salicylates and 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic-acid

A concise synthesis of cajaninstilbene acid was achieved in 7 steps from (E)-3,5-dimethoxystilbene in 8.6% overall yield via the Claisen rearrangement of an aryl reverse-prenyl ether as the key step. Cytotoxic activities against human pancreatic carcinoma PANC-1 cells of cajaninstilbene acid and amorfrutins A-D were also evaluated.

Topics: Cytotoxins; Humans; Pancreatic Neoplasms; Salicylates; Stilbenes

Topics: Binding Sites; Cajanus; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Models, Molecular; Molecular Docking Simulation; Neoplasms; Protein Binding; Protein Conformation; Protein Kinases; Protein Serine-Threonine Kinases; Salicylates; Stilbenes; Survival Analysis

Cajaninstilbene acid (CSA), an active stilbene isolated from the leaves of pigeon pea (

Topics: Animals; Caco-2 Cells; Cajanus; Humans; Intestinal Absorption; Rats; Salicylates; Stilbenes

The Staphylococcus aureus can switch to a transient genotype-invariant dormancy, known as a persister, to survive treatment with high doses of antibiotics. This transient persister is an important reason underlying its resistance. There is an urgent need to find new antibacterial agents capable of eradicating methicillin-resistant S. aureus (MRSA) persisters. In this study, 37 new derivatives of cajaninstilbene acid (CSA) were designed and synthesized, and their biological activity against MRSA persisters was evaluated. Most of the newly synthesized derivatives exhibit more potent antimicrobial properties against S. aureus and MRSA than CSA itself, and 23 of the 37 derivatives show a tendency to eradicate MRSA persisters. A representative compound (A6) was demonstrated to target bacterial cell membranes. It eradicated the adherent biofilm of MRSA in a concentration dependent manner, and showed a synergistic antibacterial effect with piperacilin. In a model mouse abscess caused by MRSA persisters, A6 effectively reduced the bacterial load in vivo. These results indicate that A6 is a potential candidate for treatment of MRSA persister infections.

Topics: Animals; Anti-Bacterial Agents; Biofilms; Cell Survival; Cell Wall; Disease Models, Animal; Methicillin-Resistant Staphylococcus aureus; Mice; Microbial Sensitivity Tests; RAW 264.7 Cells; Salicylates; Skin Diseases; Staphylococcal Infections; Staphylococcus aureus; Stilbenes; Structure-Activity Relationship

Cajaninstilbene acid (CSA), a bioactive constituent isolated from pigeon pea leaves, exhibited neuroprotective activities in previous studies. The present study aims to evaluate the antidepressant effects of CSA by using behavioral despair models of tail suspension test (TST) and forced swimming test (FST), and a chronic unpredictable mild stress (CUMS) model. CSA (30 or 60 mg/kg), intragastrically administrated for 7 days, could significantly reduce the immobility time of mice in TST and FST. CSA treatment (15 or 30 mg/kg) significantly reversed the depressive-like behavioral changes of mice induced by 3 or 6 weeks CUMS that caused the decrease of sucrose preference, the increase of latency to feed in the novelty-suppressed feeding test, and the increase of immobility time in TST of mice. Furthermore, the related mechanisms of the effect were explored by accessing the metabolite levels of kynurenine pathway of tryptophan metabolism and the expression of some related proteins in cerebral cortex of CUMS mice. Our results showed that the kynurenine pathway was upregulated after CUMS, while the alteration could be significantly reversed by CSA. CSA also reversed the CUMS-induced decrease in the levels of BDNF, PSD-95, p-Akt/Akt and p-mTOR/mTOR. Therefore, the antidepressant-like effects of CSA might be achieved through regulating tryptophan metabolism, promoting BDNF and PSD-95 expression, and activating Akt/mTOR pathway in the cerebral cortex.

Topics: Animals; Antidepressive Agents; Brain-Derived Neurotrophic Factor; Cajanus; Gene Expression Regulation; Male; Mice; Mice, Inbred BALB C; Molecular Structure; Plant Leaves; Salicylates; Stilbenes

Methicillin-resistant Staphylococcus aureus and the formation of persistent nongrowing subpopulations (persisters) is a serious threat to human. Our previous studies have proved that two cajaninstilbene acid (CSA) analogues, compound 5b and 5j display remarkable antibacterial activities, especially overcoming drug resistance of methicillin-resistant Staphylococcus aureus (MRSA). Present study found that 5b and 5j are capable of eradicating MRSA persisters. However, their underlying antibacterial mechanism is still obscure. In this study, biological evaluation was performed by transmission electron micrograph, membrane permeability and membrane depolarization experiment to reveal the effects of drugs on bacteria. Further, affinity-based protein profiling and transcriptional profiling were performed to characterise the protein targets in bacterial. Biological evaluation suggested that 5b has an effect on bacterial membrane, affinity-based protein profiling identified that 5b targets membrane associated protein PgsA and verified by in vitro labelling profile. Transcriptional profiling indicated that 5b interferes in phosphatidylglycerol (PG) synthesis pathway. This study identified a novel antibacterial target PgsA and it might be a potential target to combat the resistant bacteria.

Topics: Anti-Bacterial Agents; Dose-Response Relationship, Drug; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Molecular Structure; Proteomics; Salicylates; Stilbenes; Structure-Activity Relationship

Cajaninstilbene acid (CSA) exerts wide pharmacological activities, such as anti-inflammation, hypoglycaemic activity, analgesic effect and cognition improvement. However, it underwent severe phase II metabolism mediated by UDP-glucuronosyltransferase (UGT) in the gastrointestinal (GI) tract after oral administration, affecting its oral bioavailability. In the present study, we utilize UGT inhibitory excipient containing self-microemulsion (SME) delivery system to reduce the production of glucuronide metabolites and increase its oral bioavailability. The present results showed that although similar properties in physiochemical, cytotoxicity, cellular uptake, absorption and transport across rat everted gut sacs between SME-1 (inhibitory excipient containing SME) and SME-2 (control SME, without inhibitory excipient), an improved absolute bioavailability of 57.3 % was conferred by SME-1, significantly higher than the value of 35.4 % by SME-2 and 34.0 % by free CSA. Noticeably, the significantly lower AUC value of CSA glucuronide was determined in rats treated with SME-1 than those either treated with SME-2 or free CSA. Thus, the ability of SME-1 to enhance oral bioavailability of CSA is mainly attributed to the inhibition of phase II metabolism in the GI tract.

Topics: Administration, Oral; Animals; Biological Availability; Emulsions; Enzyme Inhibitors; Glucuronosyltransferase; Humans; Male; Rats; Rats, Wistar; Salicylates; Stilbenes; Tumor Cells, Cultured

Biofilm formation and virulence factor secretion in opportunistic pathogen Pseudomonas aeruginosa are essential for establishment of chronic and recurrent infection, which are regulated by quorum sensing (QS) system. In this study, a set of cajaninstilbene acid analogues were designed and synthesized, and their abilities to inhibit QS and biofilm formation were investigated. Among all the compounds, compounds 3g, 3m and 3o showed potent anti-biofilm activity, especially 3o exhibited promising biofilm inhibitory activity with biofilm inhibition ratio of 49.50 ± 1.35% at 50 μM. Three lacZ reporter strains were constructed to identify the effects of compound 3o on different QS systems. Compound 3o showed the suppression on the expression of lasB-lacZ and pqsA-lacZ as well as on the production of their corresponding virulence factors. Therefore, compound 3o is expected to be generated as a lead compound with inhibition of biofilm formation and QS of Pseudomonas aeruginosa.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Pseudomonas aeruginosa; Quorum Sensing; Salicylates; Stilbenes; Virulence Factors

Osteoporosis is a form of osteolytic disease caused by an imbalance in bone homeostasis, with reductions in osteoblast bone formation, and augmented osteoclast formation and resorption resulting in reduced bone mass. Cajaninstilbene acid (CSA) is a natural compound derived from pigeon pea leaves. CSA possesses beneficial properties as an anti-inflammatory, antibacterial, antihepatitis, and anticancer agent; however, its potential to modulate bone homeostasis and osteoporosis has not been studied. We observed that CSA has the ability to suppress RANKL-mediated osteoclastogenesis, osteoclast marker gene expression, and bone resorption in a dose-dependent manner. Mechanistically, it was revealed that CSA attenuates RANKL-activated NF-κB and nuclear factor of activated T-cell pathways and inhibited phosphorylation of key signaling mediators c-Fos, V-ATPase-d2, and ERK. Moreover, in osteoclasts, CSA blocked RANKL-induced ROS activity as well as calcium oscillations. We further evaluated the therapeutic effect of CSA in a preclinical mouse model and showed that in vivo treatment of ovariectomized C57BL/6 mice with CSA protects the mice from osteoporotic bone loss. Thus, this study demonstrates that osteolytic bone diseases can potentially be treated by CSA.

Topics: Animals; Bone Resorption; Calcium; Gene Expression Regulation; Mice, Inbred C57BL; NF-kappa B; NFATC Transcription Factors; Osteoblasts; Osteoclasts; Osteogenesis; Osteoporosis; Ovariectomy; RANK Ligand; Reactive Oxygen Species; Salicylates; Signal Transduction; Stilbenes

A valid and reliable method was established to separate six compounds from pigeon pea leaves via elution-extrusion counter-current chromatography. A solvent system composed of n-hexane/methanol/formic acid aqueous solution with pH = 3 (10:6:4, v/v) was screened to achieve satisfactory isolation from the ethanol extract of pigeon pea leaves. Four compounds, 9.2 mg of compound 1 (96.8%), 3.2 mg of 2 (88.0%), 6.2 mg of 4 (94.2%) and 25.2 mg of 5 (94.2%), were obtained by conventional elution from 100 mg of the precipitation fraction, respectively. Two compounds, 14.4 mg of 3 (96.3%) and 28.1 mg of 6 (96.6%), with high K values were obtained by the subsequent extrusion procedure. The compounds 1-6 were identified as 3-methoxy-5-(2-phenylethenyl)-phenol, pinostrobin chalcone, pinostrobin, 2-hydroxy-4-methoxy-6-(2-phenylvinyl)-benzoic acid, longistylin C and cajaninstilbene acid by quadrupole time-of-flight mass spectrometry, and

Topics: Antineoplastic Agents, Phytogenic; Benzoic Acid; Cell Proliferation; Countercurrent Distribution; Drug Screening Assays, Antitumor; Flavanones; Hep G2 Cells; Humans; Molecular Structure; Phenols; Pisum sativum; Plant Leaves; Salicylates; Stilbenes

The prevalence of multidrug resistance among clinically significant bacterial pathogens underlines a critical need for the development of new classes of antibacterial agents with novel structural scaffolds. Cajaninstilbene acid (CSA), which is isolated from pigeonpea leaves, has shown potent antibacterial activity. In this study, a series of 2-hydroxyl-4-methoxyl-3-(3-methylbut-2-en-1-yl)-6-(4-phenylbenzoylamino)benzoic acid derivatives derived from CSA were designed and synthesized, and their antibacterial activities were evaluated. Several synthesized compounds exhibit better antibacterial activity than CSA against Staphylococcus aureus, Staphylococcus epidermidis, and two strains of methicillin-resistant S. aureus. Meanwhile, the results of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assays illustrate the good selectivity between bacteria and normal cells of the most active compounds 6u and 6v. Furthermore, well combinations with bacterial RNA polymerase of 6u arising from docking study imply the possible mechanism of antibacterial activity of these synthetic compounds.

Topics: Animals; Anti-Bacterial Agents; Benzoates; Cell Line; Cell Survival; Chlorocebus aethiops; Microbial Sensitivity Tests; Salicylates; Staphylococcus aureus; Staphylococcus epidermidis; Stilbenes

The quorum sensing (QS) system inhibitors of Pseudomonas aeruginosa are thought to attenuate bacterial pathogenicity and drug resistance by inhibiting biofilm formation and the production of virulence factors. In this study, a synthetic approach to the natural products cajaninstilbene acid (1) and amorfrutins A (2) and B (3) has been developed and was characterized by the Heck reaction, which was used to obtain the stilbene core and a Pinick oxidation to give the O-hydroxybenzoic acid. The biological activities of these compounds against the P. aeruginosa quorum sensing systems were evaluated. Amorfrutin B (3) showed promising antibiofilm activity against P. aeruginosa PAO1 with a biofilm inhibition ratio of 50.3 ± 2.7. Three lacZ reporter strains were constructed to identify the effects of compound 3 on different QS systems. Suppression efficacy of compound 3 on the expression of lasB-lacZ and pqsA-lacZ as well as on the production of their corresponding virulence factors elastase and pyocyanin was observed.

Topics: Gene Expression; Genes, Bacterial; Molecular Structure; Pseudomonas aeruginosa; Quorum Sensing; Salicylates; Stilbenes; Virulence Factors

As a major active stilbene from the leaves of pigeon pea (Cajanus cajan), cajaninstilbene acid (CSA) exerts various pharmacological activities. The present study aimed to investigate the pharmacokinetics of CSA and one of its main metabolites (M1) to explore their fate in the body and provide a pharmacokinetic foundation for their in vivo biological activities and functional food or complementary medicine application. M1 was characterized as CSA-3-O-glucuronide using the multiple reaction monitoring-information-dependent acquisition-enhanced product ion technique. After oral and intravenous administration, plasma, urine, and bile were collected and analyzed to estimate pharmacokinetic properties of CSA and M1 and to explore the main excretion route. The oral bioavailability of CSA was estimated to be 44.36%. This study first reported that CSA is mainly metabolized to CSA-3-O-glucuronide via the first-pass effect to limit its oral bioavailability and excreted predominantly through the biliary route, while the enterohepatic circulation, extravascular distribution, and renal reabsorption characteristics of CSA might delay its elimination.

Topics: Animals; Biological Availability; Cajanus; Glucuronides; Male; Molecular Structure; Plant Extracts; Rats; Rats, Sprague-Dawley; Salicylates; Stilbenes; Tissue Distribution

In the present study, we investigated the activity and modes of action of cajanin stilbene acid (CSA) and its derivatives in terms of cytotoxicity, gene expression profile, and transcription factor activity. XTT assays on MCF7 cells were performed upon treatment with CSA or derivatives. After the determination of IC50 values, gene expression profiling was performed with Agilent microarray experiments. Deregulated genes were determined with Chipster software, pathway and functional analyses were performed with Ingenuity pathway software. In order to identify the potential upstream regulators, MatInspector software was used to perform transcription factor binding motif search in the promoter regions of the deregulated genes. Molecular docking on MYC/MAX complex and reporter cell line experiments were performed to validate the MYC inhibitory activity of CSA and its derivatives. Two known MYC inhibitors: 10058-F4 and 10074-G5 were used as positive control. All compounds showed cytotoxicities in the micromolar range. Microarray analyses pointed to cell cycle, DNA damage, and DNA repair as mainly affected cellular functions. Promoter motif analysis of the deregulated genes further supported the microarray gene expression analysis results emphasizing the relevance of transcription factors regulating cell cycle and proliferation, with MYC as being the most pronounced one. Luciferase-based reporter cell line experiments and molecular docking studies yielded supportive results emphasizing the inhibitory activity of CSA and its derivatives on MYC. CSA and its derivatives are shown to be promising anticancer compounds with low toxicity. They inhibit MYC activity comparable to 10058-F4 and 10074-G5. Further studies are warranted to analyze the therapeutic applicability of these compounds in more detail.

Topics: Antineoplastic Agents; Breast Neoplasms; Diethylstilbestrol; Drug Screening Assays, Antitumor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; MCF-7 Cells; Molecular Docking Simulation; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Salicylates; Stilbenes

Cajaninstilbene acid (CSA) is one of the active components isolated from pigeon pea leaves. In this study, anti-inflammatory effects of CSA and its synthesized derivatives were fully valued with regard to their activities on the production of nitric oxide (NO) and pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in vitro cell model, as well as their impacts on the migration of neutrophils and macrophages in fluorescent protein labeled zebrafish larvae model by live image analysis. Furthermore, the anti-inflammatory mechanism of this type of compounds was clarified by western-blot and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that CSA, as well as its synthesized derivatives 5c, 5e and 5h, exhibited strong inhibition activity on the release of NO and inflammatory factor TNF-α and IL-6 in lipopolysaccharides (LPS)-stimulated murine macrophages. CSA and 5c greatly inhibited the migration of neutrophils and macrophages in injury zebrafish larvae. CSA and 5c treatment greatly inhibited the phosphorylation of proteins involved in nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Moreover, we found that peroxisome proliferator-activated receptor gamma (PPARγ) inhibitor GW9662 could reverse partly the roles of CSA and 5c, and CSA and 5c treatment greatly resist the decrease of PPARγ mRNA and protein induced by LPS stimulation. Our results identified the promising anti-inflammatory effects of CSA and its derivatives, which may serve as valuable anti-inflammatory lead compound. Additionally, the mechanism studies demonstrated that the anti-inflammatory activity of CSA and its derivative is associated with the inhibition of NF-κB and MAPK pathways, relying partly on resisting the LPS-induced decrease of PPARγ through improving its expression.

Topics: Animals; Anti-Inflammatory Agents; Cajanus; Interleukin-6; Macrophages; Mice; Mitogen-Activated Protein Kinases; Molecular Structure; Plant Extracts; Salicylates; Stilbenes; Tumor Necrosis Factor-alpha; Zebrafish

A common building block for the synthesis of amorfrutin and cajaninstilbene acid derivatives has been developed. The library of synthesized compounds has enabled identification of new nontoxic ligands of peroxisome proliferator-activated receptors (PPAR) and potential inhibitors of the transcriptional corepressor protein NCoR. The biological data holds promise in identification of new potential leads for the antidiabetic drug discovery process.

Topics: Hypoglycemic Agents; Ligands; Molecular Structure; Peroxisome Proliferator-Activated Receptors; Salicylates; Stilbenes

Although cancer stem-like cells (CSCs) are rare, they can enter a non-proliferative or dormant state and resist therapy. Furthermore, quiescent CSCs are responsible for metastases that can appear after curative surgical treatment of a primary tumor. Because of drug resistance of CSCs, the development of novel therapies is urgently required that specifically target CSCs.. The aim of the present study was to investigate the potential of a panel of natural products and derivatives to inhibit CSC-enriched mammospheres of MCF-7 breast cancer cells.. CD44(high)/CD24(low) cells were identified by flow cytometry and maintained as mammospheres. As a control, we used two clinically established anticancer drugs (5-fluorouracil and docetaxel). A panel of natural products, shikonin, two cajanin stilbene acid (CSA) derivatives and artesunate were tested by resazurin assay on CSC-enriched mammospheres and MCF-7 monolayer cells. Besides, cellular shikonin uptake experiments were performed by flow cytometry.. We found two CSA derivatives (Nos. 6 and 19) to be active cancer stem-like MCF-7 mammospheres. Especially, CSA derivative No. 19 clearly showed collateral sensitivity in mammospheres compared to monolayer cells.. Phytochemicals which provoke collateral sensitivity in cancer-stem like cells are worth more detailed investigations in the future, since there is a great potential for improved chemotherapy to eradicate tumors and prolong cancer patients' survival times.

Topics: Antineoplastic Agents; Artemisinins; Artesunate; Humans; MCF-7 Cells; Naphthoquinones; Neoplastic Stem Cells; Phytochemicals; Salicylates; Spheroids, Cellular; Stilbenes

The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet.. We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis.. CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways.. CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; Cajanus; Caspase 3; Cell Cycle Checkpoints; DNA Damage; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Proto-Oncogene Proteins c-bcl-2; Salicylates; Signal Transduction; Stilbenes

Discovery of novel antibacterial agents with new structural scaffolds that combat drug-resistant pathogens is an urgent task. Cajaninstilbene acid, which is isolated from pigeonpea leaves, has shown antibacterial activity. In this study, a series of cajaninstilbene acid derivatives were designed and synthesized. The antibacterial activities of these compounds against gram-negative and gram-positive bacteria, as well as nine strains of methicillin-resistant staphylococcus aureus (MRSA) bacteria are evaluated，and the related structure-activity relationships are discussed. Assays suggest that some of the synthetic cajaninstilbene acid derivatives exhibit potent antibacterial activity against gram-positive bacterial strains and MRSA. Among these compounds, 5b, 5c, 5j and 5k show better antibacterial activity than the positive control compounds. The results of MTT assays illustrate the low cytotoxicity of the active compounds.

Topics: Animals; Anti-Bacterial Agents; Cell Line; Dose-Response Relationship, Drug; Gram-Negative Bacteria; Gram-Positive Bacteria; Macrophages; Mice; Microbial Sensitivity Tests; Molecular Structure; Salicylates; Stilbenes; Structure-Activity Relationship

Antiestrogenic therapy is a mainstay for estrogen receptor (ERα)-positive breast cancer. Due to the development of resistance to established antihormones such as tamoxifen, novel compounds are required. The low abundant cajanin stilbene acid (CSA) recently isolated by us from Pigeon Pea (Cajanus cajan) has structural similarities with estrogen. We analyzed the cytotoxic and anticancer activity of CSA in ERα-positive and -negative human breast cancer cells in vitro, in vivo and in silico. CSA exerts anticancer and antiestrogenic activities towards ERα-positive breast cancer, and it showed cytotoxicity towards tamoxifen-resistant MCF-7 cells, implying that CSA may be active against tamoxifen-resistant breast cancer cells. CSA showed low cytotoxicity in ERα-negative breast tumor cells as expected. Comparable cytotoxicity was observed towards p53 negative MCF-7 cells, implying that CSA is effective independent of the p53 status. Xenografted MCF-7 cells in nude mice were better inhibited by CSA than by cyclophosphamide. Testing of 8 primary cell cultures derived from human breast cancer biopsies showed that cell cultures from ER-positive tumors were more sensitive than from ER-negative ones. Dose-dependent decrease in ERα protein levels was observed upon CSA treatment. Synergistic effect with tamoxifen was observed in terms of increased p53 protein level. CSA affected pathways related to p53, cancer and cell proliferation. Gene promoter analyses supported the ERα regulation. CSA bound to the same site as 17β-estradiol and tamoxifen on ERα. In conclusion, CSA exerts its anticancer effects in ERα-positive breast cancer cells by binding and inhibiting ERα.

Topics: Adult; Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Estrogen Antagonists; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice, Nude; Middle Aged; Promoter Regions, Genetic; Receptors, Estrogen; Salicylates; Stilbenes; Tamoxifen; Xenograft Model Antitumor Assays

It has been reported that high corticosterone level could damage the normal hippocampal neurons both in vitro and in vivo. Furthermore, high concentration of corticosterone induced impair in PC12 cells has been widely used as in vitro model to screen neuroprotective agents. Cajaninstilbene acid (CSA), a natural stilbene isolated from Cajanus cajan leaves, has various activities. In present study, we investigated the effect of CSA on corticosterone-induced cell apoptosis and explored its possible signaling pathways in PC12 cells. We demonstrated that pretreatment with CSA at the concentrations of 1-8 μmol/L remarkably reduced the cytotoxicity induced by 200 μmol/L of corticosterone in PC12 cells by MTT, and further confirmed the neuroprotection by Hoechst 33342 and PI double staining and lactate dehydrogenase release (LDH) assay at the concentration of 8 μmol/L. Moreover, the cytoprotection of CSA was proved to be associated with the homeostasis of intracellular Ca(2+), relieving corticosterone-induced oxidative stress by decreasing the contents of ROS and malondialdehyde (MDA), increasing the activities of superoxide dismutase (SOD) and catalase (CAT), and the stabilization of ER stress via down-regulating the expression of ER chaperone protein glucose-regulated protein 78 (GRP78), ER stress associated transcription factor C/EBP homologous protein (CHOP/GADD153), and the X box-binding protein-1 (XBP-1), as well as the expression of ER stress-specific protein caspase-12 and its downstream protein caspase-9. Considering all the findings, it is suggested that the neuroprotective activity of CSA against the impairment induced by corticosterone in PC12 cells was through the inhibition of oxidative stress and ER stress-mediated pathway.

Topics: Animals; Apoptosis; Corticosterone; Cytoprotection; Dose-Response Relationship, Drug; Endoplasmic Reticulum Stress; Oxidative Stress; PC12 Cells; Rats; Salicylates; Stilbenes

Cajaninstilbene acid (3-hydroxy-4-prenyl-5-methoxystilben-2 -carboxylic acid, CSA), a natural stilbene isolated from the leaves of Cajanus cajan, has attracted considerable attention for its wide range of pharmacological activities. This study investigated whether CSA protects against corticosterone (CORT)-induced injury in PC12 cells and examined the potential mechanisms underlying this protective effect.. Cell viability and cytotoxicity were detected using a 3-(4,5-desethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) assay kit, respectively. PC12 cell apoptosis was measured using Hoechst 33342 staining and a DNA fragmentation assay kit, and intracellular Ca(2+) concentrations were assessed by fluorescent labelling. Next, the mitochondrial permeability transition pores (mPTPs) and mitochondrial membrane potentials (∆Ψm) were detected using a colorimetric mPTP detection kit and a 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) kit, respectively. Finally, cytochrome c, caspase-3 and inhibitor of caspase-activated deoxyribonuclease (ICAD) expression levels were monitored by western blot analysis.. Treatment with 100 µmol/l CORT induced cytotoxicity in PC12 cells. However, CSA dose-dependently increased cell viability and decreased LDH release as well as CORT-induced apoptosis. Mechanistically, compared with the CORT-treated group, CSA strongly attenuated intracellular Ca(2+) overload and restored mitochondrial functions, including mPTPs and ∆Ψm. Furthermore, the down-regulation of cytochrome c and ICAD protein expression and the blockage of caspase-3 activity were observed upon CSA treatment.. In summary, our data are the first to show that the in vitro antidepressant-like effect of CSA may be attributed to the cytoprotection of neurons and that such neuroprotective mechanisms are correlated with intracellular Ca(2+) homeostasis and mitochondrial apoptotic pathways.

Topics: Animals; Apoptosis; Corticosterone; DNA Fragmentation; Mitochondria; PC12 Cells; Rats; Salicylates; Stilbenes

Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid), an active constituent of pigeonpea leaves, an important tropical crop, is known for its clinical effects in the treatment of diabetes, hepatitis, and measles and its potential antitumor effect. In this study, the effect of the cytochrome P450 isozymes on the activity of CSA was investigated. Two hydroxylation metabolites were identified in the study. The reaction phenotype study showed that CYP3A4, CYP2C9, and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA. The metabolic food-drug interaction potential was also evaluated in vitro. The effect of CSA inhibition/induction of enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques. CSA showed different inhibitory effects on different isozymes. CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with IC50 values of 28.3 and 31.3 μM, respectively, but exhibited no inhibition activities to CYP1A2, CYP2A6, CYP2C19, CYP2D6, and CYP2E1. CSA showed a weak effect on CYP450 enzymes in a time-dependent manner. CSA did not substantially induce CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 at concentrations up to 30 μM in primary human hepatocytes. The results of our experiments may be helpful to predict clinically significant food-drug interactions when other drugs are administered in combination with CSA.

Topics: Cells, Cultured; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Female; Hepatocytes; Humans; In Vitro Techniques; Kinetics; Microsomes, Liver; Middle Aged; Molecular Structure; Oxidation-Reduction; Salicylates; Stilbenes

Cajaninstilbene acid (CSA), an active compound separated from pigeon pea leaves, possesses the highly efficient antioxidant activities. Transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important regulator of cellular oxidative stress. This study examined the role of Nrf2 in CSA-mediated antioxidant effects on human hepatocarcinoma (HepG2) cell line. The generation of reactive oxygen species (ROS) upon H2O2 and CSA treatment was lower than that of H2O2 alone. CSA activated Nrf2 as evaluated by Western blotting. A luciferase reporter assay also demonstrated that CSA-activated signaling resulted in the increased transcriptional activity of Nrf2 through binding to the antioxidant response element (ARE) enhancer sequence. Our study indicated that treatment of HepG2 cells with CSA induces Nrf2-dependent ARE activity and gene expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase modifier subunits by activation of PI3K/AKT, ERK and JNK signaling pathways. Inhibition of Nrf2 by siRNA reduced CSA-induced upregulation of these Nrf2-related enzymes. These results suggest that the Nrf2/ARE pathway plays an important role in the regulation of CSA-mediated antioxidant effects in HepG2 cells.

Topics: Antioxidant Response Elements; Antioxidants; Blotting, Western; Cytoprotection; Heme Oxygenase-1; Hep G2 Cells; Humans; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Oxidative Stress; Salicylates; Signal Transduction; Stilbenes

In this study, novel endophytic fungi producing cajaninstilbene acid (CSA) from pigeon pea [ Cajanus cajan (L.) Millsp.] were investigated and screened. CSA has prominent pharmacological activities. A total of 110 endophytic fungi isolates were grouped into 8 genera on the basis of morphological characteristics, and CSA-producing fungi were screened by liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to ITS-rDNA sequences analysis, the CSA-producing fungi were identified as Fusarium solani (ERP-07), Fusarium oxysporum (ERP-10), and Fusarium proliferatum (ERP-13), respectively. The amount of CSA produced by the ERP-13 reached 504.8 ± 20.1 μg/L or 100.5 ± 9.4 μg/g dry weight of mycelium. In a DPPH radical-scavenging assay, when the concentration of fungal CSA was 500 μg/mL, inhibition percentage could reach 80%, which was almost the same as that of standard CSA. This study first reported the natural antioxidant CSA from endophytic fungi F. solani and F. proliferatum isolated from pigeon pea.

Topics: Antioxidants; Cajanus; DNA, Fungal; Fusarium; Phylogeny; Salicylates; Sequence Analysis, DNA; Stilbenes

Cajaninstilbene acid (CSA) is a major active component present in the leaves of Cajanus cajan (L.) Millsp. The present study explores the underlying cellular mechanisms for CSA-induced relaxation in rat renal arteries. Vascular reactivity was examined in arterial rings that were suspended in a Multi Myograph System and the expression of signaling proteins was assessed by Western blotting method. CSA (0.1-10 µM) produced relaxations in rings pre-contracted by phenylephrine, serotonin, 9, 11-dideoxy-9α, 11α-epoxymethanoprostaglandin F(2α) (U46619), and 60 mM KCl. CSA-induced relaxations did not show difference between genders and were unaffected by endothelium denudation, nor by treatment with N(G)-nitro-L-arginine methyl ester, indomethacin, ICI-182780, tetraethylammonium ion, BaCl(2), glibenclamide, 4-aminopyridine or propranolol. CSA reduced contraction induced by CaCl(2) (0.01-5 mM) in Ca(2+)-free 60 mM KCl solution and by 30 nM (-)-Bay K8644 in 15 mM KCl solution. CSA inhibited 60 mM KCl-induced Ca(2+) influx in smooth muscle of renal arteries. In addition, CSA inhibited contraction evoked by phorbol 12-myristate 13-acetate (PMA, protein kinase C agonist) in Ca(2+)-free Krebs solution. Moreover, CSA reduced the U46619- and PMA-induced phosphorylation of myosin light chain (MLC) at Ser19 and myosin phosphatase target subunit 1 (MYPT1) at Thr853 which was associated with vasoconstriction. CSA also lowered the phosphorylation of protein kinase C (PKCδ) at Thr505. In summary, the present results suggest that CSA relaxes renal arteries in vitro via multiple cellular mechanisms involving partial inhibition of calcium entry via nifedipine-sensitive calcium channels, protein kinase C and Rho kinase.

Topics: Animals; Calcium; Estradiol; Female; Fulvestrant; Glyburide; Male; Muscle, Smooth, Vascular; Phosphorylation; Potassium Chloride; Protein Kinase C; Rats; Renal Artery; Salicylates; Stilbenes; Tetradecanoylphorbol Acetate; Tetraethylammonium; Vasoconstriction

The antioxidant properties, DNA damage protective activities, and xanthine oxidase (XOD) inhibitory effect of cajaninstilbene acid (CSA) derived from pigeon pea leaves were studied in the present work. Compared with resveratrol, CSA showed stronger antioxidant properties, DNA damage protective activity, and XOD inhibition activity. The IC(50) values of CSA for superoxide radical scavenging, hydroxyl radical scavenging, nitric oxide scavenging, reducing power, lipid peroxidation, and XOD inhibition were 19.03, 6.36, 39.65, 20.41, 20.58, and 3.62 μM, respectively. CSA possessed good protective activity from oxidative DNA damage. Furthermore, molecular docking indicated that CSA was more potent than resveratrol or allopurinol to interact with the active site of XOD (calculated free binding energy: -229.71 kcal mol(-1)). On the basis of the results, we conclude that CSA represents a valuable natural antioxidant source and may potentially be applicable in health food industry.

Topics: Antimutagenic Agents; Antioxidants; Cajanus; DNA Damage; DNA, Bacterial; Enzyme Inhibitors; Humans; Plant Extracts; Plant Leaves; Salicylates; Stilbenes; Xanthine Oxidase

Five fungal endophytes (K4, K5, K6, K9, K14) producing Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) were isolated from the roots of pigeon pea [Cajanus cajan (L.) Millsp.]. CSA is responsible for the prominent pharmacological activities in pigeon pea. The amount of CSA in culture solution varied among the five fungal endophytes. K4 produced the highest levels of CSA (1037.13 µg/L) among the endophytes tested after incubation for five days. Both morphological characteristics and molecular methods were used for species identification of fungal endophytes. The five endophytic isolates were characterized by analyzing the internal transcribed spacer (ITS) rRNA and β-tubulin genes. The K4, K5, K9 and K14 strains isolated from pigeon pea roots were found to be closely related to the species Fusarium oxysporum. K6 was identified as Neonectria macrodidym. The present study is the first report on the isolation and identification of fungal endophytes producing CSA in pigeon pea. The study also provides a scientific base for large scale production of CSA.

Topics: Cajanus; Endophytes; Fusariosis; Fusarium; Plant Roots; Salicylates; Stilbenes; Tubulin

Cajaninstilbene acid (CSA; 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) is a major active constituent of pigeonpea leaves, has been proven to be effective in clinical treatment of diabetes, hepatitis, measles and dysentery. A rapid and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of CSA in rat plasma and various tissues (brain, heart, lung, liver, spleen, small intestine and kidney) of rat for the first time. Rat plasma and tissue distribution pre-treated by protein precipitation with acetoacetate was analyzed using LC-MS/MS with an electrospray ionization (ESI) interface, and isoliquiritigenin was used as an internal standard. Chromatographic separation was achieved on a HIQ Sil C(18) column with the mobile phase of water and methanol (9:91, v/v) containing 0.1% formic acid and resulted in a total run time of 10 min. The isocratic elution mode pumped at a flow rate of 1.0 mL/min. The lower limit of quantification (LLOQ) which was 10 ng/mL. The calibration curve was linear from 10 to 6000 ng/mL (R=0.9967) for plasma samples and 10 to 6000 ng/mL (R>or=0.9974) for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 0.6% to 6.1% and 1.5% to 6.6%, respectively, and the intra- and inter-day assay accuracy was 93.5-104.6% and 93.3-107.5%, respectively. Recoveries in plasma and tissues ranged from 95.0% to 106.8%. The method was successfully applied in pharmacokinetic and tissue distribution studies of CSA after oral administration to rats. The pharmacokinetics of CSA showed rapid absorption and elimination (T(max), 10.7+/-0.31 min; t(1/2), 51.40+/-6.54 min). After oral administration in rats, CSA was rapidly and widely distributed in tissues. High concentrations were found in liver and kidney indicating that CSA was possibly absorbed by liver and eliminated by kidney.

Topics: Animals; Area Under Curve; Cajanus; Calibration; Carboxylic Acids; Chromatography, Liquid; Drug Stability; Female; Freezing; Half-Life; Metabolic Clearance Rate; Molecular Structure; Quality Control; Rats; Rats, Inbred Strains; Reference Standards; Reproducibility of Results; Salicylates; Sensitivity and Specificity; Stilbenes; Tandem Mass Spectrometry; Temperature; Time Factors; Tissue Distribution