sapogenins and protopanaxadiol

Ginsenoside Compound K (CK) has anti-cancer and anti-inflammatory pharmacological activities. It has not been isolated from natural ginseng and is mainly prepared by deglycosylation of protopanaxadiol. Compared with the traditional physicochemical preparation methods, the preparation of CK by hydrolysis with protopanaxadiol-type (PPD-type) ginsenoside hydrolases has the advantages of high specificity, environmental-friendliness, high efficiency and high stability. In this review, the PPD-type ginsenoside hydrolases were classified into three categories based on the differences in the glycosyl-linked carbon atoms of the hydrolase action. It was found that most of the hydrolases that could prepare CK were PPD-type ginsenoside hydrolase type Ⅲ. In addition, the applications of hydrolases in the preparation of CK were summarized and evaluated to facilitate large-scale preparation of CK and its development in the food and pharmaceutical industries.

Topics: Ginsenosides; Hydrolases; Sapogenins

Based on the definite therapeutic benefits, such as neuroprotective, cardioprotective, anticancer, anti-diabetic and so on, the Panax genus which contains many valuable plants, including ginseng (Panax ginseng C.A. Meyer), notoginseng (Panax notoginseng) and American ginseng (Panax quinquefolius L.), attracts research focus. Actually, the biological and pharmacological effects of the Panax genus are mainly attributed to the abundant ginsenosides. However, the low membrane permeability and the gastrointestinal tract influence seriously limit the absorption and bioavailability of ginsenosides. The acid or base hydrolysates of ginsenosides, 20 (R,S)-panaxadiol and 20 (R,S)-protopanaxadiol showed improved bioavailability and diverse pharmacological activities. Moreover, relative stable skeletons and active hydroxyl group at C-3 position and other reactive sites are suitable for structural modification to improve biological activities. In this review, the pharmacological activities of panaxadiol, protopanaxadiol and their structurally modified derivatives are comprehensively summarized.

Topics: Antineoplastic Agents; Dammaranes; Drug Discovery; Ginsenosides; Humans; Neuroprotective Agents; Panax; Phytochemicals; Radiation-Protective Agents; Sapogenins; Triterpenes

Asian ginseng, American ginseng, and notoginseng are three major species in the ginseng family. Notoginseng is a Chinese herbal medicine with a long history of use in many Oriental countries. This botanical has a distinct ginsenoside profile compared to other ginseng herbs. As a saponin-rich plant, notoginseng could be a good candidate for cancer chemoprevention. However, to date, only relatively limited anticancer studies have been conducted on notoginseng. In this paper, after reviewing its anticancer data, phytochemical isolation and analysis of notoginseng is presented in comparison with Asian ginseng and American ginseng. Over 80 dammarane saponins have been isolated and elucidated from different plant parts of notoginseng, most of them belonging to protopanaxadiol or protopanaxatriol groups. The role of the enteric microbiome in mediating notoginseng metabolism, bioavailability, and pharmacological actions are discussed. Emphasis has been placed on the identification and isolation of enteric microbiome-generated notoginseng metabolites. Future investigations should provide key insights into notoginseng's bioactive metabolites as clinically valuable anticancer compounds.

Topics: Animals; Antineoplastic Agents, Phytogenic; Biological Availability; Gastrointestinal Microbiome; Ginsenosides; Humans; Molecular Conformation; Neoplasms; Panax notoginseng; Phytotherapy; Sapogenins; Saponins

Reviewed here is the existing evidence for the effects of ginseng extracts and isolated ginsenosides relevant to cognition in humans. Clinical studies in healthy volunteers and in patients with neurological disease or deficit, evidence from preclinical models of cognition, and pharmacokinetic data are considered. Conditions under which disease modification may indirectly benefit cognition but may not translate to cognitive benefits in healthy subjects are discussed. The number of chronic studies of ginseng effects in healthy individuals is limited, and the results from acute studies are inconsistent, making overall assessment of ginseng's efficacy as a cognitive enhancer premature. However, mechanistic results are encouraging; in particular, the ginsenosides Rg3 , Rh1 , Rh2 , Rb1 , Rd, Rg2 , and Rb3 , along with the aglycones protopanaxadiol and protopanaxatriol, warrant further attention. Compound K has a promising pharmacokinetic profile and can affect neurotransmission and neuroprotection. Properly conducted trials using standardized tests in healthy individuals reflecting the target population for ginseng supplementation are required to address inconsistencies in results from acute studies. The evidence summarized here suggests ginseng has potential, but unproven, benefits on cognition.

Topics: Cognition; Cognition Disorders; Ginsenosides; Humans; Neuroprotective Agents; Panax; Phytotherapy; Plant Extracts; Sapogenins

Panax ginseng is used in traditional Chinese medicine to enhance stamina and capacity to cope with fatigue and physical stress. Major active components are the ginsenosides, which are mainly triterpenoid dammarane derivatives. The mechanisms of ginseng actions remain unclear, although there is an extensive literature that deals with effects on the CNS (memory, learning, and behavior), neuroendocrine function, carbohydrate and lipid metabolism, immune function, and the cardiovascular system. Reports are often contradictory, perhaps because the ginsenoside content of ginseng root or root extracts can differ, depending on the method of extraction, subsequent treatment, or even the season of its collection. Therefore, use of standardized, authentic ginseng root both in research and by the public is to be advocated. Several recent studies have suggested that the antioxidant and organ-protective actions of ginseng are linked to enhanced nitric oxide (NO) synthesis in endothelium of lung, heart, and kidney and in the corpus cavernosum. Enhanced NO synthesis thus could contribute to ginseng-associated vasodilatation and perhaps also to an aphrodisiac action of the root. Ginseng is sold in the U.S. as a food additive and thus need not meet specific safety and efficacy requirements of the Food and Drug Administration. Currently, such sales amount to over $300 million annually. As public use of ginseng continues to grow, it is important for this industry and Federal regulatory authorities to encourage efforts to study the efficacy of ginseng in humans by means of appropriately designed double-blind clinical studies.

Topics: Animals; Aphrodisiacs; Cardiovascular Agents; Drugs, Chinese Herbal; Endothelium, Vascular; Food Additives; Humans; Nitric Oxide; Panax; Plants, Medicinal; Sapogenins; Triterpenes

20(S)-Protopanaxadiol (PPD) has a higher anti-wrinkle effect than the other glycone forms of ginsenosides. However, as PPD has low solubility in water and a high molecular weight, it cannot easily penetrate the stratum corneum layer, which is the rate-limiting step of topical skin delivery. Thus, the objective was to enhance the topical skin deposition of PPD using an optimized nanostructured lipid carriers (NLC) formulation. NLC formulations were optimized using a Box-Behnken design.. NLC formulations were optimized using a Box-Behnken design, where the amount of PDD (X. An NLC was successfully prepared based on the optimized formulation determined using the Box-Behnken design. The particle size, PDI, and EE of the NLC showed less than 5% difference from the predicted values. The in vitro deposition of PPD after the application of the NLC formulation on a Strat-M. An NLC formulation for the topical delivery of PPD was successfully optimized using the Box-Behnken design, and could be further developed to enhance the topical skin deposition of PPD.

Topics: Adult; Animals; Drug Carriers; Female; Humans; Lipids; Male; Middle Aged; Nanostructures; Particle Size; Sapogenins; Skin; Skin Irritancy Tests; Solubility; Surface-Active Agents; Young Adult

A highly sensitive HPLC-MS/MS assay method was established to quantify 20(S)-protopanaxadiol (PPD) in human plasma with dexamethasone as an internal standard. The electrospray ion mass spectrometry (ESI-MS) was operated under the multiple reactions monitoring mode (MRM) using positive ion mode. PPD was extracted from 500μL plasma samples by liquid-liquid extraction then separated by a C18 analytical column with gradient elution. The concentration of PPD could be determined by this HPLC-MS/MS method over the range of 0.05-20ng/mL with the lower limit of quantification (LLOQ) of 0.05ng/mL. The method was successfully applied to phase IIa clinical trial of Yuxintine (PPD capsule) in which plasma samples of 87 subjects were analyzed following 6 weeks of oral administration of placebo or PPD capsules in 5 different doses. In this study, the measured concentration was linearly related to the oral dosage with R=0.9901. The minimum and maximum values of measured concentration were 0.06 and 11.60ng/mL, respectively. In addition, plasma concentrations of PPD in depression patients were reported for the first time in our study.

Topics: Antidepressive Agents; Chromatography, High Pressure Liquid; Humans; Limit of Detection; Quality Control; Reference Standards; Reproducibility of Results; Sapogenins; Tandem Mass Spectrometry

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh(2), were extracted from plasma by liquid-liquid extraction and separated on a Zorbax extend C(18) analytical column using methanol-acetonitrile-10mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1-100.0ng/ml with a limit of detection of 0.05ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25mg capsule.

Topics: Adult; Chromatography, Liquid; Female; Humans; Male; Mass Spectrometry; Reproducibility of Results; Sapogenins; Sensitivity and Specificity; Time Factors

20(S)-protopanaxadiol (PPD), a metabolite of

Topics: Apoptosis; Autophagy; Cell Line, Tumor; Ginsenosides; Humans; Panax; Sapogenins; Stomach Neoplasms

TGFβ1 signaling pathway is associated with many diseases, which can induce the activation of hepatic stellate cells (HSCs) and induce liver fibrosis. Studies have shown that 20S-protopanaxadiol (PPD) has a therapeutic effect on liver fibrosis, but the target is unknown. In this study, we confirmed that PPD reduced the mRNA expression of downstream genes of the TGFβ1 pathway, which suggesting PPD is associated with the TGFβ1 pathway. The protein dissociation temperature and dissociation constant (Kd) of PPD on TGFβR1 and TGFβR2 were determined, which showed that PPD combined with TGFβR1 (Kd = 1.54 μM). The docking and simulation methods were used to find their binding sites. Site mutations, protein expression and in vitro binding experiments were performed to demonstrated these sites. In particular, these sites of TGFβR1 were also the active sites of TGFβR2. Therefore, we speculated that PPD blocked the combination of TGFβR1 and TGFβR2 by binding to the D57, R58, P59, and N78 of the TGFβR1 extracellular domain. Thus, PPD could block the transmission of TGFβ1 pathway and inhibit the activation of HSCs, and treating fibrosis. Our studies showed that PPD has the potential to treat diseases related to the TGFβ1 pathway and broadens its clinical application.

Topics: Cell Line; Ginsenosides; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Sapogenins; Transforming Growth Factor beta1

20(S)-Protopanaxadiol (PPD) and 20(S)-Protopanaxatriol (PPT) are major metabolites of ginseng in humans and are considered to have estrogenic activity in cellular bioassays. In this study, we conducted in silico analyses to determine whether PPD and PPT interact with estrogen receptor alpha (ERα) and compared them with ERα agonists, partial agonists, and antagonists to identify their ERα activity. The transcriptome profile of 17β-estradiol (E2), PPD, and PPT in MCF-7 cells expressing ERα was further compared to understand the ERα activity of ginsenoside metabolites. The results showed that PPD and PPT interacted with the 1ERE, 1GWR, and 3UUD ERα proteins in the E2 interaction model, the 3ERD protein in the diethylstilbestrol (DES) interaction model, and the 1X7R protein in the genistein (GEN) interaction model. Conversely, neither the 4PP6 protein of the interaction model with the antagonist resveratrol (RES) nor the 1ERR protein of the interaction model with the antagonist raloxifene (RAL) showed the conformation of amino acid residues. When E2, PPD, and PPT were exposed to MCF-7 cells, cell proliferation and gene expression were observed. The transcriptomic profiles of E2, PPD, and PPT were compared using a knowledge-based pathway. PPD-induced transcription profiling was similar to that of E2, and the neural transmission pathway was detected in both compounds. In contrast, PPT-induced transcription profiling displayed characteristics of gene expression associated with systemic lupus erythematosus. These results suggest that ginsenoside metabolites have ERα agonist activity and exhibit neuroprotective effects and anti-inflammatory actions. However, a meta-analysis using public microarray data showed that the mother compounds GRb1 and GRg1 of PPD and PPT showed metabolic functions in insulin signaling pathways, condensed DNA repair and cell cycle pathways, and immune response and synaptogenesis. These results suggest that the ginsenoside metabolites have potent ERα agonist activity; however, their gene expression profiles may differ from those of E2.

Topics: Cell Proliferation; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Gene Expression; Genistein; Ginsenosides; Humans; MCF-7 Cells; Molecular Docking Simulation; Resveratrol; Sapogenins; Signal Transduction; Transcriptome; Triterpenes

Protopanaxadiol (PPD), a main ginseng metabolite, exerts powerful anticancer effects against multiple types of cancer; however, its cellular targets remain elusive. Here, we synthesized a cell-permeable PPD probe via introducing a bifunctional alkyne-containing diazirine photo-crosslinker and performed a photoaffinity labeling-based chemoproteomic study. We identified retinoblastoma binding protein 4 (RBBP4), a chromatin remodeling factor, as an essential cellular target of PPD in HCT116 colorectal cancer cells. PPD significantly decreased RBBP4-dependent trimethylation at lysine 27 of histone H3 (H3K27me3), a crucial epigenetic marker that correlates with histologic signs of colorectal cancer aggressiveness, and PPD inhibition of proliferation and migration of HCT116 cells was antagonized by RBBP4 RNA silencing. Collectively, our study highlights a previously undisclosed anti-colorectal cancer cellular target of the ginseng metabolite and advances the fundamental understanding of RBBP4 functions via a chemical biology strategy.

Topics: Colorectal Neoplasms; HCT116 Cells; Humans; Panax; Retinoblastoma-Binding Protein 4; Sapogenins; Transcription Factors

20(S)-Protopanaxadiol [20(S)-PPD] is a fully deglycosylated ginsenoside metabolite produced by the gut microbiota in the gastrointestinal tract. Although diverse pharmacological effects have been reported, information on the pharmacokinetic interactions of 20(S)-PPD with cytochrome P450s (CYPs) remains limited. Therefore, the inhibitory potential of 20(S)-PPD on CYP enzymes, which mainly contribute to drug pharmacokinetics, was investigated in this study. The inhibitory effect of 20(S)-PPD was strong for CYP3A4 and moderate for CYP2B6 in human liver microsomes. 20(S)-PPD inhibited Cyp3a and Cyp2b in mouse liver microsomes with a potency similar to that in humans. The solubility of 20(S)-PPD in the artificial intestinal fluid was close to IC

Topics: Animals; Cytochrome P-450 CYP2B6; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Ginsenosides; Humans; Mice; Sapogenins

Protopanaxadiol (PPD), an aglycon found in several dammarene-type ginsenosides, has high potency as a pharmaceutical. Nevertheless, application of these ginsenosides has been limited because of the high production cost due to the rare content of PPD in

Topics: CRISPR-Cas Systems; Metabolic Engineering; Metabolic Networks and Pathways; Saccharomyces cerevisiae; Sapogenins

Glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferases (UGT) contributes to the chemical and functional diversity of a number of natural products. Bacillus subtilis Bs-YjiC is a robust and versatile UGT that holds potentials in the biosynthesis of unnatural bioactive ginsenosides. To understand the molecular mechanism underlying the substrate promiscuity of Bs-YjiC, we solved crystal structures of Bs-YjiC and its binary complex with uridine diphosphate (UDP) at resolution of 2.18 Å and 2.44 Å, respectively. Bs-YjiC adopts the classical GT-B fold containing the N-terminal and C-terminal domains that accommodate the sugar acceptor and UDP-glucose, respectively. Molecular docking indicates that the spacious sugar-acceptor binding pocket of Bs-YjiC might be responsible for its broad substrate spectrum and unique glycosylation patterns toward protopanaxadiol-(PPD) and PPD-type ginsenosides. Our study reveals the structural basis for the aglycone promiscuity of Bs-YjiC and will facilitate the protein engineering of Bs-YjiC to synthesize novel bioactive glycosylated compounds.

Topics: Bacillus subtilis; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Ginsenosides; Glycosylation; Glycosyltransferases; Models, Molecular; Molecular Docking Simulation; Protein Domains; Sapogenins; Substrate Specificity; Uridine Diphosphate; Uridine Diphosphate Glucose

New remedies are required for the treatment of neuropathic pain due to insufficient efficacy of available therapies. This study provides a novel approach to develop painkillers for chronic pain treatment.. The rat formalin pain test and spinal nerve ligation model of neuropathic pain were used to evaluate antinociception of protopanaxadiol. Primary cell cultures, immunofluorescence staining, and gene and protein expression were also performed for mechanism studies.. Gavage protopanaxadiol remarkably produces pain antihypersensitive effects in neuropathic pain, bone cancer pain and inflammatory pain, with efficacy comparable with gabapentin. Long-term PPD administration does not induce antihypersensitive tolerance, but prevents and reverses the development and expression of morphine analgesic tolerance. Oral protopanaxadiol specifically stimulates spinal expression of dynorphin A in microglia but not in astrocytes or neurons. Protopanaxadiol gavage-related pain antihypersensitivity is abolished by the intrathecal pretreatment with the microglial metabolic inhibitor minocycline, dynorphin antiserum or specific κ-opioid receptor antagonist GNTI. Intrathecal pretreatment with glucocorticoid receptor)antagonists RU486 and dexamethasone-21-mesylate, but not GPR-30 antagonist G15 or mineralocorticoid receptor antagonist eplerenone, completely attenuates protopanaxadiol-induced spinal dynorphin A expression and pain antihypersensitivity in neuropathic pain. Treatment with protopanaxadiol, the glucocorticoid receptor agonist dexamethasone and membrane-impermeable glucocorticoid receptor agonist dexamethasone-BSA in cultured microglia induces remarkable dynorphin A expression, which is totally blocked by pretreatment with dexamthasone-21-mesylate.. All the results, for the first time, indicate that protopanaxadiol produces pain antihypersensitivity in neuropathic pain probably through spinal microglial dynorphin A expression after glucocorticoid receptor activation and hypothesize that microglial membrane glucocorticoid receptor/dynorphin A pathway is a potential target to discover and develop novel painkillers in chronic pain.

Topics: Animals; Dynorphins; Glucocorticoids; Hyperalgesia; Microglia; Neuralgia; Rats; Rats, Wistar; Receptors, Glucocorticoid; Sapogenins; Spinal Cord

Ginsenosides Rb1, Rb2, Rb3 and Rc, four major protopanaxadiol (PPD)-type ginsenosides, can be metabolized by gut microbiota. The composition of gut microbiota varies in different species. Existing publications have reported the metabolite fates of ginsenosides by gut microbiota from single species. However, their microbiota-related metabolic species differences have not been evaluated yet. In current study, in vitro anaerobic incubations of PPD-type ginsenosides with gut microbiota from humans, rabbits and rats were conducted. The metabolites of each ginsenoside were then identified by LC-MS. A total of 15 metabolites from the four ginsenosides were identified. The major metabolic pathways were stepwise removals of the C-20 and C-3 sugar moieties to obtain aglycone PPD. The results showed that the hydrolysis rate of C-20 terminal β-D-glucopyranosyl was significantly higher than those of α-L-arabinopyranosyl, β-D-xylopyranosyl and α-L-arabinofuranosyl in different species. The activity of β-glucosidase, the metabolic rates of parent compounds and the formation rates of their metabolites were significantly higher in gut microbiota from rabbits than from humans and rats. Our research draws researchers' attention to the species differences of microbiota-related drug metabolism.

Topics: Adult; Animals; Chromatography, Liquid; Gastrointestinal Microbiome; Ginsenosides; Humans; Male; Mass Spectrometry; Metabolome; Rabbits; Rats; Rats, Sprague-Dawley; Sapogenins; Young Adult

We explored the inhibitory effect of ginsenoside compound K (CK), 20(

Topics: Ginsenosides; Glucuronosyltransferase; Humans; Microsomes, Liver; Sapogenins; Uridine

This study aimed to investigate the effects of protopanaxadiol and protopanaxatriol ginsenosides on aconitine-induced cardiomyocyte injury and their regulatory mechanisms. The effects of ginsenosides on aconitine-induced cardiomyocyte damage were initially evaluated using H9c2 cells, and the molecular mechanisms were elucidated using molecular docking and western blotting. The changes in enzyme content, reactive oxygen species (ROS), calcium (Ca2+) concentration, and apoptosis were determined. Furthermore, an aconitine-induced cardiac injury rat model was established, the cardiac injury and serum physiological and biochemical indexes were measured, and the effects of ginsenoside were observed. The results showed that ginsenoside Rb1 significantly increased aconitine-induced cell viability, and its binding conformation with protein kinase B (AKT) protein was the most significant. In vitro and in vivo, Rb1 protects cardiomyocytes from aconitine-induced injury by regulating oxidative stress levels and maintaining Ca2+ concentration homeostasis. Moreover, Rb1 activated the PI3K/AKT pathway, downregulated Cleaved caspase-3 and Bax, and upregulated Bcl-2 expression. In conclusion, Rb1 protected H9c2 cells from aconitine-induced injury by maintaining Ca2+ homeostasis and activating the PI3K/AKT pathway to induce a cascade response of downstream proteins, thereby protecting cardiomyocytes from damage. These results suggested that ginsenoside Rb1 may be a potential cardiac protective drug.

Topics: Aconitine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Calcium; Cardiotoxicity; Cell Line; Disease Models, Animal; Ginsenosides; Heart Diseases; Homeostasis; Male; Molecular Docking Simulation; Myocytes, Cardiac; Oxidative Stress; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sapogenins; Signal Transduction

Inefficient diabetic ulcer healing and scar formation remain a challenge worldwide, owing to a series of disordered and dynamic biological events that occur during the process of healing. A functional wound dressing that is capable of promoting ordered diabetic wound recovery is eagerly anticipated. In this study, we designed a silicone elastomer with embedded 20(S)-protopanaxadiol-loaded nanostructured lipid carriers (PPD-NS) to achieve ordered recovery in scarless diabetic ulcer healing. The nanostructured lipid carriers were prepared through an emulsion evaporation-solidification method and then incorporated into a network of silicone elastomer to form a unique nanostructured lipid carrier-enriched gel formulation. Interestingly, the PPD-NS showed excellent in vitro anti-inflammatory and proangiogenic activity. Moreover, in diabetic mice with full-thickness skin excision wound, treatment with PPD-NS significantly promoted in vivo scarless wound healing through suppressing inflammatory infiltration in the inflammatory phase, promoting angiogenesis during the proliferation phase, and regulating collagen deposition in the remodeling phase. Hence, this study demonstrates that the developed PPD-NS could facilitate ordered diabetic wound recovery via multifunctional improvement during different wound-healing phases. This novel approach could be promising for scarless diabetic wound healing.

Topics: Angiogenesis Inhibitors; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Movement; Cell Survival; Cells, Cultured; Diabetic Foot; Drug Carriers; Drug Liberation; Gels; Humans; Inflammation; Lipids; Lipopolysaccharides; Male; Mice; Nanostructures; Neovascularization, Pathologic; Nitric Oxide; Particle Size; RAW 264.7 Cells; Sapogenins; Silicone Elastomers; Surface Properties; Wound Healing

Ginsenoside Rh2 (3β-O-Glc-protopanaxadiol), a trace but an important pharmacological component of ginseng, has exhibited excellent medicinal potential. Many studies have found that the synthesis of Rh2 by UDP-glucosyltransferase (UGT) is an alternative production strategy. In this study, Yjic from B. subtilis 168 was found to synthesize ginsenoside F12 (3β,12β-Di-O-Glc-protopanaxadiol) and Rh2 at a ratio of 7:3. Yjic regioselectivity toward Rh2 synthesis was successfully improved using a semi-rational design including structure-guided alanine scanning and saturation mutations. As a result, mutant M315F was found to efficiently synthesize Rh2 (~99%) and block the further glycosylation of C12-OH. The circulation of UDPG was achieved by combining M315F with AtSuSy through a cascade reaction. Furthermore, an extraordinarily high yield of Rh2 (3.7 g/L) was attained in an aqueous solvent system with 17% DMSO (v/v) through the fed-batch feeding of PPD. This study presents the high potential for the oriented preparation of ginsenoside Rh2.

Topics: Amino Acid Substitution; Bacillus subtilis; Biocatalysis; Ginsenosides; Glucosyltransferases; Glycosylation; Mutant Proteins; Mutation; Sapogenins

20(S)-Protopanaxadiol [20(S)-PPD], one of the metabolites of ginsenosides, was investigated to determine its potential mechanism for targeting to epidermal growth factor receptor (EGFR) pathway in lung cancer cell A549. Results of kinase inhibitory assay showed that 20(S)-PPD was an EGFR tyrosine kinase inhibitor. By binding to EGFR, 20(S)-PPD disrupted the EGFR/MAPK signaling. The expression of genes in the pathway was altered and the upregulation of Ras and MEK1 was extremely notable. The accumulation and phosphorylation of EGFR, Ras, BRAF, Raf-1, MEK, and ERK were variously altered. The above alteration subsequently resulted in cell cycle arrest. 20(S)-PPD interfered the cell cycle regulation network and eventually blocked cell cycle progression at G0/G1 phase, which may be the key reason for proliferation inhibition. Although some apoptosis related genes and proteins were influenced, apoptosis was not the main reason for proliferation inhibition. The cell wound healing assay confirmed that the inhibition of 20(S)-PPD to A549 cells could suppress the migration and invasion thereof. The results of molecular docking and molecular dynamics simulation provide a possible interaction mechanism between EGFR and 20(S)-PPD. The results described above suggested that 20(S)-PPD could block cell cycle progression by targeting the EGFR/MAPK signaling pathway.

Topics: A549 Cells; Apoptosis; Cell Cycle; Cell Movement; ErbB Receptors; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Protein Binding; Sapogenins

Ginsenoside Rh2, a rare protopanaxadiol (PPD)-type triterpene saponin isolated from Panax ginseng, exhibits notable anticancer and immune-system-enhancing activities. Glycosylation catalyzed by uridine diphosphate-dependent glucosyltransferase (UGT) is the final biosynthetic step of ginsenoside Rh2. In this study, UGT73C5 isolated from Arabidopsis thaliana was demonstrated to selectively transfer a glucosyl moiety to the C3 hydroxyl group of PPD to synthesize ginsenoside Rh2. UGT73C5 was coupled with sucrose synthase (SuSy) from A. thaliana to regenerate costly uridine diphosphate glucose (UDPG) from cheap sucrose and catalytic amounts of uridine diphosphate (UDP). The UGT73C5/SuSy ratio, temperature, pH, cofactor UDP, and PPD concentrations for UGT73C5-SuSy coupled reactions were optimized. Through the stepwise addition of PPD, the maximal ginsenoside Rh2 production was 3.2 mg mL

Topics: Arabidopsis; Batch Cell Culture Techniques; Biocatalysis; Biosynthetic Pathways; Drugs, Chinese Herbal; Ginsenosides; Glucosyltransferases; Panax; Sapogenins; Saponins; Triterpenes; Uridine Diphosphate

Chirality is a common phenomenon, and it is meaningful to explore interactions between stereoselective bio-macromolecules and chiral small molecules with preclinical and clinical significance. Protopanaxadiol-type ginsenosides are main effective ingredients in ginseng and are prone to biotransformation into a pair of ocotillol C20-24 epoxide epimers, namely, (20

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Catalytic Domain; Cytochrome P-450 CYP3A; Dammaranes; Epoxy Compounds; Glucuronides; Glucuronosyltransferase; Humans; Molecular Docking Simulation; Oxidation-Reduction; Panax; Sapogenins; Stereoisomerism; Triterpenes

Ginsenoside compound K (CK), one of the primary active metabolites of protopanaxadiol-type ginsenosides, is produced by the intestinal flora that degrade ginseng saponins and exhibits diverse biological properties such as anticancer, anti-inflammatory, and anti-allergic properties. However, it is less abundant in plants. Therefore, enabling its commercialization by construction of a Saccharomyces cerevisiae cell factory is of considerable significance.. We induced overexpression of PGM2, UGP1, and UGT1 genes in WLT-MVA5, and obtained a strain that produces ginsenoside CK. The production of CK at 96 h was 263.94 ± 2.36 mg/L, and the conversion rate from protopanaxadiol (PPD) to ginsenoside CK was 64.23 ± 0.41%. Additionally, it was observed that the addition of glycerol was beneficial to the synthesis of CK. When 20% glucose (C mol) in the YPD medium was replaced by the same C mol glycerol, CK production increased to 384.52 ± 15.23 mg/L, which was 45.68% higher than that in YPD medium, and the PPD conversion rate increased to 77.37 ± 3.37% as well. As we previously observed that ethanol is beneficial to the production of PPD, ethanol and glycerol were fed simultaneously in the 5-L bioreactor fed fermentation, and the CK levels reached 1.70 ± 0.16 g/L.. In this study, we constructed an S. cerevisiae cell factory that efficiently produced ginsenoside CK. Glycerol effectively increased the glycosylation efficiency of PPD to ginsenoside CK, guiding higher carbon flow to the synthesis of ginsenosides and effectively improving CK production. CK production attained in a 5-L bioreactor was 1.7 g/L after simultaneous feeding of glycerol and ethanol.

Topics: Ethanol; Fermentation; Ginsenosides; Glycerol; Metabolic Engineering; Saccharomyces cerevisiae; Sapogenins

20(S)‑Protopanaxadiol (PPD) is an active ginseng metabolite and is the final form of protopanaxadiol saponins metabolized by human intestinal microflora. The neuroprotective effects and mechanisms underlying PPD on neural stem cells (NSCs) are not completely understood. The aim of the present study was to assess the effects of PPD on the proliferation and differentiation of neural stem cells. In the present study, following treatment with different concentrations of PPD for 24 h, the percentage of BrdU‑positive cells decreased significantly with increasing concentrations of PPD. Moreover, flow cytometric analysis results indicated that PPD treatment increased the proportion of cells in the G0/G1 and G2/M phase and decreased the proportion of cells in the S phase. The activation of autophagy, determined by an increased number of autophagic vacuoles and light chain 3 lipidation, was associated with an increase in the expression of the neuronal marker tubulin‑β3 following PPD treatment. PPD also partially rescued NSCs from the inhibitory effects of the autophagic inhibitor wortmannin, suggesting that the effect of PPD on NSC differentiation was associated with autophagy. Collectively, the results indicated that PPD promoted the transition of NSCs from a state of proliferation to differentiation through the induction of autophagy and cell cycle arrest. Therefore, the present study may provide a basis for the development of regenerative therapies based on ginsenoside, an approved and safe drug.

Topics: Animals; Autophagy; Cell Cycle Checkpoints; Cell Differentiation; Cell Proliferation; Cells, Cultured; Ginsenosides; Neural Stem Cells; Panax; Rats, Sprague-Dawley; Sapogenins

(20S)-Protopanaxadiol ginsenosides Rg3, Rh2 and PPD have been demonstrated for their anticancer activity. However, the underlying mechanism of their antitumor activity remains unclear. In the present study, we investigated the role of these three ginsenosides on cell proliferation and death of human gastric cancer cells (HGC-27 cells). The sulforhodamine B (SRB) assay, Western blot analysis, fluorescence microscopy, confocal microscopy, high performance liquid chromatography (HPLC) analysis, flow cytometry, and transmission electron microscopy (TEM) were used to evaluate cell proliferation, apoptosis, and autophagy. The results showed that both Rh2 and PPD were more effective than Rg3 in inhibiting HGC-27 cell proliferation and inducing cytoplasmic vacuolation, while no significant changes in apoptosis were observed. Interestingly, cytoplasmic vacuolation and blockade of autophagy flux were observed after treatment with Rh2 and PPD. Rh2 obviously up-regulated the expression of the LC3II and p62. Furthermore, the increase in lysosomal pH and membrane rupture was observed in Rh2-treated and PPD-treated cells. When HGC-27 cells were pretreated with bafilomycin A1, a specific inhibitor of endosomal acidification, cellular vacuolization was increased, and the cell viability was significantly decreased, which indicated that Rh2-induced lysosome-damage accelerated cell death. Furthermore, data derived from mitochondrial analysis showed that excessive mitochondrial reactive oxygen species (ROS) and dysregulation of mitochondrial energy metabolism were caused by Rh2 and PPD treatment in HGC-27 cells. Taken together, these phenomena indicated that Rh2 and PPD inhibited HCG-27 cells proliferation by inducing mitochondria damage, dysfunction of lysosomes, and blockade of autophagy flux. The number of glycosyl groups at C-3 position could have an important effect on the cytotoxicity of Rg3, Rh2 and PPD.

Topics: Antineoplastic Agents; Autophagy; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Ginsenosides; Humans; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Reactive Oxygen Species; Sapogenins; Structure-Activity Relationship; Tumor Cells, Cultured

Neuroprotective strategies in the treatment of stroke have been attracting a great deal of attentions. Our previous clinical and basic studies have demonstrated that protopanaxadiol ginsenoside-Rd (Rd), a monomer compound extracted from Panax ginseng or Panax notoginseng, has neuroprotective effects against ischemic stroke, probably due to its ability to block Ca

Topics: Animals; Brain Ischemia; Calcineurin; Death-Associated Protein Kinases; Ginsenosides; Male; Neurons; Neuroprotective Agents; Panax; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sapogenins; Signal Transduction; Stroke

Through the combination of various chromatographies, 11 new 20(S)-protopanaxadiol (PPD) type saponins, named as notoginsenosides NL-E

Topics: Animals; Anti-Inflammatory Agents; Mice; Models, Molecular; Molecular Conformation; Panax notoginseng; Plant Leaves; RAW 264.7 Cells; Sapogenins; Structure-Activity Relationship

Topics: Aspergillus niger; beta-Glucosidase; Biotransformation; Food Microbiology; Ginsenosides; Hydrolysis; Panax; Plant Extracts; Sapogenins

Panax notoginseng is one of the most widely used traditional Chinese herbs with particularly valued roots. Triterpenoid saponins are mainly specialized secondary metabolites, which medically act as bioactive components. Knowledge of the ginsenoside biosynthesis in P. notoginseng, which is of great importance in the industrial biosynthesis and genetic breeding program, remains largely undetermined. Here we combined single molecular real time (SMRT) and Second-Generation Sequencing (SGS) technologies to generate a widespread transcriptome atlas of P. notoginseng. We mapped 2,383 full-length non-chimeric (FLNC) reads to adjacently annotated genes, corrected 1,925 mis-annotated genes and merged into 927 new genes. We identified 8,111 novel transcript isoforms that have improved the annotation of the current genome assembly, of which we found 2,664 novel lncRNAs. We characterized more alternative splicing (AS) events from SMRT reads (20,015 AS in 6,324 genes) than Illumina reads (18,498 AS in 9,550 genes), which contained a number of AS events associated with the ginsenoside biosynthesis. The comprehensive transcriptome landscape reveals that the ginsenoside biosynthesis predominantly occurs in flowers compared to leaves and roots, substantiated by levels of gene expression, which is supported by tissue-specific abundance of isoforms in flowers compared to roots and rhizomes. Comparative metabolic analyses further show that a total of 17 characteristic ginsenosides increasingly accumulated, and roots contained the most ginsenosides with variable contents, which are extraordinarily abundant in roots of the three-year old plants. We observed that roots were rich in protopanaxatriol- and protopanaxadiol-type saponins, whereas protopanaxadiol-type saponins predominated in aerial parts (leaves, stems and flowers). The obtained results will greatly enhance our understanding about the ginsenoside biosynthetic machinery in the genus Panax.

Topics: Alternative Splicing; Exome Sequencing; Flowers; Gene Expression Profiling; Genes, Plant; Ginsenosides; Molecular Sequence Annotation; Panax; Panax notoginseng; Plant Leaves; Plant Roots; Rhizome; RNA-Seq; Sapogenins; Saponins; Transcriptome

Colorectal carcinoma (CRC) recurrence is often accompanied by metastasis. Most metastasis undergo through epithelial-mesenchymal transition (EMT). Studies showed that retinol X receptor alpha (RXRα) and 20(S)-Protopanaxadiol (PPD) have anti-tumour effects. However, the anti-metastasis effect of 20(S)-PPD and the effect of RXRα on EMT-induced metastasis are few studies on. Therefore, the role of RXRα and 20(S)-PPD in CRC cell metastasis remains to be fully elucidated. RXRα with clinicopathological characteristics and EMT-related expression in clinical samples were examined. Then, RXRα and EMT level in SW480 and SW620 cells, overexpressed and silenced RXRα in SW620 cells and SW480 cells, respectively, were evaluated. Finally, 20(S)-PPD effect on SW620 and SW480 cells was evaluated. The results showed that a lower RXRα expression in cancer tissues, and a moderate negative correlation between RXRα and N stage, and tended to higher level of EMT. SW480 and SW620 cells had the highest and lowest RXRα expression among four CRC cell lines. SW480 had lower EMT level than SW620. Furthermore, 20(S)-PPD increased RXRα and inhibited EMT level in SW620 cell. Finally, 20(S)-PPD cannot restore SW480 cells EMT level to normal when RXRα silencing. These findings suggest that 20(S)-PPD may inhibit EMT process in CRC cells by regulating RXRα expression.

Topics: Adult; Aged; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Middle Aged; Retinoid X Receptor alpha; Sapogenins

Diol-type ginsenosides, such as protopanaxadiol (PPD), exhibit antioxidation, anti-inflammation, and antitumor effects. However, the antitumor effect of these ginsenosides and the mechanism of PPD remain unclear. In this work, the antitumor effects of several derivatives, including PPD, Rg5, Rg3, Rh2, and Rh3, were evaluated in five different cancer cell lines. PPD demonstrated the best inhibitory effects on the proliferation and migration of the five cancer cell lines, especially the hepatocellular carcinoma (HCC) cell lines. Therefore, the mechanism of action of PPD in HCC cells was elucidated. PPD inhibited the proliferation, migration, and invasion ability of HepG2 and PLC/PRF/5 cells in a dose-dependent manner. Western blot and immunofluorescence assay showed that PPD can alter the expression of epithelial-mesenchymal transition markers, increase E-cadherin expression, and decrease vimentin expression. Docking and biacore experiments revealed that STAT3 is the target protein of PPD, which formed hydrogen bonds with Gly583/Leu608/Tyr674 at the SH2 domain of STAT3. PPD inhibited the phosphorylation of STAT3 and its translocation from the cytosol to the nucleus, thereby inhibiting the expression of Twist1. PPD also inhibited tumor volume and tumor lung metastasis in PLC/PRF/5 xenograft model. In conclusion, PPD can inhibit the proliferation and metastasis of HCC cells through the STAT3/Twist1 pathway.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Female; Hep G2 Cells; Humans; Liver Neoplasms; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Sapogenins; Signal Transduction; STAT3 Transcription Factor; Transfection; Xenograft Model Antitumor Assays

P2Y. To explore stereoselective effect of naturally abundant ginsenoside isomers, including the C-20 epimers of protopanaxadiol (PPD), protopanaxatriol (PPT), and their glycosides Rg2, Rg3, Rh1, Rh2 on P2Y. Both in vitro assay and in silico molecular docking study were performed to investigate the stereoselective effects.. In vitro assay using washed rat platelets revealed differential effects of ginsenoside isomers on ADP-induced platelet aggregation with the direction and degree of action varying with chemical structures. More to the point, the ginsenoside 20S-Rh2 but not its 20R-epimer was found to be the only one that could significantly promote in vitro platelets aggregation induced by ADP. The correlation analysis demonstrated that ginsenosides may have impact on P2Y. Ginsenosides are potent P2Y

Topics: Animals; Blood Platelets; Fibrinolytic Agents; Ginsenosides; Glycosides; Humans; Male; Molecular Docking Simulation; Panax; Plants, Medicinal; Platelet Activation; Platelet Aggregation; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2Y12; Sapogenins; Stereoisomerism

The aim of this study was to prepare a 20(S)-protopanaxadiol nanocrystalline suspension and enhance the bioavailability of 20(S)-protopanaxadiol by intramuscular injection. 20(S)-Protopanaxadiol nanocrystalline suspension was prepared using an anti-solvent combined with ultrasonic approach, in which meglumine and bovine serum albumin were screened as the optimized stabilizer and the coating agent during spray drying process, respectively. The optimal nanocrystallines were nearly spherical with a uniform particle size distribution, the mean particle size, polydispersity index, and drug loading of which were 151.20 ± 2.54 nm, 0.11 ± 0.01, and 47.15% (w/w), respectively. Sterile 20(S)-protopanaxadiol nanocrystalline suspension was obtained by passing through a 0.22-μm membrane, and the average filtration efficiency (FE%) was 99.96%. The cumulative release percentage of 20(S)-protopanaxadiol nanocrystalline suspension was 92.36% 20(S)-protopanaxadiol within 60 min in vitro, which was relatively rapid compared with that of the physical mixture for 12.51% and the 20(S)-protopanaxadiol bulk powder for 9.71% during the same time interval. The sterile 20(S)-protopanaxadiol nanocrystalline suspension caused minimal irritation responses by histological examination, indicating a good biocompatibility between the 20(S)-protopanaxadiol nanocrystalline suspension and muscle tissues. In pharmacokinetic study, the absolute bioavailability of 20(S)-protopanaxadiol nanocrystalline suspension for intramuscular injection and for oral gavage was 5.99 and 0.03, respectively. In summary, the 20(S)-protopanaxadiol nanocrystalline via intramuscular injection is an efficient drug delivery system to improve its bioavailability.

Topics: Administration, Oral; Animals; Biological Availability; Drug Delivery Systems; Injections, Intramuscular; Male; Nanoparticles; Rats; Rats, Sprague-Dawley; Sapogenins; Serum Albumin, Bovine; Suspensions

20(S)-protopanaxadiol (PPD)-type ginsenosides are generally believed to be the most pharmacologically active components of Panax ginseng. These compounds induce apoptotic cell death in various cancer cells, which suggests that they have anti-cancer activity. Anti-angiogenesis is a promising therapeutic approach for controlling angiogenesis-related diseases such as malignant tumors, age-related macular degeneration, and atherosclerosis. Studies showed that 20(S)-PPD at low concentrations induces endothelial cell growth, but in our present study, we found 20(S)-PPD at high concentrations inhibited cell growth and mediated apoptosis in human umbilical vein endothelial cells (HUVECs). The mechanism by which high concentrations of 20(S)-PPD mediate endothelial cell apoptosis remains elusive. The present current study investigated how 20(S)-PPD induces apoptosis in HUVECs for the first time. We found that caspase-9 and its downstream caspase, caspase-3, were cleaved into their active forms after 20(S)-PPD treatment. Treatment with 20(S)-PPD decreased the level of Bcl-2 expression but did not change the level of Bax expression. 20(S)-PPD induced endoplasmic reticulum stress in HUVECs and stimulated UPR signaling, initiated by protein kinase R-like endoplasmic reticulum kinase (PERK) activation. Total protein expression and ATF4 nuclear import were increased, and CEBP-homologous protein (CHOP) expression increased after treatment with 20(S)-PPD. Furthermore, siRNA-mediated knockdown of PERK or ATF4 inhibited the induction of CHOP expression and 20(s)-PPD-induced apoptosis. Collectively, our findings show that 20(S)-PPD inhibits HUVEC growth by inducing apoptosis and that ATF4 expression activated by the PERK-eIF2α signaling pathway is essential for this process. These findings suggest that high concentrations of 20(S)-PPD could be used to treat angiogenesis-related diseases.

Topics: Activating Transcription Factor 4; Apoptosis; Caspase 3; Cell Proliferation; Down-Regulation; eIF-2 Kinase; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Human Umbilical Vein Endothelial Cells; Humans; Models, Biological; Proto-Oncogene Proteins c-bcl-2; Sapogenins; Signal Transduction

The rare ginsenoside Rg3 is attracting more attention because of its good physiological activity and urgent need. There are many pathways to obtain ginsenoside Rg3, including chemical and biological methods. Among these, the conversion of the protopanaxadiol-type ginsenosides by microbial hydrolysis is a trend due to its high efficiency and mild conditions. For effectively extracting from the other panaxadiol saponins, the conversion process for ginsenoside Rg3 was investigated using β-glycosidase-producing endophytic fungus in Panax ginseng in this study. The metabolic pathways are as follows: ginsenoside Rb1 → Gyp-XVII and ginsenoside Rb1 → ginsenoside Rd → ginsenoside Rg3. Phylogenetic analysis of 16S rDNA gene sequence, showed that GE 32 strain belonged to Flavobacterium species. These results suggest that the process of rare ginsenoside Rg3 production by endophytic bacteria GE 32 is efficient for the industrial production and application. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on cultivable β-glycosidase-producing endophytic bacteria from Panax ginseng. Flavobacterium sp. GE32 could convert major ginsenoside Rb1 into Gyp-XVII and minor ginsenoside Rg3. Strain GE 32 has potential to be applied on the preparation for minor ginsenoside Rg3 in pharmaceutical industry.

Topics: Biotransformation; DNA, Ribosomal; Flavobacterium; Ginsenosides; Glycoside Hydrolases; Hydrolysis; Panax; Phylogeny; Sapogenins; Saponins

Protopanaxadiol (PPD), a ginseng metabolite generated by the gut bacteria, was shown to induce colorectal cancer cell death and enhance the anticancer effect of chemotherapeutic agent 5-FU. However, the mechanism by which PPD promotes cancer cell death is not clear. In this manuscript, we showed that PPD activated p53 and endoplasmic reticulum (ER) stress and induced expression of BH3-only proteins Puma and Noxa to promote cell death. Induction of Puma by PPD was p53-dependent, whereas induction of Noxa was p53-independent. On the other hand, PPD also induced prosurvival mechanisms including autophagy and expression of Bcl2 family apoptosis regulator Mcl-1. Inhibition of autophagy or knockdown of Mcl-1 significantly enhanced PPD-induced cell death. Interestingly, PPD inhibited expression of genes involved in fatty acid and cholesterol biosynthesis and induced synergistic cancer cell death with fatty acid synthase inhibitor cerulenin. As PPD-induced ER stress was not significantly affected by inhibition of new protein synthesis, we suggest PPD may induce ER stress directly through causing lipid disequilibrium.

Topics: Apoptosis; Autophagy; Endoplasmic Reticulum Stress; HCT116 Cells; Humans; Lipid Metabolism; Panax; Sapogenins; Tumor Suppressor Protein p53

Transcriptome analysis revealed high expression of saponin biosynthetic genes may account for highly accumulated saponins in 3-year-old Panax notoginseng roots and DS and CYP716A47 - like were functionally verified by transgenic tobacco. Panax notoginseng is a well-known traditional medical herb that contains bioactive compounds known as saponins. Three major dammarene-type triterpene saponins including R1, Rb1, and Rg1 were found to be highly accumulated in the roots of 3-year-old plants when compared to those of 1-year-old plants. However, the underlying cellular mechanism is poorly understood. In this study, transcriptome analysis revealed that most genes involved in saponin biosynthesis in P. notoginseng roots augmented during their growth periods. The analysis of the KEGG pathway indicated that the primary metabolism, cell growth, and differentiation were less active in the roots of 3-year-old plant; however, secondary metabolisms were enhanced, thus providing molecular evidence for the harvesting of P. notoginseng roots in the 3rd year of growth. Furthermore, the functional role of DS and CYP716A47-like, two of the candidate genes involved in saponin biosynthesis isolated from P. notoginseng, were verified via overexpression in cultivated tobacco. Approximately, 0.325 µg g

Topics: Chromatography, High Pressure Liquid; Gene Expression Profiling; Genes, Plant; Nicotiana; Panax notoginseng; Plant Roots; Plants, Genetically Modified; Real-Time Polymerase Chain Reaction; Sapogenins; Saponins; Sequence Analysis, RNA; Triterpenes

Ginsenosides have previously been demonstrated to effectively inhibit cancer cell growth and survival in both animal models and cell lines. However, the specific ginsenoside component that is the active ingredient for cancer treatment through interaction with a target protein remains unknown. By an integrated quantitative proteomics approach via affinity mass spectrum (MS) technology, we deciphered the core structure of the ginsenoside active ingredient derived from crude extracts of ginsenosides and progressed toward identifying the target protein that mediates its anticancer activity. The Tandem Mass Tag (TMT) labeling quantitative proteomics technique acquired 55620 MS/MS spectra that identified 5499 proteins and 3045 modified proteins. Of these identified proteins, 224 differentially expressed proteins and modified proteins were significantly altered in nonsmall cell lung cancer cell lines. Bioinformatics tools for comprehensive analysis revealed that the Ras protein played a general regulatory role in many functional pathways and was probably the direct target protein of a compound in ginsenosides. Then, affinity MS screening based on the Ras protein identified 20(s)-protopanaxadiol, 20(s)-Ginsenoside Rh2, and 20(s)-Ginsenoside Rg3 had affinity with Ras protein under different conditions. In particular, 20(s)-protopanaxadiol, whose derivatives are the reported antitumor compounds 20(s)-Ginsenoside Rh2 and 20(s)-Ginsenoside Rg3 that have a higher affinity for Ras via a low KD of 1.22 μM and the mutation sites of G12 and G60, was demonstrated to play a core role in those interactions. Moreover, the molecular mechanism and bioactivity assessment results confirmed the identity of the chemical ligand that was directly acting on the GTP binding pocket of Ras and shown to be effective in cancer cell bioactivity profiles.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cell Survival; Ginsenosides; Guanosine Triphosphate; Humans; Lung Neoplasms; Molecular Docking Simulation; Neoplasm Proteins; Protein Binding; Protein Conformation; Proteomics; ras Proteins; Sapogenins

Ginsenoside compound K (CK) is considered to be a potential therapeutic drug for rheumatoid arthritis because of its good anti-inflammatory activity. The purpose of this work was to establish a rapid, sensitive and specific method for determination of CK and its active metabolite 20(S)-protopanaxadiol (20(S)-PPD). Materials & methods: The analytes and internal standards were extracted by liquid-liquid extraction. Then, were separated by high performance liquid phase and determined by triple quadrupole mass spectrometry.. A LC-MS/MS using liquid-liquid extraction was developed for determining CK over the concentration range 1.00-1002.00 ng/ml and 0.15-54.30 ng/ml for 20(S)-PPD. The lower limits of quantification for CK and 20(S)-PPD were 1.00 and 0.15 ng/ml, respectively.. This method was successfully validated for detecting both CK and 20(S)-PPD in the human plasma and urine, and was proved to be suitable for the pharmacokinetic study of CK in healthy Chinese volunteers.. ChiCTR-TRC-14004824.

Topics: Arthritis, Rheumatoid; Chromatography, Liquid; Female; Ginsenosides; Humans; Male; Panax; Sapogenins; Tandem Mass Spectrometry

Ginseng is one of the most commonly used herbs that is believed to have a variety of biological activities, including reducing blood sugar and cholesterol levels, anti-cancer, and anti-diabetes activities. However, little is known about the molecular mechanisms involved. In this study, we showed that protopanaxadiol (PPD), a metabolite of the protopanaxadiol group ginsenosides that are the major pharmacological constituents of ginsengs, significantly altered the expression of genes involved in metabolism, elevated Sestrin2 (Sesn2) expression, activated AMPK, and induced autophagy. Using CRISPR/CAS9-mediated gene editing and shRNA-mediated gene silencing, we demonstrated that Sesn2 is required for PPD-induced AMPK activation and autophagy. Interestingly, we showed that PPD-induced Sesn2 expression is mediated redundantly by the GCN2/ATF4 amino acid-sensing pathway and the PERK/ATF4 endoplasmic reticulum (ER) stress pathway. Our results suggest that ginseng metabolite PPD modulates the metabolism of amino acids and lipids, leading to the activation of the stress-sensing kinases GCN2 and PERK to induce Sesn2 expression, which promotes AMPK activation, autophagy, and metabolic health.

Topics: Activating Transcription Factor 4; Amino Acids; AMP-Activated Protein Kinases; Animals; Autophagy; eIF-2 Kinase; Endoplasmic Reticulum Stress; Fibroblasts; Ginsenosides; HCT116 Cells; HEK293 Cells; Humans; Mice; Nuclear Proteins; Panax; Protein Serine-Threonine Kinases; Sapogenins; Signal Transduction

Obesity is one of the most important health problems worldwide. Panax ginseng has been reported to exert anti-obesity effect. However, the active constituents and the underlying mechanism remained uncertain. This study uncovered the anti-obesity effect of protopanaxadiol (PPD) and its potential mechanism. To investigate the anti-obesity effect of PPD, high-fat diet induced obesity (DIO) C57BL/6 mice were treated with PPD by both intraperitoneal injection (i.p.) and oral administration. Body weight and food intake were recorded. Energy expenditure was measured by CLAMS metabolic cages. For mechanism study, C-Fos in the hypothalamus of the mice was stained following the intracerebroventricular (i.c.v.) injection of PPD. Our results showed that with both injection and feeding, PPD reduced body weight, inhibited food intake, increased energy expenditure and improved liver damage in DIO mice. Mechanistically, i.c.v. injection of PPD inhibited feeding and increased C-Fos expression in paraventricular nucleus of the hypothalamus (PVH). The results suggest that PPD may reduce body weight of DIO mice via the activation of PVH neurons and PPD is a potential therapeutic candidate for the treatment of obesity.

Topics: Animals; Body Weight; Diet, High-Fat; Energy Metabolism; Liver; Mice; Mice, Inbred C57BL; Neurons; Obesity; Paraventricular Hypothalamic Nucleus; Sapogenins

As renewable biomass, lignocellulose remains one of the major choices for most countries in tackling global energy shortage and environment pollution. Efficient utilization of xylose, an important monosaccharide in lignocellulose, is essential for the production of high-value compounds, such as ethanol, lipids, and isoprenoids. Protopanaxadiol (PPD), a kind of isoprenoids, has important medical values and great market potential.. The engineered protopanaxadiol-producing Yarrowia lipolytica strain, which can use xylose as the sole carbon source, was constructed by introducing xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis, overexpressing endogenous xylulose kinase (ylXKS) and heterologous PPD synthetic modules, and then 18.18 mg/L of PPD was obtained. Metabolic engineering strategies such as regulating cofactor balance, enhancing precursor flux, and improving xylose metabolism rate via XR (K270R/N272D) mutation, the overexpression of tHMG1/ERG9/ERG20 and transaldolase (TAL)/transketolase (TKL)/xylose transporter (TX), were implemented to enhance PPD production. The final Y14 strain exhibited the greatest PPD titer from xylose by fed-batch fermentation in a 5-L fermenter, reaching 300.63 mg/L [yield, 2.505 mg/g (sugar); productivity, 2.505 mg/L/h], which was significantly higher than the titer of glucose fermentation [titer, 167.17 mg/L; yield, 1.194 mg/g (sugar); productivity, 1.548 mg/L/h].. The results showed that xylose was more suitable for PPD synthesis than glucose due to the enhanced carbon flux towards acetyl-CoA, the precursor for PPD biosynthetic pathway. This is the first report to produce PPD in Y. lipolytica with xylose as the sole carbon source, which developed a promising strategy for the efficient production of high-value triterpenoid compounds.

Topics: Aldehyde Reductase; Biomass; Biosynthetic Pathways; D-Xylulose Reductase; Fermentation; Metabolic Engineering; Metabolic Networks and Pathways; Organisms, Genetically Modified; Sapogenins; Xylose; Yarrowia

Topics: Alkyl and Aryl Transferases; Biosynthetic Pathways; Dammaranes; Gene Expression; Ginsenosides; Oryza; Panax; Plants, Genetically Modified; Sapogenins; Saponins; Triterpenes

Although glucocorticoids (GCs) are widely used as anti-inflammatory drugs, they are often accompanied by adverse effects, which are mainly due to the transactivation of glucocorticoid receptor (GR) target genes. In order to screen novel plant-derived GR ligands (phytocorticoids) capable of separating transrepression from transactivation, this work focuses on the estimation of 20(R, S)-protopanaxadiol [PPD(R, S)] and 20(R, S)-protopanaxatriol [PPT(R, S)] for their dissociated characteristics. The reporter gene assay shows that ginsenosides cannot enhance glucocorticoid-responsive element-driven genes. The cytotoxicity assay shows that PPT(S), PPT(R), and PPD(S) can inhibit cell proliferation while PPD(R) does not suppress cell growth at available concentration. Further analysis of transactivation and transrepression activities indicates that PPD(R) can repress the transcription of GR target transrepressed gene without activating the expression of the GR target transactivated gene. Results of molecular docking suggest that PPD(R) yields more hydrogen bond interactions and a lower binding energy than its counterparts, resulting in tighter binding between PPD(R) and GR. In addition, PPD(R) achieves stability in the pocket after 2 ns, thereby facilitating exerting its regulatory role of GR target genes. By contrast, other ginsenosides fluctuate drastically during the simulations. In conclusion, PPD(R) may serve as a potential selective GR modulator (SEGRM).

Topics: Binding Sites; Cell Line, Tumor; Cell Proliferation; Ginsenosides; Humans; Ligands; Molecular Docking Simulation; Molecular Dynamics Simulation; Receptors, Glucocorticoid; Sapogenins; Stereoisomerism; Structure-Activity Relationship; Transcriptional Activation

The ginsenoside 20-

Topics: Bacterial Proteins; beta-Glucosidase; Biotransformation; Escherichia coli; Firmicutes; Genetic Engineering; Ginsenosides; Molecular Chaperones; Panax; Sapogenins

20(S)-Protopanaxadiol (PPD) is a basic aglycone of the dammarane triterpenoid saponins and exerts antidepressant-like effects on behaviour in the forced swimming test (FST) and tail suspension test (TST) and in rat olfactory bulbectomy depression models. However, the antidepressant effects of PPD have not been studied thoroughly. The objective of the present study was first to investigate the effect of PPD on depression behaviours induced by chronic social defeat stress (CSDS) in mice. The results showed that CSDS was effective in producing depression-like behaviours in mice, as indicated by decreased responses in the social interaction test, sucrose preference test, TST, and FST, and that this effect was accompanied by noticeable alterations in the levels of oxidative markers (superoxide dismutase, catalase, and lipid peroxidation) and monoamines (5-HT and NE) in the hippocampus and serum corticosterone levels. Additionally, western blot analysis revealed that CSDS exposure significantly downregulated BDNF, p-TrkB/TrkB, p-Akt/Akt, and p-mTOR/mTOR protein expression in the hippocampus. Remarkably, chronic PPD treatment significantly ameliorated these behavioral and biochemical alterations associated withCSDS-induced depression. Our results suggest that PPD exerts antidepressant-like effects in mice with CSDS-induced depression and that this effect may be mediated by the normalization of neurotransmitter and corticosterone levels and the alleviation of oxidative stress, as well as the enhancement of the PI3K/Akt/mTOR-mediated BDNF/TrkB pathway.

Topics: Animals; Antidepressive Agents; Chronic Disease; Corticosterone; Depression; Disease Models, Animal; Hippocampus; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Rats; Sapogenins; Stress, Psychological

To improve the absorption of poorly water-soluble 20(S)-protopanaxadiol (20(S)-PPD), novel 20(S)-PPD-loaded redispersible dry suspension and dry emulsion were developed in this study. 20(S)-PPD dry suspension (PPD-DS) was prepared by enabling drug fully dispersed with suspending agent Avicel CL611 and solubilizer Poloxamer 188. 20(S)-PPD dry emulsion (PPD-DE) was prepared by employing oleic acid as oil phase, Cremophor RH-40 as surfactant, and n-butyl alcohol as co-surfactant. Both PPD-DS and PPD-DE were evaluated for their physicochemical characterization after being dispersed in distilled water. The in vivo pharmacokinetics was evaluated by UPLC-MS/MS. The droplet size of PPD-DS and PPD-DE was in the scope of 1446-1653 nm and 652.8-784.5 nm. The sedimentation volume ratios of PPD-DS and PPD-DE were both at value of 1. The zeta potential of PPD-DS and PPD-DE were from - 53.7 to - 70.4 mV and - 27.5 to - 34.5 mV, respectively, which indicated stable systems. PPD-DS and PPD-DE both achieved dramatically enhanced aqueous solubility and higher perfusion of 20(S)-PPD in rats' intestine. Although statistically, no oral bioavailability enhancements of 20(S)-PPD were achieved in PPD-DE and PPD-DS, there were some improvements in the pharmacokinetic behaviors. Especially, PPD-DS could be a promising drug delivery carrier for 20(S)-PPD with the advantages of long-term stability, dosing flexibility, and the convenience of administering to infants and to those who have difficulty swallowing tablets or capsules.

Topics: Administration, Oral; Animals; Biological Availability; Chromatography, Liquid; Desiccation; Drug Carriers; Emulsions; Male; Rats; Rats, Sprague-Dawley; Sapogenins; Solubility; Surface-Active Agents; Suspensions; Tandem Mass Spectrometry

20(S)-protopanaxadiol (PPD) possesses various biological properties, including anti-inflammatory, antitumor and anti-fatigue properties. Recent studies found that PPD functioned as a neurotrophic agent to ameliorate the sensory deficit caused by glutamate-induced excitotoxicity through its antioxidant effects and exhibited strong antidepressant-like effects in vivo. The objective of the present study was first to investigate the effect of PPD in scopolamine (SCOP)-induced memory deficit in mice and the potential mechanisms involved. In this study, mice were pretreated with PPD (20 and 40 μmol/kg) and donepezil (1.6 mg/kg) intraperitoneally (i.p) for 14 days. Then, open field test was used to assess the effect of PPD on the locomotor activity and mice were daily injected with SCOP (0.75 mg/kg) to induce cognitive deficits and then subjected to behavioral tests by object location recognition (OLR) experiment and Morris water maze (MWM) task. The cholinergic system function, oxidative stress biomarkers and protein expression of Egr-1, c-Fos, and c-Jun in mouse hippocampus were examined. PPD was found to significantly improve the performance of amnesia mice in OLR and MWM tests. PPD regulated cholinergic function by inhibiting SCOP-induced elevation of acetylcholinesterase (AChE) activity, decline of choline acetyltransferase (ChAT) activity and decrease of acetylcholine (Ach) level. PPD suppressed oxidative stress by increasing activities of antioxidant enzymes such as superoxide dismutase (SOD) and lowering maleic diadehyde (MDA) level. Additionally, PPD significantly elevated the expression of Egr-1, c-Fos, and c-Jun in hippocampus at protein level. Taken together, all these results suggested that 20(S)-protopanaxadiol (PPD) may be a candidate compound for the prevention against memory loss in some neurodegenerative diseases such as Alzheimer's disease (AD).

Topics: Animals; Antioxidants; Dose-Response Relationship, Drug; Drug Administration Schedule; Early Growth Response Protein 1; Gene Expression Regulation; Hippocampus; JNK Mitogen-Activated Protein Kinases; Maze Learning; Memory Disorders; Mice; Parasympathetic Nervous System; Proto-Oncogene Proteins c-fos; Sapogenins; Scopolamine

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Autophagy; Cell Line; Cognitive Dysfunction; eIF-2 Kinase; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; HEK293 Cells; Humans; Male; Mice; Mice, Transgenic; Nesting Behavior; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Sapogenins; Signal Transduction; Spatial Navigation; TOR Serine-Threonine Kinases

Sleep deprivation (SD) is associated with oxidative stress that causes learning and memory impairment. 20(S)-Protopanaxadiol (PPD), one of the protopanaxadiol-type saponins, has antioxidant and neuroprotective effect. This study was designed to research the protective effect of PPD against cognitive deficits induced by chronic sleep deprivation (CSD) in mice. The CSD model was induced by subjecting the mice to our self-made Sleep Interruption Apparatus (SIA) continuously for 14 days. The memory enhancing effects of PPD were evaluated by behavioral tests and the related mechanism was further explored by observing the oxidative stress changes in the cortex and hippocampus of mice. The results revealed that PPD (20 and 40 μmol/kg, i.p.) administration significantly improved the cognitive performance of CSD model mice in object location recognition experiment, novel object recognition task and Morris water maze test. Furthermore, PPD effectively restored the levels/activities of antioxidant defense biomarkers in the cortex and hippocampus, including the superoxide dismutase (SOD) enzyme activity, catalase (CAT) enzyme activity, glutathione (GSH), and lipid peroxidation (LPO). In conclusion, PPD could attenuate cognitive deficits induced by CSD, and the neuroprotective effect of PPD might be mediated by alleviation of oxidative stress. It was assumed that PPD has the potential to be a neuroprotective substance for cognition dysfunction.

Topics: Animals; Brain; Chronic Disease; Dose-Response Relationship, Drug; Lipid Peroxidation; Male; Maze Learning; Memory Disorders; Mice, Inbred ICR; Motor Activity; Neuroprotective Agents; Nootropic Agents; Oxidative Stress; Random Allocation; Recognition, Psychology; Sapogenins; Sleep Deprivation; Spatial Memory

The ginsenoside 20- O-β-glucopyranosyl-20( S)-protopanaxadiol, compound K, has attracted much attention in functional food, traditional medicine, and cosmetic industries because of diverse pharmaceutical activities. The effective production of compound K from ginseng extracts has been required. However, an enzyme capable of completely converting all protopanaxadiol (PPD)-type ginsenosides to compound K has not been reported until now. In this study, unlike other enzymes, β-glucosidase from Caldicellulosiruptor bescii was able to hydrolyze sugar moieties such as l-arabinofuranose as well as d-glucose and l-arabinopyranose as the C-20 outer sugar in ginsenosides. Thus, ginsenoside Rc containing l-arabinofuranose can be converted to compound K by only this enzyme. Under the optimized reaction conditions, the enzyme completely converted PPD-type ginsenosides in ginseng extracts to compound K with the highest productivity among the reported results. This is the first report of the enzyme capable of completely converting all PPD-type ginsenosides into compound K.

Topics: Bacterial Proteins; beta-Glucosidase; Biocatalysis; Biotransformation; Enzyme Stability; Firmicutes; Hot Temperature; Molecular Structure; Panax; Plant Extracts; Sapogenins

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Female; Hemolysis; Hydrogen-Ion Concentration; Kinetics; Mice, Inbred C57BL; Nanoparticles; Panax; Plant Extracts; Sapogenins; Tissue Distribution

Topics: Animals; Chromatography, Liquid; Drug Stability; Limit of Detection; Linear Models; Male; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sapogenins; Tandem Mass Spectrometry

20(S)-Protopanaxadiol (PPD) is one of the major active metabolites of ginseng. It has been reported that 20(S)-PPD shows a broad spectrum of antitumor effects. Our research study aims were to investigate whether apoptosis of human breast cancer MCF-7 cells could be induced by 20(S)-PPD by targeting the Phosphatidylinositol 3-kinase/Protein kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway in vitro and in vivo. Cell cycle analysis was performed by Propidium Iodide (PI) staining. To overexpress and knock down the expression of mTOR, pcDNA3.1-mTOR and mTOR small interfering RNA (siRNA) transient transfection assays were used, respectively. Cell viability and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-test and Annexin V /PI double-staining after transfection. The antitumor effect in vivo was determined by the nude mice xenograft assay. After 24 h of incubation, treatment with 20(S)-PPD could upregulate phosphorylated-Phosphatase and tensin homologue deleted on chromosome 10 (p-PTEN) expression and downregulate PI3K/AKT/mTOR-pathway protein expression. Moreover, G0/G1 cell cycle arrest in MCF-7 cells could be induced by 20(S)-PPD treatment at high concentrations. Furthermore, overexpression or knockdown of mTOR could inhibit or promote the apoptotic effects of 20(S)-PPD. In addition, tumor volumes were partially reduced by 20(S)-PPD at 100 mg/kg in a MCF-7 xenograft model. Immunohistochemical staining indicated a close relationship between the inhibition of tumor growth and the PI3K/AKT/mTOR signal pathway. PI3K/AKT/mTOR pathway-mediated apoptosis may be one of the potential mechanisms of 20(S)-PPD treatment.

Topics: Apoptosis; Cell Line, Tumor; Female; Humans; MCF-7 Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Sapogenins; Signal Transduction; TOR Serine-Threonine Kinases

Endometriosis (EMS) is an estrogen-dependent gynecological disease with a low autophagy level of ectopic endometrial stromal cells (eESCs). Impaired NK cell cytotoxic activity is involved in the clearance obstruction of the ectopic endometrial tissue in the abdominopelvic cavity. Protopanaxadiol (PPD) and protopanaxatriol (PPT) are two metabolites of ginsenosides, which have profound biological functions, such as anti-cancer activities. However, the role and mechanism of ginsenosides and metabolites in endometriosis are completely unknown. Here, we found that the compounds PPD, PPT, ginsenoside-Rg3 (G-Rg3), ginsenoside-Rh2 (G-Rh2), and esculentoside A (EsA) led to significant decreases in the viability of eESCs, particularly PPD (IC50 = 30.64 µM). In vitro and in vivo experiments showed that PPD promoted the expression of progesterone receptor (PR) and downregulated the expression of estrogen receptor α (ERα) in eESCs. Treatment with PPD obviously induced the autophagy of eESCs and reversed the inhibitory effect of estrogen on eESC autophagy. In addition, eESCs pretreated with PPD enhanced the cytotoxic activity of NK cells in response to eESCs. PPD decreased the numbers and suppressed the growth of ectopic lesions in a mouse EMS model. These results suggest that PPD plays a role in anti-EMS activation, possibly by restricting estrogen-mediated autophagy regulation and enhancing the cytotoxicity of NK cells. This result provides a scientific basis for potential therapeutic strategies to treat EMS by PPD or further structural modification.

Topics: Autophagy; Endometriosis; Endometrium; Female; Ginsenosides; Humans; Killer Cells, Natural; Receptors, Estrogen; Sapogenins; Stromal Cells

Panax ginseng C.A. Meyer (Araliaceae), mainly cultivated in Korea and Northeast China, is processed before use based on its long history of ethnopharmacological evidence. Ginsenosides have been regarded as the main active components responsible for the pharmacological activities of ginseng. Although the Maillard reaction is known as a major source of compounds related to enhanced antioxidant activity by heat treatment in various crude drugs or foods, the chemical and free radical-scavenging activity changes of ginsenosides brought about by the Maillard reaction have not yet been elucidated. This paper gives a review of our recent findings, with emphasis on the hydroxyl radical (•OH)-scavenging activity changes of ginsengs and ginsenosides by heat-processing using an electron spin resonance spectrometer. 20(S)- Rg3 showed the strongest activity, and the next was in the decreasing order of Rb1, Rg1, Rc, Rb2, and Rd. The •OH-scavenging activities of ginsenosides were related to the ferrous metal ion-chelating activities of their aglycone, 20(S)-protopanaxadiol. In addition, the ferrous metal ion-chelating activities of ginsenosides were thought to be influenced by their types of hydrophilic sugar moieties. Moreover, Rb1 was changed into 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rg5 by heat-processing, and the sugar moieties at carbon-20 were separated. The generated amount of 20(S)-Rg3 was higher than when Rb1 was heat-processed without amino acids, and a significant increase in Maillard reaction products was noted. Based upon chemical and •OH-scavenging activity tests using Maillard reaction model experiments, the scientific evidence underlying the increase in free radical-scavenging activity of ginseng induced by heat-processing was elucidated.

Topics: Drug Discovery; Electron Spin Resonance Spectroscopy; Ferrous Compounds; Free Radical Scavengers; Ginsenosides; Hot Temperature; Hydroxyl Radical; Iron Chelating Agents; Maillard Reaction; Panax; Sapogenins

Topics: Chromatography, High Pressure Liquid; Computational Biology; Cytochrome P-450 Enzyme System; Gene Expression Profiling; Gene Expression Regulation, Plant; Panax notoginseng; Plant Extracts; Sapogenins; Saponins; Transcriptome

Ginsenosides are dammarane-type or triterpenoidal saponins that contribute to the various pharmacological activities of the medicinal herb Panax ginseng. The putative biosynthetic pathway for ginsenoside biosynthesis is known in P. ginseng, as are some of the transcripts and enzyme-encoding genes. However, few genes related to the UDP-glycosyltransferases (UGTs), enzymes that mediate glycosylation processes in final saponin biosynthesis, have been identified. Here, we generated three replicated Illumina RNA-Seq datasets from the adventitious roots of P. ginseng cultivar Cheongsun (CS) after 0, 12, 24, and 48 h of treatment with methyl jasmonate (MeJA). Using the same CS cultivar, metabolomic data were also generated at 0 h and every 12-24 h thereafter until 120 h of MeJA treatment. Differential gene expression, phylogenetic analysis, and metabolic profiling were used to identify candidate UGTs. Eleven candidate UGTs likely to be involved in ginsenoside glycosylation were identified. Eight of these were considered novel UGTs, newly identified in this study, and three were matched to previously characterized UGTs in P. ginseng. Phylogenetic analysis further asserted their association with ginsenoside biosynthesis. Additionally, metabolomic analysis revealed that the newly identified UGTs might be involved in the elongation of glycosyl chains of ginsenosides, especially of protopanaxadiol (PPD)-type ginsenosides.

Topics: Gene Expression Regulation, Plant; Ginsenosides; Panax; Phylogeny; Plant Proteins; Sapogenins

Melanoma is one of the most lethal skin malignancies in the world. Interferons (IFNs) have been also demonstrated in response to tumor cell and IFNs such as IFN-α have been used for melanoma treatment. The long chain double-stranded RNA (dsRNA) (from a variety of nonviral sources) is a potent activator of the IFN system and an inducer of cell apoptosis. Panaxadiolsaponins (PDS) is a major Panax ginseng-derived active component with known antitumor activity and immune modulation. Here, we investigated a hypothesis that the combination of PDS and total natural dsRNA (as opposed to the synthetic dsRNA) will suppress tumor growth better than the individual agents. We have evaluated the antitumor and immunostimulatory effects of the combination of natural long chain dsRNA (derived from yeast) and PDS on melanoma cell line B16 and mice xenograft model. The underlying mechanisms of growth suppression were investigated by analyzing dsRNA-activated pathways, apoptosis, and cell cycle. Natural dsRNA and PDS exert superior anticancer effects than either agent alone. Natural dsRNA and PDS combination might be a promising strategy for treating malignancies, including melanoma.

Topics: Animals; Antineoplastic Agents; Biological Products; Cell Proliferation; Drug Screening Assays, Antitumor; Melanoma, Experimental; Mice; Panax; RNA, Double-Stranded; Sapogenins; Tumor Cells, Cultured

Protopanaxadiol (PPD), an active triterpene compound, is the precursor of high-value ginsenosides. In this study, we report a strategy for the enhancement of PPD production in Saccharomyces cerevisiae by cofermentation of glucose and xylose. In mixed sugar fermentation, strain GW6 showed higher PPD titer and yield than that obtained from glucose cultivation. Then, engineering strategies were implemented on GW6 to enhance the PPD yields, such as adjustment of the central carbon metabolism, optimization of the mevalonate pathway, reinforcement of the xylose assimilation pathway, and regulation of cofactor balance, namely, overexpression of xPK/PTA, ERG10/ERG12/ERG13, XYL1/XYL2/TAL1, and POS5, respectively. In particular, the final obtained strain GW10, harboring overexpressed POS5, exhibited the highest PPD yield, which was 2.06 mg of PPD/g of mixed sugar. In a 5-L fermenter, PPD titer reached 152.37 mg/L. These promising results demonstrate the great advantages of mixed sugar over glucose for high-yield production of PPD.

Topics: Aldehyde Reductase; D-Xylulose Reductase; Ethanol; Fermentation; Glucose; Metabolic Engineering; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sapogenins; Xylose

Ginseng (Panax ginseng) and its bioactive components, ginsenosides, are popular medicinal herbal products, exhibiting various pharmacological effects. Despite their advocated use for medication, the long cultivation periods of ginseng roots and their low ginsenoside content prevent mass production of this compound. Yeast Saccharomyces cerevisiae was engineered for production of protopanaxadiol (PPD), a type of aglycone characterizing ginsenoside. PPD-producing yeast cell factory was further engineered by obtaining a balance between enzyme expressions and altering cofactor availability. Different combinations of promoters (P

Topics: Biosynthetic Pathways; Carbon; Metabolic Engineering; NADP; Oxidation-Reduction; Promoter Regions, Genetic; Saccharomyces cerevisiae; Sapogenins

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist.

Topics: Animals; Apoptosis; Binding Sites; Cell Line, Tumor; Cell Nucleus; Chlorocebus aethiops; COS Cells; HEK293 Cells; Humans; Mitochondria; Molecular Docking Simulation; NF-kappa B; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Receptors, Glucocorticoid; Sapogenins; Transcriptional Activation

Topics: Antineoplastic Agents, Phytogenic; Cell Cycle; Cell Proliferation; Cell Survival; Down-Regulation; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Microscopy; Sapogenins; Signal Transduction; Stochastic Processes; TOR Serine-Threonine Kinases

Topics: Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Enzymologic; Ginsenosides; Humans; Keratinocytes; MAP Kinase Signaling System; Matrix Metalloproteinase 1; Sapogenins; Ultraviolet Rays

Ginsenosides are believed to be the principal components behind the pharmacological actions of ginseng, and their bioactive properties are closely related to the type, position, and number of sugar moieties attached to the aglycone; thus, modification of the sugar chains may markedly change their biological activities. In this study, major protopanaxadiol type ginsenosides (PD) Rb1, Rc, and Rb2 were isolated from Panax ginseng and were transformed using two probiotic strains namely Bifidobacterium lactis Bi-07 and Lactobacillus rhamnosus HN001 to obtain specific deglycosylated ginsenosides. It was demonstrated that B. lactis transformed ginsenosides Rb1, Rc, and Rb2 to Rd within 1 h of fermentation and rare ginsenoside F2 by the conversion of Rd after 12-h fermentation. The maximum Rd concentration was 147.52 ± 1.45 μg/mL after 48-h fermentation as compared to 45.85 ± 0.71 μg/mL before fermentation. In contrast, L. rhamnosus transformed Rb1, Rc, and Rb2 into Rd as the final metabolite after 72-h fermentation. B. lactis displayed significantly (p < 0.05) higher β-glucosidase activity against p-nitrophenyl-β-glucopyranoside than L. rhamnosus and higher bioconversion efficiency during fermentation. The present study suggests that the fermentation of major PD type ginsenosides with B. lactis Bi-07 may serve as an effective means to afford bioactive deglycosylated ginsenosides and to create novel ginsenoside extracts.

Topics: beta-Glucosidase; Bifidobacterium animalis; Fermentation; Ginsenosides; Lacticaseibacillus rhamnosus; Panax; Probiotics; Sapogenins

Topics: Animals; Cell Survival; Diabetes Mellitus, Experimental; Female; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MAP Kinase Signaling System; Mice; Mice, Transgenic; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Small Interfering; Sapogenins; Signal Transduction; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A; Wound Healing

Laryngeal carcinoma (LC) is one of the most prevalent malignant tumors in the head and neck area. Due to its high morbidity and mortality, LC poses a serious threat to human life and health. Even with surgical removal, some patients were not sensitive to radiotherapy or experienced transfer or recurrence. 20(s)-Protopanaxadiol (PPD), a natural product from Panax ginseng, has been reported to have cytotoxic effects against several cancer cell lines. However, whether it can improve the radiation sensitivity and the underlying mechanism of PPD's sensitization effect is still unknown. Herein, from in vitro and in vivo experiments, we found that the combination of PPD and radiation not only significantly inhibited proliferation and induced apoptosis, but also suppressed the tumor growth in mouse models. These findings confirmed the role of PPD in enhancing the sensitivity of radiotherapy. Moreover, our work showed that the expression levels of mTOR and its downstream effectors decreased remarkably after PPD addition when compared to radiation only. This result suggested that PPD's excellent synergistic effects with radiation might be associated with the down-regulation of the mTOR signaling pathway in Hep-2 cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma; Cell Line, Tumor; Cell Proliferation; Combined Modality Therapy; Down-Regulation; Female; Humans; Laryngeal Neoplasms; Mice; Mice, Nude; Panax; Radiation-Sensitizing Agents; Sapogenins; TOR Serine-Threonine Kinases

Protopanaxatriol and protopanaxadiol exhibit limited oral bioavailability due to the poor solubility and intestinal cytochromes P450-mediated metabolism. This study set out to develop a novel cytochromes P450 inhibitory excipient(s)-based self-microemulsion to encapsulate protopanaxatriol and protopanaxadiol so as to enhance the

Topics: Animals; Biological Availability; Caco-2 Cells; Cytochrome P-450 Enzyme Inhibitors; Emulsions; Excipients; Humans; Intestinal Mucosa; Intestines; Microsomes, Liver; Rats; Sapogenins

SHENMAI injection (SMI), derived from famous Shen Mai San, is a herbal injection widely used in China. Ginsenosides are the major components of SMI. To monitor the exposure level of SMI during long-term treatment, a 6-month toxicokinetic experiment was performed. Twenty-four beagle dogs were dived into four groups (n = 6 in each group): a control group (0.9% NaCl solution) and three SMI groups (2, 6 or 3 mg/kg). The dogs were i.v. infused with vehicle or SMI daily for 180 d. Blood samples for analysis were collected at specific time points as follows: pre-dose (0 h); at 10, 30, and 60 min during infusion; and at 10, 30, 60, 90, 120, 240, and 300 min post-administration. Concentrations of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 in the plasma were determined simultaneously by liquid chromatography-tandem mass spectrometry. Non-compartmental parameters were further calculated and analyzed. Significant differences were found between the kinetic behavior of 20(S)-protopanaxadiol-type (PPD-type) and 20(S)-protopanaxatriol-type (PPT-type) ginsenosides. Increasing in the exposure level of PPD-type ginsenosides was observed in dogs during the experiment. Therefore, PPD-type ginsenosides are closely related to the immunity modulation effect of SMI. Increased PPD-type ginsenoside exposure level may present potential toxicity and induce drug-drug interaction risks during SMI administration. As such, PPD-type ginsenoside accumulation must be carefully monitored in future SMI research.

Topics: Animals; Body Burden; Chromatography, High Pressure Liquid; Dogs; Drug Combinations; Drugs, Chinese Herbal; Female; Ginsenosides; Infusions, Intravenous; Male; Models, Biological; Reproducibility of Results; Sapogenins; Tandem Mass Spectrometry; Time Factors; Toxicokinetics

Ilyonectria mors-panacis belongs to I. radicicola species complex and causes root rot and replant failure of ginseng in Asia and North America. The aims of this work were to identify I. mors-panacis that infect Korean ginseng using molecular approaches and to investigate whether their aggressiveness depends on their ability to metabolize ginseng saponins (ginsenosides) by their β-glucosidases, in comparison with other identified Ilyonectria species. Fourteen isolates were collected from culture collections or directly isolated from infected roots and mainly identified based on histone H3 (HIS H3) sequence. Among them, six isolates were identified as I. mors-panacis while others were identified as I. robusta and I. leucospermi. The pathogenicity tests confirmed that the isolates of I. mors-panacis were significantly more aggressive than I. robusta and I. leucospermi. The major ginsenosides in I. mors-panacis-infected roots were significantly reduced while significantly increased in those infected with other species. In vitro, the isolates were tested for their sensitivity and ability to metabolize the total major ginsenosides (Total MaG), protopanaxadiol-type major ginsenosides (PPD-type MaG), and protopanaxatriol-type major ginsenosides (PPT-type MaG). Unexpectedly, the growth rate and metabolic ability of I. mors-panacis isolates were significantly low on the three different ginsenoside fractions while those of I. robusta and I. leucospermi were significantly reduced on PPT-type MaG and Total MaG fractions and not affected on PPD-type MaG fraction. Our results indicate that major ginsenosides, especially PPT-type, have an antifungal effect and may intervene in ginseng defense during Ilyonectria species invasion, in particular the weak species. Also, the pathogenicity of I. mors-panacis may rely on its ability to reduce saponin content; however, whether this reduction is caused by detoxification or another method remains unclear.

Topics: Antifungal Agents; Ginsenosides; Hypocreales; Panax; Plant Diseases; Plant Roots; Sapogenins; Virulence

Protopanaxadiol (PPD) is an active compound in Panax ginseng. Recently, an optimized PPD synthesis pathway contained a ROS releasing step (a P450-type PPD synthase, PPDS) was introduced into Saccharomyces cerevisiae. Here reported a synergistic effect of PPDS-CPR (CPR, cytochrome P450 reductase) uncoupling and ethanol stress on ROS releasing, which reduced cells viability. To build a robust strain, a cell wall integrity associated gene SSD1 was high-expressed to improve ethanol tolerance, and ROS level decreased for 24.7%. Then, regulating the expression of an oxidative stress regulation gene YBP1 decreased 75.2% of ROS releasing, and improved cells viability from 71.3±1.3% to 88.3±1.4% at 84h. Increased cells viability enables yeast to produce more PPD through feeding additional ethanol. In 5L fermenter, PPD production of W3a-ssPy reached to 4.25±0.18g/L (19.48±0.28mg/L/OD600), which is the highest yield reported so far. This work makes the industrial production of PPD possible by microbial fermentation.

Topics: Fermentation; Reactive Oxygen Species; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sapogenins

To explore the antitumour mechanism of 20(S)-protopanaxadiol (PPD) while maintaining its uncovered pharmacological active site 3-hydroxyl, 28-hydroxy protopanaxadiol (17), a small molecular probe template of PPD was first designed and synthesised based on the Baldwin's reaction. Thus, 28-hydroxyl of 17 was built successfully as a derivatized site of molecular probe's functional and report groups. The important intermediates and final product were confirmed by ESI-MS and nuclear magnetic resonance spectra with good yield. These studies provided a valuable basis for probe research of PPD.

Topics: Antineoplastic Agents; Catalytic Domain; Molecular Probes; Sapogenins; Spectrum Analysis

In this study, we prepared solid dispersions (SDs) of 20(

Topics: Calorimetry, Differential Scanning; Ligands; Magnetic Resonance Spectroscopy; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Structure; Polymers; Sapogenins; Solubility; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

Ginsenosides are used as existing markers of red ginseng (RG) quality, and ginsenoside ratios are also indicative of the different components of red ginseng. For the analysis and classification of ginsenoside content, red ginseng was separated into three parts, namely, main roots, lateral roots, and fine roots, and each extract was subjected to ultra-performance liquid chromatography quadruple time-of-flight mass spectrometry (UPLC-QToF-MS) with multivariate statistical analysis. Principal component analysis (PCA) showed a clear discrimination between the extracts of main roots and fine roots and suggested discrimination markers (four for the main roots and five for the fine roots). The fine root markers were identified as ginsenoside. We identified two markers for the main roots of red ginseng in this study. Moreover, the contents of 22 ginsenosides were analyzed in all three components of red ginseng. Fine roots have the highest protopanaxadiol (PPD)/protopanaxatriol (PPT) ratio. The PPD group of ginsenosides, which is quantitatively dominant in fine roots, clearly distinguishes the main roots from the other parts.

Topics: Chromatography, High Pressure Liquid; Ginsenosides; Metabolomics; Panax; Plant Roots; Principal Component Analysis; Sapogenins; Tandem Mass Spectrometry

Panax notoginseng is a commonly used traditional Chinese medicine with blood activating effect while has continuous cropping obstacle problem in planting process. In present study, a semimicroextraction method with water-saturated n-butanol on 0.1 g notoginseng sample was established with good repeatability (RSD<2.5%) and 9.6%-20.6% higher extraction efficiency of seven saponins than the conventional method. A total of 16 characteristic peaks were identified by LC-MS-IT-TOF, including eight 20(S)-protopanaxatriol (PPT) type saponins and eight 20(S)-protopanaxadiol (PPD) type saponins. The established method was utilized to evaluate the quality of notoginseng samples cultivated by manual intervened methods to overcome continuous cropping obstacles.As a result, HPLC fingerprint similarity, content of Fa and ratio of notoginsenoside K and notoginsenoside Fa (N-K/Fa) were found out to be as valuatable markers of the quality of samples in continuous cropping obstacle research, of which N-K/Fa could also be applied to the analysis of notoginseng samples with different growth years.Notoginseng samples with continuous cropping obstacle had HPLC fingerprint similarity lower than 0.87, in consistent with normal sample, and had significant lower content of notoginsenoside Fa and significant higher N-K/Fa (2.35-4.74) than normal group (0.45-1.33). All samples in the first group with manual intervention showed high similarity with normal group (>0.87), similar content of common peaks and N-K/Fa (0.42-2.06). The content of notoginsenoside K in the second group with manual intervention was higher than normal group. All samples except two displayed similarity higher than 0.87 and possessed content of 16 saponins close to normal group. The result showed that notoginseng samples with continuous cropping obstacle had lower quality than normal sample. And manual intervened methods could improve their quality in different levels.The method established in this study was simple, fast and accurate, and the markers may provide new guides for quality control in continuous cropping obstacle research of notoginseng.

Topics: Agriculture; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Medicine, Chinese Traditional; Panax notoginseng; Sapogenins; Saponins

Topics: Administration, Oral; Animals; Antineoplastic Agents; Biological Availability; Drug Delivery Systems; Hep G2 Cells; Humans; Mice; Nanoparticles; Sapogenins; Suspensions

Ginsenoside Rb3 has been proved to have antidepressant-like effects, which possesses 1 xylose and 3 glucose moieties with 20(S)-protopanaxadiol (PPD) as the aglycone. However, it is commonly accepted that orally ingested ginsenosides can be deglycosylated or partially deglycosylated into active derivatives by the intestinal bacteria. To identify potential antidepressant drug candidates, we compared the antidepressant-like activities between ginsenoside Rb3 and its four deglycosylated derivatives, Rg3, Rh2, compound K (C-K), and PPD. Effects of acute (1-day), short chronic (7-days), and longer chronic treatments (14-days) with these ginsenosides (50 and 100mg/kg, p.o.) on the behavioral changes in the forced swim test (FST), tail suspension test (TST) and open field test were investigated. Serum corticosterone and adrenocorticotropic hormone (ACTH) levels and mouse brain monoamine neurotransmitters 5-HT, NA and DA levels were measured using commercially available competitive enzyme-linked immunosorbent assay (ELISA) kits. Interestingly, C-K showed antidepressant-like activities similar to that of Rb3, and Rg3 displayed antidepressant-like effects at lower dosage and faster time, indicating it has better effects than Rb3, whereas Rh2 and PPD failed to show any effect. Our results also showed, unlike the positive control fluoxetine, Rb3, Rg3 and C-K significantly increased the NA levels in the brain regions of mice exposed to FST but did not affect the 5-HT and DA levels. Moreover, treatment with Rg3 could reverse swim stress-induced increased levels of serum ACTH and corticosterone. These results suggest that C-K and Rg3 are the active deglycosylated derivatives, especially the latter compound, which is more potent than Rb3 and exerts antidepressant-like effects by regulating NA, ACTH and corticosterone levels.

Topics: Adrenocorticotropic Hormone; Animals; Antidepressive Agents; Brain Chemistry; Corticosterone; Depressive Disorder; Ginsenosides; Hindlimb Suspension; Male; Mice; Motor Activity; Neurotransmitter Agents; Sapogenins; Swimming

Ginsenosides are triterpenoid saponins, which is an active compound responsible for most of the pharmacological effects of ginseng (Panax ginseng Meyer). It is known to have numerous structural and pharmacological properties. However, aqueous solubility and delivery of ginsenosides in targeted region by avoiding undesirable toxicity to normal cell is also of prime importance. The aim of this study was to obtain amphiphilic ginsenoside derivatives in which hydrophilic polymers were conjugated to ginsenosides to enhance the water solubility and targeted delivery. To this end, the hydrophobic protopanaxadiol ginsenoside aglycone (aPPD) was covalently conjugated to the backbone of hydrophilic polyethylene glycol (PEG) through a pH sensitive ester linkage, which was confirmed by

Topics: Cell Line, Tumor; Drug Screening Assays, Antitumor; Ginsenosides; Humans; Polyethylene Glycols; Sapogenins

The aim of this study was to fabricate 20(S)-protopanaxadiol (PPD) nanocrystals to improve PPD's oral bioavailability and brain delivery. PPD nanocrystals were fabricated using an anti-solvent precipitation approach where d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) was optimized as the stabilizer. The fabricated nanocrystals were nearly spherical with a particle size and drug loading of 90.44 ± 1.45 nm and 76.92%, respectively. They are in the crystalline state and stable at 4°C for at least 1 month. More than 90% of the PPD could be rapidly released from the nanocrystals, which was much faster than the physical mixture and PPD powder. PPD nanocrystals demonstrated comparable permeability to solution at 2.52 ± 0.44×10(-5)cm/s on MDCK monolayers. After oral administration of PPD nanocrystals to rats, PPD was absorbed quickly into the plasma and brain with significantly higher Cmax and AUC0-t compared to those of the physical mixture. However, no brain targeting was observed, as the ratios of the plasma AUC0-t to brain AUC0-t for the two groups were similar. In summary, PPD nanocrystals are a potential oral delivery system to improve PPD's poor bioavailability and its delivery into the brain for neurodegenerative disease and intracranial tumor therapies in the future.

Topics: Administration, Oral; Animals; Biological Availability; Brain; Chemistry, Pharmaceutical; Dogs; Drug Carriers; Drug Liberation; Madin Darby Canine Kidney Cells; Male; Nanoparticles; Particle Size; Rats; Sapogenins; Solubility

A series of oxidized products have been systematically semisynthesized from 20(S)-ginsenoside Rg3, Rh2, 20(S)-protopanaxadiol (PPD) and their 20(R)-epimers and the majority of these products were evaluated for their cytotoxic activity against HeLa cells and HepG2 cells by MTT assay for the first time. Twenty-two products were obtained and elucidated based on comprehensive (1)H NMR, (13)C NMR, two-dimensional (2D) NMR, and mass spectral data and the results reported in previous literature. All the four ocotillol type saponins (20S,24R(δ86, δ85); 20S,24S(δ87, δ88); 20R,24R(δ86, δ86); 20R,24S(δ86, δ87) were obtained. In addition, eight compounds (3, 8, 9, 10, 15, 16, 19 and 22) with the cyclized side chain were firstly identified. Most of the tested compounds possessed cytotoxicity to a certain degree against the two types of cells which implied these oxidized products could play a certain role on anti-cancer functions of the raw materials in vivo. Meanwhile, the results proved that the configurations at C-20 or C-24 and the number of glycosyl at C-3 have important influence on the cytotoxicity. The products 1, 2, 11-17, 20 and 22 should possess great activities and deserved further investigation as potential cytotoxic agents.

Topics: Antineoplastic Agents; Chemistry Techniques, Synthetic; Ginsenosides; HeLa Cells; Hep G2 Cells; Humans; Oxidation-Reduction; Sapogenins; Stereoisomerism; Structure-Activity Relationship; Triterpenes

The potential cancer preventive roles of calcitriol, the dihydroxylated metabolite of Vitamin D3, as well as 20(S)-protopanaxadiol (aPPD), the aglycone of the protopanaxadiol family of ginsenosides, have gained much attention in recent years for the prevention/treatment of prostate cancer (PCa). In the present study, we evaluated the anticancer and chemosensitization effects of calcitriol at clinically relevant concentrations and aPPD, either alone or in combination, in two well-characterized human PCa cell lines: androgen-sensitive non-metastatic LNCaP cells and androgen-independent metastatic C4-2 cells. The effects of the treatments on PCa cell viability and proliferation rates were evaluated by MTS and Brdu assays, respectively. Combination Indices (CI) and Dose Reduction Indices (DRI) were estimated to assess synergistic anticancer activity using Calcusyn software (Biosoft, Cambridge, UK). Then, we determined the potential Pharmacodynamic interaction mechanisms as follows: The protein expression levels of the genes those are known to control cell cycle (cyclin D1 and cdk2); apoptosis (Bcl-2, Bax, and Capspases 3), androgen receptor and Vitamin D receptors were examined upon combinational treatment. The cell viability assay data show that addition of 10nM calcitriol to aPPD significantly lowered its IC50 values from the range of 41-53μM to 13-23μM, in LNCaP and C4-2 prostate cancer cells. The cell proliferation rate was significantly lower for combination treatments compared to the cells treated with aPPD alone. Similarly, Western blot results indicate that aPPD significantly upregulated Vitamin D receptor (VDR) expression, while calcitriol further enhanced the ability of aPPD to induce pro-apoptotic BAX, increased cleaved caspase-3 and downregulate cdk2 protein levels. Thus, the pharmacodynamic interaction between aPPD and calcitriol in impacting growth inhibition and apoptosis appears to be synergistic in nature. In conclusion, calcitriol sensitizes PCa cells to aPPD-mediated anticancer effects by enhancing its ability to induce apoptosis and reduce cell proliferation, and this synergism may limit calcitriol toxicity by facilitating the use of lower calcitriol doses. The associated increase in VDR expression and calcitriol half-life may be mechanistically associated with this sensitization effect.

Topics: Antineoplastic Agents; Apoptosis; Calcitriol; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Humans; Intracellular Signaling Peptides and Proteins; Kallikreins; Male; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Receptors, Calcitriol; Sapogenins

Ginseng (Panax ginseng C.A. Mey.) is commonly used in traditional oriental medicine for its wide spectrum of medicinal properties, including anti-inflammatory, antitumorigenic, adaptogenic and anti-aging properties. 20(S)-Protopanaxadiol (PPD), the main intestinal metabolite of ginsenosides, is one of the active ingredients in ginseng. In this study, we aimed to investigate the neuroprotective effects of PPD on PC12 cells; however, the underlying mechanisms remain elusive. We examined cell viability by MTT assay and the morphological changes of PC12 cells following glutamate‑induced cell damage and evaluated the anti‑apoptotic effects of PPD using Hoechst 33258 staining, western blot analysis and Muse™ Cell Analyzer and the antioxidant effects of PPD using FACS analysis and immunofluorescence. Furthermore, PPD exerted protective effects on PC12 cells via the inhibition of mitochondrial damage against glutamate-induced excitotoxicity using immunofluorescence, electron microscopy and FACS analysis. We demonstrate that treatment with PPD suppresses apoptosis, which contributes to the neuroprotective effects of PPD against glutamate‑induced excitotoxicity in PC12 cells. Treatment with PPD inhibited nuclear condensation and decreased the number of Annexin V-positive cells. In addition, PPD increased antioxidant activity and mitochondrial homeostasis in the glutamate-exposed cells. These antioxidant effects were responsible for the neuroprotection and enhanced mitochondrial function following treatment with PPD. Furthermore, PD inhibited the glutamate-induced morphological changes in the mitochondria and scavenged the mitochondrial and cytosolic reactive oxygen species (ROS) induced by glutamate. In addition, mitochondrial function was significantly improved in terms of mitochondrial membrane potential (MMP) and enhanced mitochondrial mass compared with the cells exposed to glutamate and not treated with PPD. Taken together, the findings of our study indicate that the antioxidant effects and the enhanced mitochondrial function triggered by PPD contribute to the inhibition of apoptosis, thus leading to a neuroprotective response, as a novel survival mechanism.

Topics: Animals; Apoptosis; Cytosol; Glutamic Acid; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neuroprotective Agents; Panax; PC12 Cells; Rats; Reactive Oxygen Species; Sapogenins

Ginsenosides, the major bioactive components of Panax ginseng, are regarded as promising high-value pharmaceutical compounds. In ginseng, ginsenosides are produced from their precursor protopanaxadiol. Recently, an artificial biosynthetic pathway of protopanaxadiol was built in Saccharomyces cerevisiae by introducing a P. ginseng dammarenediol-II synthase, a P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS), and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). In this engineered yeast strain, however, the low metabolic flux through PPDS resulted in a low productivity of protopanaxadiol. Moreover, health of the yeast cells was significantly affected by reactive oxygen species released by the pool coupling between PPDS and ATR1. To overcome the obstacles in protopanaxadiol production, PPDS was modified through transmembrane domain truncation and self-sufficient PPDS-ATR1 fusion construction in this study. The fusion enzymes conferred approximately 4.5-fold increase in catalytic activity, and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. Our in vivo experiment indicated that the engineered yeast carrying fusion protein effectively converted 96.8% of dammarenediol-II into protopanaxadiol. Protopanaxadiol production in a 5 L bioreactor in fed-batch fermentation reached 1436.6 mg/L. Our study not only improved protopanaxadiol production in yeast, but also provided a generic method to improve activities of plant cytochrome P450 monooxygenases. This method is promising to be applied to other P450 systems in yeast. Biotechnol. Bioeng. 2016;113: 1787-1795. © 2016 Wiley Periodicals, Inc.

Topics: Cytochrome P-450 Enzyme System; Fermentation; Metabolic Engineering; Saccharomyces cerevisiae; Sapogenins; Saponins; Synthetic Biology; Triterpenes

In cancer cells, failure of chemotherapy is often caused by the ATP-binding cassette subfamily B member 1 (ABCB1), and few drugs have been successfully developed to overcome ABCB1-mediated multi-drug resistance (MDR). To suppress ABCB1 activity, we previously designed and synthesized a new series of derivatives based on 20(S)-protopanoxadiol (PPD). In the present study, we investigated the role of PPD derivatives in the function of ABC transporters. Non-toxic concentrations of the PPD derivative PPD12 sensitized ABCB1-overexpressing cells to their anti-cancer substrates better than either the parental PPD or inactive PPD11. PPD12 increased intracellular accumulation of adriamycin and rhodamine123 in resistant cancer cells. Although PPD12 did not suppress the expression of ABCB1 mRNA or protein, it stimulated the activity of ABCB1 ATPase. Because PPD12 is a competitive inhibitor, it was predicted to bind to the large hydrophobic cavity of homology-modeled human ABCB1. PPD12 also enhanced the efficacy of adriamycin against ABCB1-overexpressing KB/VCR xenografts in nude mice. In conclusion, PPD12 enhances the efficacy of substrate drugs in ABCB1-overexpressing cancer cells. These findings suggest that a combination therapy consisting of PPD12 with conventional chemotherapeutic agents may be an effective treatment for ABCB1-mediated MDR cancer patients.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; Cell Line, Tumor; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; HEK293 Cells; HL-60 Cells; Humans; Male; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Rhodamines; Sapogenins; Xenograft Model Antitumor Assays

20(S)- and 20(R)-ginsenoside Rg3 are a pair of epimers which could be deglycosylated to ginsenoside Rh2 and protopanaxadiol (PPD) in vivo. To better understand the differences of pharmacokinetic parameters and metabolism behaviors of Rg3 epimers in rat plasma, a sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and fully validated. This chromatographic method separate 20(S)-/20(R)-Rg3, 20(S)-/20(R)-Rh2 and 20(S)-/20(R)-PPD by gradient elution of 10 mM ammonium acetate solution (pH 5.0) and acetonitrile on a C18 column with a total run time of 15 min. 20(S)-protopanaxatriol (PPT) was used as internal standard, and multiple reaction monitoring (MRM) mode with negative electrospray ionization were performed. The lower limit of quantitations (LLOQs) were between 4.2 and 4.8 ng/ml, and the accuracies were between 91.7% and 112.2% with intra- and inter-day precisions less than 11.6%. This method was successfully applied to a pharmacokinetic study of intravenous and intra-gastric administration of 20(S)-Rg3 and 20(R)-Rg3 to rats. It has been found that both epimers can be deglycosylated to their corresponding chiral metabolites, i.e., Rh2 and PPD, with different extents. However, 20(R)-Rg3 underwent single direction chiral inversion to 20(S)-Rg3 in rats. Stereoselective pharmacokinetic parameters, metabolic degrees and chiral inversion extents of Rg3 epimers in rats were also discussed for the first time.

Topics: Animals; Chromatography, Liquid; Ginsenosides; Male; Plasma; Rats; Rats, Sprague-Dawley; Sapogenins; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Tandem Mass Spectrometry

This paper, for the first time, reported a speedy hyphenated technique of low toxic dual ultrasonic-assisted dispersive liquid-liquid microextraction (dual-UADLLME) coupled with microwave-assisted derivatization (MAD) for the simultaneous determination of 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT). The developed method was based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection using multiple-reaction monitoring (MRM) mode. A mass spectrometry sensitizing reagent, 4'-carboxy-substituted rosamine (CSR) with high reaction activity and ionization efficiency was synthesized and firstly used as derivatization reagent. Parameters of dual-UADLLME, MAD and UHPLC-MS/MS conditions were all optimized in detail. Low toxic brominated solvents were used as extractant instead of traditional chlorinated solvents. Satisfactory linearity, recovery, repeatability, accuracy and precision, absence of matrix effect and extremely low limits of detection (LODs, 0.010 and 0.015ng/mL for PPD and PPT, respectively) were achieved. The main advantages were rapid, sensitive and environmentally friendly, and exhibited high selectivity, accuracy and good matrix effect results. The proposed method was successfully applied to pharmacokinetics of PPD and PPT in rat plasma.

Topics: Animals; Blood Chemical Analysis; Chromatography, High Pressure Liquid; Limit of Detection; Liquid Phase Microextraction; Microwaves; Rats; Sapogenins; Solvents; Tandem Mass Spectrometry; Ultrasonics

Peroxisome proliferator activated receptor (PPAR) is a nuclear receptor that is one of the transcription factors regulating lipid and glucose metabolism. Fermented ginseng (FG) is a ginseng fermented by Lactobacillus paracasei A221 containing minor ginsenosides and metabolites of fermentation. DNA microarray analysis of rat liver treated with FG indicated that FG affects on lipid metabolism are mediated by PPAR-α. To identify a PPAR-α agonist in FG, PPAR-α transcription reporter assay-guided fractionation was performed. The fraction obtained from the MeOH extract of FG, which showed potent transcription activity of PPAR-α, was fractionated by silica gel column chromatography into 16 subfractions, and further separation and crystallization gave compound 1 together with four known constituents of ginseng, including 20(R)- and 20(S)-protopanaxadiol, and 20(R)- and 20(S)-ginsenoside Rh1. The structure of compound 1 was identified as 10-hydroxy-octadecanoic acid by (1)H- and (13)C-NMR spectra and by EI-MS analysis of the methyl ester of 1. Compound 1 demonstrated much higher transcription activity of PPAR-α than the other isolated compounds. In addition, compound 1 also showed 5.5-fold higher transcription activity of PPAR-γ than vehicle at the dose of 20 μg/mL. In the present study, we identified 10-hydroxy-octadecanoic acid as a dual PPAR-α/γ agonist in FG. Our study suggested that metabolites of fermentation, in addition to ginsenosides, contribute to the health benefits of FG.

Topics: Animals; Cell Line; Chlorocebus aethiops; Fermentation; Ginsenosides; Glucose; Lacticaseibacillus paracasei; Lipid Metabolism; Male; Molecular Structure; Panax; Plant Extracts; PPAR alpha; PPAR gamma; Rats, Wistar; Sapogenins; Stearic Acids; Transcription, Genetic

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Topics: Area Under Curve; Calorimetry, Differential Scanning; Chemistry, Pharmaceutical; Microscopy, Electron, Scanning; Molecular Docking Simulation; Molecular Structure; Phospholipids; Powder Diffraction; Sapogenins; Solubility; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

A novel strategy for the qualitative and quantitative determination of 20(S)-protopanaxatriol saponins (PTS) and 20(S)-protopanaxadiol saponins (PDS) in Panax notoginseng, Panax ginseng and Panax quinquefolium, based on the overlapping peaks of main components of PTS (calibrated by ginsenoside Rg1) and PDS (calibrated by ginsenoside Rb1), was proposed. The analysis was performed by using high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD). Under specific chromatographic conditions, all samples showed two overlapping peaks containing several main ginsenosides belonging to PTS and PDS, respectively. The overlapping peaks were also identified by using HPLC-MS. Based on the sum and ratio of PTS and PDS, 60 tested Panax samples were divided into three main clusters according to their species. The findings suggested that this strategy provides a simple and rapid approach to quantify PTS and PDS in Panax herbs.

Topics: Algorithms; Chromatography, High Pressure Liquid; Ginsenosides; Mass Spectrometry; Panax; Sapogenins; Saponins

This study aimed to assess the skin-related anti-photoaging activities of the 2 epimeric forms of protopanaxadiol (PPD), 20(S)-PPD and 20(R)-PPD, in cultured human keratinocytes (HaCaT cells). The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), as well as cell viability for HaCaT cells under UV-B irradiation. The activities for MMP-2 and -1 in conditioned medium were determined using gelatin zymography, and MMP-2 protein in the conditioned medium was detected using Western blot analysis. 20(S)-PPD, but not 20(R)-PPD, suppressed UV-B-induced ROS elevation. Neither of the epimers, at the concentrations used, exhibited cytotoxicity, irrespective of UV-B irradiation. 20(S)-PPD, but not 20(R)-PPD, exhibited an inhibitory effect on UV-B-induced MMP-2 activity and expression in HaCaT cells. In brief, only 20(S)-PPD, a major metabolic product of PPD-type ginsenosides, inhibits UV-B-induced ROS and MMP-2 elevation, implying its stereospecific anti-photoaging activity on the skin.

Topics: Cell Line, Transformed; Cell Survival; Humans; Keratinocytes; Matrix Metalloproteinase 2; Reactive Oxygen Species; Sapogenins; Stereoisomerism; Ultraviolet Rays

(20S,24S)-epoxy-dammarane-3,12,25-triol (24S-epimer) and (20S,24R)-epoxy- dammarane-3,12,25-triol (24R-epimer), a pair of ocotillol type epimers, were identified as the main metabolites of 20(S)-protopanaxadiol (PPD). The aim of this study was to systematically investigate the formation and metabolism of this pair of epimers in vivo and in vitro and to elucidate the isoforms of cytochrome P450 enzymes responsible for the stereoselective metabolism of both epimers. The result showed that 24S-epimer was a more predominant ingredient in rat plasma after oral administration of PPD with higher area under the curve (AUC) values. Both the enzyme kinetic evaluations of the formation and elimination of 24S-epimer and 24R-epimer in rat liver microsomes (RLM) and human liver microsomes (HLM) indicated that 24S-epimer had a higher formation rate and a lower oxygenation metabolism rate than 24R-epimer, and the stereoselective differences were more obvious in HLM than in RLM. The chemical inhibition and recombinant human P450 isoforms assay showed that CYP3A4 was the predominant isoform responsible for the further metabolism of 24R-epimer in HLM. The biliary excretion ratio of the 24S-epimer glucuronide was more than 28-fold higher than that of 24R-epimer glucuronide after intravenous administration to rats, which also indicated 24S-epimer was more preferential to be metabolized as the glucuronide conjugate than 24R-epimer.

Topics: Administration, Oral; Animals; Bile; Cytochrome P-450 Enzyme System; Ginsenosides; Humans; Male; Microsomes, Liver; Rats, Sprague-Dawley; Sapogenins; Saponins; Species Specificity; Stereoisomerism

A β-glucosidase from Gordonia terrae was cloned and expressed in Escherichia coli. The recombinant enzyme with a specific activity of 16.4 U/mg for ginsenoside Rb1 was purified using His-trap chromatography. The purified enzyme specifically hydrolyzed the glucopyranosides at the C-20 position in protopanaxadiol (PPD)-type ginsenosides and hydrolyzed the glucopyranoside at the C-6 or C-20 position in protopanaxatriol (PPT)-type ginsenosides. The reaction conditions for the high-level production of Rg3 from Rb1 by the enzyme were pH 6.5, 30°C, 20 mg/ml enzyme, and 4 mg/ml Rb1. Under these conditions, G. terrae β-glucosidase completely converted Rb1 and Re to Rg3 and Rg2, respectively, after 2.5 and 8 h, respectively. Moreover, the enzyme converted Rg1 to Rh1 at 1 h with a molar conversion yield of 82%. The enzyme at 10 mg/ml produced 1.16 mg/ml Rg3, 1.47 mg/ml Rg2, and 1.17 mg/ml Rh1 from Rb1, Re, and Rg1, respectively, in 10% (w/v) ginseng root extract at pH 6.5 and 30°C after 33 h with molar conversion yields of 100%, 100%, and 77%, respectively. The combined molar conversion yield of Rg2, Rg3, and Rh1 from total ginsenosides in 10% (w/v) ginseng root extract was 68%. These above results suggest that this enzyme is useful for the production of ginsenosides Rg3, Rg2, and Rh1.

Topics: beta-Glucosidase; Escherichia coli; Ginsenosides; Gordonia Bacterium; Hydrogen-Ion Concentration; Molecular Weight; Panax; Plant Extracts; Plant Roots; Sapogenins; Substrate Specificity; Temperature

In this study, we evaluated the effects of protopanaxadiol (PPD), a gut microbiome induced ginseng metabolite, in increasing the anticancer effects of a chemotherapeutic agent fluorouracil (5-FU) on colorectal cancer. An in vitro HCT-116 colorectal cancer cell proliferation test was conducted to observe the effects of PPD, 5-FU and their co-administration and the related mechanisms of action. Then, an in vivo xenografted athymic mouse model was used to confirm the in vitro data. Our results showed that the human gut microbiome converted ginsenoside compound K to PPD as a metabolite. PPD and 5-FU significantly inhibited HCT-116 cell proliferation in a concentration-dependent manner (both p<0.01), and the effects of 5-FU were very significantly enhanced by combined treatment with PPD (p<0.01). Cell cycle evaluation demonstrated that 5-FU markedly induced the cancer cell S phase arrest, while PPD increased arrest in G1 phase. Compared to the control, 5-FU and PPD increased apoptosis, and their co-administration significantly increased the number of apoptotic cells (p<0.01). Using bioluminescence imaging, in vivo data revealed that 5-FU significantly reduced the tumor growth up to Day 20 (p<0.05). PPD and 5-FU co-administration very significantly reduced the tumor size in a dose-related manner (p<0.01 compared to the 5-FU alone). The quantification of the tumor size and weight changes for 43 days supported the in vivo imaging data. Our results demonstrated that the co-administration of PPD and 5-FU significantly inhibited the tumor growth, indicating that PPD significantly enhanced the anticancer action of 5-FU, a commonly used chemotherapeutic agent. PPD may have a clinical value in 5-FU's cancer therapeutics.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Cell Proliferation; Colonic Neoplasms; Disease Models, Animal; Fluorouracil; HCT116 Cells; Humans; Mice; Mice, Nude; Panax; Sapogenins; Treatment Outcome; Xenograft Model Antitumor Assays

Among important components of American ginseng, protopanaxadiol (PPD) showed more active anticancer potential than other triterpenoid saponins. In this study, we determined the in vivo effects of PPD in a mouse cancer model first. Then, using human colorectal cancer cell lines, we observed significant cancer cell growth inhibition by promoting G1 cell cycle redistribution and apoptosis. Subsequently, we characterized the downstream genes targeted by PPD in HCT-116 cancer cells. Using Affymetrix high density GeneChips, we obtained the gene expression profile of the cells. Microarray data indicated that the expression levels of 76 genes were changed over two-fold after PPD, of which 52 were upregulated while the remaining 24 were downregulated. Ingenuity pathway analysis of top functions affected was carried out. Data suggested that by regulating the interactions between p53 and DR4/DR5, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway played a key role in the action of PPD, a promising colon cancer inhibitory compound.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Profiling; Humans; Mice; Panax; Sapogenins; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Xenograft Model Antitumor Assays

The aim of the present study was to determine the mechanisms through which 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (20GPPD) promotes the production of hyaluronic acid (HA) in human keratinocytes. 20GPPD is the primary bioactive metabolite of Rb1, a major ginsenoside found in ginseng (Panax ginseng). We sought to elucidate the underlying mechanisms behind the 20GPPD-induced production of HA. We found that 20GPPD induced an increase in HA production by elevating hyaluronan synthase 2 (HAS2) expression in human keratinocytes. The phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was also enhanced by 20GPPD in a dose-dependent manner. The pharmacological inhibition of ERK (using U0126) or Akt (using LY294002) suppressed the 20GPPD-induced expression of HAS2, whereas treatment with an epidermal growth factor receptor (EGFR) inhibitor (AG1478) or an intracellular Ca2+ chelator (BAPTA/AM) did not exert any observable effects. The increased Src phosphorylation was also confirmed following treatment with 20GPPD in the human keratinocytes. Following pre-treatment with the Src inhibitor, PP2, both HA production and HAS2 expression were attenuated. Furthermore, the 20GPPD-enhanced ERK and Akt signaling decreased following treatment with PP2. Taken together, our results suggest that Src kinase plays a critical role in the 20GPPD-induced production of HA by acting as an upstream modulator of ERK and Akt activity in human keratinocytes.

Topics: Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Ginsenosides; Humans; Hyaluronic Acid; Keratinocytes; Phosphorylation; Proto-Oncogene Proteins c-akt; Sapogenins; src-Family Kinases

Panax ginseng C.A. MEYER (Araliaceae), which contains ginsenosides as its main components, has been shown to have various biological effects, including anti-inflammatory, anxiolytic, anti-stress, and anti-tumor effects. Orally administered ginsenoside Rb1 and Re are metabolized to 20(S)-protopanaxadiol (PPD) and compound K via ginsenoside Rd and 20(S)-protopanaxatriol (PPT) and ginsenoside Rh1 via ginsenoside Rg1 by gut microbiota, respectively. Therefore, we investigated the anti-stress effects of these metabolites, PPD and PPT, by measuring their anxiolytic and anti-inflammatory effects in immobilized mice. Treatment with PPD and PPT prior to immobilization stress increased the time spent in open arms and open arm entries in the elevated plus-maze (EPM) test. The anxiolytic effects of PPD (10 mg/kg) and PPT (10 mg/kg) were comparable to that of buspirone (1 mg/kg). This observed anxiolytic effect of PPD was significantly blocked by flumazenil or bicuculline, and the effect of PPT was blocked by WAY-100635. Treatment with PPD also potently suppressed immobilization stress-induced serum levels of corticosterone and interleukin (IL)-6 by the enzyme-linked immunosorbent assay. However, PPT treatment did not suppress them. Based on these findings, PPD and PPT may exhibit the anxiolytic effect via γ-aminobutyrateA (GABAA) receptor(s) and serotonergic receptor(s), respectively, and PPD may have an anti-inflammatory effect that is more potent than that of PPT.

Topics: Animals; Anti-Anxiety Agents; Anti-Inflammatory Agents; Bicuculline; Corticosterone; Flumazenil; GABA Modulators; GABA-A Receptor Antagonists; Interleukin-6; Male; Mice, Inbred ICR; Piperazines; Pyridines; Receptors, GABA-A; Receptors, Serotonin; Restraint, Physical; Sapogenins; Serotonin Antagonists; Stress, Psychological

The P2X7 receptor is an ATP-gated ion channel predominantly expressed in immune cells and plays a key role in inflammatory processes. Ginseng is a well-known Chinese herb with both pro- and anti-inflammatory properties and many of its actions have been ascribed to constituent ginsenosides. We screened a number of ginsenoside compounds for pharmacological activity at P2X7 receptors, that might contribute to the reported immunomodulatory actions of ginseng.. We used several assays to measure responses of P2X7 receptors, ATP-mediated dye uptake, intracellular calcium measurement and whole-cell patch-clamp recordings. HEK-293 cells stably expressing human P2X7 receptors were used in addition to mouse macrophages endogenously expressing P2X7 receptors.. Four ginsenosides of the protopanaxdiol series, Rb1, Rh2, Rd and the metabolite compound K (CK) potentiated the dye uptake responses of P2X7 receptors, whereas other ginsenosides tested were ineffective (1-10 μM). The potentiation was rapid in onset, required a threshold concentration of ATP (>50 μM) and had an EC50 of 1.08 μM. CK markedly enhanced ATP-activated P2X7 currents, probably via an extracellular site of action. One of the consequences of this potentiation effect is a sustained rise in intracellular Ca(2+) that could account for the decrease in cell viability in mouse macrophages after a combination of 500 μM ATP and 10 μM CK that are non-toxic when applied alone.. This study identifies selected ginsenosides as novel potent allosteric modulators of P2X7 channels that may account for some of the reported immune modulatory actions of protopanaxdiol ginsenosides in vivo.

Topics: Adenosine Triphosphate; Animals; Benzoxazoles; Calcium; Cell Line; Fluorescent Dyes; Ginsenosides; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Patch-Clamp Techniques; Quinolinium Compounds; Receptors, Purinergic P2X7; Sapogenins

Nine new minor dehydrogenated and cleavaged dammarane-type triterpenoid saponins, namely notoginsenosides ST6-ST14 (1-9) were isolated from the steamed roots of Panax notoginseng, together with 14 known ones. Among them, 5-7 and 21-22 were protopanaxadiol type and the left 18 compounds, including 1-4, 8-20, and 23 were protopanaxatriol type saponins. Their structures were identified by extensive analysis of MS, 1D and 2D NMR spectra, and acidic hydrolysis. Resulted from the side chain cleavage, the new saponins 1 and 2 featured in a ketone group at C-25, and 3-5 had an aldehyde unit at C-23. The known saponins 12, 16 and 18 displayed the enhancing potential of neurite outgrowth of NGF-mediated PC12 cells at a concentration of 10 μM, while 20 exhibited acetyl cholinesterase inhibitory activity, with IC50 value of 13.97 μM.

Topics: Animals; Cholinesterase Inhibitors; Dammaranes; Inhibitory Concentration 50; Molecular Structure; Panax notoginseng; PC12 Cells; Plant Roots; Rats; Sapogenins; Saponins; Triterpenes

Protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has high medicinal values. In this work, we reported the PPD production in transgenic tobacco co-overexpressing PgDDS and CYP716A47. PPD is an aglycone of ginsenosides produced by Panax species and has a wide range of pharmacological activities. PPD is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme. Here, we established a PPD production system via cell suspension culture of transgenic tobacco co-overexpressing the genes for PgDDS and CYP716A47. The concentration of PPD in transgenic tobacco leaves was 2.3-5.7 µg/g dry weight (DW), depending on the transgenic line. Leaf segments were cultured on medium with various types of hormones to induce callus. Auxin treatment, particularly 2,4-D, strongly enhanced the production of DD (783.8 µg g(-1) DW) and PPD (125.9 µg g(-1) DW). Treatment with 2,4-D enhanced the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes. PPD production reached 166.9 and 980.9 µg g(-1) DW in a 250-ml shake flask culture and in 5-l airlift bioreactor culture, respectively.

Topics: 2,4-Dichlorophenoxyacetic Acid; Alkyl and Aryl Transferases; Bioreactors; Biosynthetic Pathways; Cells, Cultured; Cytochrome P-450 Enzyme System; Gas Chromatography-Mass Spectrometry; Genes, Plant; Ginsenosides; Mevalonic Acid; Nicotiana; Panax; Plant Proteins; Plants, Genetically Modified; Sapogenins; Saponins; Triterpenes

20(S)-Protopanaxadiol (20(S)-PPD) is one type of sapogenin of protopanaxadiols and has a variety of pharmacological activities. In order to improve the dissolution of 20(S)-PPD as well as its oral bioavailability, a self-microemulsifying drug delivery system (SMEDDS) was utilized for 20(S)-PPD preparation. Following the preparation of the 20(S)-PPD SMEDDS, its dissolution, stability, and intestinal absorption in rats were studied, and the pharmacokinetics and optimal dosage after oral administration were evaluated. The dissolution tendency of the SMEDDS in phosphate buffered saline (PBS), 0.1 M HCl and distilled water was consistent. SMEDDS was stable under a condition of high temperature (40°C), high humidity or with strong light irradiation, or within 6 h in artificial digestive tracts. 20(S)-PPD SMEDDS was well-absorbed in all intestinal segments in rats. When the drug concentration was higher than 200 µg/mL or the perfusion flow was faster than 0.5 mL/min, passive diffusion of drug in the duodenum reached a saturated level. In addition, P-glycoprotein inhibitor did not affect the intestinal absorption of 20(S)-PPD SMEDDS. Pharmacokinetic study showed that Tmax in male rats was shortened significantly, while Cmax and area under the curve (AUC(0-t)) were remarkably increased. The relative oral bioavailability of 20(S)-PPD SMEDDS was increased approximately three fold compared with the 20(S)-PPD carboxy methyl cellulose (CMC). 20(S)-PPD SMEDDS (100 mg/mL) was administered by gastric infusion to both mice and rats for 14 d. SMEDDS improved the oral bioavailability of 20(S)-PPD and reduced the necessary drug dosage. 20(S)-PPD SMEDDS could become a promising clinical alternative as an anti-tumor or antidepressant drug.

Topics: Administration, Oral; Animals; Biological Availability; Drug Delivery Systems; Female; Intestinal Absorption; Male; Mice; Mice, Inbred Strains; Particle Size; Rats; Rats, Wistar; Sapogenins; Surface Properties; Tissue Distribution

Ginseng (Panax ginseng C.A. MEYER, Araliaceae), which contains protopanaxadiol-type and protopanaxatriol-type ginsenosides, has been used for inflammation, fatigue, stress, and tumor in Asian countries. Orally administered ginsenosides are metabolized to their aglycones 20(S)-protopanaxadiol (PPD) and 20(S)-protopanaxatriol (PPT) by gut microbiota. However, their anti-fatigue effects have not been studied thoroughly. Therefore, we investigated the anti-fatigue activities of PPD and PPT in mice, using the weight-loaded swimming (WLS) and the rota-rod tests. Ginseng water extract (GW), ginseng saponin fraction (GWS) and ginseng polysaccharide fraction (GWP) at concentrations of 50 and 100 mg/kg and PPD and PPT at 5 and 10 mg/kg were orally administered to mice once daily for 5 d. GW, GWS, and PPT significantly increased the WLS time, however, GWP and PPD did not cause any significant change. PPT induced the most significant increase in WLS time. PPD (10 mg/kg) and PPT (5 and 10 mg/kg) inhibited the WLS-induced increase in corticosterone, lactate, lactate dehydrogenase (LDH), and creatinine levels as well as the reduction in glucose level. PPT increased the riding time in the rota-rod test, and also inhibited corticosterone, lactate, and creatinine levels. These findings suggest that the anti-fatigue effect of ginseng may be attributable to its saponins, particularly PPT, rather than to its polysaccharides.

Topics: Animals; Corticosterone; Creatinine; Fatigue; Fatty Acids, Nonesterified; Lactic Acid; Male; Mice; Mice, Inbred ICR; Rotarod Performance Test; Sapogenins; Swimming

Gypenoside LVI and gypenoside XLVI are the major bioactive dammarane saponins from Gynostemma pentaphyllum. Gypenoside LVI, gypenoside XLVI, and their metabolite 2α-OH-protopanaxadiol (2α-OH-PPD) possess potent non-small cell lung carcinoma A549 cell inhibitory activity. A sensitive liquid chromatography tandem mass spectrometry method was developed and validated to study the pharmacokinetics of gypenoside LVI and XLVI, 2α-OH-PPD, metabolite 1 (M1), and metabolite 2 (M2) after administration of gypenosides or 2α-OH-PPD. Plasma samples from rats were protein precipitated with methanol. Analytes were detected by triple quadrupole MS/MS with an electrospray ionization source in the positive multiple reaction monitoring mode. The transition m/z 441.4→109.2 was selected to quantify gypenoside LVI and XLVI, and 2α-OH-PPD, because of the extensive conversion of the gypenosides to aglycone in the ionization source. M1 and M2 are isomers that shared the transition m/z 493.4→143.1. To avoid interference, the baseline separation of each analyte was performed on a SunFire C18 column with a gradient of acetonitrile (0.1% formic acid, v/v) and water (0.1% formic acid, v/v). The chromatographic run time was 10min. The linearity was validated over a plasma concentration range from 2.00 to 2000ng/mL for M1 and M2, and from 10.0 to 2000 for gypenosides LVI and XLVI, and 2α-OH-protopanaxadiol. The lower limits of quantification were 10.0, 10.0, 10.0, 2.00, and 2.00ng/mL for gypenoside LVI, gypenoside XLVI, 2α-OH-PPD, M1, and M2, respectively, with acceptable intra-/inter-day precision and accuracy. The extraction recovery rates were >86.9% for each compound. No apparent matrix effect or instability was observed during each step of the bioanalysis. After full validation, this method was proved to be simple, fast, and efficient in analyzing large batches of plasma samples for the analytes.

Topics: Animals; Chromatography, High Pressure Liquid; Gynostemma; Limit of Detection; Linear Models; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sapogenins; Tandem Mass Spectrometry

Chemical and pharmacological studies of Panax vietnamensis (Vietnamese ginseng; VG) have been reported since its discovery in 1973. However, the content of each saponin in different parts of VG has not been reported. In this study, 17 ginsenosides in the different underground parts of P. vietnamensis were analyzed by HPLC/evaporative light scattering detector (ELSD). Their contents in the dried rhizome, radix, and fine roots were 195, 156, and 139 mg/g, respectively, which were extremely high compared to other Panax species. The content of protopanaxatriol (PPT)-type saponins were not much different among underground parts; however, the content of protopanaxadiol (PPD)- and ocotillol (OCT)-type saponins were greatly different. It is noteworthy that the ginsenoside pattern in the fine roots is different from other underground parts. In particular, despite the content of PPD-type saponins being the highest in the fine roots, which is similar to other Panax species, the total content of saponins was the lowest in the fine roots, which is different from other Panax species. The ratios of PPT : PPD : OCT-type saponins were 1 : 1.7 : 7.8, 1 : 1.6 : 5.5, and 1 : 4.8 : 3.3 for the rhizome, radix, and fine roots, respectively. OCT-type saponins accounted for 36-75% of total saponins and contributed mostly to the difference in the total saponin content of each part.

Topics: Chromatography, High Pressure Liquid; Ginsenosides; Panax; Plant Roots; Sapogenins; Saponins

20(S)-Protopanaxadiol (aPPD), a ginseng sapogenin, has been shown to be a promising anti-cancer compound and anti-depressant agent. Although the bacterial biotransformation of ginsenosides has been studied thoroughly, few have reported on the cytochrome P450 (P450) mediated metabolism of aPPD. Taken orally, aPPD must first undergo absorption and metabolism in the intestine before further metabolism in the liver. The present study investigated the comparative biotransformation profile of aPPD in human intestinal microsomes (HIM) and human liver microsomes (HLM) and characterized the human P450 enzymes involved in aPPD metabolism. Three major monooxygenated metabolites and five minor dioxygenated metabolites were identified as the predominant products in aPPD incubations with HIM and HLM using liquid chromatography-mass spectrometry. Reaction phenotyping studies were performed with a panel of specific P450 chemical inhibitors, antibody inhibition and human recombinant P450 enzymes. Ketoconazole, a CYP3A inhibitor, blocked the formation of oxygenated metabolites of aPPD in both HIM and HLM in a concentration dependent manner. Among the human recombinant P450 enzymes assayed, CYP3A4 exhibited the highest activity towards aPPD oxidative metabolite formation, followed by CYP3A5. In summary, the results have shown that aPPD is extensively metabolized by HIM and the metabolite profile following in vitro incubations is similar in HIM and HLM. CYP3A4 and CYP3A5 isoforms are the predominant enzymes responsible for oxygenation of aPPD in HIM and HLM. The characterization of aPPD as a CYP3A substrate may facilitate better prediction of drug-herb interactions when aPPD is taken concomitantly with other therapeutic agents.

Topics: Biotransformation; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Intestinal Mucosa; Isoenzymes; Liver; Microsomes; Sapogenins

A recombinant β-glucosidase from Sphingopyxis alaskensis with a specific activity of 233.3Umg(-1) was purified by His-trap chromatography. The native enzyme was a 206kDa tetramer. The maximum enzyme activity was observed at pH 5.5 and 50°C. However, above 40°C, the enzyme stability significantly decreased. The enzyme hydrolyzed only the outer glucose at the C-3 position in protopanaxadiol-type ginsenosides without further hydrolysis. Because of the narrow substrate specificity, the enzyme completely converted ginsenosides Rb1, Rb2, Rc, and Rd as substrates to gypenoside XVII, compound O, compound Mc1, and F2, respectively, and it converted ginsenoside Rg3 to Rh2 with a molar conversion yield of 89%. These results suggest that the recombinant β-glucosidase from S. alaskensis is a potential producer of the rare ginsenosides gypenoside XVII, compound O, compound Mc1, F2, and Rh2. Among ginsenoside substrates, Rb1 was used for the high-level production of the rare ginsenoside gypenoside XVII. The optimum reaction conditions were pH 5.5, 40°C, 0.5mgml(-1) (116.7Uml(-1)) enzyme, and 8.0gl(-1) ginsenoside Rb1. Under these conditions, 6.8gl(-1) gypenoside XVII was produced by the enzyme after 1h with a molar conversion yield of 100% and a productivity of 6.8gl(-1)h(-1).

Topics: Bacterial Proteins; beta-Glucosidase; Cloning, Molecular; Genes, Bacterial; Genome, Bacterial; Ginsenosides; Glucose; Hydrolysis; Recombinant Proteins; Sapogenins; Sphingomonadaceae; Substrate Specificity

Ginsenosides are the primary bioactive components of ginseng, which is a popular medicinal plant that exhibits diverse pharmacological activities. Protopanaxadiol, protopanaxatriol and oleanolic acid are three basic aglycons of ginsenosides. Producing aglycons of ginsenosides in Saccharomyces cerevisiae was realized in this work and provides an alternative route compared to traditional extraction methods. Synthetic pathways of these three aglycons were constructed in S. cerevisiae by introducing β-amyrin synthase, oleanolic acid synthase, dammarenediol-II synthase, protopanaxadiol synthase, protopanaxatriol synthase and NADPH-cytochrome P450 reductase from different plants. In addition, a truncated 3-hydroxy-3-methylglutaryl-CoA reductase, squalene synthase and 2,3-oxidosqualene synthase genes were overexpressed to increase the precursor supply for improving aglycon production. Strain GY-1 was obtained, which produced 17.2 mg/L protopanaxadiol, 15.9 mg/L protopanaxatriol and 21.4 mg/L oleanolic acid. The yeast strains engineered in this work can serve as the basis for creating an alternative way for producing ginsenosides in place of extractions from plant sources.

Topics: Ginsenosides; Oleanolic Acid; Panax; Plant Proteins; Plant Roots; Saccharomyces cerevisiae; Sapogenins

The beneficial effects of vitamin D3 are exerted through 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the dihydroxy metabolite of vitamin D3. Hepatic and intestinal biotransformation of 1α,25(OH)2D3 and modifiers of metabolic capacity could be important determinants of bioavailability in serum and tissues. Ginsenosides and their aglycones, mainly 20(S)-protopanaxadiol (aPPD) and 20(S)-protopanaxatriol (aPPT), are routinely ingested as health supplements. The purpose of the present study was to investigate the potential of ginsenosides and their aglycones to block hepatic and intestinal inactivation of 1α,25(OH)2D3, which is the most potent ligand of vitamin D receptor. In vitro biotransformation reactions were initiated with NADPH regenerating solutions following initial preincubation of pooled human hepatic or intestinal microsomal protein or human recombinant CYP3A4 supersomes with 1α,25(OH)2D3 or midazolam. Formation of hydroxylated metabolites of 1α,25(OH)2D3 or midazolam was analyzed using liquid chromatography-mass spectrometry. Co-incubation of 1α,25(OH)2D3 with various ginsenosides (Rg1, Rh2, aPPD, aPPT and total ginsenosides) led to differential inhibition (30-100%) of its hydroxylation. Results suggest that aPPD, aPPT and Rh2 strongly attenuated the hydroxylation of 1α,25(OH)2D3. Follow up inhibition studies with aPPD and aPPT at varying concentrations (0.5-100μM) led to up to 91-100% inhibition of formation of hydroxylated metabolites of 1α,25(OH)2D3 thus preventing inactivation of active vitamin D3. The IC50 values of aPPD or aPPT for the most abundant hydroxylated metabolites of 1α,25(OH)2D3 ranged from 3.3 to 9.0μM in human microsomes. The inhibitory mechanism of aPPD or aPPT for CYP3A4-mediated biotransformation of 1α,25(OH)2D3 was competitive in nature (apparent Ki: 1.7-2.9μM). Similar inhibitory effects were also observed upon addition of aPPD or aPPT into midazolam hydroxylation assay. In summary, our results suggest that ginsenosides, specifically aPPD and aPPT, inhibit the CYP3A4-mediated catabolism of active vitamin D3 in human liver and intestine, potentially providing additional vitamin D-related benefits to patients with cancer, neurodegenerative and metabolic diseases.

Topics: Biotransformation; Calcitriol; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Humans; Intestines; Kinetics; Microsomes, Liver; Midazolam; Sapogenins

20(S)-Protopanaxadiol (PPD), a dammarane-type triterpenoid sapogenin, acts as the pharmacophore of ginsenosides which are considered as the principal bioactive components in Chinese ginseng. To fully understand the mechanism of action of PPD, it is important to study its metabolic profiles in vivo.. Plasma, urine, fece and bile were collected after administration of PPD formulated in 0.5% aqueous Tween-80 to rats (150 mg/kg). Samples were analyzed by using a sensitive and reliable method based on ultra-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS/MS) in both positive and negative ion mode. The chemical structures of metabolites were elucidated by comparing the retention time, accurate molecular mass, and fragmentation patterns of analytes with those of PPD.. In total 29 metabolites, including 10 new metabolites (M20-M29), were tentatively identified and characterized. Among them, two metabolites (M3 and M4) were unambiguously identified by matching their retention times and fragmentation patterns with their standards. Principal metabolites, namely, 20, 24-oxide metabolites (M3 and M4), 26/27-carboxylic acid derivatives (M22 and M23) and a glucuronidated product (M28), were found in the rat plasma.. The results showed that phase I metabolites are monooxygenation, dioxygenation and oxidative dehydrogenation metabolites, and phase II metabolic pathways were demonstrated to be cysteine conjugation and glucuronidation. The newly identified metabolites are useful to understand the mechanism of elimination of PPD and, in turn, its effectiveness and toxicity.

Topics: Administration, Oral; Animals; Bile; Chromatography, High Pressure Liquid; Feces; Ions; Male; Rats; Rats, Sprague-Dawley; Sapogenins; Tandem Mass Spectrometry

Adenosine 5'-monophosphate-activated protein kinase (AMPK) has been demonstrated as a promising drug target due to its regulatory function in glucose and lipid metabolism. 20(S)-protopanoxadiol (PPD) was firstly identified from high throughput screening as a small molecule activator of AMPK subtype α2β1γ1. In order to enhance its potency on AMPK, a series of PPD derivatives were synthesized and evaluated. Structure-activity relationship study showed that the amine derivatives at the 24-position (groups I-VI) can improve the potency (EC50: 0.7-2.3 μM) and efficacy (fold: 2.5-3.8). Among them, compounds 12 and 13 exhibited the best potency (EC50: 1.2 and 0.7 μM) and efficacy (fold: 3.7 and 3.8). Further study suggested the mechanism of AMPK activation may functioned at the allosteric position, resulting the inhibition of the lipid synthesis in HepG2 cell model.

Topics: AMP-Activated Protein Kinases; Dose-Response Relationship, Drug; Drug Discovery; Enzyme Activation; Enzyme Activators; Hep G2 Cells; Humans; Molecular Conformation; Phosphorylation; Sapogenins; Structure-Activity Relationship; Tumor Cells, Cultured

Panax quinquefolius is one of perennial herbs and well known for its outstanding pharmacological activity. Ginsenosides are thought to be the main active ingredients in Panax quinquefolius and exist in many kinds of plant genus Panax (ginseng). Dammarenediol synthase, which is considered as a key enzyme in ginsenoside biosynthesis pathway can convert 2, 3-oxidosqualene into dammarenediol-II. However, the dammarenediol synthase gene in Panax quinquefolius has not been identified. Here, we cloned and identified a dammarenediol synthase gene from Panax quinquefolius (PqDS, GenBank accession No. KC316048) at the first time, and reverse transcription-PCR (RT-PCR) analysis also showed an obvious transcription increase of PqDS in the methyl jasmonate (MeJA)-induced hairy roots. Ectopic expression of PqDS in yeast resulted in the production of dammarenediol-II was confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Moreover, overexpression of PqDS in transgenic hairy roots could increase the transcription of gene PqDS and another P450 gene PqD12H (encoding protopanaxadiol synthase in Panax quinquefolius), the accumulation of ginsenosides also increased at the same time. In addition, both PqDS and PqD12H gene co-expressed in recombinant yeast result in the production of protopanaxadiol was detected by LC/APCIMS; this result also provides a new strategy for the abundant production of protopanaxadiol in vitro.

Topics: Amino Acid Sequence; Cytochrome P-450 Enzyme System; Gene Expression; Molecular Sequence Data; Panax; Phylogeny; Plant Proteins; Plant Roots; Saccharomyces cerevisiae; Sapogenins; Saponins; Triterpenes

Panax quinquefolius is one of perennial herbs and well known for its outstanding pharmacological activity. Ginsenosides are thought to be the main active ingredients in P. quinquefolius and exist in many kinds of plant genus Panax (ginseng). Protopanaxatriol synthase, which is considered cytochrome P450 (CYP450) in ginsenoside biosynthesis pathway can convert protopanaxadiol into protopanaxatriol. However, the protopanaxatriol synthase gene in P. quinquefolius has not been identified. Here, we cloned and identified a protopanaxatriol synthase gene from P. quinquefolius (CYP6H, GenBank accession no. KC190491) at the first time, reverse transcription-PCR (RT-PCR) analysis showed no obvious transcription change of CYP6H in methyl jasmonate (MeJA)-induced hairy roots. Ectopic expression of CYP6H in Saccharomyces cerevisiae resulted in the production of protopanaxatriol with added exogenous protopanaxadiol and confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Moreover, high-performance liquid chromatography (HPLC) analysis shows that RNA interferences of CYP6H in transgenic hairy roots could increase the accumulation of protopanaxadiol-type ginsenosides and decrease the accumulation of protopanaxatriol-type ginsenosides, whereas the effect of overexpression CYP6H in transgenic hairy roots was contrary. Our study indicated that CYP6H is a gene encoding protopanaxadiol 6-hydroxylase which could convert protopanaxadiol into protopanaxatriol in P. quinquefolius ginsenoside biosynthesis, we also have confirmed the function of CYP6H on effect accumulation of ginsenosides.

Topics: Amino Acid Sequence; Aryl Hydrocarbon Hydroxylases; Cloning, Molecular; Ginsenosides; Molecular Sequence Data; Panax; Phylogeny; Plant Proteins; Plant Roots; Saccharomyces cerevisiae; Sapogenins; Transcription, Genetic

20(S)-Protopanaxadiol (PPD), a ginsenoside isolated from Pananx quinquefolium L., has been shown to inhibit growth and proliferation in several cancer cell lines. The aim of this study was to evaluate its anticancer activity in human breast cancer cells. MCF-7 cells were incubated with different concentrations of 20(S)-PPD and cytotoxicity was evaluated by MTT assay. Occurrence of apoptosis was detected by DAPI and Annexin V-FITC/PI double staining. Mitochondrial membrane potential was measured with Rhodamine 123. The Bcl-2 and Bax expression were determined by Western blot analysis. Caspase activity was measured by colorimetric assay. 20(S)-PPD dose-dependently inhibited cell proliferation in MCF-7 cells, with an IC50 value of 33.3 μM at 24h. MCF-7 cells treated with 20(S)-PPD presented typical apoptosis, as observed by morphological analysis in cell stained with DAPI. The percentages of annexin V-FITC positive cells were 8.92%, 17.8%, 24.5% and 30.5% in MCF-7 cells treated with 0, 15, 30 and 60μM of 20(S)-PPD, respectively. Moreover, 20(S)-PPD could induce mitochondrial membrane potential loss, up-regulate Bax expression and down-regulate Bcl-2 expression. These events paralleled activation of caspase-9, -3 and PARP cleavage. Apoptosis induced by 20(S)-PPD was blocked by z-VAD-fmk, a pan-caspase inhibitor, suggesting induction of caspase-mediated apoptotic cell death. In conclusion, the 20(S)-PPD investigated is able to inhibit cell proliferation and to induce cancer cell death by a caspase-mediated apoptosis pathway.

Topics: Antidepressive Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cell Proliferation; Cytochromes c; Female; Humans; Membrane Potential, Mitochondrial; Mitochondria; Sapogenins; Signal Transduction; Tumor Cells, Cultured

Ginseng is a medicinal herb that requires cultivation under shade conditions, typically for 4-6 years, before harvesting. The principal components of ginseng are ginsenosides, glycosylated tetracyclic terpenes. Dammarene-type ginsenosides are classified into two groups, protopanaxadiol (PPD) and protopanaxatriol (PPT), based on their hydroxylation patterns, and further diverge to diverse ginsenosides through differential glycosylation. Three early enzymes, dammarenediol-II synthase (DS) and two P450 enzymes, protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), have been reported, but glycosyltransferases that are necessary to synthesize specific ginsenosides have not yet been fully identified. To discover glycosyltransferases responsible for ginsenoside biosynthesis, we sequenced and assembled the ginseng transcriptome de novo and characterized two UDP-glycosyltransferases (PgUGTs): PgUGT74AE2 and PgUGT94Q2. PgUGT74AE2 transfers a glucose moiety from UDP-glucose (UDP-Glc) to the C3 hydroxyl groups of PPD and compound K to form Rh2 and F2, respectively, whereas PgUGT94Q2 transfers a glucose moiety from UDP-Glc to Rh2 and F2 to form Rg3 and Rd, respectively. Introduction of the two UGT genes into yeast together with PgDS and PgPPDS resulted in the de novo production of Rg3. Our results indicate that these two UGTs are key enzymes for the synthesis of ginsenosides and provide a method for producing specific ginsenosides through yeast fermentation.

Topics: Ginsenosides; Glycosyltransferases; Molecular Sequence Data; Panax; Plant Proteins; Plant Roots; Plants, Medicinal; Sapogenins

The aim of this study was to systematically evaluate the effect of the cultivation year on the quality of different ginseng tissues. Qualitative and quantitative analyses of ginsenosides were conducted using a UPLC-UV-MS method. Eight main ginsenosides in three tissues (leaf, rhizome and main root) and four parts (periderm, phloem, cambium and xylem) of ginseng aged from 1 to 13 years were determined using a UPLC-PDA method. Additionally, the antioxidant capacities of ginseng leaves were analyzed by the DPPH, ABTS and HRSA methods. It was found that the contents of ginsenosides increased with cultivation years, causing a sequential content change of ginsenosides in an organ-specific manner: leaf > rhizome > main root. The ratio between protopanaxatriol (PPT, Rg1, Re and RF) and protopanaxadiol (PPD, Rb1, Rb2, RC and Rd) in the main root remained stable (about 1.0), while it increased in leaf from 1.37 to 3.14 and decreased in the rhizome from 0.99 to 0.72. The amount of ginsenosides accumulated in the periderm was 45.48 mg/g, which was more than twice as high compared with the other three parts. Furthermore, the antioxidant activities of ginseng leaves were measured as Trolox equivalents, showing that antioxidant activity increased along with time of cultivation. The results show that the best harvest time for shizhu ginseng is the fifth year of cultivation, and the root and rhizome could be used together within seven planting years for their similar PPT/PPD level. Besides, the quality of the ginseng products would be enhanced with the periderm. The ginseng leaf is rich in ginsenosides and has potential application for its antioxidant capacity.

Topics: Antioxidants; Ginsenosides; Panax; Plant Leaves; Plant Roots; Rhizome; Sapogenins; Tissue Distribution

Castration-resistant progression of prostate cancer after androgen deprivation therapies remains the most critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor (AR) activity is an established driver of castration-resistant progression, and upregulation of the full-length AR (AR-FL) and constitutively-active AR splice variants (AR-Vs) has been implicated to contribute to the resurgent AR activity. We reported previously that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) can reduce the abundance of both AR-FL and AR-Vs. In the present study, we further showed that the effect of PPD on AR expression and target genes was independent of androgen. PPD treatment resulted in a suppression of ligand-independent AR transactivation. Moreover, PPD delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation and inhibited the growth of castration-resistant 22Rv1 xenograft tumors with endogenous expression of AR-FL and AR-Vs. This was accompanied by a decline in serum prostate-specific antigen levels as well as a decrease in AR levels and mitoses in the tumors. Notably, the 22Rv1 xenograft tumors were resistant to growth inhibition by the next-generation anti-androgen enzalutamide. The present study represents the first to show the preclinical efficacy of PPD in inhibiting castration-resistant progression and growth of prostate cancer. The findings provide a rationale for further developing PPD or its analogues for prostate cancer therapy.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Disease Progression; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Male; Mice, Nude; Neoplasm Transplantation; Prostatic Neoplasms, Castration-Resistant; Receptors, Androgen; Sapogenins

Natural products play an important role in cancer therapeutics, and lately more attentions have been paid to the prevention of major lethal malignancies, such as colorectal cancer (CRC). After oral ingestion, botanicals' parent compounds can be converted to their metabolites by the enteric microbiome, and these metabolites may have different bioactivities and variable bioavailability. In this study, we used an active ginseng metabolite, protopanaxadiol (PPD), as an example to assess its colon cancer preventive effect by comparing its effect with the treatment effect of fluorouracil (5-FU). A xenograft tumor nude mouse model with human colon cancer cell inoculation was used. After preventive PPD or treatment 5-FU administration with the same dose (30 mg/kg), tumor growth inhibition was evaluated by both a Xenogen bioluminescence imaging technique and manual tumor size measurement. Our data showed that preventive PPD very significantly inhibited the tumor growth compared to 5-FU (p < 0.01). Our data suggest that the PPD is a promising cancer prevention agent. More studies are needed to explore the chemopreventive actions of PPD and its potential clinical utility.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Biological Availability; Biological Products; Colorectal Neoplasms; Disease Models, Animal; Fluorouracil; Heterografts; Humans; Mice, Nude; Neoplasm Transplantation; Panax; Phytotherapy; Sapogenins

To study the preparation of 20(S)-protopanaxadiol-phospholipid (20(S)-PPD) complex HAP assemblies.. 20(S)-PPD phospholipid complex was assembled with the drug carriers of HAP. Effects of technological factors on the assembled amount were investigated, including HAP species, phospholipid complex concentration, ratio of HAP and assembled liquid, and then the preparation technology of 20(S)-PPD phospholipid complex HAP assemblies was determined.. The average quality of phospholipid complex assembled was 136. 26 mg/g,the average assembled rate was 5.3% for 20(S)-PPD phospholipid complex HAP assemblies prepared by the determined assembly process. FT-IR showed that 20(S)-PPD phospholipid complex was absorbed in the HAP assemblies, and the hydrogen-bonding effect was the main mechanism of HAP assemblies to assemble and adsorb phospholipid complex. The cumulative release rate in pH 7.4 phosphate buffer of the HAP assemblies indicated that the assemblies had the effect of sustained release.. The phospholipid complex HAP assembles have the advantages of simple preparation process, and sustaining release effect, which can provide preliminary research foundation for research and development of 20(S)-PPD sustained-release preparations.

Topics: Adsorption; Delayed-Action Preparations; Drug Carriers; Phospholipids; Sapogenins; Spectroscopy, Fourier Transform Infrared

20(S)-protopanaxadiol (PPD), similar to several other anticancer agents, has low oral absorption and is extensively metabolized. These factors limit the use of PPD for treatment of human diseases.. In this study, we used cubic nanoparticles containing piperine to improve the oral bioavailability of PPD and to enhance its absorption and inhibit its metabolism. Cubic nanoparticles loaded with PPD and piperine were prepared by fragmentation of glyceryl monoolein (GMO)/poloxamer 407 bulk cubic gel and verified using transmission electron microscopy and differential scanning calorimetry. We evaluated the in vitro release of PPD from these nanoparticles and its absorption across the Caco-2 cell monolayer model, and subsequently, we examined the bioavailability and metabolism of PPD and its nanoparticles in vivo.. The in vitro release of PPD from these nanoparticles was less than 5% at 12 hours. PPD-cubosome and PPD-cubosome loaded with piperine (molar ratio PPD/piperine, 1:3) increased the apical to basolateral permeability values of PPD across the Caco-2 cell monolayer from 53% to 64%, respectively. In addition, the results of a pharmacokinetic study in rats showed that the relative bioavailabilities of PPD-cubosome [area under concentration-time curve (AUC)(0-∞)] and PPD-cubosome containing piperine (AUC(0-∞)) compared to that of raw PPD (AUC(0-∞)) were 166% and 248%, respectively.. The increased bioavailability of PPD-cubosome loaded with piperine is due to an increase in absorption and inhibition of metabolism of PPD by cubic nanoparticles containing piperine rather than because of improved release of PPD. The cubic nanoparticles containing piperine may be a promising oral carrier for anticancer drugs with poor oral absorption and that undergo extensive metabolism by cytochrome P450.

Topics: Absorption; Administration, Oral; Alkaloids; Animals; Area Under Curve; Benzodioxoles; Biological Availability; Caco-2 Cells; Humans; Male; Nanoparticles; Particle Size; Piperidines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Sapogenins

Mixed micelles are widely used to increase solubility and bioavailability of poorly soluble drugs. One promising antitumor drug candidate is 20(S)-protopanaxadiol (PPD), although its clinical application is limited by low water solubility and poor bioavailability after oral administration. In this study, we developed mixed micelles consisting of PPD-phospholipid complexes and Labrasol(®) and evaluated their potential for oral PPD absorption. Micelles were prepared using a solvent-evaporation method, and their physicochemical properties, including particle size, zeta potential, morphology, crystal type, drug loading, drug entrapment efficiency, and solubility, were characterized. Furthermore, in vitro release was investigated using the dialysis method, and transport and bioavailability of the mixed micelles were investigated through a Caco-2 cell monolayer and in vivo absorption studies performed in rats. Compared with the solubility of free PPD (3 μg/mL), the solubility of PPD in the prepared mixed micelles was 192.41 ± 1.13 μg/mL in water at room temperature. The in vitro release profiles showed a significant difference between the more rapid release of free PPD and the slower and more sustained release of the mixed micelles. At the end of a 4-hour transport study using Caco-2 cells, the apical-to-basolateral apparent permeability coefficients (P(app)) increased from (1.12 ± 0.21) × 10(6) cm/s to (1.78 ± 0.16) × 10(6) cm/s, while the basolateral-to-apical P(app) decreased from (2.42 ± 0.16) × 10(6) cm/s to (2.12 ± 0.32) × 10(6). In this pharmacokinetic study, compared with the bioavailability of free PPD (area under the curve [AUC](0-∞)), the bioavailability of PPD from the micelles (AUC(0-∞)) increased by approximately 216.36%. These results suggest that novel mixed micelles can significantly increase solubility, enhance absorption, and improve bioavailability. Thus, these prepared micelles might be potential carriers for oral PPD delivery in antitumor therapies.

Topics: Administration, Oral; Animals; Area Under Curve; Biological Availability; Caco-2 Cells; Calorimetry, Differential Scanning; Drug Carriers; Glycerides; Humans; Male; Micelles; Organic Chemicals; Particle Size; Phospholipids; Rats; Rats, Sprague-Dawley; Sapogenins; Solubility

A novel, rapid and sensitive liquid chromatography/quadrupole linear ion trap mass spectrometry [LC-ESI-(QqLIT)MS/MS] method was developed and validated for the quantification of protopanaxadiol (PPD) in rat plasma. Oleanolic acid (OA) was used as internal standard (IS). A simple protein precipitation based on acetonitrile (ACN) was employed. Chromatographic separation was performed on a Sepax GP-C18 column (50 × 2.1 mm, 5 μM) with a mobile phase consisting of ACN-water and 1.5 μM formic acid and 25 mM lithium acetate (90 : 10, v/v) at a flow rate of 0.4 ml/min for 3.0 min. Multiple-reaction-monitoring mode was performed using lithium adduct ion as precursor ion of m/z 467.5/449.4 and 455.6/407.4 for the drug and IS, respectively. Calibration curve was recovered over a concentration range of 0.5-100 ng/ml with a correlation coefficient >0.99. The limit of detection was 0.2 ng/ml in rat plasma for PPD. The results of the intraday and interday precision and accuracy studies were well within the acceptable limits. The validated method was successfully applied to investigate the pharmacokinetic study of PPD after intravenous and gavage administration to rat.

Topics: Animals; Antidepressive Agents; Chromatography, High Pressure Liquid; Limit of Detection; Lithium; Male; Rats; Rats, Sprague-Dawley; Sapogenins; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Time Factors

Plant-derived active constituents and their semi-synthetic or synthetic analogs have served as major sources of anticancer drugs. 20(S)-protopanaxadiol (PPD) is a metabolite of ginseng saponin of both American ginseng (Panax quinquefolius L.) and Asian ginseng (Panax ginseng C.A. Meyer). We previously demonstrated that ginsenoside Rg3, a glucoside precursor of PPD, exhibits anti-proliferative effects on HCT116 cells and reduces tumor size in a xenograft model. Our subsequent study indicated that PPD has more potent antitumor activity than that of Rg3 in vitro although the mechanism underlying the anticancer activity of PPD remains to be defined. Here, we investigated the mechanism underlying the anticancer activity of PPD in human cancer cells in vitro and in vivo. PPD was shown to inhibit growth and induce cell cycle arrest in HCT116 cells. The in vivo studies indicate that PPD inhibits xenograft tumor growth in athymic nude mice bearing HCT116 cells. The xenograft tumor size was significantly reduced when the animals were treated with PPD (30 mg/kg body weight) for 3 weeks. When the expression of previously identified Rg3 targets, A kinase (PRKA) anchor protein 8 (AKAP8L) and phosphatidylinositol transfer protein α (PITPNA), was analyzed, PPD was shown to inhibit the expression of PITPNA while upregulating AKAP8L expression in HCT116 cells. Pathway-specific reporter assays indicated that PPD effectively suppressed the NF-κB, JNK and MAPK/ERK signaling pathways. Taken together, our results suggest that the anticancer activity of PPD in colon cancer cells may be mediated through targeting NF-κB, JNK and MAPK/ERK signaling pathways, although the detailed mechanisms underlying the anticancer mode of PPD action need to be fully elucidated.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; HEK293 Cells; Heterografts; Humans; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; NF-kappa B; Nuclear Proteins; Panax; Phospholipid Transfer Proteins; Plant Extracts; Sapogenins; Signal Transduction

Ginsenosides are medicinal ingredients of the cardiovascular herb Panax notoginseng roots (Sanqi). Here, we implemented a human study (ChiCTR-ONC-09000603; www.chictr.org) to characterize pharmacokinetics and metabolism of ginsenosides from an orally ingested Sanqi-extract (a 1:10 water extract of Sanqi) and the human plasma and urine samples were analyzed by liquid chromatography-mass spectrometry. Plasma and urinary compounds derived from ginsenosides included: 1) intestinally absorbed ginsenosides Ra3, Rb1, Rd, F2, Rg1, and notoginsenoside R1; and 2) the deglycosylated products compound-K, 20(S)-protopanaxadiol, 20(S)-protopanaxatriol, and their oxidized metabolites. The systemic exposure levels of the first group compounds increased as the Sanqi-extract dose increased, but those of the second group compounds were dose-independent. The oxidized metabolites of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol represented the major circulating forms of ginsenosides in the bloodstream, despite their large interindividual differences in exposure level. The metabolites were formed via combinatorial metabolism that consisted of a rate-limiting step of ginsenoside deglycosylation by the colonic microflora and a subsequent step of sapogenin oxidation by the enterohepatic cytochrome P450 enzymes. Significant accumulation of plasma ginsenosides and metabolites occurred in the human subjects receiving 3-week subchronic treatment with the Sanqi-extract. Plasma 20(S)-protopanaxadiol and 20(S)-protopanaxatriol could be used as pharmacokinetic markers to reflect the subjects' microbial activities, as well as the timely-changes and interindividual differences in plasma levels of their respective oxidized metabolites. The information gained from the current study is relevant to pharmacology and therapeutics of Sanqi.

Topics: Administration, Oral; Adult; Area Under Curve; Female; Ginsenosides; Humans; Male; Panax notoginseng; Plant Extracts; Plant Roots; Sapogenins

To establish a high-performance liquid chromatographic/tandem mass spectrometry (HPLC-MS/MS) method for determining 20(S)-protopanaxadiol (PPD) in rat plasma, in order to analyze pharmacokinetic characteristics of PPD and PPD cubic nanoparticles.. Sprague-Dawley rats were administered orally with PPD and PPD cubic nanoparticles, respectively. Their blood samples were obtained from fossa orbitalis at regular time points. The mobile phase was 0.05% formic acidac etonitrile-0.05% formic acidac water (95:5). Electrospray ionization (ESI) was adopted for the quadrupole tandem mass spectrum. SCAN mode was used for the quantitative analysis, with m/z 460. 4/425.3 and m/z 622.9/318.3 (Rh2, interior label) as secondary fragment ions. The concentration of PPD in plasma was analyzed. The concentration-time curve was mapped. The data were calculated by DAS program.. The linearity of the PPD plasma concentration determination method ranged between 10-1 407 microg x L(-1), with the limit of quantification of 2.5 microg x L(-1). Both of the inter-day and intra-day precisions (RSD) were less than 13.25%, and the accuracy (relative error) was between +/- 8.50%.. The method was so highly specific and sensitive with less plasma that it is suitable for pharmacokinetic studies. The prepared 20(S)-protopanaxadiol lipid cubic nanoparticles can enhance its absorption in vivo. Its relative bioavailability is 166% of the raw material.

Topics: Absorption; Administration, Oral; Animals; Antidepressive Agents; Biological Availability; Chromatography, High Pressure Liquid; Female; Lipids; Male; Nanoparticles; Rats; Rats, Sprague-Dawley; Sapogenins; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Time Factors

Novel 20(S)-protopanoxadiol (PPD) analogues were designed, synthesized, and evaluated for the chemosensitizing activity against a multidrug resistant (MDR) cell line (KBvcr) overexpressing P-glycoprotein (P-gp). Structure-activity relationship analysis showed that aromatic substituted aliphatic amine at the 24-positions (groups V) effectively and significantly sensitized P-gp overexpressing multidrug resistant (MDR) cells to anticancer drugs, such as docetaxel (DOC), vincristine (VCR), and adriamycin (ADM). PPD derivatives 12 and 18 showed 1.3-2.6 times more effective reversal ability than verapamil (VER) for DOC and VCR. Importantly, no cytotoxicity was observed by the active PPD analogues (5μM) against both non-MDR and MDR cells, suggesting that PPD analogues serve as novel lead compounds toward a potent and safe resistance modulator. Moreover, a preliminary mechanism study demonstrated that the chemosensitizing activity of PPD analogues results from inhibition of P-glycoprotein (P-gp) overexpressed in MDR cancer cells.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Docetaxel; Dose-Response Relationship, Drug; Doxorubicin; Drug Design; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Neoplasms; Sapogenins; Taxoids; Verapamil

In this study, we investigated the cardioprotective effect of Panax quinquefolium 20(S)-protopanaxadiol saponins (PQDS) both in vivo and in vitro. An animal model of acute myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery in Sparague Dawley rats. Neonatal rat cardiomycocytes were used to examine the cytoprotective effect of PQDS against H202 exposure. Pretreatment with PQDS (25 and 50 mg/kg) could significantly improve the heart function, remarkably decrease infarct size from 20.87% to 14.87% (p<0.01), decrease the levels of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and cardiac troponin T (cTnT) content in serum (p< 0.05). Meanwhile, pretreatment with PQDS (25 and 50 mg/kg) significantly increased the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-Px) in the heart, and decreased the level of malondiadehyde (MDA) in the myocardium (p<0.05). Histopathological results demonstrated the same protective effect of PQDS. Pretreatment with PQDS (200 and 400 microg/ml) prior to H202 exposure could increase cell viability of neonatal rat cardiomycocytes. Pretreatment PQDS (200 and 400 microg/ml) also increased the activity of SOD, decreased level of LDH in the cultured supernatant and the MDA level in cardiomyocytes. These results indicated that PQDS had a cardioprotective effect proven in vivo and in vitro. The mechanisms might be due to its scavenging lipid peroxidation products, increasing endogenous antioxidant defense enzymes.

Topics: Animals; Animals, Newborn; Biomarkers; Cardiotonic Agents; Cells, Cultured; Coronary Occlusion; Electrocardiography; Female; Heart Function Tests; Hydrogen Peroxide; Indicators and Reagents; Male; Myocardial Infarction; Myocytes, Cardiac; Oxidants; Panax; Rats; Rats, Sprague-Dawley; Sapogenins; Survival

In this study, the biopharmaceutical properties of 20 (S)-protopanaxadiol (PPD) were studied. Firstly, the equilibrium solubility and apparent oil/water partition coefficient of PPD were used to predict the absorption in vivo. Meanwhile the membrane permeability and absorption window were studied by Caco-2 cell model and single-pass intestinal perfusion model. Furthermore, the bioavailability and metabolism were combined to study the absorption properties and metabolic properties in vivo. All of them were used to provide theoretical and practical foundation for designing PPD preparation. The results showed that PPD is poorly water-soluble, and the equilibrium solubility in water is only 35.24 mg x L(-1). The oil-water partition coefficient is 46.21 (logP = 1.66). By Caco-2 cell model, the results showed PPD uptake in general, and it also has efflux. By in situ intestinal perfusion model, the results showed that the absorption of PPD in the intestine is good, and the effective permeability coefficient were duodenum > jejunum > ileum > colon. The oral bioavailability of PPD was 29.39%. It was not well. Metabolic studies showed PPD in vivo presented a wide spread metabolism. So the main factors that restricted oral bioavailability of PPD were the poor solubility and first-pass effect.

Topics: Administration, Oral; Animals; Area Under Curve; Biological Availability; Caco-2 Cells; Humans; Intestinal Absorption; Male; Permeability; Rats; Rats, Sprague-Dawley; Sapogenins; Solubility; Tissue Distribution

The ginsenoside compound K has pharmaceutical activities, including anti-tumor, anti-inflammatory, anti-allergic, and hepatoprotective effects. To increase the production of compound K, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase (CS-abf) and/or the α-L-arabinopyranoside-hydrolyzing β-galactosidase from Caldicellulosiruptor saccharolyticus (CS-bgal) were mixed with the β-D-glucopyranoside-hydrolyzing β-glucosidase from Sulfolobus acidocaldarius (SA-bglu). The optimum conditions for the production of ginsenoside compound K from ginsenoside Rc or Rb₂, or from major protopanaxadiol ginsenosides in ginseng root extract were determined to be pH 6.0 and 75°C with 8 mg ml⁻¹ ginsenoside Rc, 8 mg ml⁻¹ Rb₂, or 10% (w/v) ginseng root extract; and 10.5 U ml⁻¹ CS-abf or CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, or 10.5 U ml⁻¹ CS-abf and 10.5 U ml⁻¹ CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, respectively. Under optimum conditions, ginsenosides Rc and Rb2, and major protopanaxadiol ginsenosides in ginseng root extract were completely converted to compound K after 12, 14, and 20 h, respectively, with the respective productivities of 388, 328, and 144 mg l⁻¹ h⁻¹. This is the first report of the complete conversion of major protopanaxadiol ginsenosides to compound K.

Topics: Bacteria; beta-Galactosidase; beta-Glucosidase; Ginsenosides; Glycoside Hydrolases; Panax; Plant Extracts; Plant Roots; Sapogenins

Using enrichment culture, Sphingobacterium multivorum GIN723 (KCCM80060) was isolated as having activity for deglycosylation of compound K and ginsenoside F1 to produce ginsenoside aglycons such as S-protopanaxadiol (PPD(S)) and S-protopanaxatriol (PPT(S)). Through BLAST search, purified enzyme from S. multivorum GIN723 was revealed to be the outer membrane protein. The purified enzyme from S. multivorum GIN723 has unique specificity for the glucose moiety. However, it has activity with PPD and PPT group ginsenosides such as ginsenosides Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, and F1. From these results, it was predicted that the enzyme has activity on several ginsenosides. Therefore, the biotransformation pathway from Rb1, which is a major, highly glycosylated compound of ginseng, was analyzed using high-performance liquid chromatography and electrospray ionization mass spectrometry/mass spectrometry. The dominant biotransformation pathway from Rb1 to PPD(S) was determined to be Rb1 → Gp-XVII → Gp-LXXV → CK → PPD(S). S. multivorum GIN723 can be used as a whole cell biocatalyst because its activity as whole cells is nine times higher than its activity as cell extracts. The specific activity of whole cells is 2.89 nmol/mg/min in the production of PPD(S). On the other hand, the specific activity of cell extracts is 0.32 nmol/mg/min. The productivity of this enzyme in whole cell form is 500 mg/1 l of cultured cell. Its optimum reaction condition is 10 mM of calcium ions added to a phosphate buffer with a pH of 8.5.

Topics: Biotransformation; Chromatography, High Pressure Liquid; Ginsenosides; Glycosylation; Molecular Structure; Plant Extracts; Sapogenins; Sphingobacterium; Tandem Mass Spectrometry

This study aims to evaluate the bioactivity of five components of the traditional Chinese medicine complex prescription Jiangzhi granules against hepatocellular steatosis.. The five major components, including protopanaxadiol, tanshinone IIA, emodin, chlorogenic acid, and nuciferine, were extracted from Jiangzhi granules. Their cytotoxicity was assessed to determine the safe dose of each component for HepG2 cells. HepG2 cellular steatosis was induced using 1 mmol/L of free fatty acids (FFAs) for 24 h, and then treated with each component at high, intermediate, and low doses (500, 50, and 5 μmol/L), respectively for another 24 h. The effects on HepG2 steatosis were observed directly under optical phase microscopy, or through oil red O staining and Nile red assays. In addition, the levels of reactive oxygen species (ROS) in the steatotic HepG2 cells with and without high-dose protopanaxadiol treatment were measured using fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate staining.. No obvious cytotoxicity was observed in the HepG2 cells incubated with each of the five components at up to 500 μmol/L. At 24 h after incubation with FFAs, the HepG2 cells swelled and many lipid droplets accumulated. The lipid content was attenuated after 24 h of incubation with protopanaxadiol, tanshinone IIA, and emodin at 500 or 50 μmol/L (P < 0.05), especially with 500 μmol/L protopanaxadiol (P < 0.01). In addition, the ROS level was elevated in steatotic cells, but decreased after intervention with 500 μmol/L protopanaxadiol (P < 0.05).. Protopanaxadiol, tanshinone IIA, and emodin alleviate hepatocellular steatosis in a dose-dependent manner, and oxidative stress regulation may partially contribute to the effects of protopanaxadiol.

Topics: Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Fatty Liver; Hep G2 Cells; Humans; Oxidative Stress; Reactive Oxygen Species; Sapogenins

A new protopanaxadiol-type ginsenoside, 6-O-β-d-glucopyranosyl-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol-3-one (1), along with three known compounds, was isolated from the roots of Panax notoginseng. Their structures were determined based on some pieces of spectroscopic and chemical evidence. Compound 1 exhibited cytotoxic activity against five human cancer cell lines, with IC50 values ranging from 5.4 to 8.6 μg/ml.

Topics: Antineoplastic Agents, Phytogenic; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Ginsenosides; Humans; Inhibitory Concentration 50; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Panax notoginseng; Plant Roots; Sapogenins

In this study, ultra-performance liquid chromatography (UPLC)/quadrupole-time-of-flight mass spectrometry (QTOF-MS) was applied to the rapid analysis of 20(S)-protopanaxadiol (PPD) metabolites in rats after oral administration, enabling the structural characterization of 23 metabolites in plasma, bile, urine, and feces. 16 of these, including M1-M5, M9, and M11-M15, have not been previously reported. The results also indicated that demethylation, dehydration, dehydrogenation, oxidation, deoxidation, and glucuronidation were the major metabolic reactions of PPD in vivo. This study provides important information about the metabolism of PPD which will be helpful for fully understanding its mechanism of action. Furthermore, structural modification of PPD in vivo may aid in obtaining new chemical derivatives for pharmacological screening.

Topics: Animals; Bile; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Feces; Male; Mass Spectrometry; Panax; Rats; Rats, Sprague-Dawley; Sapogenins

Some of ginsenosides, root extracts from Panax ginseng, exert cytotoxicity against cancer cells through disruption of membrane subdomains called lipid rafts. Protopanaxadiol (PPD) exhibits the highest cytotoxic effect among 8 ginsenosides which we evaluated for anti-cancer activity. We investigated if PPD disrupts lipid rafts in its cytotoxic effects and also the possible mechanisms.. Eight ginsenosides were evaluated using different cancer cells and cell viability assays. The potent ginsenoside, PPD was investigated for its roles in lipid raft disruption and downstream pathways to apoptosis of cancer cells. Anti-cancer effects of PPD was also investigated in vivo using mouse xenograft model.. PPD consistently exerts its potent cytotoxicity in 2 cell survival assays using 5 different cancer cell lines. PPD disrupts lipid rafts in different ways from methyl-β-cyclodextrin (MβCD) depleting cholesterol out of the subdomains, since lipid raft proteins were differentially modulated by the saponin. During disruption of lipid rafts, PPD activated neutral sphingomyelinase 2 (nSMase 2) hydrolyzing membrane sphingomyelins into pro-apoptotic intracellular ceramides. Furthermore, PPD demonstrated its anti-cancer activities against K562 tumor cells in mouse xenograft model, confirming its potential as an adjunct or chemotherapeutic agent by itself in vivo.. This study demonstrates that neutral sphingomyelinase 2 is responsible for the cytotoxicity of PPD through production of apoptotic ceramides from membrane sphingomyelins. Thus neutral sphingomyelinase 2 and its relevant mechanisms may potentially be employed in cancer chemotherapies.

Topics: Animals; Cell Line, Tumor; Cell Membrane; Cytotoxins; Female; Ginsenosides; Humans; Mice; Mice, Inbred BALB C; Neoplasms; Panax; Sapogenins; Sphingomyelin Phosphodiesterase

Panaxadiol is a novel antitumor agent extracted from the Chinese medical herb Panax ginseng. This agent is being developed for the treatment of tumor diseases. A rapid, selective, and simple method based on ultrafast liquid chromatography-tandem mass spectrometry was established and validated to determine panaxadiol in rat plasma following oral and sublingual intravenous administration of panaxadiol. The plasma samples were pretreated with acetic ether, and chromatographic separation was achieved on a Shim-pack XR-ODS III column using isocratic elution with the mobile phase of 0.1 % formic acid and acetonitrile. Analytes and protopanaxadiol (internal standard) were analyzed and identified using electrospray positive ionization mass spectrometry in the multiple reaction-monitoring mode. The MS/MS detection was carried out by monitoring the fragmentation of m/z 461.45 → m/z 127.1 for panaxadiol and m/z 425.4 → m/z 95.1 for protopanaxadiol (internal standard) on a triple-quadrupole mass spectrometer. The result showed good linearity over a wide concentration range (0.1-20 ng/mL) (R2 > 0.999) and its lower limit of detection and quantification were 0.03 and 0.1 ng/mL, respectively. The intra- and interday precision (RSD %) was within 15 % and the accuracy ranged from 94.9 % to 112.0 %. The absolute bioavailability was 12.5 %. The method was fully validated and successfully applied to the pharmacokinetic study of a single dose of panaxadiol.

Topics: Animals; Biological Availability; Chromatography, High Pressure Liquid; Drug Stability; Drugs, Chinese Herbal; Ginsenosides; Male; Panax; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sapogenins; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

The biotransformation of 20(S)-protopanaxadiol (1) by Aspergillus niger AS 3.1858 was conducted. Seven metabolites 26-hydroxyl-20(S)-protopanaxadiol (2); 23, 24-en-25-hydroxyl-20(S)-protopanaxadiol (3); 25, 26-en-20(S)-protopanaxadiol (4); (E)-20, 22-en-25-hydroxyl-20(S)-protopanaxadiol (5); 25, 26-en-24(R)-hydroxyl-20(S)-protopanaxadiol (6); 25, 26-en-24(S)-hydroxyl-20(S)-protopanaxadiol (7); and 23, 24-en-25-ethoxyl-20(S)-protopanaxadiol (8) were afforded. Among them, 6, 7, and 8 are new compounds. The chemical structures of these metabolites were elucidated based on extensive spectral data including 2D NMR and HRMS. In addition, the cytotoxicity of substrate and all transformed products was evaluated by MTT assay using a panel of seven human tumor cell lines (Du-145, Hela, K562, K562/ADR, SH-SY5Y, HepG2, and MCF-7 cells) and one normal cell line Vero.

Topics: Animals; Antineoplastic Agents, Phytogenic; Aspergillus niger; Biological Products; Biotransformation; Chlorocebus aethiops; HeLa Cells; Hep G2 Cells; Humans; MCF-7 Cells; Molecular Structure; Neoplasms; Panax; Phytotherapy; Sapogenins; Vero Cells

20(S)-Protopanaxadiol (PPD), an aglycone saponin ginsenoside isolated from Panax quinquefolium L, has been shown to inhibit the growth and proliferation in several cancer lines. However, the underlying molecular mechanisms remain poorly understood. In this study, we investigated the apoptosis-induced effects and the mechanism of 20(S)-PPD on human lung adenocarcinoma A549 cells. 20(S)-PPD showed a potent antiproliferative activity against A549 cells by triggering apoptosis. 20(S)-PPD-induced apoptosis was characterized by a dose-dependent loss of the mitochondrial membrane, release of cytochrome c, second mitochondria-derived activator of caspase (Smac) and apoptosis-inducing factor (AIF), activation of caspase-9/-3, and cleavage of poly (ADP-ribose) polymerase (PARP). Caspase-dependence was indicated by the ability of the pan-caspase inhibitor z-VAD-fmk to attenuate 20(S)-PPD-induced apoptosis. After treatment with 20(S)-PPD, the proportion of A549 cells at the G0/G1 phase increased, while cells at the S and G2/M phases decreased. Furthermore, 20(S)-PPD also triggered down-regulation of phosphorylated Akt (Ser473/Thr308) and glycogen synthase kinase 3β (GSK 3β). Knockdown of GSK 3β with siRNA promoted the apoptotic effects of 20(S)-PPD. These results revealed an unexpected mechanism of action for this unique ginsenoside: triggering a mitochondrial-mediated, caspase-dependent apoptosis via down-regulation of the PI3K/Akt signaling pathway in A549 cells. Our findings encourage further studies of 20(S)-PPD as a promising chemopreventive agent against lung cancer.

Topics: Adenocarcinoma; Apoptosis; Caspases; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Oncogene Protein v-akt; Panax; Phosphatidylinositol 3-Kinases; Phytotherapy; Sapogenins; Signal Transduction; Tumor Cells, Cultured

(20R)-25-Methoxyl-dammarane-3β,12β,20-triol (25-OCH₃-PPD) is a dammarane-type sapogenin showing anti-proliferative potential. In our study, two series of analogs substituted at the C-3 or C-3 and C-12 positions with fatty acids were prepared conveniently. The cytotoxic activity of these compounds was evaluated using four different human tumor cell lines (A549, Hela, HT-29 and MCF-7) and a normal cell line (IOSE144). As compared with 25-OCH₃-PPD, compounds 1a, 1b, 2a and 2b showed better anti-proliferative activities against all tumor cell lines and all the derivatives, with low toxicities in the normal cell line. Compound 1a (C-3 monoformiate) exhibited the strongest activity with the IC₅₀ value of 5.2 μM towards HT29 cells. The results indicated that the difference in the substituents may affect the anti-proliferative activity of the compounds. The longer the side chain of 25-OCH₃-PPD is, the lower the anti-proliferative activity would be. This information may be useful for evaluating the structure-activity relationship of other dammarane-type sapogenins and for development of novel antineoplastic agents.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dammaranes; Drug Screening Assays, Antitumor; Ginsenosides; Humans; Inhibitory Concentration 50; Molecular Structure; Sapogenins; Structure-Activity Relationship; Triterpenes

Gambogic acid (GA) is known for its anti-cancer activity in a phase II clinical trial. However, the detailed molecular mechanisms of its anti-multidrug resistance remain unclear. The present study was designed to study the relationship between GA and multidrug-resistant protein ATP-binding cassette transporter B1 (ABCB1). GA dose dependently inhibited ABCB1 activity levels in the in vitro Pgp-Glo assay system and increased the cellular accumulation of ABCB1 substrate adriamycin. Although GA had no significant influence on ABCB1 mRNA in the real-time PCR assay, Western blot detection indicated the compound reduced ABCB1 protein levels. Further study showed the proteasome inhibitor MG-132 reversed the GA-decreased ABCB1 level and prolonged half-life of ABCB1. It was also found that GA coordinated with other anti-cancer drugs (such as adriamycin, docetaxel, verapamil and protopanaxadiol) to enhance cellular cytotoxicity on human epithelial cancer cell lines with higher ABCB1 expression levels. These data suggest that GA functions as a non-competitive inhibitor of ABCB1 by directly inhibiting and reducing its expression levels by promoting protein degradation through post-translational proteasome pathway. The results of this study will aid in the understanding of the synergistic effects of combining GA with other drugs as a new anti-multidrug-resistant agent.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Neoplasms, Glandular and Epithelial; Sapogenins; Xanthones

As a public health problem, prostate cancer engenders huge economic and life-quality burden. Developing effective chemopreventive regimens to alleviate the burden remains a major challenge. Androgen signaling is vital to the development and progression of prostate cancer. Targeting androgen signaling via blocking the production of the potent ligand dihydrotestosterone has been shown to decrease prostate cancer incidence. However, the potential of increasing the incidence of high-grade prostate cancers has been a concern. Mechanisms of disease progression after the intervention may include increased expression of androgen receptor (AR) in prostate tissue and expression of the constitutively active AR splice variants (AR-Vs) lacking the ligand-binding domain. Thus, novel agents targeting the receptor, preferentially both the full-length and AR-Vs, are urgently needed. In the present study, we show that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) effectively downregulates the expression and activity of both the full-length AR and AR-Vs. The effects of PPD on AR and AR-Vs are manifested by an immediate drop in proteins followed by a reduction in transcripts, attributed to PPD induction of proteasome-mediated degradation and inhibition of the transcription of the AR gene. We further show that although PPD inhibits the growth as well as AR expression and activity in LNCaP xenograft tumors, the morphology and AR expression in normal prostates are not affected. This study is the first to show that PPD suppresses androgen signaling through downregulating both the full-length AR and AR-Vs, and provides strong rationale for further developing PPD as a promising agent for the prevention and/or treatment of prostate cancer.

Topics: Alternative Splicing; Animals; Cell Line, Tumor; Down-Regulation; Humans; Male; Mice; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Receptors, Androgen; Sapogenins

As with many other anti-cancer agents, 20(S)-protopanaxadiol (PPD) has a low oral absorption. In this study, in order to improve the oral bioavailability of PPD, the cubic nanoparticles that it contains were used to enhance absorption. Therefore, the cubic nanoparticle loaded PPD were prepared through the fragmentation of the glyceryl monoolein (GMO)/poloxamer 407 bulk cubic gel and were verified by transmission electron microscope, small angle X-ray scattering and differential scanning calorimetry. The in vitro release of 20(S)-protopanaxadiol from these nanoparticles was less than 5% at 12h. And then Caco-2 cell monolayer model was used to evaluate the absorption of PPD in vitro. Meanwhile the rat intestinal perfusion model and bioavailability were also estimated in vivo. The results showed that, in the Caco-2 cell model, the PPD-cubosome could increase the permeability values from the apical (AP) to the basolateral (BL) of PPD at 53%. The result showed that the four-site rat intestinal perfusion model was consistent with the Caco-2 cell model. And the result of a pharmacokinetic study in rats showed that the relative bioavailability of the PPD-cubosome (AUC(0-∞)) compared with the raw PPD (AUC(0-∞)) was 169%. All the results showed that the PPD-cubosome enhanced bioavailability was likely due to the increased absorption by the cubic nanoparticles rather than by the improved release. Hence, the cubic nanoparticles may be a promising oral carrier for the drugs that have a poor oral absorption.

Topics: Administration, Oral; Animals; Antineoplastic Agents, Phytogenic; Area Under Curve; Biological Availability; Caco-2 Cells; Half-Life; Humans; Intestinal Mucosa; Liquid Crystals; Male; Molecular Structure; Nanostructures; Perfusion; Rats; Rats, Sprague-Dawley; Sapogenins

Biotransformation of 20(S)-protopanaxadiol (1) by the fungus Absidia corymbifera AS 3.3387 yielded five metabolites (2-6). On the basis of spectroscopic data analyses, the metabolites were identified as 26-hydroxyl-20(S)-protopanaxadiol (2), 23, 24-en-25-hydroxyl-20(S)-protopanaxadiol (3), 25-hydroxyl-20(S)-protopanaxadiol (4), 7β-hydroxyl-20(S)-protopanaxatriol (5), and 7-oxo-20(S)-protopanaxatriol (6), respectively. Among them, 5 and 6 are new compounds. These results indicated that A. corymbifera AS 3.3387 could catalyze the side-chain oxidation-reduction, 7β hydroxylation, and the specific C-7 dehydrogenation of derivatives of 20(S)-protopanaxadiol. The metabolites 2, 5, and 6 showed the more potent inhibitory effects against DU-145 and PC-3 cell lines than the substrate.

Topics: Absidia; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Humans; Male; Molecular Structure; Prostatic Neoplasms; Sapogenins

Microbial transformation of 20(S)-protopanaxadiol (1) by Mucor racemosus AS 3.205 yielded two novel hydroperoxylated metabolites and three known hydroxylated metabolites. The structures of the metabolites were identified as 26-hydroxyl-20(S)-protopanaxadiol (2), 23,24-en-25-hydroxyl-20(S)-protopanaxadiol (3), 25,26-en-24(R)-hydroperoxyl-20(S)-protopanaxadiol (4), 23,24-en-25-hydroperoxyl-20(S)-protopanaxadiol (5), and 25-hydroxyl-20(S)-protopanaxadiol (6). 4 and 5 are new compounds. Metabolites 2, 4, and 5 showed the more potent inhibitory effects against DU-145 and PC-3 cell lines than the substrate.

Topics: Antineoplastic Agents; Biotransformation; Cell Line, Tumor; Humans; Magnetic Resonance Spectroscopy; Male; Molecular Structure; Mucor; Prostatic Neoplasms; Sapogenins

Protopanaxadiol (PPD) is a triterpenoid that can be prepared from steamed ginseng. PPD possesses anticancer potential via caspase-dependent apoptosis. Whether paraptosis, a type of the caspase-independent cell death, is also induced by PPD has not been evaluated.. Cell death, the cell cycle and intracellular reactive oxygen species (ROS) were analyzed by flow cytometry after staining with annexin V/PI, PI/RNase or H2DCFDA. We observed morphological changes by crystal violet staining assay. Mitochondrial swelling was measured by ultraviolet-visible spectrophotometry. The activation of NF-κB was measured by luciferase reporter assay.. At comparable concentrations of 5-fluorouracil, PPD induced more cell death in human colorectal cancer cell lines HCT-116 and SW-480. We demonstrated that PPD induced paraptosis in these cancer cells. PPD treatment significantly increased the percentage of cancer cells with cytoplasmic vacuoles. After the cells were treated with PPD and cycloheximides, cytoplasmic vacuole generation was inhibited. The paraptotic induction effect of PPD was also supported by the results of the mitochondrial swelling assay. PPD induced ROS production in cancer cells, which activated the NF-κB pathway. Blockage of ROS by NAC or PS-1145 inhibited the activation of NF-κB signaling.. PPD induces colorectal cancer cell death in part by induction of paraptosis. The anticancer activity of PPD may be enhanced by antioxidants such as green tea, which also inhibit the activation of NF-κB signaling.

Topics: Anticarcinogenic Agents; Antineoplastic Agents, Phytogenic; Cell Death; Chemoprevention; Colorectal Neoplasms; HCT116 Cells; Heterocyclic Compounds, 3-Ring; Humans; Mitochondria; NF-kappa B; Panax; Phytotherapy; Plant Extracts; Pyridines; Reactive Oxygen Species; Sapogenins; Signal Transduction; Vacuoles

Dammarane Sapogenins (DS), with main ingredients of protopanaxatriol (PPT, 33%) and protopanaxadiol (PPD, 16%), is an alkaline hydrolyzed product of ginsenosides and had significant activities in improving learning and memory and decreasing chemotherapy-induced myelosuppression. In the present study, the pharmacokinetics and oral bioavailabilities of PPT and PPD were investigated when a single dose of DS was administrated orally (75 mg/kg) and intravenously (i.v., 30 mg/kg) to rats. Their in vitro stabilities in the GI tract were also investigated. PPT and PPD concentrations were measured by LC-MS. The results showed that PPT was eliminated rapidly from the body with an average t1/2, λz value of 0.80 h and CL of 4.27 l/h/kg after i.v. administration, while PPD was eliminated relatively slowly with a t1/2, λz of 6.25 h and CL of 0.98l/h/kg. After oral administration, both PPD and PPT could be absorbed into the body, but their systemic exposures were quite different. PPT was absorbed into the body quickly, with a Tmax of 0.58 h and a Cmax of 0.13 μg/ml, while PPD was absorbed relatively slowly with a Tmax of 1.82 h and a Cmax of 1.04 μg/ml. The absolute bioavailabilities of PPT and PPD were estimated as 3.69% and 48.12%, respectively. The stability test found that PPT was instable in the stomach with 40% degradation after 4h incubation at 37°C, both in pH1.2 buffer and in the stomach content solution. The instability in the stomach might be one of the reasons for PPT's poor bioavailability.

Topics: Animals; Biological Availability; Drug Administration Routes; Gastric Mucosa; Gastrointestinal Contents; Ginsenosides; Hydrogen-Ion Concentration; Intestinal Absorption; Male; Molecular Structure; Panax; Plant Extracts; Rats; Rats, Sprague-Dawley; Sapogenins

Using enrichment culture, Rhizobium sp. strain GIN611 was isolated as having activity for deglycosylation of a ginsenoside, compound K (CK). The purified heterodimeric protein complex from Rhizobium sp. GIN611 consisted of two subunits with molecular masses of 63.5 kDa and 17.5 kDa. In the genome, the coding sequence for the small subunit was located right after the sequence for the large subunit, with one nucleotide overlapping. The large subunit showed CK oxidation activity, and the deglycosylation of compound K was performed via oxidation of ginsenoside glucose by glycoside oxidoreductase. Coexpression of the small subunit helped soluble expression of the large subunit in recombinant Escherichia coli. The purified large subunit also showed oxidation activity against other ginsenoside compounds, such as Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, F1, and the isoflavone daidzin, but at a much lower rate. When oxidized CK was extracted and incubated in phosphate buffer with or without enzyme, (S)-protopanaxadiol [PPD(S)] was detected in both cases, which suggests that deglycosylation of oxidized glucose is spontaneous.

Topics: Base Sequence; Chromatography, High Pressure Liquid; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Ginsenosides; Molecular Sequence Data; Molecular Structure; Oxidoreductases; Panax; Plant Extracts; Polymerase Chain Reaction; Recombinant Proteins; Rhizobium; Sapogenins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

To study stereoselectivity in the pharmacokinetics of each epimer, a rapid, specific and reliable liquid chromatography tandem mass spectrometry method has been established for simultaneous quantitation of both epimers in rat plasma. Plasma samples were pretreated by liquid-liquid extraction. Chromatographic separations were performed on a Shim-pack XR-ODS C18 column (50 mm × 2.1mm, i.d., 2.2 μm) with an isocratic elution. Both epimers and the internal standard tanshinone II A were ionized with an ESI source operated in positive ion mode and measured by selective reactions monitoring mode. Calibration curve was linear over the concentration range of 1-1000 ng/mL with the lower limit of quantitation of 1 ng/mL for both epimers. Intra and inter-day precisions were less than 6.7% and 9.5%, and the accuracy was within ±5.8% for both epimers. The validated method has been successfully applied to a pharmacokinetic study of the two epimers in rats after oral administration.

Topics: Administration, Oral; Animals; Chromatography, Liquid; Drug Stability; Ginsenosides; Limit of Detection; Liquid-Liquid Extraction; Male; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sapogenins; Sensitivity and Specificity; Stereoisomerism; Tandem Mass Spectrometry

A ginseng pathogen, Cylindrocarpon destructans, and five nonpathogens were tested for their sensitivity to a total ginsenoside fraction (T-GF), a protopanaxadiol-type ginsenoside fraction (PPD-GF) and a protopanaxatriol-type ginsenoside fraction (PPT-GF) from the roots of Panax ginseng C.A. Meyer. The results showed that T-GF inhibited growth of the five ginseng nonpathogens, while it promoted growth of the ginseng pathogen C. destructans. PPT-GF and PPD-GF both inhibited the growth of the five ginseng nonpathogens, although the activity of PPT-GF was higher than that of PPD-GF. PPT-GF and PPD-GF exhibited different activities on C. destructans: PPT-GF inhibited its growth, whereas PPD-GF significantly enhanced its growth. The subsequent analysis of enzymatic degradation of ginsenosides by the test fungi showed that C. destructans can consecutively hydrolyze the terminal monosaccharide units from the sugar chains attached at C3 and C20 in PPD-type ginsenosides by extracellular glycosidase activity to yield four major products, gypenoside XVII (G-XVII), compound O, compound Mb and the ginsenoside F(2). By contrast, the ginseng nonpathogens Aspergillus nidulans and Cladosporium fulvum have no extracellular glycosidase activity toward sugar chains attached to C3 in PPD-type ginsenosides. These results indicated that ginsenosides might act as host chemical defenses, while the ginseng root pathogenic fungi might counter their toxicity by converting PPD-type ginsenosides into growth or host recognition factors. The ability of ginseng root pathogens to deglycosylate PPD-type ginsenosides may be a pathogenicity factor.

Topics: Antifungal Agents; Ginsenosides; Molecular Structure; Panax; Plant Roots; Sapogenins

In this paper, the kinetics of a cloned special glucosidase, named ginsenosidase type III hydrolyzing 3-O-glucoside of multi-protopanaxadiol (PPD)-type ginsenosides, were investigated. The gene (bgpA) encoding this enzyme was cloned from a Terrabacter ginsenosidimutans strain and then expressed in E. coli cells. Ginsenosidase type III was able to hydrolyze 3-O-glucoside of multi-PPD-type ginsenosides. For instance, it was able to hydrolyze the 3-O-β-D-(1-->2)-glucopyranosyl of Rb1 to gypenoside XVII, and then to further hydrolyze the 3-O-β-D-glucopyranosyl of gypenoside XVII to gypenoside LXXV. Similarly, the enzyme could hydrolyze the glucopyranosyls linked to the 3-O- position of Rb2, Rc, Rd, Rb3, and Rg3. With a larger enzyme reaction Km value, there was a slower enzyme reaction speed; and the larger the enzyme reaction Vmax value, the faster the enzyme reaction speed was. The Km values from small to large were 3.85 mM for Rc, 4.08 mM for Rb1, 8.85 mM for Rb3, 9.09 mM for Rb2, 9.70 mM for Rg3(S), 11.4 mM for Rd and 12.9 mM for F2; and Vmax value from large to small was 23.2 mM/h for Rc, 16.6 mM/h for Rb1, 14.6 mM/h for Rb3, 14.3 mM/h for Rb2, 1.81mM/h for Rg3(S), 1.40 mM/h for Rd, and 0.41 mM/h for F2. According to the Vmax and Km values of the ginsenosidase type III, the hydrolysis speed of these substrates by the enzyme was Rc>Rb1>Rb3>Rb2>Rg3(S)>Rd>F2 in order.

Topics: Actinomycetales; Bacterial Proteins; Cloning, Molecular; Ginsenosides; Glucosidases; Kinetics; Molecular Structure; Sapogenins; Substrate Specificity

This study focuses on determining the pharmacokinetics, biodistribution, and efficacy of the ginsenoside aglycone protopanaxadiol (aPPD) administered as a single agent in a novel oral dosage formulation. To obtain these data and to characterize the stability of aPPD, appropriate analytical assay development was carried out. The solubility and stability of aPPD were determined, and the compound was formulated for oral gavage. aPPD levels in blood and tissues following oral administration to nu/nu nude mice were determined using liquid chromatography-mass spectrometry/mass spectrometry. The efficacy of aPPD was determined upon oral administration to nu/nu nude mice bearing PC-3 human prostate cancer xenograft tumors. Immunohistochemical analysis of tumor tissues was performed to establish apoptotic indices and Ki-67 expression as markers of proliferation. The maximum solubility of aPPD in ethanol was 68.4 mg/ml. aPPD administered at a dose of 70 mg/kg yielded a T(max) of approximately 40 min and a C(max) value of 3.9 ± 1.4 μg/ml, and no toxicity was observed. aPPD accumulated largely in the stomach and small intestine and was also present in the brain. This dose engendered a significant delay in PC-3 tumor growth, an increase in apoptotic index, and a decrease in Ki-67 levels. We have shown that aPPD is a stable compound that can be formulated for oral gavage. Pharmacokinetic studies demonstrate the ability of this compound to be absorbed after oral administration. Future studies will assess the activity and pharmacokinetics of aPPD when administered in combination with standard chemotherapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Drug Compounding; Drug Stability; Ginsenosides; Humans; Immunohistochemistry; Male; Mass Spectrometry; Maximum Tolerated Dose; Mice; Mice, Nude; Molecular Structure; Prostatic Neoplasms; Sapogenins; Solid Phase Extraction; Solubility; Tissue Distribution; Xenograft Model Antitumor Assays

Optimal hydrolytic activity of β-glucosidase from Dictyoglomus turgidum for the ginsenoside Rd was at pH 5.5 and 80 °C, with a half-life of ~11 h. The enzyme hydrolysed β-linked, but not α-linked, sugar moieties of ginsenosides. It produced the rare ginsenosides, aglycon protopanaxadiol (APPD), compounds Y, and Mc, via three unique transformation pathways: Rb(1) → Rd → F(2) → compound K → APPD, Rb(2) → compound Y, and Rc → compound Mc. The enzyme converted 0.5 mM Rb(2) and 0.5 mM Rc to 0.5 mM compound Y and 0.5 mM compound Mc after 3 h, respectively, with molar conversion yields of 100 %.

Topics: Bacteria; beta-Glucosidase; Biotransformation; Enzyme Stability; Ginsenosides; Hydrogen-Ion Concentration; Sapogenins; Temperature; Time Factors

Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs, and the root of this plant contains pharmacologically active components, called ginsenosides. Ginsenosides, a class of tetracyclic triterpene saponins, are synthesized from dammarenediol-II after hydroxylation by cytochrome P450 (CYP) and then glycosylation by a glycosyltransferase. Protopanaxadiol synthase, which is a CYP enzyme (CYP716A47) that catalyzes the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol, was recently characterized. Here, we isolated two additional CYP716A subfamily genes (CYP716A52v2 and CYP716A53v2) and determined that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in P. ginseng. Both CYP716A47 and CYP716A53v2 mRNAs accumulated ubiquitously in all organs of ginseng plants. In contrast, CYP716A52v2 mRNA accumulated only in the rhizome. Methyl jasmonate (MeJA) treatment resulted in the obvious accumulation of CYP716A47 mRNA in adventitious roots. However, neither CYP716A52v2 nor CYP716A53v2 mRNA was affected by MeJA treatment during the entire culture period. The ectopic expression of CYP716A53v2 in recombinant WAT21 yeast resulted in protopanaxatriol production after protopanaxadiol was added to the culture medium. In vitro enzymatic activity assays revealed that CYP716A53v2 catalyzed the oxidation of protopanaxadiol to produce protopanaxatriol. The chemical structures of the protopanaxatriol products were confirmed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Our results indicate that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that produces protopanaxatriol from protopanaxadiol, which is an important step in the formation of dammarane-type triterpene aglycones in ginseng saponin biosynthesis.

Topics: Biocatalysis; Biosynthetic Pathways; Chromatography, Liquid; Cytochrome P-450 Enzyme System; DNA, Complementary; Enzyme Assays; Gene Expression Regulation, Plant; Genes, Plant; Ginsenosides; Mass Spectrometry; Panax; Phylogeny; Plant Proteins; Plant Roots; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Saccharomyces cerevisiae; Sapogenins; Transcription, Genetic

The ability of fresh lemon (Citrus limon) to convert protopanaxadiol-type saponins into ginsenoside Rg3 was investigated, and the structures of 20(S)-ginsenoside Rg3 (1) and 20(R)-ginsenoside Rg3 (2) were identified by 1H NMR and 13C NMR spectroscopy. The experiment showed that lemon possesses the strong ability to hydrolyze ginsenosides. When protopanaxadiol-type saponins (16 mg/mL) were hydrolyzed by fresh lemon juice at 80 degrees C for 3 hrs, the conversion ratios of ginsenoside Rb1, Rb2, Rc and Rd were 92.9%, 90.0%, 96.90/0 and 55.5%, respectively, and the yields of 20(S)-ginsenoside Rg3 and 20(R)-ginsenoside Rg3 were, respectively, 31.2% and 28.3%.

Topics: Citrus; Ginsenosides; Hydrolysis; Magnetic Resonance Spectroscopy; Sapogenins

Although the treatment outcome of acute lymphoblastic leukemia (ALL) has been improved in the past decades by combination chemotherapy, toxic side-effects of chemotherapeutics remain a major problem. Therefore, new alternative agents with low toxicity are urgently needed. Natural products provide a rich source of screening potential anti-cancer drugs. 20(S)-protopanoxadiol (PPD), a major gastrointestinal metabolic product of ginsenosides, exhibits promising anti-cancer activity with low toxicity. However, the anti-cancer activity of PPD against ALL has not been evaluated. In this study, we examined the anti-cancer effect of PPD on ALL cell lines Reh and RS4;11. The growth of leukemia cells and normal cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell cycle, apoptosis and differentiation was determined by flow cytometry. The results showed that PPD inhibited the growth of Reh and RS4;11 cells, but had little toxicity to peripheral blood mononuclear cells (PBMC). PPD also blocked cell cycle progression from G0/G1 phase and induced cell differentiation. However, cell apoptosis was not affected. These data indicate that PPD exerts anti-cancer effects by stimulating differentiation and inhibiting growth and cell cycle progression of ALL cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sapogenins

Protopanaxadiol (PPD), an aglycon of ginseng saponins, has shown anticancer activity in earlier studies. Here, we have reported the semisynthesis of nine PPD derivatives with acetyl substitutions. Subsequently, the antiproliferative effects of these nine analogs on different human cancer cell lines have been investigated. Compounds 1, 3, and 5 showed more significant and more potent antiproliferative activity compared with PPD and other derivatives. A flow cytometric assay indicated that compounds 1, 3, and 5 arrested cell cycle progression in the G1 phase and significantly induced apoptosis of cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Colorectal Neoplasms; Drug Screening Assays, Antitumor; Female; G1 Phase; HCT116 Cells; Humans; Magnetic Resonance Spectroscopy; Panax; Sapogenins; Saponins; Structure-Activity Relationship

The production of compound K and aglycon protopanaxadiol (APPD) from ginsenoside Rd and ginseng root extract was performed using a recombinant β-glycosidase from Pyrococcus furiosus. The activity for Rd was optimal at pH 5.5 and 95°C with a half-life of 68 h at 95°C. β-Glycosidase converted Rb(1), Rb(2), Rc, and Rd to APPD via compound K. With increases in the enzyme activity, the productivities of compound K and APPD increased. The substrate concentration was optimal at 4.0 mM Rd or 10% (w/v) ginseng root extract; 4 mM of Rd was converted to 3.3 mM compound K with a yield of 82.5% (mol/mol) and a productivity of 2,010 mg l(-1) h(-1) at 1 h and was hydrolyzed completely to APPD with 364 mg l(-1) h(-1) after 5 h. Rb(1), Rb(2), Rc, and Rd at 3.9 mM in 10% ginseng root extract were converted to 3.1 mM compound K with 79.5% and 1,610 mg l(-1) h(-1) at 1.2 h and were hydrolyzed completely to APPD with 300 mg l(-1) h(-1) after 6 h. The concentrations and productivities of compound K and APPD in the present study are the highest ever reported.

Topics: beta-Glucosidase; Enzyme Stability; Ginsenosides; Half-Life; Hydrogen-Ion Concentration; Panax; Plant Extracts; Plant Roots; Pyrococcus furiosus; Recombinant Proteins; Sapogenins; Temperature

20(S)-Protopanaxadiol (PPD, 1) is one of the aglycones of the ginsenosides and has a wide range of pharmacological activities. At present, PPD has progressed to early clinical trials as an antidepressant. In this study, its fate in mixed human liver microsomes (HLMs) and human hepatocytes was examined for the first time. By using liquid chromatography-electrospray ionization ion trap mass spectrometry, 24 metabolites were found. Four metabolites were isolated, and their structures were elucidated as (20S,24S)-epoxydammarane-3,12,25-triol (2), (20S,24R)-epoxydammarane-3,12,25-triol (3), (20S,24S)-epoxydammarane-12,25-diol-3-one (4), and (20S,24R)-epoxydammarane-12,25-diol-3-one (5) based on a detailed analysis of their spectroscopic data. The predominant metabolic pathway of PPD observed was the oxidation of the 24,25-double bond to yield 24,25-epoxides, followed by hydrolysis and rearrangement to form the corresponding 24,25-vicinal diol derivatives (M6) and the 20,24-oxide form (2 and 3). Further sequential metabolites (M2-M5) were also detected through the hydroxylation and dehydrogenation of 2 and 3. All of the phase I metabolites except for M1-1 possess a hydroxyl group at C-25 of the side chain, which was newly formed by biotransformation. Two glucuronide conjugates (M7) attributed to 2 and 3 were detected in human hepatocyte incubations, and their conjugation sites were tentatively assigned to the 25-hydroxyl group. The findings of this study strongly suggested that the formation of the 25-hydroxyl group is very important for the elimination of PPD.

Topics: Animals; Antidepressive Agents; Biotransformation; Chromatography, High Pressure Liquid; Epoxy Compounds; Feces; Ginsenosides; Glucuronides; Hepatocytes; Humans; Male; Microsomes, Liver; Molecular Structure; Rats; Rats, Sprague-Dawley; Sapogenins; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

20S-protopanaxadiol (aPPD) is a metabolite of ginseng saponins, which is reported to be pro-apoptotic in some cells but anti-apoptotic in neuronal cells by regulating Akt signaling. Owing to its cholesterol-like structure, we hypothesized that aPPD may regulate Akt signaling by interacting with lipid rafts. Here, we compared Akt signaling in glioblastoma U87MG and neuroblastoma Neuro-2a cells treated with aPPD. aPPD did not change Akt activity in the total plasma membranes of each cell type, but drastically altered the activity of raft-associated Akt. Strikingly, Akt activity was decreased in the rafts of U87MG cells but increased in N2a cells by aPPD through regulating raft-associated dephosphorylation. The bidirectional regulation of raft-associated Akt signaling by aPPD enhanced the chemotoxicity of Paclitaxel or Vinblastine in U87MG cells but attenuated the excitotoxicity of N-methyl-D-aspartate in N2a cells. Our results demonstrated that the activity of raft-associated but not total membrane Akt determines its cellular functions. Lipid rafts differ in different types of cells, which allows for the possibility of cell-type-specific targeting for which aPPD might prove to be a useful agent.

Topics: Cell Line, Tumor; Humans; Membrane Microdomains; Phosphorylation; Plant Extracts; Proto-Oncogene Proteins c-akt; Sapogenins; Signal Transduction; Species Specificity

20(S)-Protopanaxadiol (PPD), a metabolite of ginsenosides, has been demonstrated to possess cytotoxic effects on several cancer cell lines. The molecular mechanism is, however, not well understood. In this study, we have shown that PPD inhibits cell growth and induces apoptosis in human hepatocarcinoma HepG2 cells. PPD-treated cells showed a massive cytoplasmic vacuolization and a dramatic change of endoplasmic reticulum (ER) morphology. The induction of ER stress is associated with the upregulation of ER stress-associated genes and proteins. PPD activates the unfolded protein response (UPR) through the phosphorylation of PERK and eIF2α, the splicing of XBP1 mRNA, and the cleavage of AFT6. PPD also induces the intrinsic and extrinsic apoptotic pathways. It activates DR5, caspase-8, -9, -3, and promotes the cleavage of PARP while it downregulates Bcl-2, Bcl-x(L) and mitochondrial membrane potential. Knockdown of one of the three UPR limbs by specific siRNAs did not affect PPD-induced apoptosis, which was however, significantly suppressed by the downregulation of CHOP. Western blot analysis showed that PPD-stimulated downregulation of Bcl-2 protein, increase of DR5 protein, activation of caspase-8 and cleavage of PARP were significantly inhibited in CHOP siRNA-transfected cells. Taken together, we have identified ER as a molecular target of PPD and our data support the hypothesis that PPD induces HepG2 cell apoptosis through the ER stress pathway.

Topics: Apoptosis; Endoplasmic Reticulum Stress; Extracellular Signal-Regulated MAP Kinases; Ginsenosides; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitochondria; p38 Mitogen-Activated Protein Kinases; Receptors, TNF-Related Apoptosis-Inducing Ligand; Sapogenins; Transcription Factor CHOP; Unfolded Protein Response; Up-Regulation; Vacuoles

Six new protopanaxadiol-type ginsenosides, named ginsenosides Ra(4) -Ra(9) (1-6, resp.), along with 14 known dammarane-type triterpene saponins, were isolated from the root of Panax ginseng, one of the most important Chinese medicinal herbs. The structures of the new compounds were determined by spectroscopic methods, including 1D- and 2D-NMR, HR-MS, and chemical transformation as (20S)- 3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[β-D-xylopyranosyl-(1→4)-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (1), (20S)-3-O-[β-D-6-O-acetylglucopyranosyl-(1→2)-β-D-glucopyranosyl]-20-O-[β-D-xylopyranosyl-(1→4)-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (2), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (3), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L-arabinopyranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (4), (20S)-3-O-{β-D-4-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L-arabinofuranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (5), (20S)-3-O-{β-D-6-O-[(E)-but-2-enoyl]glucopyranosyl-(1→2)-β-D-glucopyranosyl}-20-O-[α-L-arabinofuranosyl-(1→6)-β-D-glucopyranosyl]protopanaxadiol (6). The sugar moiety at C(3) of the aglycone of each new ginsenoside is butenoylated or acetylated.

Topics: Acylation; Ginsenosides; Molecular Structure; Panax; Plant Roots; Plants, Medicinal; Sapogenins

Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs and contains pharmacologically active components, ginsenosides, in its roots. Ginsenosides, a class of tetracyclic triterpene saponins, are thought to be synthesized from dammarenediol-II after hydroxylation by the Cyt P450 (CYP) enzyme and then glycosylation by glycosyltransferase (GT). However, no genes encoding the hydroxylation and glycosylation in ginsenoside biosynthesis have been identified. Here, we identify protopanaxadiol synthase, which is a CYP enzyme (CYP716A47), to be involved in the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol. Nine putative full CYP sequences were isolated from the expressed sequence tags (ESTs) of methyl jasmonate (MeJA)-treated adventitious ginseng roots. The CYP716A47 gene product was selected as the putative protopanaxadiol synthase because this gene was transcriptionally activated not only by MeJA treatment but also in transgenic ginseng that overexpresses squalene synthase and overproduces ginsenosides. In vitro enzymatic activity assays revealed that CYP716A47 catalyzed the oxidation of dammarenediol-II to produce protopanaxadiol. Ectopic expression of CYP716A47 in recombinant WAT21 yeasts that were fed dammarenediol-II yielded protopanaxadiol. Furthermore, co-expression of the dammarenediol synthase gene (PgDDS) and CYP716A47 in yeast yielded protopanaxadiol without adding dammarenediol-II. The chemical structures of the protopanaxadiol products from dammarenediol-II were confirmed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Thus, CYP716A47 is a dammarenediol 12-hydroxylase that produces protopanaxadiol from dammarenediol-II.

Topics: Acetates; Biocatalysis; Biosynthetic Pathways; Chromatography, Liquid; Cyclopentanes; Cytochrome P-450 Enzyme System; DNA, Complementary; Expressed Sequence Tags; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Gene Library; Genes, Plant; Ginsenosides; Mass Spectrometry; Oxylipins; Panax; Phylogeny; Plants, Genetically Modified; RNA, Messenger; Saccharomyces cerevisiae; Sapogenins; Saponins; Sequence Analysis, DNA; Transcription, Genetic; Triterpenes

To investigate the protective effects of protopanaxadiol-type ginsenoside (PDG) and its metabolite ginsenoside M1 (G-M1) on carbon tetrachloride (CCl(4))-induced chronic liver injury in ICR mice, we carried out conversion of protopanaxadiol-type ginsenosides to ginsenoside M1 using snailase. The optimum time for the conversion was 24 h at a constant pH of 4.5 and an optimum temperature of 50 °C. The transformation products were identified by high-performance liquid chromatography and electrospray ion-mass spectrometry. Subsequently, most of PDG was decomposed and converted into G-M1 by 24 h post-reaction. During the study on hepatoprotective in a mice model of chronic liver injury, PDG or G-M1 supplement significantly ameliorated the CCl(4)-induced liver lesions, lowered the serum levels of select hepatic enzyme markers (alanine aminotransferase, ALT, and aspartate aminotransferase, AST) and malondialdehyde and increased the activity of superoxide dismutase in liver. Histopathology of the liver tissues showed that PDG and G-M1 attenuated the hepatocellular necrosis and led to reduction of inflammatory cell infiltration. Therefore, the results of this study show that PDG and G-M1 can be proposed to protect the liver against CCl(4)-induced oxidative injury in mice, and the hepatoprotective effect might be attributed to amelioration of oxidative stress.

Topics: Alanine Transaminase; Animals; Antioxidants; Aspartate Aminotransferases; Biotransformation; Body Weight; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Chromatography, High Pressure Liquid; Chronic Disease; Ginsenosides; Liver; Male; Malondialdehyde; Mice; Mice, Inbred ICR; Organ Size; Protective Agents; Sapogenins; Snails; Spectrometry, Mass, Electrospray Ionization; Superoxide Dismutase; Tissue Extracts

Panax ginseng has long been used in Asia as a herbal medicine for the prevention and treatment of various diseases, including cancer. The current study evaluated the cytotoxic potency against a variety of cancer cells by using ginseng ethanol extracts (RSE), protopanaxadiol (PPD)-type, protopanaxatriol (PPT)-type ginsenosides fractions, and their hydrolysates, which were prepared by stepwise hydrolysis of the sugar moieties of the ginsenosides. The results showed that the cytotoxic potency of the hydrolysates of RSE and total PPD-type or PPT-type ginsenoside fractions was much stronger than the original RSE and ginsenosides; especially the hydrolysate of PPD-type ginsenoside fractions. Subsequently, two derivatives of protopanaxadiol (1), compounds 2 and 3, were synthesized via hydrogenation and dehydration reactions of compound 1. Using those two derivatives and the original ginsenosides, a comparative study on various cancer cell lines was conducted; the results demonstrated that the cytotoxic potency was generally in the descending order of compound 3 > 20(S)-dihydroprotopanaxadiol (2) > PPD (1) > 20(S)-Rh2 > 20(R)-Rh2 ≈ 20(R)-Rg3 ≈ 20(S)-Rg3. The results clearly indicate the structure-related activities in which the compound with less polar chemical structures possesses higher cytotoxic activity towards cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Cell Death; Cell Line, Tumor; Cell Proliferation; Chemical Fractionation; Chromatography, High Pressure Liquid; Drug Screening Assays, Antitumor; Ginsenosides; Humans; Hydrolysis; Plant Extracts; Sapogenins; Structure-Activity Relationship; Time Factors

A lot of anti-diabetic agents using natural plants have been extensively studied. Ginsenosides are known to be used as a remedy for diabetes in Asian countries and American Societies. Diabetic nephropathy is a major complication of diabetes mellitus. Extracellular matrix in mesangial cells is mainly composed of fibronectin and the increase of fibronectin is a hallmark of diabetic nephropathy. Protopenaxadiol (PPD) is a major component of total ginseng. Thus, we examined the regulatory mechanism of PPD derivatives-induced preventive effect of fibronectin expression in mesangial cells cultivated under diabetic condition. In present study, ginsenoside Rb1 prevented the high glucose-induced increase of fibronectin expression in mesangial cells. Ginsenoside Rb2 and Rg3 also mildly inhibited it. However, ginsenoside Rc and Rd did not prevent the high glucose-induced increase of fibronectin expression in mesangial cells. In addition, ginsenoside Rb1 prevented high glucose-induced phosphorylation of p44/42 mitogen activated protein kinase (MAPK), p38 MAPK, JNK/SAPK, and Akt. These results suggest that ginsenoside Rb1 is the most powerful component of PPD derivatives. In conclusion, ginsenoside Rb1 prevented high glucose-induced increase of fibronectin expression via the inhibition of MAPK-Akt signaling cascade.

Topics: Animals; Blotting, Western; Carbohydrate Sequence; Fibronectins; Glomerular Mesangium; Glucose; Male; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Sapogenins; Signal Transduction

To prepare and evaluate in vitro the 20 (S) -Protopanaxadiol (Ppd) pharmacosome. The Ppd pharmacosome was successfully prepared by thin film-dispersion and its stability in vitro was studied. The particle size of pharmacosome was evaluated by dynamic scattering (DLS) and the encapsulation efficiency was determined by using centrifugal ultra-filtration. The encapsulation efficiency of Ppd pharmacosome was (80.84 +/- 0.53)% with the diameter of 100. 1 nm; While the encapsulation efficiency of Ppd pharmacosome that added Brij 78 added was (72.76 +/- 0.63)% with the diameter of 117. 3 nm. In addition, the effect of some factors on the encapsulation efficiency and the particles size, such as temperature, alcohol, pH and artificial gastrointestinal fluids, were investigated respectively. The selected formulation and technology are simple and practical to prepare Ppd pharmacosome and preparation properties are more stable.

Topics: Chemistry, Pharmaceutical; Drug Stability; Ethanol; Gastric Acid; Hydrogen-Ion Concentration; Light; Particle Size; Sapogenins; Scattering, Radiation; Temperature

20(S)-protopanaxadiol (PPD(S)) and 20(R)-protopanaxadiol (PPD(R)), the main metabolites of ginsenosides Rg3(S) and Rg3(R) in black ginseng, are potential candidates for anti-cancer therapy due to their pharmacological activities such as anti-tumor properties. In the present study, we report the preparation of PPD(S, R) by a combination of steaming and biotransformation treatments from ginseng. Aspergillus niger was isolated from soil and showed a strong ability to transform Rg3(S, R) into PPD(S, R) with 100% conversion. Furthermore, the enzymatic reactions were analyzed by reversed-phase HPLC, showing the biotransformation pathways: Rg3(S)-->Rh2(S)-->PPD(S) and Rg3(R)-->Rh2(R)-->PPD(R), respectively. In addition, 12 ginsenosides including 3 pairs of epimers, namely Rg3(S), Rg3(R), Rh2(S), Rh2(R), PPD(S) and PPD(R), were simultaneously determined by reversed-phase HPLC. Our study may be highly applicable for the preparation of PPD(S) and PPD(R) for medicinal purposes and also for commercial use.

Topics: Antineoplastic Agents; Aspergillus niger; Biotransformation; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Ginsenosides; Panax; Sapogenins; Stereoisomerism

Effects of protopanaxdiol (PDG) and protopanaxatriol (PTG) types of ginsenosides isolated from the leaves of American ginseng on porcine pancreatic lipase activity were determined in vitro. PDG inhibited the pancreatic lipase activity in a dose-dependent manner at the concentrations of 0.25-1mg/ml. It inhibited hydrolysis of about 83.2% of triolein at about 1mg/ml of PDG. However, PTG showed no inhibitory activity. Therefore, anti-obesity activity of PDG was evaluated in mice fed a high-fat diet. The results demonstrated that PDG was effective in preventing and healing obesity, fatty liver and hypertriglyceridemia in mice fed with a high-fat diet.

Topics: Animals; Anti-Obesity Agents; Dietary Fats; Female; Ginsenosides; Mice; Molecular Structure; Obesity; Panax; Plant Leaves; Sapogenins

An ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed to investigate 20(S)-protopanaxadiol (PPD) pharmacokinetics in rats. Rat plasma samples were treated using a solid-phase extraction with satisfactory recovery (> 81%). The method showed an excellent sensitivity that the limit of detection (LOD) and the lower limit of quantitation (LLOQ) of PPD were 0.5 and 2 ng/mL, respectively. The method was applied to the evaluation of pharmacokinetics from two types of PPD formulations. The PPD emulsion showed more rapid and efficient drug absorption, and higher and more persistent plateau concentration of PPD in plasma than PPD oil solution. PPD emulsion was demonstrated to be a promising dosage form. In spite of lower plateau plasma drug concentration, PPD oil solution was characterized by the easiness in preparation and the persistent, durative plateau plasma concentration of PPD, there is room to further improve its bioavailability.

Topics: Animals; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sapogenins; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Ginseng has been used for mood adjustment in traditional Chinese medicine for thousands of years. Our previous study has shown that, total ginsenosides, the major pharmacologically functional ingredients of ginseng, possess antidepressant activity. In the present study, we hypothesized that an intestinal metabolite of ginseng, 20(S)-protopanaxadiol (code name S111), as a post metabolism compound (PMC) of ingested ginsenosides, may be responsible for the antidepressant activity of ginseng. To test this hypothesis, antidepressant-like activity of orally given S111 was measured in animal tests including tail suspension test, forced swimming test and rat olfactory bulbectomy depression model. In all those tests, S111 demonstrated antidepressant-like activity as potent as fluoxetine. S111 treated bulbectomy animals had higher levels of monoamine neurotransmitters in the brain and in vitro reuptake assay showed that S111 had a mild inhibitory effect. Furthermore, S111 but not fluoxetine significantly reduced brain oxidative stress and down-regulated serum corticosterone concentration in bulbectomy animals. No disturbance to central nervous system (CNS) normal functions were found in S111 treated animals. These results suggest that the ginseng active metabolite S111 is a potential antidepressant. Since the monoamine reuptake activity of this compound is rather weak, it remains to be investigated whether its antidepressant-like effect is by mechanisms that are different from current antidepressants. Furthermore, this study has demonstrated that post metabolism compounds (PMCs) of herb medicines such as S111 may be a novel source for drug discovery from medicinal herbs.

Topics: Administration, Oral; Animals; Antidepressive Agents; Avoidance Learning; Depression; Disease Models, Animal; Male; Mice; Panax; Rats; Rats, Sprague-Dawley; Sapogenins

A previous study demonstrated that ginseng crude saponins prevent obesity induced by a high-fat diet in rats. Ginseng crude saponins are known to contain a variety of bioactive saponins. The present study investigated and compared the antiobesity activity of protopanaxadiol (PD) and protopanaxatriol (PT) type saponins, major active compounds isolated from crude saponins. Male 4-week-old Sprague-Dawley rats were fed with normal diet (N) or high-fat diet (HF). After 5 weeks, the HF diet group was subdivided into the control HF diet, HF diet-PD and HF diet-PT group (50 mg/kg/day, 3 weeks, i.p.). Treatment with PD and PT in the HF diet group reduced the body weight, total food intake, fat contents, serum total cholesterol and leptin to levels equal to or below the N diet group. The hypothalamic expression of orexigenic neuropeptide Y was significantly decreased with PD or PT treatment, whereas that of anorexigenic cholecystokinin was increased, compared with the control HF diet group. In addition, PD type saponins had more potent antiobesity properties than PT saponins, indicating that PD-type saponins are the major components contributing to the antiobesity activities of ginseng crude saponins. The results suggest that the antiobesity activity of PD and PT type saponins may result from inhibiting energy gain, normalizing hypothalamic neuropeptides and serum biochemicals related to the control of obesity.

Topics: Animals; Anti-Obesity Agents; Body Weight; Cholecystokinin; Cholesterol; Eating; Hypothalamus; Leptin; Male; Neuropeptide Y; Obesity; Panax; Rats; Rats, Sprague-Dawley; Sapogenins; Triglycerides

Ginsenosides are used widely for medicinal purposes, but the mechanisms of their action are still unclear, although there is some evidence that these effects are mediated by nuclear receptors. Here we examined whether two metabolites of ginsenoside, protopanaxadiol (g-PPD) and protopanaxatriol (g-PPT), could modulate endothelial cell functions through the glucocorticoid receptor (GR) and oestrogen receptor (ER). EXPERIMENT APPROACHES: The effects of g-PPD and g-PPT on intracellular calcium ion concentration ([Ca(2+)](i)) and nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) were measured using Fura-2-acetoxymethyl ester, 4-amino-5-methylamino-2',7'-difluorofluorescein and Griess reagent. Effects on expression of GR and ER isoforms in HUVECs were determined using reverse transcriptase-/real-time PCR and immunocytochemistry. Phosphorylation of endothelial NO synthase (eNOS) was assessed by Western blotting.. Ginsenoside protopanaxadiol and g-PPT increased [Ca(2+)](i), eNOS phosphorylation and NO production in HUVECs, which were inhibited by the GR antagonist, RU486, the ER antagonist, ICI 182,780 and siRNA targeting GR or ERbeta. The NO production was Ca(2+)-dependent and the [Ca(2+)](i) elevation in HUVECs resulted from both intracellular Ca(2+) release and extracellular Ca(2+) influx.. Ginsenoside protopanaxadiol and g-PPT were functional ligands for both GR and ERbeta, through which these ginsenoside metabolites exerted rapid, non-genomic effects on endothelial cells.

Topics: Blotting, Western; Calcium; Cell Line; Dose-Response Relationship, Drug; Endothelial Cells; Estrogen Receptor alpha; Estrogen Receptor beta; Humans; Immunohistochemistry; Nitric Oxide; Protein Binding; Receptors, Glucocorticoid; Reverse Transcriptase Polymerase Chain Reaction; Sapogenins; Time Factors; Transfection

One of the major causes for cancer cells to resist current chemotherapy is attributed to the over-expression of P-glycoprotein (P-gp), resulting in insufficient drug delivery to the tumor sites. Protopanaxadiol ginsenosides Rg3 and Rh2 are known to induce apoptosis and significantly enhance the tumor inhibitory effects of chemotherapeutics in a synergistic fashion. One of the possible mechanisms is by blocking P-gp activity. The final deglycosylation metabolite of protopanaxadiols (PPDs) IN VIVO is 20S-protopapanaxadiol (aglycone PPD, aPPD), which has also shown anticancer activity and synergy with chemotherapy drugs. In the present study, P-gp over-expressing cancer cells were utilized to test whether aPPD also inhibits P-gp activity. We found that aPPD caused similar cytotoxicity in P388adr cells as their parental non-MDR cells, suggesting that aPPD may not be a substrate of P-gp. On the other hand, the calcein AM efflux assay showed that aPPD was able to inhibit P-gp activity as potently as verapamil on MDR cells. The blockage of P-gp activity was highly reversible as wash-out of aPPD resulted in an immediate recovery of P-gp activity. Unlike verapamil, aPPD did not affect ATPase activity of P-gp suggesting a different mechanism of action. The above results indicate that aPPD, unlike its precursor ginsenosides Rg3 and Rh2, is not a substrate of P-gp. It is also the first time that aPPD has showed a reversible nature of its P-gp inhibition. In addition to its pro-apoptotic nature, aPPD may be a potential new P-gp inhibitor for cancer treatment.

Topics: Adenosine Triphosphatases; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Drug Resistance, Neoplasm; Mice; Sapogenins

To establish a RP-HPLC method for content and entrapment efficiency of 20 (S)-protopanaxadiol in pharmacosomes.. The separation was performed with a COSMOSIL 5 C18-MS-II column (4.6 mm x 250 mm, 5 mmicrom) using methanol-water (95:5) as the mobile phase and detected at 203 nm. The flow rate was 1.0 mL x min(-1) and 50 microL sample solution was injected for each time.. The calibration curve was linear within the range 0.1-0.5 mg x mL(-1) (r = 0. 9999) , the intra-day RSD and inter-day RSD were less than 2% and the average recovery was between 101.44%-103.11% (n = 3).. The method is simple, accurate, sensitive and applicable for determination of content and entrapment efficiency of 20 (S)-protopanaxadiol pharmacosomes.

Topics: Calibration; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Panax; Reproducibility of Results; Sapogenins

To investigate the antioxidative effects of ginsenosides [protopanaxadiol derivatives (PD):protopanaxatriol derivatives (PT) = 1:1] from the roots of Korean ginseng, cell viability, malondialdehyde (MDA) production, antioxidant enzyme activities, and expressions of apoptosis were analyzed after pretreatment of human hepatoma HepG2 cells with H(2)O(2). Cell death was increased through H(2)O(2) treatment dose dependently, and a dose of ginseng extract (PD:PT = 1:1) of 18.6 microg/mL was enough to derive it in reverse. MDA production was reduced through the administration of ginseng extracts even with more intensive H(2)O(2) treatments. Through the use of even low levels of ginseng extract (e.g., 1.86 microg/mL), catalase (CAT) activity was easily reduced from the plateau induced by H(2)O(2). The glutathione peroxidase activity was no better than that of CAT. We assume that ginseng extract acts as an antioxidant even when effective levels of ginseng differ. A ginseng extract dose of 18.6 microg/mL increased the apoptotic expression of oxidative stressed signals, such as c-Jun-N-terminal kinase and stress-activated protein kinase expressions, and mitochondrial cytochrome c released caspase-3 activation; however, these expressions changed with higher doses of ginseng.

Topics: Antioxidants; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Catalase; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Korea; Liver Neoplasms; Malondialdehyde; Mitogen-Activated Protein Kinases; Oxidants; Panax; Plant Extracts; Sapogenins; Saponins

To prepare and isolate the rare ginsenoside derivatives of anti-cancer activity.. Acid-degradation products of ginsenosides were isolated and purified by various chromatographic techniques such as silica gel and so on; compounds were identified and elucidated by spectral and chemical methods.. Eleven compounds were obtained from the products of Acid-degradation.. Compound I, II are discoverd from the roots, stems, leaves, fruits and alabastrums of Panax quinquefolium L. for the first time. Compound IX of derivate of the aglycone of protopanoxadiol ginsenoside was discovered and reported, having the conspicuous anti-cancer activity for the first time from the fruits of Panax ginseng.

Topics: Antineoplastic Agents, Phytogenic; Ginsenosides; Hydrolysis; Magnetic Resonance Spectroscopy; Molecular Structure; Panax; Plant Leaves; Plant Stems; Sapogenins

20(S)-Protopanaxadiol (PPD), the main metabolite of protopanoxadiol type ginsenosides (e.g. Rg3 and Rh2), is a very promising anti-cancer drug candidate. To evaluate the pharmacokinetic property of PPD, we reported a reliable, sensitive and simple method utilizing liquid chromatography (HPLC)-atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) to determine PPD. PPD and the internal standard, panoxadiol (PD) were extracted from plasma with acetic ether, separated on a C18 reverse column, and then analyzed by APCI-MS. Targeting fragment ion at m/z 425 for both PPD and PD was monitored in selected-ion monitoring (SIM) mode. PPD can be quantitatively determined at the concentration as low as 1 ng/mL using 200 microL plasma. And the sensitive method showed excellent linearity over a range from 1 to 1000 ng/mL, high recovery, accuracy and precision at the concentrations of 2.5, 100.0 and 1000.0 ng/mL, respectively. The method was successfully applied to pharmacokinetic study of PPD in rats. Pharmacokinetic parameters were calculated and absolute bioavailability of PPD was 36.8+/-12.4%, at least ten times higher than that of Rg3 and Rh2, indicating its good absorption in gastrointestinal tract. It was further suggested that PPD be a promising anti-cancer candidate and probably responsible for the observed pharmacological activity of Rg3 and Rh2.

Topics: Administration, Oral; Animals; Anticarcinogenic Agents; Area Under Curve; Atmospheric Pressure; Biological Availability; Calibration; Chromatography, High Pressure Liquid; Guidelines as Topic; Half-Life; Injections, Intravenous; Male; Mass Spectrometry; Metabolic Clearance Rate; Molecular Structure; Quality Control; Rats; Rats, Sprague-Dawley; Reference Standards; Reproducibility of Results; Sapogenins; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization

We recently isolated 20(S)-25-methoxyl-dammarane-3beta, 12beta, 20-triol (25-OCH3-PPD), a natural product from Panax notoginseng, and demonstrated its cytotoxicity against a variety of cancer cells. Here we report the effects of this compound in vitro and in vivo on human prostate cancer cells, LNCaP (androgen-dependent) and PC3 (androgen-independent), in comparison with three structurally related ginsenosides, ginsenoside Rh2, ginsenoside Rg3, and 20(S)-protopanaxadiol. Of the four test compounds, 25-OCH3-PPD was most potent. It decreased survival, inhibited proliferation, induced apoptosis, and led to G1 cell cycle arrest in both cell lines. It also decreased the levels of proteins associated with cell proliferation (MDM2, E2F1, cyclin D1, and cdks 2 and 4) and increased or activated pro-apoptotic proteins (cleaved PARP, cleaved caspase-3, -8, and -9). In LNCaP cells, 25-OCH3-PPD inhibited the expression of the androgen receptor and prostate-specific antigen. Moreover, 25-OCH3-PPD inhibited the growth of prostate cancer xenograft tumours. Combining 25-OCH3-PPD with conventional chemotherapeutic agents or with radiation led to potent antitumour effects; tumour regression was almost complete following administration of 25-OCH3-PPD and either taxotere or gemcitabine. 25-OCH3-PPD also demonstrated low toxicity to noncancer cells and no observable toxicity in animals. In conclusion, our preclinical data indicate that 25-OCH3-PPD is a potential therapeutic agent against both androgen-dependent and androgen-independent prostate cancer.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Cell Cycle; Cell Proliferation; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Ginsenosides; Humans; In Vitro Techniques; Male; Mice; Mice, Nude; Prostate-Specific Antigen; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sapogenins; Survival Rate; Triterpenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The ginsenoside Rh(2) and its aglycone 20(S)-protopanaxadiol are known to inhibit the binding of [(3)H]batrachotoxinin 20alpha-benzoate to site 2 on voltage-gated sodium channels and electrophysiological investigations conducted by others have shown that ginsenosides cause voltage-dependent inhibition of reconstituted forms of the sodium channel. Here we describe the actions of Rh(2) and 20(S)-protopanaxadiol on sodium channel function and release of neurotransmitters resulting from activation of native sodium channels in synaptic preparations isolated from whole mouse brain. Rh(2) and 20(S)-protopanaxadiol inhibited veratridine-dependent (tetrodotoxin-suppressible) depolarization of synaptoneurosomes as determined using the rhodamine 6G method although 20(S)-protopanaxadiol was more potent as an inhibitor than Rh(2). Veratridine- (sodium channel-) dependent release of the neurotransmitters L-glutamate and GABA was almost fully inhibited by 20(S)-protopanaxadiol, however, less complete inhibition was observed with Rh(2). At its maximum inhibitory concentration, Rh(2) also produced release of l-glutamate and GABA from synaptosomes, in contrast to 20(S)-protopanaxadiol. We conclude that low to moderate micromolar concentrations of Rh(2) and 20(S)-protopanaxadiol inhibit sodium channel function and sodium channel-activated release of neurotransmitters. Apparently the ginsenoside Rh(2) cannot achieve complete inhibition of sodium channel-activated transmitter release because at high concentrations it also stimulates release.

Topics: Animals; Brain; Dose-Response Relationship, Drug; gamma-Aminobutyric Acid; Ginsenosides; Glutamic Acid; In Vitro Techniques; Male; Membrane Potentials; Mice; Sapogenins; Sodium Channel Blockers; Sodium Channels; Synaptosomes; Tetrodotoxin; Triterpenes; Veratridine

Angiogenesis is a critical step in tumor progression and involves several steps including endothelial cell (EC) proliferation, migration, and matrix remodeling. We investigated the antiangiogenic effects of 20( S)-protopanaxadiol ( 1) and 20( S)-protopanaxatriol ( 2), the sapogenins of two major ginseng saponins, in an angiogenesis model of human umbilical vein endothelial cells (HUVECs). These compounds inhibited the proliferative activity of HUVECs in a dose-dependent manner and have potential as anticancer drug candidates. In addition, we report the complete and unambiguous assignment of (1)H NMR spectra of 1 and 2, based on analyses of 2D NMR spectra including COSY, NOESY, HSQC, and HMBC. This report is the first to completely assign the (1)H NMR signals of 2, together with correction of data for 1 from prior reports.

Topics: Angiogenesis Inhibitors; Dose-Response Relationship, Drug; Humans; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Sapogenins; Stereoisomerism; Triterpenes; Umbilical Veins

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

Protopanaxadiol saponins (Rg3, Rd, Rc, Rb1 and Rb2) and protopanaxatriol saponins (Rg1, Re and Rg2) isolated from the root of Panax ginseng C.A. Meyer were evaluated for their adjuvant effects on the immune responses to ovalbumin (OVA) in mice. BALB/c mice were subcutaneously injected twice at a 3-week interval with 10 microg of ovalbumin or 10 microg of OVA plus 50 microg of ginsenosides Rg3, Rd, Rc, Rb1, Rb2, Rg1, Re or Rg2 or Quil A (n=5). Blood samples were collected for measuring specific total-IgG, IgG1 and IgG2a, and splenocytes were harvested for determining lymphocyte proliferation as well as IFN-gamma and IL-5 production 2 weeks after the boosting. The results indicated that OVA-specific antibody responses were significantly higher in mice immunized with OVA co-administered with Rg1, Re, Rg2, Rg3 and Rb1 but not with Rd, Rc and Rb2 when compared with the control (immunized with OVA only). Significantly enhanced splenocyte proliferative responses to Con A, LPS and OVA as well as the production of both IL-5 and IFN-gamma stimulated by OVA were also detected in mice immunized with OVA co-administered with Rg1 but not with Rb1, Re and Rg3. Of the ginsenosides studied, Rg1, Re, Rg2, Rg3 and Rb1 have more potent adjuvant properties than the others, indicating that they are the major constituents contributing to the adjuvant activities of total ginseng saponins. Varieties of ginsenosides in adjuvant activity might be attributed to the varieties of molecular conformations determined by the side sugar chains attaching to their dammarane skeleton.

Topics: Adjuvants, Immunologic; Animals; Female; Ginsenosides; Immunoglobulin G; Interferon-gamma; Interleukin-5; Mice; Mice, Inbred BALB C; Ovalbumin; Panax; Sapogenins; Spleen; Triterpenes

20S-Protopanaxadiol (1) is an aglycon metabolic derivative of the protopanaxadiol-type ginseng saponins. In the present study, 1 was used to induce cytotoxicity for two human glioma cell lines, SF188 and U87MG. For the SF188 cells, 1 activated caspases-3, -8, -7, and -9 within 3 h and induced rapid apoptosis, which could be partially inhibited by a general caspase blocker and completely abolished when the caspase blocker was used in combination with an antioxidant. Compound 1 also induced cell death in U87MG cells but did not activate any caspases in these cells. Monodansylcadaverine staining showed that 1 induced dramatic autophagy in both cell lines. Elevated levels of superoxide anion in both cells and reduced levels of phosphorylated Akt in U87MG cells were also demonstrated. These results showed that 20S-protopanaxadiol (1) induces different forms of programmed cell death, including both typical apoptosis and autophagy through both caspase-dependent and -independent mechanisms.

Topics: Caspases; Cell Death; Glioma; Humans; Molecular Structure; Sapogenins; Triterpenes

In this paper, the new type ginsenosidase which hydrolyzing multi-glycosides of ginsenoside, named ginsenoside type I from Aspergillus sp.g48p strain was isolated, characterized and generally described. The enzyme molecular weight was about 80 kDa. Ginsenosidase type I can hydrolyze different glycoside of protopanaxadiol type ginsenosides (PPD); i.e., can hydrolyze the 3(carbon)-O-beta-glucoside of Rb1, Rb2, Rb3, Rc, Rd; can hydrolyze 20(carbon)-O-beta-glucoside of Rb1, 20-O-beta-xyloside of Rb3, 20-O-alpha-arabinoside(p) of Rb2 and 20-O-alpha-arabinoside(f) of Rc to produce mainly F2, compound-K (C-K) and small Rh2, but can not hydrolyze the glycosides of protopanaxatriol type ginsenoside (PPT) such as Re, Rf, Rg1. So, when the ginsenosidase type I hydrolyzed ginsenosides, the enzyme selected ginsenoside-aglycone type, can hydrolyze different glycosides of PPD type ginsenoside; however no selected glycoside type, can hydrolyze multi-glycosides of PPD type ginsenosides. These properties were novel properties, and differentiated with the other previously described glycosidases.

Topics: Aspergillus fumigatus; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Ginsenosides; Glycoside Hydrolases; Hydrogen-Ion Concentration; Hydrolysis; Molecular Weight; Plants, Medicinal; Sapogenins; Temperature; Triterpenes

20S-protopanaxadiol (aPPD) is a major gastrointestinal metabolic product of ginsenosides. The latter share structural similarity with steroids and are the main pharmacologically active component in ginseng.. The authors investigated the interaction between aPPD and estrogen receptors (ER) in human breast adenocarcinoma MCF-7 cells through receptor binding assay, ER-induced gene expression, and cell proliferation both in vitro and in vivo.. aPPD, but not its close analog ginsenosides, competed with the [(3)H]-17-beta estradiol (E2) for ER with IC(50) at 26.3 microM. aPPD alone weakly induced luciferase reporter-gene expression controlled by an estrogen-regulated element, which was completely blocked by tamoxifen. aPPD alone, or in synergy with tamoxifen, blocked E2-induced transcriptional activation. aPPD also inhibited colony formation of endometrial cancer cells. aPPD potently inhibited estrogen-stimulated MCF-7 cell proliferation and synergistically enhanced the cytotoxicity of tamoxifen on both ER+ MCF-7 and ER- MDA-MB231 cells. Furthermore, aPPD, but not tamoxifen, inhibited Akt phosphorylation. Growth of MCF-7 xenograft tumor supplemented with E2 was completely inhibited in animals treated with aPPD, tamoxifen, or aPPD plus tamoxifen.. These results suggested that aPPD inhibits estrogen-stimulated gene expression and cell proliferation in ER-positive breast cancer cells. In addition, aPPD synergistically enhances cytotoxicity of tamoxifen in an ER-independent fashion, probably by down-regulating Akt activity.

Topics: Adenocarcinoma; Animals; Blotting, Western; Breast Neoplasms; Cell Proliferation; Drug Synergism; Estrogen Antagonists; Female; Humans; Mice; Mice, SCID; Panax; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Sapogenins; Tamoxifen; Transplantation, Heterologous; Triterpenes; Tumor Cells, Cultured

The hydrolysis of protopanaxadiol-type saponin mixture by various glycoside hydrolases was examined. Among these enzymes, crude preparations of lactase from Aspergillus oryzae, beta-galactosidase from A. oryzae, and cellulase from Trichoderma viride were found to produce ginsenoside F(2) [3-O-(beta-D-glucopyranosyl)-20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol], compound K [20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol], and ginsenoside Rd {3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl]-20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol}, respectively, from protopanaxadiol-type saponin mixture in large quantities. Moreover, the crude preparation of lactase from Penicillium sp. having a high producing activity of ginsenoside Rh(1) (6-O-beta-D-glucopyranosyl-20(S)-protopanaxatriol) from protopanaxatriol-type saponin mixture gave ginsenoside Rd as a main product, ginsenoside Rg(3) {3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, and compound K from protopanaxadiol-type saponin mixture. The hydrolytic pathways of ginsenosides Rb(1), Rb(2), and Rc to ginsenosides Rd, Rg(3), and F(2), and compound K by crude preparations of four glycoside hydrolases were also studied. This is the first report on the enzymatic preparation of an intestinal bacterial metabolite, ginsenoside F(2), in quantity, and a considerable amount of a minor saponin, ginsenoside Rg(3), from a protopanaxadiol-type saponin mixture.

Topics: Aspergillus oryzae; beta-Galactosidase; Cellulase; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Ginsenosides; Glycoside Hydrolases; Lactase; Plants, Medicinal; Sapogenins; Saponins; Substrate Specificity; Trichoderma; Triterpenes

We previously showed that 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (M1), a metabolite of protopanaxadiol-type ginseng saponins by intestinal bacteria had axonal extension activity in degenerated neurons, and improved memory disorder and synaptic loss induced by an active fragment of amyloid beta, Abeta(25-35). It is unknown how M1 shows these effects in neurons. To clarify the signal transduction mechanism of M1-induced axonal extension, phosphorylated proteins by M1 stimulation were identified because most cellular signal pathways are regulated by phosphorylation/dephosphorylation. The combination of immunoprecipitation and MALDI-TOF-MS revealed that teneurin-2 and mPar3 were specifically phosphorylated by M1 stimulation. Because mPar3 is known as an axonal specifying molecule and to be regulated by phosphatidylinositol 3-kinase (PI3-kinase), the involvement of teneurin-2 and PI3-kinase in the M1 signal was studied. In teneurin-2-deficient cortical neurons, M1-induced axonal extension and PI3-kinase activation were significantly inhibited. In addition, treatment with PI3-kinase inhibitor also reduced M1-induced axonal extension. These results suggest that M1 induces axonal outgrowth through the teneurin-2-PI3-kinase cascade.

Topics: Amyloid beta-Peptides; Animals; Cells, Cultured; Cerebral Cortex; Chromones; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Immunohistochemistry; Immunoprecipitation; Membrane Proteins; Microtubule-Associated Proteins; Morpholines; Neurites; Neurons; Peptides; Phosphatidylinositol 3-Kinases; Phosphorylation; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sapogenins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transfection; Triterpenes

A [(3)H]batrachotoxinin A-20alpha-benzoate ([(3)H]BTX-B) binding assay was used to investigate the interaction of two ginseng aglycones (20(S)protopanaxadiol and 20(S)protopanaxatriol) and Rh(2) (a monoglucoside of 20(S)protopanaxadiol) with voltage-gated sodium channels in mouse brain. All compounds inhibited the binding of [(3)H]BTX-B and IC(50)s were established at 42 microM (20(S)protopanaxadiol), 79 microM (20(S)protopanaxatriol) and 162 microM (Rh(2)). Scatchard analysis confirmed that 20(S)protopanaxadiol and Rh-2 reduced the B(max) of [(3)H]BTX-B binding while Rh(2) also increased the K(d). At IC(50) concentrations and above, 20(S)protopanaxadiol and Rh(2) increased the dissociation of the [(3)H]BTX-B:sodium channel complex above that produced by a saturating concentration of veratridine, but failed to reduce the rate of association of [(3)H]BTX-B with sodium channels. Reversal of the inhibition of [(3)H]BTX-B binding by 20(S)protopanaxadiol and Rh(2) occurred slowly. We conclude that the 20(S)protopanaxadiol and the less potent inhibitor Rh(2) destabilize BTX-B-activated sodium channels through non-covalent allosteric modification of neurotoxin binding site 2.

Topics: Animals; Batrachotoxins; Binding, Competitive; Cell Membrane; Cerebral Cortex; Dose-Response Relationship, Drug; Ginsenosides; Kinetics; Male; Mice; Molecular Structure; Neurons; Panax; Plant Extracts; Radioligand Assay; Sapogenins; Sodium Channels; Solubility; Synaptosomes; Triterpenes; Tritium

There is still an argument about ginseng-prescription drug interactions. To evaluate the influence on cytochrome P450 (P450) activities of ginseng in the present study, the influence on P450 activities of naturally occurring ginsenosides and their degradation products in human gut lumen was assayed by using human liver microsomes and cDNA-expressed CYP3A4. The results showed that the naturally occurring ginsenosides exhibited no inhibition or weak inhibition against human CYP3A4, CYP2D6, CYP2C9, CYP2A6, or CYP1A2 activities; however, their main intestinal metabolites demonstrated a wide range of inhibition of the P450-mediated metabolism. There was no mechanism-based inhibition found on these P450 isoforms. It is noteworthy that Compound K, protopanaxadiol (Ppd), and protopanaxatriol (Ppt) all exhibited moderate inhibition against CYP2C9 activity, and Ppd and Ppt also exhibited potent competitive inhibition against CYP3A4 activity. We suggest that after oral administration, naturally occurring ginsenosides might influence hepatic P450 activity in vivo via their intestinal metabolites.

Topics: Animals; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Ginsenosides; Humans; Intestinal Mucosa; Microsomes, Liver; Models, Biological; Rats; Sapogenins; Triterpenes

Ginsenoside 20(S)-protopanaxadiol, one of metabolites of ginseng saponins, has been well characterized to possess the pleiotropic anticancer capabilities in several cancer cell lines. The object of this study was to investigate the effects of ginsenoside 20(S)-protopanaxadiol on the invasion in vitro and the expression of matrix metalloproteinase-2 in human fibrosarcoma HT1080 cells in absence of cytotoxicity. Our results showed that ginsenoside 20(S)-protopanaxadiol exerted a concentration-dependent inhibitory effect on the proliferation of HT1080 cells (IC50 was 76.78+/-2.24 microM, 48 hr). Treatment with 20(S)-protopanaxadiol significantly declined the invasive capacity of HT1080 cells compared to the control cells (P<0.01) in the in vitro invasion assay. Further analysis with gelatin zymography and western blotting revealed that both the activity and the expression of matrix metalloproteinase-2 decreased dramatically in a concentration-dependent manner (P<0.01). Taken together, these results indicated that ginsenoside 20(S)-protopanaxadiol is able to inhibit the invasiveness of HT1080 cells significantly in vitro and this action may be primarily due to down-regulating the expression of matrix metalloproteinase-2. Ginsenoside 20(S)-protopanaxadiol, a metabolite of ginseng, may be applied as a potential therapeutic agent in the prevention and treatment of cancer.

Topics: Antineoplastic Agents, Phytogenic; Cell Death; Cell Proliferation; Fibrosarcoma; Ginsenosides; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Sapogenins; Triterpenes; Tumor Cells, Cultured

The uptake and metabolism profiles of ginsenoside Rh2 and its aglycon protopanaxadiol (ppd) were studied in the human epithelial Caco-2 cell line. High-performance liquid chromatography-mass spectrometry was applied to determine Rh2 and its aglycon ppd concentration in the cells at different pH, temperature, concentration levels and in the presence or absence of inhibitors. Rh2 uptake was time and concentration dependent, and its uptake rates were reduced by metabolic inhibitors and influenced by low temperature, thus indicating that the absorption process was energy-dependent. Drug uptake was maximal when the extracellular pH was 7.0 for Rh2 and 8.0 for ppd. Rh2 kinetic analysis showed that a non-saturable component (Kd 0.17 nmol x h(-1) x mg(-1) protein) and an active transport system with a Km of 3.95 micromol x l(-1) and a Vmax of 4.78 nmol x h(-1) x mg(-1)protein were responsible for the drug uptake. Kinetic analysis of ppd showed a non-saturable component (Kd 0.78 nmol x h(-1) x mg(-1) protein). It was suggested that active extrusion of P-glycoprotein and drug degradation in the intestine may influence Rh2 bioavailability.

Topics: Caco-2 Cells; Ginsenosides; Humans; Sapogenins; Triterpenes

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of 20(R)-ginsenoside Rg3 in dog. The plasma samples were pretreated by liquid-liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. Dioscin was used as the internal standard. The method had a lower limit of quantitation of 0.5 ng/mL for Rg3 in 200 microL of plasma or 2 ng/mL in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of Rg3 in plasma. The intra- and inter-day precisions were measured to be below 8% and accuracy between -1.5 and 1.4% for all quality control samples. This quantitation method was successfully applied to pharmacokinetic studies of Rg3 after both an oral and an intravenous administration to beagle dogs. No Rh2 and protopanaxadiol were detected in plasma.

Topics: Administration, Oral; Animals; Blood Chemical Analysis; Chromatography, Liquid; Dogs; Female; Ginsenosides; Injections, Intra-Arterial; Male; Mass Spectrometry; Metabolic Clearance Rate; Reproducibility of Results; Sapogenins; Sensitivity and Specificity; Triterpenes

To support pharmacokinetic studies of ginsenosides, a novel method to quantitatively analyze ginsenoside Rg3 (Rg3), its prosapogenin ginsenoside Rh2 (Rh2) and aglycone 20(S)-protopanaxadiol (ppd) in rat plasma was developed and validated. The method was based on gradient separation of ginsenosides present in rat plasma using high performance liquid chromatography (HPLC), followed by detection with electrospray ionization(ESI) mass spectrometry (MS) in negative ion mode with the mobile phase additive, ammonium chloride (500 microM). Differentiation of ginsenosides was achieved through simultaneous detection of the [M(+)Cl(-)] adduct of ginsenoside Rg3 and [M(+)Cl(-)] adducts of its deglycosylated metabolites Rh2 and ppd, and other ions after solid phase extraction (SPE). The /specific ions monitored were m/z 819.50 for Rg3, m/z 657.35 for Rh2, m/z 495.40 for ppd and m/z 799.55 for the internal standard (digitoxin). The mean recoveries for Rg3, Rh2 and ppd were 77.85, 82.65 and 98.33%, respectively using 0.1 ml plasma for extraction. The lower limits of quantification were 10.0, 2.0 and 8.0 ng/ml (equivalent to 0.1, 0.02 and 0.08 ng in each 10 microl injection onto the HPLC column) for Rg3, Rh2 and ppd, respectively. The method has been demonstrated to be highly sensitive and accurate for the determination of Rg3 and its metabolites in rat plasma.

Topics: Animals; Chromatography, High Pressure Liquid; Drug Stability; Freezing; Ginsenosides; Rats; Rats, Sprague-Dawley; Sapogenins; Spectrometry, Mass, Electrospray Ionization; Triterpenes

We showed recently that ginsenosides inhibit the activity of various types of ion channel. Here we have investigated the role of the carbohydrate component of ginsenoside Rg3 in the inhibition of Na+ channels. The channels were expressed in Xenopus oocytes by injecting cRNAs encoding rat brain Nav1.2 alpha and beta1 subunits, and analyzed by the two-electrode voltage clamp technique. Treatment with Rg3 reversibly inhibited the inward Na+ peak current (INa) with an IC50 of 32.2 +/- 4.5 microM, and the inhibition was voltage-dependent. To examine the role of the sugar moiety, we prepared a straight chain form of the second glucose and a conjugate of this glucose with 3-(4-hydroxyphenyl) propionic acid hydrazide (HPPH). Neither derivative inhibited INa. Treatment with the carbohydrate portion of ginsenoside Rg3, sophorose [beta-D-glucopyranosyl (1-->2)- beta-glucopyranoside], or the aglycone (protopanaxadiol), on their own or in combination had no effect on INa. These observations indicate that the carbohydrate portion of ginsenoside Rg3 plays an important role in its effect on the Na+ channel.

Topics: Animals; Ginsenosides; Glucans; NAV1.2 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Neurons; Oocytes; Patch-Clamp Techniques; Sapogenins; Sodium Channels; Structure-Activity Relationship; Triterpenes; Xenopus

20( S)-Protopanaxadiol (PPD) is one of the metabolites of ginsenosides from Panax ginseng. In this study, we demonstrate that PPD inhibits the increase in lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression through inactivation of nuclear factor-kappaB by preventing degradation of inhibitory factor-kappaBalpha. PPD also induces heme oxygenase 1 (HO-1) expression in RAW 264.7 cells, at the mRNA and protein levels, in the presence and absence of LPS. This effect is associated with suppression of LPS-induced nitric oxide (NO) production and iNOS expression. The HO-1 inducer hemin is associated with the suppression of LPS-induced NO production in a dose-dependent manner, and the HO-1 inhibitor tin protoporphyrin attenuates the inhibitory activity of PPD on LPS-induced NO production. These results provide evidence for the role of HO-1 in the inhibition of LPS-induced NO production by PPD.

Topics: Anti-Inflammatory Agents; Cell Line; Enzyme Induction; Heme Oxygenase-1; Lipopolysaccharides; Macrophages; Nitric Oxide; Sapogenins; Triterpenes

To study the alkaline-degradation products of ginsenosides from leaves and stems of Panax quinquefolium L.. Isolation and purification were carried out on silica gel and HPLC; the structures of chemical constituents were elucidated by spectral analysis.. From the alkaline-degradation products, nine compounds were identified as: 20 (S) -protopanaxadiol (I), 20 (S) -dammar-25 (26)-ene-3beta, 12beta, 20-triol (II), 24 (R) -ocotillol (III), 20 (S) -protopanaxatriol (IV), 20 (S) -dammar-25 (26)-ene-3beta, 6alpha, 12beta, 20-tetrol (V), dammar-20 (21), 24-diene-3beta, 12beta-diol (VI), dammar-20(21), 24-diene-3beta, 6alpha, 12beta-triol (VII), 20 (S), 24 (S) -dammar-25 (26) -ene-3beta, 6alpha, 12beta, 20, 24-pentanol (VIII), 20 (S) -dammar-23-ene-25-hydroperoxyl-3beta, 6alpha, 12beta, 20-tetrol (IX).. The configuration of C20 position of ginsenosides was not changed by alkaline-degradation. The complete assignments of 1H and 13C NMR chemical shifts of four new compounds V, VII, VIII, IX, were acquired by means of 2D NMR spectra. Compound I showed antitumor effect on human colon carcinoma cells in vitro.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Ginsenosides; Humans; Molecular Conformation; Molecular Structure; Panax; Plant Leaves; Plant Stems; Plants, Medicinal; Sapogenins; Triterpenes

We previously screened neurite outgrowth activities of several Ginseng drugs in human neuroblastoma, and demonstrated that protopanaxadiol (ppd)-type saponins were active constituents. Since ppd-type saponins are known to be completely metabolized to 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (M1) by intestinal bacteria when taken orally, M1 and ginsenoside Rb1, as a representative of ppd-type saponins, were examined for cognitive disorder. In a mouse model of Alzheimer's disease (AD) by Abeta(25-35) i.c.v. injection, impaired spatial memory was recovered by p.o. administration of ginsenoside Rb1 or M1. Although the expression levels of phosphorylated NF-H and synaptophysin were reduced in the cerebral cortex and the hippocampus of Abeta(25-35)-injected mice, their levels in ginsenoside Rb1- and M1-treated mice were almost completely recovered up to control levels. Potencies of the effects were not different between ginsenoside Rb1 and M1 when given orally, suggesting that most of the ginsenoside Rb1 may be metabolized to M1, and M1 is an active principal of ppd-type saponins for the memory improvement. In cultured rat cortical neurons, M1 showed extension activity of axons, but not dendrites. The axon-specific outgrowth was seen even when neuritic atrophy had already progressed in response to administration of Abeta(25-35) as well as in the normal condition. These results suggest that M1 has axonal extension activity in degenerated neurons, and improve memory disorder and synaptic loss induced by Abeta(25-35). M1 was shown to be effective in vitro and in vivo, indicating that Ginseng drugs containing ppd-type saponins may reactivate neuronal function in AD by p.o. administration.

Topics: Amyloid beta-Peptides; Animals; Atrophy; Axons; Female; Ginsenosides; Image Processing, Computer-Assisted; Immunohistochemistry; Male; Maze Learning; Memory Disorders; Mice; Motor Activity; Neurites; Peptide Fragments; Pregnancy; Rats; Rats, Sprague-Dawley; Sapogenins; Saponins; Synapses; Triterpenes

Ginsenosides derived from 20(S)-protopanaxatriol (PT) and 20(S)-protopanaxadiol (PD) groups had similar characteristic cytotoxic effects on the growth of two intestinal cells lines, Int-407 and Caco-2. Pure Rh2, a ginsenoside structurally related to PD, inhibited intestinal cell growth at greater than twice the concentration of PD, while Rh1, a ginsenoside structurally related to aglycone PT, had no cytotoxic effect. Concentrations causing growth inhibition of 50% of cells (LC50) for the compounds PD, PT, and Rh2 were 23, 26, and 53 microg/mL, respectively, for Int-407 cells. In comparison, the LC50 for PD and PT was determined to be 24 microg/mL, and that for Rh2 was 55 microg/mL in Caco-2 cells. A standardized North American ginseng extract with a known ginsenosides composition did not induce cytotoxicity in either of the intestinal cell lines. Cell cycle analysis showed characteristically different (P = 0.05) effects of ginsenosides PD, Rh2, and PT in both cell lines. Rh2 treatment of Int-407 caused a significantly (P = 0.05) higher production of sub-G1 (apoptotic) cells (35% +/- 1%) compared with untreated cells (14% +/- 0.3%) after 24 h. PD and Rh2 treatments were both significantly (P < 0.05) higher in apoptotic cells than in untreated cells after 48 and 72 h. Similar results were obtained for treatment of Caco-2 cells. Lactate dehydrogenase (LDH) activity in both cell lines was similar for PD and Rh2 and higher (P = 0.05) than for PT treatment at most time periods. These results show a specific structure-function relationship for bioactive ginsenosides in two contrasting intestinal cell types.

Topics: Apoptosis; Caco-2 Cells; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; G1 Phase; Ginsenosides; Humans; L-Lactate Dehydrogenase; Plant Roots; Sapogenins; Structure-Activity Relationship; Time Factors; Triterpenes; United States

Certain ginsenosides, also known as triterpene glycosides, have been recently reported to have a characteristic effect on cultured intestinal and leukemia cell growth. Ginsenoside aglycones 20(S)-protopanaxadiol (PD), 20(S)-protopanaxatriol (PT), and ginsenoside Rh2 have been identified as having a strong effect on reducing cell viability. Furthermore, ginsenoside Rh2 is thought to be a rare ginsenoside not found in all ginseng products. Rather, Rh2 has been recently reported to be a breakdown product of thermal processing of North American ginseng. In this study, pure ginsenosides PD, PT, Rh2 standards and an enriched Rh2 fraction derived from ginseng leaf were tested in cultured Caco-2 cells for relative cytotoxic potency. PD and Rh2 LC50 were similar after 24 to 72 h, whereas a drop in PT LC50 occurred later at 48 and 72 h. Furthermore, PD and Rh2 affected membrane integrity as indicated by LDH secretion earlier than PT and the enriched Rh2 fraction (P < or = 0.05). Ginsenoside Rh2 showed the greatest (P < or = 0.05) build up of necrotic cells (18.3 +/- 0.1%) at the respective LC50 after 24 h and PD (21.3 +/- 0.3%) showed the largest effect after 44 h of exposure. The effect on apoptotic cells at 44 h of treatment were significantly different (P < or = 0.05) for Rh2 (21 +/- 0.4%), PD (14.6 +/- 0.1%), enriched Rh2 leaf fraction (9.9 +/- 0.6%), and PT (2.3 +/- 0.1%) treatments. Caco-2 caspase-3 activity was different between ginsenoside exposure; Rh2 (10.6 +/- 0.3 nM pNA) had the greatest (P < or = 0.05) activity followed by the enriched Rh2 leaf fraction (8.3 +/- 0.2 nM pNA), PT (7.3 +/- 0.3 nM pNA). The PD (4.8 +/- 0.04 nM pNA) treatment was similar to untreated cells (4.3 +/- 0.05 nM pNA) in caspase-3 activity. These results show variable bioactive response in cultured intestinal cell to specific ginsenosides and an enriched Rh2 North American ginseng extract which may be explained on basis of hydrophobic/hydrophilic balance.

Topics: Annexin A5; Apoptosis; Caco-2 Cells; Caspases; Cell Fractionation; Cell Survival; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Ginsenosides; Humans; Isomerism; L-Lactate Dehydrogenase; Mass Spectrometry; Molecular Structure; Panax; Plant Extracts; Propidium; Sapogenins; Spectrometry, Mass, Electrospray Ionization; Time Factors; Triterpenes

The intestinal bacterial metabolites of ginsenosides are responsible for the main pharmacological activities of ginseng. The purpose of this study was to find whether these metabolites influence hepatic metabolic enzymes and to predict the potential for ginseng-prescription drug interactions. Utilizing the probe reaction of CYP3A activity, testosterone 6beta-hydroxylation, the effects of derivatives of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol families on CYP3A activity in rat liver microsomes were assayed. Our results showed that ginsenosides from the 20(S)-protopanaxadiol and 20(S)-protopanaxatriol family including Rb1, Rb2, Rc, Compound-K, Re, and Rg1 had no inhibitory effect, whereas Rg2, 20(S)-panaxatriol and 20(S)-protopanaxatriol exhibited competitive inhibitory activity against CYP3A activity in these microsomes with the inhibition constants (Ki) of 86.4+/-0.8 microM, 1.7+/-0.1 microM, and 3.2+/-0.2 microM, respectively. This finding demonstrates that differences in their chemical structure might influence the effects of ginsenosides on CYP3A activity and that ginseng-derived products might have potential for significant ginseng-drug interactions.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Bacteria; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP3A; Drugs, Chinese Herbal; Ginsenosides; Hydroxylation; Hydroxytestosterones; In Vitro Techniques; Intestinal Mucosa; Intestines; Male; Microsomes, Liver; Oxidoreductases, N-Demethylating; Rats; Rats, Sprague-Dawley; Sapogenins; Triterpenes

Even though the degradation of ginsenosides has been thoroughly studied in animals and in vitro using acids, enzymes, and intestinal bacteria, knowledge concerning the systemic availability of ginsenosides and their degradation products in humans is generally lacking. Therefore, the attention in this article is focused on the identification of ginsenosides and their hydrolysis products reaching the systemic circulation in man. This is of great importance in understanding clinical effects, preventing herb-drug interactions, and optimizing the biopharmaceutical properties of ginseng preparations. Using a sensitive mass spectrometric method, which is specific for the identification of ginsenosides in complex biological matrices, the degradation pathway of ginsenosides in the gastrointestinal tract of humans could be elucidated following the oral administration of ginseng. Within the frame of a pilot study, human plasma and urine samples of two subjects were screened for ginsenosides and their possible degradation products. In general, the urine data coincided well with the plasma data. In both volunteers the same hydrolysis products, which are not originally present in the Ginsana extract (Pharmaton S.A., Lugano, Switzerland) ingested, were identified in plasma and urine. It was shown that two hydrolysis products of the protopanaxatriol ginsenosides, namely G-Rh1 and G-F1 may reach the systemic circulation. In addition, compound-K, the main intestinal bacterial metabolite of the protopanaxadiol ginsenosides, was detected in plasma and urine. These products are probably responsible for the action of ginseng in humans. In opposition to previous reports, G-Rb1 was identified in plasma and urine of one subject.

Topics: Administration, Oral; Biotransformation; Capsules; Ginsenosides; Humans; Mass Spectrometry; Sapogenins; Triterpenes

Oxidative stress has been implicated as a primary cause of renal failure in certain renal diseases. Indeed, renal proximal tubule is a very sensitive site to oxidative stress and retains functionally fully characterized transporters. It has been reported that ginsenosides have a beneficial effect on diverse diseases including oxidative stress. However, the protective effect of ginsenosides on oxidative stress has not been elucidated in renal proximal tubule cells. Thus, we examined the effect of ginsenosides on oxidative stress-induced alteration of apical transporters and its related mechanism in renal proximal tubule cells. In the present study, hydrogen peroxide (H(2)O(2)) (>10(-5) M) inhibited alpha-methyl-D-glucopyranoside uptake in a dose-dependent manner (p < 0.05). It also inhibited Pi and Na(+) uptake. At a concentration of 20 microg/ml, total ginsenosides significantly reduced H(2)O(2)-induced inhibition of apical transporters. In contrast, protopanaxadiol (PD) and protopanaxatriol (PT) saponins exhibited a less preventive effect than total ginsenosides (p < 0.05). Furthermore, we examined its action mechanism. H(2)O(2) increased lipid peroxide formation, arachidonic acid (AA) release, and Ca(2+) uptake. These effects on H(2)O(2) were significantly prevented by total ginsenosides and PD or PT sanponins. However, total ginsenosides appear to be more protective than PD and PT saponins (p < 0.05). In conclusion, ginsenosides prevented H(2)O(2)-induced inhibition of apical transporters via a decrease in oxidative stress, AA release, and Ca(2+) uptake in primary cultured renal proximal tubule cells.

Topics: Animals; Arachidonic Acid; Calcium; Dose-Response Relationship, Drug; Ginsenosides; Hydrogen Peroxide; Kidney Tubules, Proximal; Lipid Peroxides; Male; Membrane Transport Proteins; Methylglucosides; Oxidative Stress; Phosphorus; Rabbits; Sapogenins; Sodium; Triterpenes

Extension of axons and dendrites in neurons may compensate for and repair damaged neuronal networks in the dementia brain. To find out drugs capable of regenerating the neuronal network, we focused on several herbal drugs belonging to the genus Panax, kinds of Ginseng, and investigated neurite outgrowth activity of their extracts and compounds. We found that the methanol extracts of Ginseng (root of P. ginseng), Notoginseng (root of P. notoginseng) and Ye-Sanchi in Chinese (rhizome of a relative to P. vietnamensis) increased neurite outgrowth in SK-N-SH cells. The protopanaxadiol-type saponins, ginsenosides Rb(1) and Rb(3), and notoginsenosides R(4) and Fa isolated from Ye-Sanchi extract extended neurites, while protopanaxatriol-, ocotillol- and oleanane-type saponins had no effect on the neurite outgrowth. The percentage of cells with multipolar neurites and number of varicosities were intensely high in cells treated with the methanol extract of Ye-Sanchi as well as ginsenosides Rb(1) and Rb(3), and notoginsenosides R(4) and Fa. Both phosphorylated NF-H-expressing neurites and MAP2-expressing ones were extended by treatment with those saponins and the extract. Especially, longer neurites were mainly positive for phosphorylated NF-H. These results suggest that protopanaxadiol-type saponins enhance axonal and dendritic formation activity.

Topics: Axons; Dendrites; Drugs, Chinese Herbal; Humans; Immunohistochemistry; Methanol; Neurites; Panax; Rhizome; Sapogenins; Saponins; Triterpenes; Tumor Cells, Cultured

The aim of the present study was to characterize the endothelium-dependent relaxation elicited by ginsenosides, a mixture of saponin extracted from Panax ginseng, in isolated rat aorta. Relaxations elicited by ginsenosides were mimicked by ginsenoside Rg1 and ginsenoside Rg1, two major ginsenosides of the protopanaxatriol group. Ginsenoside Rg3 was about 100-fold more potent than ginsenoside Rg1. The endothelium-dependent relaxation in response to ginsenoside Rg3 was associated with the formation of cycle GMP. These effects were abolished by N(G)-nitro-L-arginine and methylene blue. Relaxations in response to ginsenoside Rg3 were unaffected by atropine, diphenhydramine, [D-Pro2, D-Trp7,9]substance P, propranolol, nifedipine, verapamil and glibenclamide but were markedly reduced by tetraethylammonium. Tetraethylammonium modestly reduced the relaxation induced by sodium nitroprusside. These findings indicate that ginsenoside Rg3 is a major mediator of the endothelium-dependent nitric oxide-mediated relaxation in response to ginsenosides in isolated rat aorta, possibly via activation of tetraethylammonium-sensitive K+ channels.

Topics: Adrenergic beta-Antagonists; Animals; Antineoplastic Agents, Phytogenic; Aorta, Thoracic; Atropine; Calcium Channel Blockers; Chromatography, High Pressure Liquid; Diphenhydramine; Dose-Response Relationship, Drug; Endothelium, Vascular; Ginsenosides; Glyburide; Histamine H1 Antagonists; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Relaxation; Nitroprusside; Potassium Channel Blockers; Potassium Channels; Propranolol; Rats; Rats, Sprague-Dawley; Sapogenins; Saponins; Tetraethylammonium; Triterpenes; Vasodilator Agents; Vasomotor System

The effects of two ginseng saponins having a different ratio of protopanaxadiol (PD) and protopanaxatriol saponins (PT) on the learning impairment induced by scopolamine, and learning and memory in mice were investigated in a passive avoidance task and a Morris water maze task. The ratio of PD and PT was 1.24 and 1.46, respectively. Before training, the ginseng saponins were administered intraperitoneally at doses of 50 and 100 mg/kg. The two saponins improved the scopolamine-induced learning impairment at different dosages in mice, 50 and 100 mg/kg, respectively. However, the two saponins did not show a favorable effect on learning and memory in normal mice. Korean red ginseng saponin with a low PD/PT ratio had an improving effect on spatial working memory, but the saponin with a high PD/PT ratio did not. This finding suggests that the PD/PT ratio of the ginseng saponins may be an important factor in the pharmacological role of red ginseng as a medicinal herb.

Topics: Animals; Avoidance Learning; Drugs, Chinese Herbal; Injections, Intraperitoneal; Learning Disabilities; Male; Maze Learning; Memory, Short-Term; Mice; Mice, Inbred ICR; Muscarinic Antagonists; Panax; Plants, Medicinal; Sapogenins; Saponins; Scopolamine; Space Perception; Triterpenes

An improved gas chromatographic-mass spectrometric method (GC-MS) with a fast solid-phase extraction on a newly introduced C18 microcolumn, was applied to study the urinary excretion 20(S)-protopanaxadiol and 20(S)-protopanaxatriol glycosides in man after oral administration of ginseng preparations. Using panaxatriol as internal standard, 20(S)-protopanaxadiol and 20(S)-protopanaxatriol (the aglycones of ginsenosides) could be determined at a detection level of a few ng per ml urine by GC-MS with selected-ion monitoring after their release from glycosides which occur in urine. The extraction recovery of ginsenosides from urine was more than 80% and the intra-assay coefficient of variation was less than 5.0%. The results after intake of single doses of ginseng preparations demonstrated a linear relation between the amounts of ginsenosides consumed and the 20(S)-protopanaxatriol glycosides excreted in urine. About 1.2% of the dose was recovered in five days.

Topics: Gas Chromatography-Mass Spectrometry; Ginsenosides; Humans; Panax; Plants, Medicinal; Sapogenins; Saponins; Triterpenes

Antitumor and cytotoxic activity of monoglucosides such as 3-0-panaxadiol (1), 12-0-panaxadiol (2) and 20-0-panaxadiol (3) and 3-0-betulafolientriol (4), 12-0-betulafolientriol (5) and 20-0-betulafolientriol (6) was studied. It was found that in concentrations of 10 to 50 micrograms/ml the above monosides induced marked impairment of the selective permeability of the tumor cells and the inhibition of the labeled precursor inclusion into the macromolecule biosynthesis. Administration of the monosides in a single dose of 100 mg/kg 24 hours after the inoculation of the Ehrlich tumor cells resulted in prolongation of the mean life-span of the mice by 144 per cent (1), 153 per cent (2), 144 per cent (3), 125 per cent (4), 133 per cent (5) and 178 per cent (6). A significant reduction of the tumor mass was observed at the early stages of the tumor development and later the tumor progress intensively resumed. The tests for the effect of the monoside-activated macrophages on the growth of the tumor cells showed that production of the growth factors by the macrophages was stable and had a negative action on the efficacy of the chemotherapy with the monoglucosides.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Drugs, Chinese Herbal; Glycosides; Macrophages; Mice; Molecular Structure; Sapogenins; Triterpenes; Tumor Cells, Cultured

To facilitate studies on the possible presence of ginseng products in serum, tissues, and excretions, a procedure to optimize the analysis of the ginseng specific products, i.e., ginsenosides, had to be worked out. With the present method the two sapogenins, 20S-protopanaxadiol and 20S-protopanaxatriol, can be produced from ginsenosides Rb1, Rc, Rd, Re, and Rg1 in 80% yield by using an improved alkaline cleavage procedure. In contrast to previously described acid hydrolysis procedures for ginsenosides, our alkaline conditions caused no epimerization, no hydroxylation, and no cyclization of the side chain. Furthermore, no unchanged ginsenosides were recovered. The products of alkaline and acidic cleavage were separated, identified, and characterized by GC, GC-MS, and HPLC. In contrast to alkaline cleavage, treatment with acid afforded a number of side products. The C-20S-epimers of the ginseng sapogenins could be distinguished from C-20R epimers by difference in mass spectra and retention time after trimethylsilylation.

Topics: Chromatography, Gas; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Ginsenosides; Hydrogen-Ion Concentration; Hydrolysis; Panax; Plants, Medicinal; Sapogenins; Saponins; Triterpenes

The uptake and metabolism of ginsenoside Rh2 (Rh2) by B16 melanoma cells were studied. In a medium containing 2% fetal calf serum, the uptake of Rh2 reached a maximum of 3 nmol/10(6) cells at 3-6 h after Rh2 (12.5 microM) was added, but gradually decreased to 0.8 nmol/10(6) cells. In these cells, protopanaxadiol (PPD), which is an aglycon of Rh2, increased inversely with the decrease in Rh2 as a result of deglycosylation by the cells. When PPD (8 microM) was added to the medium, the uptake reached a plateau of 2.4 nmol/10(6) cells, within 0.5 h. The association constant of Rh2 (1.74 +/- 1.08 x 10(6) M-1) for bovine serum albumin (BSA) was significantly higher than that of PPD (9.90 +/- 1.10 x 10(4) M-1). In a serum-free medium, both Rh2 and PPD were incorporated within 1.5 h. The uptake rate constant of Rh2 (1.20 +/- 0.20 h-1) was not significantly different from that of PPD (1.02 +/- 0.15 h-1), but the release rate constant of PPD (2.12 +/- 0.38 h-1) was significantly lower than that of Rh2 (3.03 +/- 0.57 h-1). These differences in affinity for BSA and the release rate constants were thought to be the cause of the difference in uptake kinetics between these drugs. The effects of Rh2 and PPD on the cells were identical, and there was no difference in the lag periods before the appearance of their effects, despite their differing rates of uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Cycle; Ginsenosides; Glycosylation; Melanoma, Experimental; Mice; Molecular Structure; Panax; Plants, Medicinal; Sapogenins; Saponins; Serum Albumin, Bovine; Triterpenes; Tumor Cells, Cultured

Two minor compounds isolated from the leaves of Panax ginseng C. A. Meyer were characterized as 20(R)-protopanaxadiol (I) and 3 beta, 6 alpha, 12 beta-trihydroxydammar-20 (22), 24-diene-6-O-alpha-L-rhamnosyl-(1----2)-beta-D-glucopyranoside (II) on the basis of spectral analysis and chemical evidences. I was isolated for the first time from the leaves; II was shown to be a new saponin and was named as ginsenoside-Rg4.

Topics: Chemical Phenomena; Chemistry; Ginsenosides; Panax; Plants, Medicinal; Sapogenins; Saponins; Triterpenes