Shen Yuan extract exerts a hypnotic effect via the tryptophan/5-hydroxytryptamine/melatonin pathway in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sleep plays a critical role in several physiologic processes, and sleep disorders increase the risk of depression, dementia, stroke, cancer, and other diseases. Stress is one of the main causes of sleep disorders. Ginseng Radix et Rhizoma and Polygalae Radix have been reported to have effects of calming the mind and intensifying intelligence in Chinese Pharmacopoeia. Traditional Chinese medicine prescriptions composed of Ginseng Radix et Rhizoma and Polygalae Radix (Shen Yuan, SY) are commonly used to treat insomnia, depression, and other psychiatric disorders in clinical practice. Unfortunately, the underlying mechanisms of the SY extract's effect on sleep are still unknown. AIM OF THE STUDY: This study aimed to investigate the hypnotic effect of the SY extract in normal mice and mice with chronic restraint stress (CRS)-induced sleep disorders and elucidate the underlying mechanisms. MATERIALS AND METHODS: The SY extract (0.5 and 1.0 g/kg) was intragastrically administered to normal mice for 1, 14, and 28 days and to CRS-treated mice for 28 days. The open field test (OFT) and pentobarbital sodium-induced sleep test (PST) were used to evaluate the hypnotic effect of the SY extract. Liquid chromatography-tandem mass spectrometry and enzyme-linked immunosorbent assay were utilized to detect the levels of neurotransmitters and hormones. Molecular changes at the mRNA and protein levels were determined using real-time quantitative polymerase chain reaction and Western blot analysis to identify the mechanisms by which SY improves sleep disorders. RESULTS: The SY extract decreased sleep latency and increased sleep duration in normal mice. Similarly, the sleep duration of mice subjected to CRS was increased by administering SY. The SY extract increased the levels of tryptophan (Trp) and 5-hydroxytryptamine (5-HT) and the expression of tryptophan hydroxylase 2 (TPH2) in the cortex of normal mice. The SY extract increased the Trp level, transcription and expression of estrogen receptor beta and TPH2 in the cortex in mice with sleep disorders by decreasing the serum corticosterone level, which promoted the synthesis of 5-HT. Additionally, the SY extract enhanced the expression of arylalkylamine N-acetyltransferase, which increased the melatonin level and upregulated the expressions of melatonin receptor-2 (MT2) and Cryptochrome 1 (Cry1) in the hypothalamus of mice with sleep disorders. CONCLUSIONS: The SY extract exerted a hypnotic effect via the Trp/5-HT/melatonin pathway, which augmented the synthesis of 5-HT and melatonin and further increased the expressions of MT2 and Cry1.

Antidepressant effects of Parishin C in chronic social defeat stress-induced depressive mice.

ETHNOPHARMACOLOGY RELEVANCE: Parishin C (Par), a prominent bioactive compound in Gastrodia elata Blume with little toxicity and shown neuroprotective effects. However, its impact on depression remains largely unexplored. AIM OF THE STUDY: This study aims to investigate the antidepressant effects of Par using a chronic social defeat stress (CSDS) mouse model and elucidate its molecular mechanisms. MATERIALS AND METHODS: The CSDS-induced depression mouse model was used to evaluate the therapeutic efficacy of Par. The social interaction test (SIT) and sucrose preference test (SPT), tail suspension test (TST) and forced swim test (FST) were conducted to assess the effects of Par on depressive-like behaviours. The levels of corticosterone, neurotransmitters (5-HT, DA and NE) and inflammatory cytokines (IL-1ß, TNF-α, and IL-6) were evaluated by enzyme-linked immunosorbent assay (ELISA). Activation of a microglia was assessed by immunofluorescence labeling Iba-1. The protein expressions of NLRP3, ASC, caspase-1, and IL-6 verified by Western blot. RESULT: Oral administration of Par (4 and 8 mg/kg) and fluoxetine (10 mg/kg, administration significantly ameliorate depression-like behaviors induced by CSDS, as shown by the increase social interaction in SIT, increase sucrose preference in SPT and the decrease immobility in TST and FST. Par administration decreased serum corticosterone level and increased the 5-HT, DA and NE concentration in the hippocampus and prefrontal cortex. Furthermore, Par treatment suppressed microglial activation (Iba1) as well as reduced levels of IL-1ß, TNF-α, and IL-6) with decreased protein expressions of NLRP3, ASC, caspase-1, and IL-6. CONCLUSIONS: our study provides the first evidence that Par exerts antidepressant-like effects in mice with CSDS-induced depression. This effect appears to be mediated by the normalization of neurotransmitter and corticosterone levels, inhibition of NLRP3 inflammasome activation. This newfound antidepressant property of Par offers a novel perspective on its pharmacological effects, providing valuable insights into its potential therapeutic and preventive applications in depression treatment.

Undescribed diphenyl ethers betaethrins A-I from a desert plant endophytic strain of the fungus Phoma betae A.B. Frank (Didymellaceae).

Ten diphenyl ethers (DPEs), including nine undescribed analogs named betaethrins A-I, were isolated from the desert plant endophytic fungus Phoma betae A.B. Frank (Didymellaceae). Their structures were determined mainly by NMR, HR-ESI-MS spectral and X-ray diffraction experiments. Betaethrins D-I possessed different fatty acid chains connected with the B-ring, which was the first report in all DPEs. The shielding effect of the B-ring on H-6 (A-ring) in methyl barceloneate, betaethrin A and betaethrins D-F (asterric acid analogs) was first observed and analyzed, which could differentiate the 1H-NMR chemical shift values of H-4/H-6 without the assistance of 3-OH. An empirical rule was then suggested: the steric hindrance between the A- and B-rings in asterric acid analogs might prevent these two aromatic rings from rotating freely, which led to the 1H-NMR chemical shift value of H-6 being in the high field zone due to the shielding effect of the B-ring on H-6. Based on the empirical rule, the chemical shift values of the A-ring in methyl barceloneate were revised. The possible biosynthesis of these isolates was postulated. Betaethrin H showed moderate cytotoxicity against MCF-7 and HepG2 cancer cell lines. Betaethrins A-F, H and I displayed strong antioxidant activities. These results further implied that endophytic fungi from unique environments, such as desert plants, with few chemical studies are an important resource of undescribed and bioactive metabolites.

Chronic restraint stress induced changes in colonic homeostasis-related indexes and tryptophan-kynurenine metabolism in rats.

Chronic stressors represented risk factors for the etiology or exacerbation of several gastrointestinal diseases. The goal of the present study was to examine whether chronic restraint stress (CRS) could initiate and aggravate colonic inflammation, integrity damage and metabolic disturbance of rats. Firstly, increased inflammatory cytokines (interferon-γ (IFN-γ), tumor necrosis factor-α(TNF-α) and interleukin-10(IL-10)) and decreased tight junction (TJ) proteins (occludin and zonula occludins-1 (ZO-1)) in rat colon were observed. Secondly, untargeted metabolomics based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass (UPLC-Q-TOF/MS) revealed that TRP metabolism was the most prominently affected. Thirdly, quantification of TRP and its metabolites via prominence ultrafast liquid chromatography coupled with a QTRAP 5500 mass (UFLC-QTRAP-5500/MS) showed that TRP, kynurenine (KYN), kynurenic acid (KA) and 3-hydroxykynurenine (3-HK) were significantly increased. At the same time, 5-hydroxytryptamine (5-HT) was unchanged and 5-hydroxyindolacetic acid (5-HIAA) was significantly decreased in the colon of CRS rats. Besides, TRP metabolic enzyme changes were with the same trends as the corresponding metabolites. Thus, our data showed that CRS could initiate colonic inflammation, integrity damage and colonic metabolism disturbance, especially TRP-KYN metabolism pathway of rats, which may provide an experimental background for future research on stress-related gastrointestinal dysfunction. SIGNIFICANCE: Chronic exposure to psychological stress could induce metabolic imbalance of the body, and stressful life events were intimately correlated with frequent relapses in patients with intestinal disorders. The present study showed that chronic restraint stress (CRS) could initiate and aggravate colonic inflammation, integrity damage and metabolic disturbance, especially tryptophan-kynurenine metabolism of rats. Tryptophan-kynurenine pathway may be involved in the initiation and development of diseases induced by chronic stress. This research may shed light on future research on stress-related gastrointestinal dysfunction.

Absorption, Metabolism, and Excretion of Cajaninstilbene Acid.

Cajaninstilbene acid (CSA), an active stilbene isolated from the leaves of pigeon pea (Cajanus cajan), exhibits several bioactivities. To develop CSA as a potential nutraceutical and provide pharmacokinetic foundations for its further in vivo bioactivity studies, this study aims to explore its absorption, metabolism, and excretion systematically. Human colon adenocarcinoma (Caco-2) cell monolayers were utilized to investigate the CSA transport mechanism. CSA metabolites were identified in rat biological samples and quantified to explore their excretion routes. CSA exhibited a high permeability and was transported across Caco-2 monolayers mainly by passive transport via the transcellular process. Four new CSA metabolites were found in vivo, namely, CSA-2-COO-glucuronide, 6,12-dihydroxy CSA, 3-hydroxy-5-methoxystilbene-3-O-glucuronide, and 6-hydroxy CSA-3-O-glucuronide, in addition to our previously reported metabolite CSA-3-O-glucuronide. These metabolites were mainly excreted in bile. Our results indicate that metabolism but not absorption is the major barrier limiting the oral bioavailability of CSA.

Enhancing in vivo oral bioavailability of cajaninstilbene acid using UDP-glucuronosyl transferase inhibitory excipient containing self-microemulsion.

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.

Hippocampus Metabolic Disturbance and Autophagy Deficiency in Olfactory Bulbectomized Rats and the Modulatory Effect of Fluoxetine.

An olfactory bulbectomy (OBX) rodent is a widely-used model for depression (especially for agitated depression). The present study aims to investigate the hippocampus metabolic profile and autophagy-related pathways in OBX rats and to explore the modulatory roles of fluoxetine. OBX rats were given a 30-day fluoxetine treatment after post-surgery rehabilitation, and then behavioral changes were evaluated. Subsequently, the hippocampus was harvested for metabonomics analysis and Western blot detection. As a result, OBX rats exhibited a significantly increased hyperemotionality score and declined spatial memory ability. Fluoxetine reduced the hyperemotional response, but failed to restore the memory deficit in OBX rats. Sixteen metabolites were identified as potential biomarkers for the OBX model including six that were rectified by fluoxetine. Disturbed pathways were involved in amino acid metabolism, fatty acid metabolism, purine metabolism, and energy metabolism. In addition, autophagy was markedly inhibited in the hippocampus of OBX rats. Fluoxetine could promote autophagy by up-regulating the expression of LC3 II, beclin1, and p-AMPK/AMPK, and down-regulating the levels of p62, p-Akt/Akt, p-mTOR/mTOR, and p-ULK1/ULK1. Our findings indicated that OBX caused marked abnormalities in hippocampus metabolites and autophagy, and fluoxetine could partly redress the metabolic disturbance and enhance autophagy to reverse the depressive-like behavior, but not the memory deficits in OBX rats.

Study on the Material Basis of Neuroprotection of Myrica rubra Bark.

Background: Increasing attention has been given to the search for neuroprotective ingredients from natural plants. Myrica rubra bark (MRB) has been used in traditional oriental medicine for over thousand years and has potential neuroprotection. Methods and Results: Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to identify the compounds in MRB extract, and the MTT assay was performed to evaluate the neuroprotection of six major compounds from MRB against glutamate-induced damage in PC12 cells. The result displayed nineteen compounds were identified, and myricitrin and myricanol 11-sulfate were shown to have neuroprotection, which prevented cell apoptosis through alleviating oxidative stress by reducing the levels of reactive oxygen species and methane dicarboxylic aldehyde, as well as by enhancing the activities of superoxide dismutase. Conclusions: Several active compounds from MRB may offer neuroprotection and have the potential for the development of new drugs against central nervous system diseases.

Protective effects of Myrica rubra flavonoids against hypoxia/reoxygenation-induced cardiomyocyte injury via the regulation of the PI3K/Akt/GSK3ß pathway.

Myrica rubra is well known for its delicious taste and high nutritional value. The present study investigated the potential protective effects and mechanisms of M. rubra flavonoids (MRF) extract on isoproterenol (ISO)­induced myocardial injury in rats and hypoxia/reoxygenation (H/R) injury in H9c2 cardiomyocytes. An in vivo study revealed that MRF decreased serum cardiac enzyme levels, ameliorated pathological heart alterations and increased the antioxidant potential. The in vitro investigation demonstrated that MRF inhibited cell death, reactive oxygen species (ROS) accumulation, mitochondrial membrane depolarization, apoptosis rate and caspase­3 activation and enhanced the Bcl­2/Bax ratio during H/R injury. These effects were accompanied by the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase (GSK)­3ß. Further mechanism studies demonstrated that LY294002, a specific inhibitor of phosphoinositide 3­kinase (PI3K), abolished the MRF­mediated cardioprotection against H/R­induced apoptosis and ROS overproduction. Collectively, these results suggested that MRF exerts cardioprotective effects by attenuating oxidative damage and cardiomyocyte apoptosis most likely via a PI3K/Akt/GSK3ß­dependent mechanism.

Myricanol rescues dexamethasone-induced muscle dysfunction via a sirtuin 1-dependent mechanism.

BACKGROUND: Muscle atrophy and weakness are adverse effects of high dose or the sustained usage of glucocorticoids. Loss of mitochondria and degradation of protein are highly correlated with muscle dysfunction. The deacetylase sirtuin 1 (SIRT1) plays a vital role in muscle remodelling. The current study was designed to identify myricanol as a SIRT1 activator, which could protect skeletal muscle against dexamethasone-induced wasting. METHODS: The dexamethasone-induced atrophy in C2C12 myotubes was evaluated by expression of myosin heavy chain, muscle atrophy F-box (atrogin-1), and muscle ring finger 1 (MuRF1), using western blots. The mitochondrial content and oxygen consumption were assessed by MitoTracker staining and extracellular flux analysis, respectively. Muscle dysfunction was established in male C57BL/6 mice (8-10 weeks old, n = 6) treated with a relatively high dose of dexamethasone (25 mg/kg body weight, i.p., 10 days). Body weight, grip strength, forced swimming capacity, muscle weight, and muscle histology were assessed. The expression of proteolysis-related, autophagy-related, apoptosis-related, and mitochondria-related proteins was analysed by western blots or immunoprecipitation. RESULTS: Myricanol (10 µM) was found to rescue dexamethasone-induced muscle atrophy and dysfunction in C2C12 myotubes, indicated by increased expression of myosin heavy chain (0.33 ± 0.14 vs. 0.89 ± 0.21, *P < 0.05), decreased expression of atrogin-1 (2.31 ± 0.67 vs. 1.53 ± 0.25, *P < 0.05) and MuRF1 (1.55 ± 0.08 vs. 0.99 ± 0.12, **P < 0.01), and elevated ATP production (3.83 ± 0.46 vs. 5.84 ± 0.79 nM/mg protein, **P < 0.01), mitochondrial content (68.12 ± 10.07% vs. 116.38 ± 5.12%, *P < 0.05), and mitochondrial oxygen consumption (166.59 ± 22.89 vs. 223.77 ± 22.59 pmol/min, **P < 0.01). Myricanol directly binds and activates SIRT1, with binding energy of -5.87 kcal/mol. Through activating SIRT1 deacetylation, myricanol inhibits forkhead box O 3a transcriptional activity to reduce protein degradation, induces autophagy to enhance degraded protein clearance, and increases peroxisome proliferator-activated receptor γ coactivator-1α activity to promote mitochondrial biogenesis. In dexamethasone-induced muscle wasting C57BL/6 mice, 5 mg/kg myricanol treatment reduces the loss of muscle mass; the percentages of quadriceps and gastrocnemius muscle in myricanol-treated mice are 1.36 ± 0.02% and 0.87 ± 0.08%, respectively (cf. 1.18 ± 0.06% and 0.78 ± 0.05% in dexamethasone-treated mice, respectively). Myricanol also rescues dexamethasone-induced muscle weakness, indicated by improved grip strength (70.90 ± 4.59 vs. 120.58 ± 7.93 g, **P < 0.01) and prolonged swimming exhaustive time (48.80 ± 11.43 vs. 83.75 ± 15.19 s, **P < 0.01). Myricanol prevents dexamethasone-induced muscle atrophy and weakness by activating SIRT1, to reduce muscle protein degradation, enhance autophagy, and promote mitochondrial biogenesis and function in mice. CONCLUSIONS: Myricanol ameliorates dexamethasone-induced skeletal muscle wasting by activating SIRT1, which might be developed as a therapeutic agent for treatment of muscle atrophy and weakness.

Rapid Determination of Active Compounds and Antioxidant Activity of Okra Seeds Using Fourier Transform Near Infrared (FT-NIR) Spectroscopy.

Okra seeds (OSD) have been proved to possess significantly anti-fatigue activity and due to their high contents of flavonoids and polyphenols. While, the quality of OSD is easily affected by harvest time, region and other factors. In this research, the rapid method based on Fourier transform near infrared (FT-NIR) spectroscopy was developed for quality assessment of okra seeds. Firstly, 120 samples' spectra were acquired, and quantification of isoquercitrin, quercetin-3-O-gentiobioside, total phenols (TP) and antioxidant assays including 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging, ferric reducing antioxidant power (FRAP) were conducted. Next, partial least squares (PLS) regression and full cross-validation were applied to develop calibration models for these data, and external validation was used to determine models' quality. The coefficient of determination for calibration ( R c 2 ), the root mean square error of cross validation (RMSECV) and the corresponding determination coefficients for cross-validation ( R cv 2 ) proved all these models have excellent precision. Besides, the residual predictive deviation (RPD) of models (4.07 for isoquercitrin, 4.04 for quercetin-3-O-gentiobioside, 9.79 for TP, 4.58 for DPPH and 4.12 for FRAP) also demonstrated that these models possessed good predicative ability. All these results showed that FT-NIR spectroscopy could be used to rapidly determine active compounds and antioxidant activity of okra seeds.

Longistyline C acts antidepressant in vivo and neuroprotection in vitro against glutamate-induced cytotoxicity by regulating NMDAR/NR2B-ERK pathway in PC12 cells.

Depressive disorder is a common psychiatric disease which ranks among the leading cause of disability worldwide. The antidepressants presently used had low cure rate and caused a variety of side-effects. The screening of antidepressant drugs is usually used classic behavioural tests and neuroprotective strategy. Longistyline C, a natural stilbene isolated from the leaves of Cajanuscajan (L.) Millsp, was firstly investigated the antidepressant effect using animal behavioural tests, and studied the neuroprotection and its possible signaling pathways on glutamate-induced injury in PC12 cells. The results of animal test demonstrated that longistyline C had the antidepressant activity, which the effect is similar to the positive control. In current study, we investigated the effect of longistyline C on glutamate-induced injury in PC12 cells and explored its possible signaling pathways. The results demonstrated that pretreatment with longistyline C at the concentrations of 2-8 µmol/L for 24 h had a significant reduction of the cytotoxicity induced by glutamate (15 mmol/L) in PC12 cells using MTT, lactate dehydrogenase (LDH) release assay and Annexin V-PI double staining. Subsequently, we found that pretreatment with longistyline C (8 µmol/L) could drastically down-regulate the over-expression of NMDAR/NR2B and Ca2+/calmodulin-dependent protein kinase II (CaMKII), up-regulate the expressions of p-ERK and p-CREB and alleviate ER stress. In conclusison, longistyline C is most possibly through regulating NMDAR/NR2B-ERK1/2 related pathway and restoring endoplasmic reticulum function to exert neuroprotective effect against glutamate-induced injury in PC12 cells.

Plasma and brain pharmacokinetics of ganoderic acid A in rats determined by a developed UFLC-MS/MS method.

Ganoderic acid A (GAA), an active triterpenoid of the traditional Chinese herbal medicine Lingzhi, has been reported to exhibit antinociceptive, antioxidative, and anti-cancer activities. The present study aims to establish a sensitive and rapid UPLC-MS/MS method for studying the plasma and brain pharmacokinetics of GAA in rats. The analytes were separated on a C18 column eluted with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at 0.3mL/min. The eluate was monitored by a mass detector using an MRM (m/z, 515.3-285.1) model in negative electrospray ionization. The calibration curve showed good linearity (r2>0.99), with limits of detection and quantification of 0.25 and 2.00 nmol/L, respectively. The intra- and inter-day precision and accuracy were less than 9.99% and ranged from 97.45% to 114.62%, respectively. The extraction recovery from plasma was between 92.89% and 98.87%. GAA was found to be stable in treated samples at room temperature (22°C) for 12h and in plasma at -20°C for 7d. The developed method was successfully applied to a pharmacokinetic study of GAA in rats. GAA could be rapidly absorbed into the circulation (Tmax, 0.15h) and eliminated relatively slowly (t1/2, 2.46h) after orally dosing, and could also be detected in the brain lateral ventricle (Tmax, 0.25h and t1/2, 1.40h) after intravenously dosing. The absolute oral bioavailability and brain permeability of GAA were estimated to be 8.68% and 2.96%, respectively.

Effects of phenolic constituents of daylily flowers on corticosterone- and glutamate-treated PC12 cells.

BACKGROUND: Daylily flowers, the flower and bud parts of Hemerocallis citrina or H. fulva, are well known as Wang-You-Cao in Chinese, meaning forget-one's sadness plant. However, the major types of active constituents responsible for the neurological effects remain unclear. This study was to examine the protective effects of hydroalcoholic extract and fractions and to identify the active fractions. METHODS: The extract of daylily flowers was separated with AB-8 resin into different fractions containing non-phenolic compounds, phenolic acid derivatives and flavonoids as determined using UPLC-DAD chromatograms. The neuroprotective activity was measured by evaluating the cell viability and lactate dehydrogenase release using PC12 cell damage models induced by corticosterone and glutamate. The neurological mechanisms were explored by determining their effect on the levels of dopamine (DA), 5-hydroxy tryptamine (5-HT), γ-aminobutyric acid (GABA), noradrenaline (NE) and acetylcholine (ACh) in the cell culture medium measured using an LC-MS/MS method. RESULTS: Pretreatment of PC12 cells with the extract and phenolic fractions of daylily flowers at concentrations ranging from 0.63 to 5 mg raw material/mL significantly reversed corticosterone- and glutamate-induced neurotoxicity in a dose-dependent manner. The fractions containing phenolic acid derivatives (0.59% w/w in the flowers) and/or flavonoids (0.60% w/w) exerted similar dose-dependent neuroprotective effect whereas the fractions with non-phenolic compounds exhibited no activity. The presence of phenolic acid derivatives in the corticosterone- and glutamate-treated PC12 cells elevated the DA level in the cell culture medium whereas flavonoids resulted in increased ACH and 5-HT levels. CONCLUSION: Phenolic acid derivatives and flavonoids were likely the active constituents of daylily flowers and they conferred a similar extent of neuroprotection, but affected the release of neurotransmitters in a different manner.

Antioxidant and Anti-Fatigue Constituents of Okra.

Okra (Abelmoschus esculentus (L.) Moench), a healthy vegetable, is widely spread in tropical and subtropical areas. Previous studies have proven that okra pods possess anti-fatigue activity, and the aim of this research is to clarify the anti-fatigue constituents. To achieve this, we divided okra pods (OPD) into seeds (OSD) and skins (OSK), and compared the contents of total polysaccharides, total polyphenols, total flavonoids, isoquercitrin, and quercetin-3-O-gentiobiose and the antioxidant activity in vitro and anti-fatigue activity in vivo between OSD and OSK. The contents of total polyphenols and total polysaccharides were 29.5% and 14.8% in OSD and 1.25% and 43.1% in OSK, respectively. Total flavonoids, isoquercitrin and quercetin-3-O-gentiobiose (5.35%, 2.067% and 2.741%, respectively) were only detected in OSD. Antioxidant assays, including 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging, ferric reducing antioxidant power (FRAP) and reducing power test, and weight-loaded swimming test showed OSD possessed significant antioxidant and anti-fatigue effects. Moreover, biochemical determination revealed that that anti-fatigue activity of OSD is caused by reducing the levels of blood lactic acid (BLA) and urea nitrogen (BUN), enhancing hepatic glycogen storage and promoting antioxidant ability by lowering malondialdehyde (MDA) level and increasing superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) levels. These results proved okra seeds were the anti-fatigue part of okra pods and polyphenols and flavonoids were active constituents.

Myricitrin attenuates endothelial cell apoptosis to prevent atherosclerosis: An insight into PI3K/Akt activation and STAT3 signaling pathways.

Blood vessel endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) has been implicated in the pathogenesis of atherosclerosis and vasculopathy. The ox-LDL-elicited reactive oxygen species (ROS) release has been assumed to serve a critical function in endothelial damage. Myricitrin (from Myrica cerifera) is a natural antioxidant that has strong anti-oxidative, anti-inflammatory, and anti-nociceptive activities. However, the protective effect of myricitrin on ROS-induced endothelial cell injury and its related molecular mechanisms have never been investigated. This study demonstrates that myricitrin can inhibit ox-LDL-induced endothelial apoptosis and prevent plaque formation at an early stage in an atherosclerotic mouse model. The administration of myricitrin in vivo decreases the thickness of the vascular wall in the aortic arch of ApoE-/- mice. In vitro study shows that ox-LDL-induced human umbilical vein endothelial cell apoptosis can be reduced upon receiving myricitrin pre-treatment. Treatment with myricitrin significantly attenuated ox-LDL-induced endothelial cell apoptosis by inhibiting LOX-1 expression and by increasing the activation of the STAT3 and PI3K/Akt/eNOS signaling pathways. At the same time, our result demonstrates that myricitrin treatment optimizes the balance of pro/anti-apoptosis proteins, including Bax, Bad, XIAP, cIAP-2, and survivin. Our study suggests that myricitrin treatment can effectively protect cells from ox-LDL-induced endothelial cell apoptosis, which results in reduced atherosclerotic plaque formation. This result indicates that myricitrin can be used as a drug candidate for the treatment of cardiovascular diseases.

Tenuifolin, a secondary saponin from hydrolysates of polygalasaponins, counteracts the neurotoxicity induced by Aß25-35 peptides in vitro and in vivo.

Alzheimer's disease (AD) is associated with damage to hippocampal neurons and declines in cognitive functions. The accumulation of amyloid peptides is regarded as a crucial event in the initiation of AD. The neurotoxicity induced by Aß25-35 peptides was used to screen for cytoprotective factors in vitro, and the cognitive deficits induced by the injection of Aß25-35 into the hippocampus were used to evaluate effect on learning and memory. Our previous study revealed that hydrolysate of polygalasaponins (HPS) clearly improve the cognitive deficits induced by the injection of Aß25-35 in mice, but the potential active constituent of HPS remains unclear. The purposes of this study were to separate and purify the secondary saponins of HPS, screen for neuroprotective effects of the constituents in vitro, and to evaluate the effect of cognition in vivo. Various chromatographic methods were used to separate and purify the HPS. The neuroprotective effects were examined in Aß25-35-damage-induced PC12 cells. The protective effect of tenuifolin on the cognitive impairments induced by Aß25-35 injection was assessed using the Morris water maze and step-through passive avoidance tests. Tenuifolin and fallaxsaponin A were isolated from the HPS. Tenuifolin possessed neuroprotective effects against Aß25-35-induced apoptosis in PC12 cells and significantly improved the cognitive deficits induced by the intrahippocampal injection of Aß25-35 in mice. Thus, tenuifolin is one of the active constituents of HPS against the neurotoxicity induced by Aß25-35 peptides in vitro and in vivo.

Neuroprotective effect of water extract of Panax ginseng on corticosterone-induced apoptosis in PC12 cells and its underlying molecule mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Panax ginseng C.A. Meyer (Family Araliaceae) is an important medicinal plant which has been employed as a panacea for more than 2,000 years in China. It has the actions of invigorating primordial qi, recovering pulse and desertion, engendering liquid, and calming spirit. The water extract of Panax ginseng (WEG) has been used to treat kinds of central nervous system disorders, such as depression, insomnia, Alzheimer׳s disease and Parkinson׳s disease. Our previous work has demonstrated that WEG possessed antidepressant-like activities in both acute and chronic stress models of depression. Nevertheless, there are no studies on the cytoprotection and potential mechanisms of WEG on corticosterone-induced apoptosis. The present study focuses on cytoprotection against corticosterone-induced neurotoxicity in PC12 cells and its underlying molecule mechanisms of the antidepressant-like effect of WEG. MATERIALS AND METHODS: The PC12 cells were treated with 250 µmol/L corticosterone in the absence or presence of WEG for 24h, then 3-(4,5-dimethy thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) detection, Hoechst33342 staining and TUNEL staining were investigated to confirm the neuroprotection of WEG. Then, mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), intracellular Ca(2+) ([Ca(2+)]i), reactive oxygen species (ROS) concentration, and the expression level of glucocorticoid receptor (GR), heat shock protein 90 (Hsp90), histone deactylase 6 (HDAC6), glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible protein 153 (GADD153), X-box DNA-binding protein-1 (XBP-1), caspase-12, cytochrome C, inhibitor of caspase-activated deoxyribonuclease (ICAD), caspase-3 and caspase-9 were assessed by Western Blot analysis to understand the molecule mechanisms of neuroprotection of WEG. RESULTS: WEG partly reversed corticosterone-induced damage in PC12 cells, which increased cell viability, decreased LDH release, and attenuated corticosterone-induced apoptosis as compared with the corticosterone-treated group. Mechanistically, compared with the corticosterone-treated group, WEG strongly attenuated [Ca(2+)]i overload and ROS level, and restored mitochondrial function, including mPTP and MMP. Furthermore, WEG strongly up-regulated the expression of GR and HDAC6, and down-regulated the expression of Hsp90, cytochrome C, ICAD, caspase-3, caspase-9 as well as endoplasmic reticulum (ER) stress-related proteins, such as GADD153, GRP78, XBP-1, and caspase-12. CONCLUSION: WEG possessed neuroprotection against corticosterone-induced damage in PC12 cells, and the underlying molecule mechanisms was depended on the intervening of HDAC6 and HSP90 of the GR-related function proteins, and subsequent restoration of ER and mitochondria functions.

Cajaninstilbene acid prevents corticosterone-induced apoptosis in PC12 cells by inhibiting the mitochondrial apoptotic pathway.

BACKGROUND/AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Cajaninstilbene acid protects corticosterone-induced injury in PC12 cells by inhibiting oxidative and endoplasmic reticulum stress-mediated apoptosis.

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.