Chinese Herbal; Liver; Plasminogen Activator Inhibitor 1; Rats; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad3 Protein; Time Factors; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

To study the expression of vascular endothelial growth factor (VEGF) in U937 foam cells and the inhibitory effect of salvianolic acid B and Ginkgo biloba extract in vitro.. U937 cells were incubated with 80 mg.L-1 oxidized low density lipoprotein (OX-LDL) for 48 h and a macrophage-derived foam cell model was established. The VEGF concentration in the media was determined by ELISA; the VEGF protein expression in cells was measured with immunohistochemistry; the VEGF mRNA level in cells was measured by in situ hybridization; the positive ratio detected by a morphometrical analysis system was used as the amount of the VEGF protein expression and the mRNA level.. After U937 cells were incubated with OX-LDL, VEGF expression level increased greatly both in the cells and in the media. Salvianolic acid B and Ginkgo biloba extract were shown to remarkably inhibit the increase of VEGF. After treated with 10 micrograms/L-1 salvianolic acid B and Ginkgo biloba extract, the VEGF protein concentration in the media and positive ratio in the cells decreased compared with foam cells. After treated with 10 micrograms.L-1 salvianolic acid B and 100 micrograms.L-1 Ginkgo biloba extract, the VEGF mRNA level decreased measured by in situ hybridization.. A high VEGF expression level was determined in U937 foam cells. Salvianolic acid B and Ginkgo biloba extract were found to inhibit VEGF expression significantly in U937 foam cells in vitro.

Topics: Benzofurans; Foam Cells; Gene Expression; Ginkgo biloba; Humans; Plant Extracts; RNA, Messenger; U937 Cells; Vascular Endothelial Growth Factor A

High-speed counter-current chromatography was applied to the isolation and purification of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude salvianolic acid B was obtained by extraction with ethanol-water from S. miltiorrhiza Bunge. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (3:7:1:9, v/v) was successfully performed yielding 342 mg salvianolic acid B at 98% purity from 500 mg of the crude extract in a one-step separation.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Countercurrent Distribution; Salvia

To observe the effect of salvianolic acid-B (SalB) against the cytoxicity of amyloid beta peptide (A-beta)(25-35) to PC12 cells, the cells were incubated with A-beta, and the cytoxicity was investigated by MTT, flow cytometry and a cell free apoptotic system. The expression of prostate apoptotic response-4 (Par-4) was detected by Western blot. Aged A-beta 10 micromol/L significantly inhibited the MTT reduction of PC12 cells, SalB1 micromol/L inhibited the toxicity induced by A-beta. In flow cytometric analysis, PC12 cells treated with A-beta exhibited degraded DNA content characteristic of apoptosis cells (1.53% vs 19.9%). PC12 cells pretreated with SalB (10 nmol/L, 100 nmol/L, 1 micromol/L) manifested relatively low proportion of apoptosis (15.7%, 13.5%, 11.8%, respectively). SalB (10 nmol/L - 1 micromol/L) when added at the beginning of the cell free apoptotic reaction had no apparent effect on the nuclei apoptosis. Pretreatment of PC12 cells with SalB largely prevented the increase in Par-4 expression of the cells when they were exposed to A-beta. The results suggest that Par-4 is involved in the protective effect of SalB against A-beta-induced damage in PC12 cells.

Topics: Amyloid beta-Peptides; Animals; Apoptosis; Apoptosis Regulatory Proteins; Benzofurans; Carrier Proteins; Cell-Free System; Drugs, Chinese Herbal; Flow Cytometry; Intracellular Signaling Peptides and Proteins; Male; Mice; Neuroprotective Agents; PC12 Cells; Peptide Fragments; Rats

Water soluble extracts of the herbal plant, Salvia miltiorrhiza (Danshen) exhibited potent effect against HIV-1 integrase activity in vitro and viral replication in vivo. We have developed an extensive purification scheme to isolate effective, non-toxic inhibitors against human immunodeficiency virus type 1 (HIV-1) using the 3'-processing activity of integrase as a purification guide and assay. Two water soluble compounds, M(5)22 and M(5)32, have been discovered by isolating them from S. miltiorrhiza roots in purities of >99.5% as shown by NMR spectral analysis with yields of 0.018 and 0.038%, respectively. Structural determination revealed that M(5)22 is lithospermic acid and M(5)32 is lithospermic acid B. These two structurally related compounds are potent anti-HIV inhibitors and showed no cytotoxicity to H9 cells at high concentrations (CC(100)>297 microM for M(5)22 and >223 microM for M(5)32). The IC50 for inhibition of 3'-processing by HIV-1 integrase was found to be 0.83 microM for M(5)22 and 0.48 microM for M(5)32. In addition, M(5)22 and M(5)32 inhibited HIV-1 integrase catalytic activities of 3'-joining to the target DNA with IC50 of 0.48 microM for M(5)22 and 0.37 microM for M(5)32. Furthermore, kinetic and mechanistic studies suggested that drug binding to HIV-1 integrase and inhibition of enzymatic activity occur at a fast rate. Both M(5)22 and M(5)32 do not prevent HIV entry in H9 cells. They also show no inhibition of reverse transcriptase activity in infected cells. The levels of intracellular strong stop and full-length viral DNA remained unchanged following drug treatment. However, both inhibitors strongly suppressed the acute HIV-1 infection of H9 cells with IC50 values of 2 and 6.9 microM for M(5)22 and M(5)32, respectively. Thus these two selective integrase inhibitors hold promise as a novel class of therapeutic drugs for AIDS based on their high potencies and absence of cytotoxicity.

Topics: Benzofurans; Cell Line; Depsides; DNA, Viral; Dose-Response Relationship, Drug; Enzyme Inhibitors; HIV Integrase; HIV-1; Humans; Magnetic Resonance Spectroscopy; Plant Extracts; Plant Roots; Salvia miltiorrhiza; Virus Replication

To examine the effect of recombinant human vascular endothelial growth factor (VEGF) on the low density lipoprotein (LDL) permeability of bovine aortic endothelial cells (BAEC) and the inhibitory effect of salvianolic acid B in vitro.. The confluence BAEC monolayers were cultured with normal medium and with medium containing VEGF or salvianolic acid B at various concentrations and for various time periods. The iodine labeled LDL flux across the monolayers was then performed, and radioactivity was measured by SN-695 automatic liquid scintillation counter.. Addition of purified human recombinant VEGF to the BAEC monolayers could significantly increase the permeability of the monolayer to 125I-LDL (P < 0.01). The permeability-increasing activity of VEGF on the BAEC monolayers was both dose and time dependent. Salvianolic acid B could markedly inhibit the VEGF-induced hyperpermeability in BAECs (P < 0.01).. VEGF plays a role in the formation and development of atherosclerosis, and salvianolic acid B has inhibitory effect on VEGF-induced hyperpermeability in BAEC.

Topics: Animals; Aorta; Benzofurans; Capillary Permeability; Cattle; Cells, Cultured; Drugs, Chinese Herbal; Endothelial Growth Factors; Endothelium, Vascular; Intercellular Signaling Peptides and Proteins; Lipoproteins, LDL; Lymphokines; Recombinant Proteins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To develop an HPLC method for determining salvianolic acid B in radix of Salvia miltiorrhiza.. The sample was extracted with 75% methanol. ODS column was used with methanol-5% acetic acid solution (35:65) as mobile phase. Detection wavelength was 281 nm.. Salvianolic acid B was separated well. Good linearity of salvianolic acid B was obtained (r = 0.9997) within the range of 0.17-1.7 micrograms. The average recovery was 98.9%. Repeatability was good, and RSD was 1.82%.. This method can be used for quality control of radix of Salvia miltiorrhiza.

Topics: Benzofurans; Chromatography, High Pressure Liquid; Plant Roots; Plants, Medicinal; Quality Control; Salvia miltiorrhiza

Apoptosis has been suggested to participate in stabilizing cell number in restenosis. Salvia miltiorrhiza (SM) Bunge which is a Chinese herb widely used for the treatment of cardiovascular disorders contains a potent antioxidant, Salvianolic acid B. To determine whether the antioxidant affects vascular apoptosis, the present study examined the frequency of apoptotic cell death in atherosclerotic plaques and in restenotic lesions of cholesterol-fed rabbits. New Zealand White rabbits were treated with a normal diet (normal), a 2% cholesterol diet (HC), a 2% cholesterol diet and endothelial denudation (HC-ED), a 2% cholesterol diet with 5% water-soluble extract of SM (4.8 g/Kg B.W./day) and endothelial denudation (HC-ED-SM), or with a 2% cholesterol diet containing probucol (0.6 g/kg B.W./day) and endothelial denudation (HC-ED-probucol). Apoptosis and associated cell types were examined in serial paraffin sections by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry. The expression of p53, an apoptosis-related protein, was also examined. Apoptosis was mainly detected in the neointima of the three groups with endothelial denudation. The percentage of apoptotic cells in SM-treated group (68.5+/-5.9%) was significantly higher than that of normal (0%), HC (1.9+/-1.2%), HC-ED (46.1+/-5.4%), and probucol-treated (32.8+/-3.9%) groups. The SM treatment markedly reduced the thickness of the neointima which was mainly composed of smooth muscle cells with few macrophages. In accordance with the apoptotic cell counts, positive immunoreactivity for p53 was observed in restenotic lesions from HC-ED, SM-treated and probucol-treated groups but not in the intima of the other two groups. These results suggest that the treatment with salvianolic acid B-rich fraction of SM induces apoptosis in neointima which in turn may help prevent the neointimal thickening.

Topics: Angioplasty; Animals; Antioxidants; Aorta; Apoptosis; Benzofurans; Cell Count; Cholesterol, Dietary; DNA; Electrophoresis, Polyacrylamide Gel; Hyperplasia; Immunohistochemistry; In Situ Nick-End Labeling; Microscopy, Electron; Plant Extracts; Plants, Medicinal; Rabbits; Spectrophotometry, Ultraviolet; Tumor Suppressor Protein p53

Attachment to, and migration of leukocytes into the vessel wall is an early event in atherogenesis. Expression of cell adhesion molecules by the arterial endothelium may play a major role in atherosclerosis. It has been suggested that antioxidants inhibit the expression of adhesion molecules and may thus attenuate the processes leading to atherosclerosis. In the present study, the effects of a potent water-soluble antioxidant, salvianolic acid B (Sal B), and an aqueous ethanolic extract (SME), both derived from a Chinese herb, Salvia miltiorrhiza, on the expression of endothelial-leukocyte adhesion molecules by tumor necrosis factor-alpha (TNF-alpha)-treated human aortic endothelial cells (HAECs) were investigated. When pretreated with SME (50 and 100 microg/ml), the TNF-alpha-induced expression of vascular adhesion molecule-1 (VCAM-1) was notably attenuated (77.2 +/- 3.2% and 80.0 +/- 2.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 microg/ml), 84.5 +/- 1.9%, 78.8 +/- 1.2%, 58.9 +/- 0.4%, 58.7 +/- 0.9%, and 57.4 +/- 0.3%, respectively. Dose-dependent lowering of expression of intercellular cell adhesion molecule-1 (ICAM-1) was also seen with SME or Sal B. In contrast, the expression of endothelial cell selectin (E-selectin) was not affected. SME (50 microg/ml) or Sal B (5 microg/ml) significantly reduced the binding of the human monocytic cell line, U937, to TNF-alpha-stimulated HAECs (45.7 +/- 2.5% and 55.8 +/- 1.2%, respectively). SME or Sal B significantly inhibited TNF-alpha-induced activation of nuclear factor kappa B (NF-kappaB) in HAECs (0.36- and 0.48-fold, respectively). These results demonstrate that SME and Sal B have anti-inflammatory properties and may explain their anti-atherosclerotic properties. This new mechanism of action of Sal B and SME, in addition to their previously reported inhibition of LDL, may help explain their efficacy in the treatment of atherosclerosis.

Topics: Antioxidants; Aorta; Benzofurans; Blotting, Western; Cell Adhesion; Cell Line; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; E-Selectin; Endothelium, Vascular; Gene Expression Regulation; Humans; Intercellular Adhesion Molecule-1; Microscopy, Fluorescence; NF-kappa B; Plant Extracts; Probucol; Salvia miltiorrhiza; Time Factors; Transcription Factor RelA; Tumor Necrosis Factor-alpha; U937 Cells; Vascular Cell Adhesion Molecule-1

To observe the effect of salvianolic acid-B (SalB) on amyloid beta-protein (A-beta) fibril formation and its toxicity towards PC12 cells.. A-beta (1 - 40) was incubated with or without SalB at 25 degrees C for 30 h, 48 h, and 100 h. Fibril formation was then viewed under an electron microscope. Toxicity of the A-beta (1 - 40) towards PC12 cells was measured with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). A-beta (25 - 35) was aged by incubating at 25 degrees C for 7 d, then the peptide was incubated with PC12 cells with or without SalB. Toxicity of A-beta (25-35) towards PC12 cells was observed with MTT.. Following incubation at 25 degrees C for 30 h, A-beta (1 - 40) (100 mg/L) aggregated and formed fibrils. SalB 10-100 nmol/L completely prevented the fibril formation within 30 h. Extension of amyloid fibrils increased with prolonging the incubation time. SalB inhibited the fibril formation process during this period. In the MTT assay A-beta (1 - 40) incubated with SalB manifested significantly lower toxicity to PC12 cells compared with that without SalB. Besides, SalB 1 micromol/L significantly attenuated the toxicity of aged A-beta (25 - 35) to PC12 cells.. SalB could inhibit A-beta aggregation and fibril formation, as well as directly inhibit the cellular toxicity of aged A-beta towards PC12 cells.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Benzofurans; Cell Culture Techniques; Cell Division; Drugs, Chinese Herbal; PC12 Cells; Rats

To study the roles of nitric oxide (NO) and nitric oxide synthase (NOS) on the glutamate (Glu) and beta-amyloid peptide [beta-AP (1-40)] mediated neurotoxicity in primary cultured fetal rat cortical neuron and the neuroprotective effects of salvianolic acid B (Sal B) against the beta-AP (1-40) and its mechanism of action.. With application of specific agonist and antagonist of NOS, establishment of the sodium nitroprusside (SNP), Glu and beta-AP (1-40) neurotoxicity model, the cell viability, lactate dehydrogenase (LDH) efflux and NO release were detected by using morphological observation, MTT stain, spectrophotometric measurement and Griess method, respectively, in primary cultured fetal rat cortical neurons.. Glu and beta-AP (1-40) were shown to increase the NO release of the neuron. Furthermore, nNOS was found to play an important role in the neurotoxicity of glutamate, iNOS may probably be involved in the neurotoxicity of beta-AP (1-40). Sal B (0.01, 0.10, 1.00 microgram.L(-1)) was shown to increase the cell viability, decrease the LDH release rate and inhibit NO release in a dose-dependent manner.. These results suggest that the neurotoxicity of Glu and beta-AP(1-40) may be partly mediated through different types of NOS. Sal B was found to prevent the beta-AP(1-40) toxicity by directly or indirectly decreasing NO release.

Topics: Amyloid beta-Peptides; Animals; Benzofurans; Cell Survival; Cells, Cultured; Cerebral Cortex; Drugs, Chinese Herbal; Female; Fetus; L-Lactate Dehydrogenase; Neurons; Neuroprotective Agents; Nitric Oxide; Rats; Rats, Wistar

This paper deals with the effects of 2,4-D, BA and GA3 on the growth and content of two depsides (rosmarinic acid and lithospermic acid B) in suspension cells of Salvia miltiorrhiza. The results showed cell growth and rosmarinic acid content reached the maximum on the 12th day and lithospermic acid B content on the 16th day after incubating cells in the subculture medium MS + 2, 4-D 1 mg/L + KT 0.1 mg/L. This cell line was a growth-product-associated. With the same concentration (1 mg/L), 2, 4-D stimulated the cell growth but prohibited the formation of lithospermic acid B; GA3 inhabited the cell growth but stimulated the formation of two depsides; The effects of BA is between 2, 4-D and GA3. The concentration optimum of phytohormones tested displayed 3 mg/L for BA and 1 mg/L for GA3. For the optimum time of adding BA and GA3 was in med-term (the 8th day) and early term (at the beginning) of culture period respectively. The synergiatic function of BA and GA3 on the depside formation was also showed that adding GA3 (1 mg/L) was favour for the formation of lithospermic acid B of the suspension cell cultured in the medium containing BA 3 mg/L. The suitable time of adding GA3 was the 6the day after incubating cells in the medium of MS + BA 3 mg/L.

Topics: Benzofurans; Cell Division; Cells, Cultured; Cinnamates; Depsides; Plant Growth Regulators; Plant Leaves; Plants, Medicinal; Rosmarinic Acid; Salvia miltiorrhiza

A high-performance liquid chromatography (HPLC) analysis system based on a water-acetonitrile gradient program was established for simultaneous quantification of shikimate-derived secondary metabolites in cultured cells of Lithospermum erythrorhizon. The cells cultured in pigment production medium (M-9) are capable of producing five highly hydrophilic compounds such as p-hydroxybenzoic acid-O-glucoside and lithospermic acid B, as well as eleven lipophilic compounds including echinofuran B and acetylshikonin. In addition to the wide polarities of those compounds, many of them are unstable under light, dryness, oxygen and heating. Thus, a new extraction procedure for all these compounds was also established by use of ultrasonication under ice-water chilling with MeOH as the solvent. This procedure was applied to the quantitative analyses of these compounds in cell cultures and hairy root cultures of Lithospermum, and in the intact plants as well.

Topics: Anthraquinones; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Benzofurans; Cells, Cultured; Chromatography, High Pressure Liquid; Cold Temperature; Depsides; Furans; Glucosides; Naphthoquinones; Parabens; Plant Roots; Plants, Medicinal; Shikimic Acid; Solvents; Sonication

The objective of this work was to study the protective effects of salvianolic acid B (Sal B) on the dysfunctions of learning and memory induced by transient cerebral ischemia in mice. The mechanisms of its actions also were researched both in vivo and in vitro. The model of dysfunction of learning and memory induced by transient cerebral ischemia in mice was used. One trail passive avoidance tests were used to evaluate the learning and memory functions and experiments in vitro were employed to observe the antioxidative effects of Sal B. Cerebral transient ischemia would impair the function of memory in mice. In step down test. the error number and latency were 2.63 and 120.5 in control group and were 1.35 and 234.4 respectively in sham operated group (p < 0.05). In Sal B treated groups, the error number was less and latency was longer significantly than those of control group. Meanwhile. 3 and 10 mgkg(-1) of Sal B iv. reduced the malondialdehyde contents in cortex, hippocampus and striatum of cerebral transient ischemia rat ion vivo. Sal B 10--100 nmol L(-1) also inhibited lipid-peroxidation and scavenged free hydroxyl radicals in vitro. As conclusion. Sal B ameliorated learning and memory dysfunctions induced by cerebral transient ischemia. Its actions might be related to its antioxidant activity.

Topics: Animals; Antioxidants; Avoidance Learning; Benzofurans; Brain; Free Radical Scavengers; Hydroxyl Radical; Ischemic Attack, Transient; Learning; Lipid Peroxidation; Male; Malondialdehyde; Memory; Mice; Rats; Rats, Wistar

To study the protective effects of salvianolic acid B (Sal B) against the ischemia-reperfusion induced rat brain injury.. Focal cerebral ischemia-reperfusion model in rats was employed to study the protective effects of Sal B. The behavioural tests were used to evaluate the damage to the central nervous system. Spectrophotometric assay methods were used to measure the activity of superoxide dismutase (SOD), contents of reduced glutathione (GSH), malondialdehyde (MDA), adenosine 5-triphosphorate (ATP), and lactate acid (LA) in experimental rats' brain homogenate.. Focal cerebral ischemia-reperfusion resulted in abnormal behavior which could be alleviated by Sal B 10 mg.kg-1 i.v., and nimodipine (Nim) 4 mg.kg-1 i.p. At the same time, Sal B 10 mg.kg-1 and Nim 4 mg.kg-1 could inhibit the decrease in SOD, GSH, and ATP levels and the increase in MDA and LA levels caused by ischemia-reperfusion in brain.. Sal B showed a protective action against the ischemia-reperfusion induced injury in rat brain by reducing lipid peroxides, scavenging free radicals and improving the energy metabolism.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Benzofurans; Brain; Brain Ischemia; Lactic Acid; Male; Malondialdehyde; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase

The dry root and rhizome of Salvia miltiorhiza (Lamiaceae) are used as a crude drug Danshen, while those of S. deserta (Xinjiang-Danshen) are mixed in Danshen at Xinjiang province when the former is in short supply. The water and MeOH extracts of S. deserta showed strong aldose reductase (AR) inhibitory activity, and their active constituents were determined to be polar compounds different from "tanshinones" of S. miltiorhiza, i.e., lithospermic acid B (1), salvianolic acid K (2), salviaflaside (3), and rosmarinic acid (4) (IC50, 2.63-3.91 microM). We also examined the AR inhibitory activity of water and MeOH extracts of seventeen Salvia plants, including ten species of Danshen resources (S. bowleyana, S. deserta, S. miltiorhiza, S. miltiorhiza var. miltiorhiza f. alba, S. paramiltiorhiza, S. paramiltiorhiza f. purpureo-rubra, S. przewalskii, S. przewalskii var. mandarinorum, S. sinica f. purpurea, S. trijuga), and their water extracts were also analyzed by liquid chromatography-mass spectrometry (LC-MS). The results indicated that there were four types with regard to the AR inhibitory activity and three types with regard to the amount of 1. Ten species used as Danshen resources showed good correlation between the AR inhibitory activity and the morphological classification. However, the intensities of their AR inhibitory activity varied, and they contained 1 in varying amounts. These facts suggested that the ten species were not the same, and thus their use as a Danshen resource should be based on their activity and/or active constituents.

Topics: Aldehyde Reductase; Benzofurans; Chromatography, Liquid; Cinnamates; Depsides; Enzyme Inhibitors; Glucosides; Lamiaceae; Mass Spectrometry; Phenylpropionates; Plant Extracts; Plants, Medicinal; Rosmarinic Acid

The incorporation of [3H]thymidine obviously tends to decrease in the primary cultured hepatocytes of rats when the cells are treated with carbon tetrachloride. Meanwhile the activity of ALT markedly increases in the medium. But these pathologic alterations are inhibited by Salvianic acid B(SA-B) and the effect of 10(-6) mol/L SA-B is the best.

Topics: Alanine Transaminase; Animals; Benzofurans; Carbon Tetrachloride; Cells, Cultured; Liver; Male; Rats; Rats, Wistar

The antioxidative effect of three water-soluble components isolated from Salvia miltiorrhiza has been investigated. All the three components were found to inhibit both NADPH-vit C and Fe(2+)-cysteine induced lipid peroxidation (malondialdehyde formation) in rat brain, liver and kidney microsomes in vitro. The order of their inhibitory effect is as follows: salvianolic acid A, salvianolic acid B and rosmarinic acid. The inhibitory effect on lipid peroxidation induced by NADPH-Vit C was more than that induced by Fe(2+)-cysteine. In addition, the three compounds lowered the production of superoxide anion radical (O2-) in xanthine-xanthine oxidase system. The order of their potency was similar to that in antilipoperoxidation. The above results suggest that the three components have strong antilipoperoxidant activity in vitro, which may be partly through scavenging O2-..

Topics: Animals; Antioxidants; Benzofurans; Brain; Caffeic Acids; Cinnamates; Depsides; Drugs, Chinese Herbal; In Vitro Techniques; Kidney; Lactates; Lipid Peroxidation; Male; Malondialdehyde; Microsomes; Microsomes, Liver; Rats; Rats, Inbred Strains; Rosmarinic Acid