Mechanism of gypenoside ⅩⅦ against cerebral ischemia/reperfusion injury based on nuclear factor erythroid 2-related factor 2/antioxidant responsive element signaling pathway / 中华危重病急救医学

Objective:To explore the mechanism of gypenoside ⅩⅦ against cerebral ischemia/reperfusion (I/R) through nuclear factor erythroid 2-related factor 2/antioxidant responsive element (Nrf2/ARE) signaling pathway.Methods:Forty SPF Sprague Dawley (SD) rats were randomly divided into sham operated group, I/R model group, 25, 50 and 100 mg/kg gypenoside ⅩⅦ groups ( n = 8). Gypenoside ⅩⅦ groups were administered 25, 50 or 100 mg/kg (0.01 mL/g) gypenoside ⅩⅦ by intragastric administration for 14 days; the other two groups received the same dose of saline. Rat cerebral I/R model was established by modified line bolt method; rats in the sham operated group underwent the same procedure without producing substantial embolization. After 24 hours of reperfusion, the neurological deficit scores of the rats in each group were assessed. Rat abdominal aortic whole blood was collected and the serum reactive oxygen species (ROS), heme oxygenase-1 (HO-1), γ-glutamylcysteine synthase (γ-GCS), superoxide dismutase (SOD), quinone NADH oxidoreductase 1 (NQO1), and malondialdehyde (MDA) were detected. Then whole brain tissue was harvested and penumbra tissue was isolated from cerebral cortex, the general condition of rat brain tissue and the volume of cerebral infarction were evaluated, the histopathological changes in the brain were observed under light microscopy, the mRNA expressions of Nrf2 and Keap1 were measured by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR), the protein expressions of Nrf2 and Keap1 were determined by Western blotting. Results:After 24 hours of reperfusion, compared with the sham operated group, the score of neurological deficit and infarct volume were significantly increased, the NQO1, SOD and γ-GCS levels in serum were significantly decreased, MDA, HO-1 and ROS levels in serum were significantly increased, the Nrf2 and Keap1 mRNA and protein expressions in the ischemic penumbra were significantly increased in rats from I/R model group. Compared with the I/R model group, the neurological deficit scores (1.50±0.53, 1.37±0.52 vs. 2.75±0.46) and brain infarct volume [(19.8±5.1)%, (21.4±6.4)% vs. (42.3±5.8)%] were significantly reduced, serum NQO1, SOD, HO-1 and γ-GCS were significantly increased [NQO1 (ng/L): 186.05±10.38, 220.75±16.22 vs. 131.36±5.95, SOD (kU/L): 63.23±5.30, 72.70±8.62 vs. 36.75±6.55, HO-1 (ng/L): 60.57±7.93, 60.35±4.72 vs. 42.72±4.95, γ-GCS (kU/L): 8.81±0.53, 8.72±0.69 vs. 6.80±0.56], serum MDA and ROS levels were significantly reduced [MDA (μmol/L): 5.94±0.66, 5.61±0.53 vs. 10.88±1.34, ROS (kU/L): 69.11±4.23, 67.12±4.52 vs. 104.43±7.54], the mRNA and protein expressions of Nrf2 and Keap1 in the ischemic penumbra were significantly increased in rats from 50 mg/kg and 100 mg/kg gypenoside ⅩⅦ groups [Nrf2 mRNA (2 -△△Ct): 1.90±0.13, 2.13±0.18 vs. 1.48±0.11, Keap1 mRNA (2 -△△Ct): 1.78±0.11, 1.85±0.10 vs. 1.43±0.10, Nrf2/β-actin: 0.73±0.04, 0.79±0.03 vs. 0.60±0.03, Keap1/β-actin: 0.71±0.01, 0.76±0.03 vs. 0.61±0.01], all the comparative differences were statistically significant (all P < 0.01); 25 mg/kg gypenoside ⅩⅦ had no significant effect. Conclusion:Gypenoside ⅩⅦ (50 mg/kg and 100 mg/kg) may play a role in anti-cerebral I/R injury by regulating NQO1, SOD, HO-1, γ-GCS, ROS and MDA through Nrf2/ARE signaling pathway.

Dammarane-type triterpenoid saponins isolated from Gynostemma pentaphyllum ameliorate liver fibrosis via agonizing PP2Cα and inhibiting deposition of extracellular matrix / 中国天然药物

Gypenosides, structurally analogous to ginsenosides and derived from a sustainable source, are recognized as the principal active compounds found in Gynostemma pentaphyllum, a Chinese medicinal plant used in the treatment of the metabolic syndrome. By bioactive tracking isolation of the plants collected from different regions across China, we obtained four new gypenosides (1-4), together with nine known gypenosides (5-13), from the methanol extract of the plant. The structures of new gypenosides were elucidated by one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectra, complemented by chemical degradation experiments. Through comprehensive evaluation involving COL1A1 promoter assays and PP2Cα activity assays, we established a definitive structure-activity relationship for these dammarane-type triterpenoids, affirming the indispensability of the C-3 saccharide chain and C-17 lactone ring in effectively impeding extracellular matrix (ECM) deposition within hepatic stellate cells. Further in vivo study on the CCl4-induced liver damage mouse model corroborated that compound 5 significantly ameliorated the process of hepatic fibrosis by oral administration. These results underscore the potential of dammarane-type triterpenoids as prospective anti-fibrotic leads and highlight their prevalence as key molecular frameworks in the therapeutic intervention of chronic hepatic disorders.

Conversion of gypenosides by enzymatic hydrolysis with β-glucanase / 国际药学研究杂志

Objective: To investigate the enzymatic hydrolysis of gypenosides by β-glucanase 26130 CN to prepare some hydrolyzed secondary saponins, so as to provide material basis for further biological studies. Methods: Using β-glucanase 26130 CN, the total saponins from Herba Gynostemmatis were hydrolyzed with the enzyme catalysis, and the hydrolytic products were analyzed by ultra high- performance liquid chromatography coupled with quadrupole time- of- flight mass spectrometry(UHPLC- Q- TOF/MSE)to identify the converted products. Then, the main components of Herba Gynostemmatis, gypenosides XLIX and A, were used as substrates of the β-glucanase 26130 CN for convertsion to secondary saponin products. The products were separated by preparative highperformance liquid chromatography(HPLC)and identified by NMR and MS. Results: Twenty eight triterpenoid saponins were identified in the total saponin hydrolysate on the basis of their high-resolution MS data, by comparison with the data in the literature, and seven of them were validated to be the converted products. It was found that the β-glucanase 26130 CN could hydrolyze the glycosidic bond of terminal glucose or xylose in the molecule of gypenosides. By the enzymatic hydrolysis of gypenoside XLIX and gypenoside A, gypenoside I(the one glucosyl-lost gypenoside XLIX)and gypenoside UL1(the one xylosyl-lost gypenoside A)were obtained via the preparative HPLC separation of the gypenoside XLIX and gypenoside A hydrolysates, respectively. Conclusion: β-glucanase 26130 CN could effectively catalyze the hydrolysis of terminal glucosyl and xylosyl groups in gypenosides, with a relatively high hydrolytic conversion rate, which could be used to prepare some secondary saponins or aglycones.

Study on quality standard of gypenosides extract and Gypenosides Tablets / 中国中药杂志

In response to no national standard for Gynostemma pentaphyllum, a market survey was carried out, and 17 batches of gypenosides extract and 29 batches of Gypenosides Tablets on the market were collected. With gypenoside A as an index, the TLC qualitative identification and HPLC quantitative evaluation method of gypenosides extract and tablets was established. Based on the determination results of 17 batches of gypenosides extract and 29 batches of Gypenosides Tablets, the quality standards of gypenosides extract and tablets were formulated respectively, so as to give suggestions for improving the quality standards of gypenosides extract and tablets. Compared with the existing ministerial standards, the qualitative identification and quantitative detection of specific components were added, in order to provide scientific basis and suggestions for the revision of the quality standard of gypenosides extract and tablet preparation.

Simultaneous determination of five constituents in Jiangzhi Granules by HPLC / 中成药

AIM To establish an HPLC method for the simultaneous content determination of five constituents in Jiangzhi Granules (Salviae miltiorrhizae Radix et Rhizoma,Polygoni cuspidati Rhizoma et Radix,Artemisiae scopariae Herba,etc.).METHODS The analysis of methanol extract of this drug was performed on a 30 ℃ thermostatic Hanbon-C18 column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile (A) 0.1％ phosphoric acid (B) flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelengths were set at 286 nm (0-30,40-50 min) and 203 nm (30-40 min).RESULTS Gypenoside A,chlorogenic acid,polydatin,salvianolic acid B and emodin showed good linear relationships within the ranges of 0.176-2.46,0.056-0.84,0.36-5.40,0.192-2.88 and 0.12-1.80 μg (r ＞0.999 0),respectively,whose average recoveries were 97.17％-102.59％ with the RSDs of 1.9％-2.9％.CONCLUSION This accurate and simple method can be used for the quality control of Jiangzhi Granules.

Effect of gypenoside on lipopolysaccharide-mediated microglial inflammatory response / 国际脑血管病杂志

Objective To investigate the effect of gypenoside on lipopolysaccharide (LPS)-mediated inflammatory response. Methods The BV2 microglia cell line was cultured in vitro. The BV2 microglia cells were divided into four groups: normal control, LPS (10 ng/ml), GP + LPS (GP 20 μg/ml, LPS 10 ng/ml), and GP (20 μg/ml). After 24 h cultivation, ELISA was used to detect the levels of tumor necrosis factor α(TNF-α), interleukin (IL)-1β, and IL-6. Immunocytochemistry staining and Western blot were used to detect the expression levels of nuclear factor (NF-κB) and suppressor of cytokine signaling 1 (SOCS-1). Results Compared with the normal control group, the release of TNF-α, IL-1β and IL-6, as well as the expression level of NF-κB in the LPS group were increased significantly (all P 0. 05 ). Conclusions GP can significantly inhibit the LPS-mediated microglial inflammatory response. SOCS-1 protein may be involved in GP inhibiting LPS-mediated microglial inflammatory response.

The anti-proliferation effect of gypenosides on cervical cancer HeLa cells and its molecular mechanism / 肿瘤

Objective: To investigate the inhibitory effect of gypenosides on proliferation of human uterine cervical cancer HeLa cells in vitro, and to explore its possible mechanism. Methods: The HeLa cells were treated with different concentrations of gypenosides (low-dose group, 4.5 μg/mL; middle-dose group, 45 μg/mL; high-dose group, 450 μg/mL). The cells treated with 0.9% sodium chloride solution was designed as a control group. The inhibitory effect of gypenosides on the proliferation of HeLa cells was detected by MTT assay and BrdU (5-bromo-2-deoxyuridine) incorporation experiment. The effect of gypenosides on apoptosis of HeLa cells was detected by FCM (flow cytometry). The expression levels of Bcl-2, Bax, ERK1/2 (extracellular regulated protein kinase 1/2), p-ERK1/2 (phospho-ERK1/2), MEK1/2 (mitogen-activated protein kinase kinase) and p-MEK1/2 were detected by Western blotting. Results: The survival rate of HeLa cells was decreased significantly after treatment with gypenosides (45 μg/mL) at 24 h (P < 0.01). The survival rate of HeLa cells was decreased significantly after treatment with gypenosides (4.5 μg/mL) at 48 h (P < 0.01). The inhibitory effect of gypenosides (450 μg/mL) on the cell proliferation was confirmed by BrdU incorporation experiment. The apoptotic rate of HeLa cells was increased significantly after treatment with different concentrations of gypenosides. Gypenosides could down-regulate the expression levels of Bcl-2 and p-ERK1/2 and up-regulate the expression level of Bax. Conclusion: Gypenosides can significantly inhibit the proliferation of cervical cancer HeLa cells and also markedly induce the apoptosis. This effect may related to the up-regulation of Bax expression and down-regulation of Bcl-2 and p-ERK1/2 expressions. Copyright © 2013 by TUMOR.

Effects of gypenosides on nuclear factor κB and p38 mitogen activated protein kinase signaling pathways in photodamaged skin of mice / 中华皮肤科杂志

Objective To observe the effects of gypenosides (GP) on nuclear factor κB (NF-κB) and p38 mitogen activated protein kinase (p38MAPK) signaling pathways in photodamaged skin of mice,and to explore the mechanisms underlying the protective effects of GP against photodamage.Methods Eighty Balb/C female mice were randomly divided into 8 groups: blank control group receiving no treatment,ultraviolet B (UVB) model group receiving UVB irradiation for 60 seconds,GP group Ⅰ receiving topical GP treatment followed by UVB irradiation,GP group Ⅱ receiving UVB irradiation followed by topical GP treatment,VitE group Ⅰ receiving topical VitE treatment followed by UVB irradiation,VitE group Ⅱ receiving UVB irradiation followed by topical VitE treatment,matrix group Ⅰ receiving topical matrix treatment followed by UVB irradiation,matrix group Ⅱ receiving UVB irradiation followed by topical matrix treatment.UVB irradiation lasted 60 seconds at one time,and was given once every other day for 7 times to establish a skin model of photodamage.The interval between irradiation and topical treatment was 30 minutes in all the groups except the control group and UVB model group.After the last treatment,mice were sacrificed.Western blot was performed to measure the protein expressions of inhibitor of NF-κB(IκB),inhibitor of NF-κB kinase (IKK),p38MAPK as well as phosphorylated p38MAPK (pp38) in skin tissue from the mice.Results No expressions of IκB or IKK were observed in the blank control group.The expression level of IκB was 0.40 ± 0.07 in UVB model group,significantly lower than that in GP group Ⅰ (1.63 ± 0.85,P ＜ 0.05) and GP group Ⅱ (0.90 ± 0.40,P ＜ 0.05),whereas the level of IKK protein was higher in UVB model group than in the GP group Ⅰ and Ⅱ (2.01 ± 1.75vs.0.23 ± 0.12 and 0.45 ± 0.29,both P ＜ 0.05).No significant difference was observed in the expression of IκB or IKK proteins between the GP group Ⅰ,Ⅱ,VitE group Ⅰ and Ⅱ or in the expression of p38MAPK between the 8 groups.The phosphorylated p38MAPK expression in UVB model group was significantly higher than that in GP group Ⅰ and Ⅱ (0.835 ± 0.049 vs.0.425 ± 0.054 and 0.571 ± 0.090,both P＜ 0.05),but similar to that in VitE groups.Conclusions UVB can activate NF-κB and phosphorylated p38MAPK signaling pathways; GP 1.5％ cream can inhibit UVB-induced activation of NF-κB and p38MAPK pathways,which may be one of the mechanisms underlying its protective effects against inflammation and photodamage.

A New Triterpene Saponin from Gynostemma pentaphyllum / 中草药·英文版

Objective To study the triterpene saponins from Gynostemma pentaphyllum with antitumor activities.Methods The 75% EtOH extract of G.pentaphyllum was used for isolation by silica gel column chromatography and preparative HPLC.The structures of pure compounds isolated were identified by the spectral analysis and chemical evidence.Results Two compounds were isolated and identified as 23(S)-3β,20ξ,21ξ-trihydroxy-19-oxo-21,23-epoxydammar-24-ene 3-O-α-L-rhanmopyranosyl(1→2)-[β-D-xylopyranosyl(1→β)]-β-D-arabinopyranoside(1)and23(S)-21(R)-O-n-butyl-3β,20ξ-dihydroxy-21,23-epoxydammar-24-ene 3-O-α-L-rhamnopyranosyl(1→2)-[β-D-xylo-pyranosyl(1→3)]-β-D-arabinopyrannside(2).Conclusion Compound 2 is a new triterpene saponin with moderate antitumor activities against the HL-60,Colon205,and Du145 cell lines.

Regulation of gypenosides on TGF-?/Smad signaling pathway and expression of connective tissue growth factor in unilateral ureteral obstruction rats with renal tubulointerstitial fibrosis / 中国医师杂志

Objective To investigate the expression transforming growth factor?1(TGF-?1),Smad7 and connective tissue growth factor(CTGF) in rats with renal tubulointerstitial fibrosis and the effects of Gypenosides(GPs) treatment on expression of TGF-?,Smad7 and CTGF.Methods Unilateral ureteral Obstruction(UUO) rat animal models were used and the rats were divided into GPs group(Gypenosides 200 mg?Kg~(-1)?d~(-1) by gastric gavage from 3d before the obstruction to 14 days after the induction),model group and sham operated group.The expression of TGF-?1,Smad7 and CTGF in the obstructed kidneys was assessed by RT-PCR,immunohistochemistry and HE staining methods after 3,7,and 14d respectively.The degree of tubulointerstitial damage was scored by Masson staining methods.Results Compared with the sham operated group,the expressions of TGF-?1,CTGF mRNA and protein were markedly increased in UUO group,and the expression of Smad7 mRNA and protein was markedly decreased(all P

Technology of extracting gypenoside from crude Herba Gynostemmae Pentaphylli solution by foaming separation / 中草药

Objective To study the method of extracting the gypenoside from crude Herba Gynostemmae Pentaphylli solution.Methods The gypenoside was extracted from crude Herba Gynostemmae Pentaphylli solution by foaming separation.The optimum conditions were obtained through calculating the recovery rate and enrichment ratio.Results Under the optimum conditions:the concentration of the gypenoside in crude Herba Gynostemmae Pentaphylli solution was 3.21 ?g/mL,the gas flow rate was 30 mL/min,the volume of the solution was 110 mL,and pH value was 9.0 as well,the recovery rate of gypenoside was 49.20% and the enrichment ratio could reach 4.511.Conclusion Foam separation is a simple and effective method of extracting gypenoside from crude Herba Gynostemmae Pentaphylli solution.

Protective effect of gypenoside on brainstem ischemic injuries in dogs / 中国药理学通报

The brainstem ischemic injuries were produced by the occlusion of the basilar artery for 6 h in dogs. Gypenoside (150 mg ?kg-1)were intradudinally given at 3 h before the occlusion. We discovered that gypenoside alleviated the ischemic neuronal damage (histopathologically evaluated by the light and electronic microscope), ameliorated the abnormal changes of brainstem auditory evoked potentials (BAEP) (F

PROTECTIVE EFFECTS OF GYPENOSIDES ON RAT HEPATIC LIPID PEROXIDATION AND MEMBRANE FLUIDITY DAMAGE: IN VITRO STUDIES / 中国药理学通报

The present in vitro study on rat liver microsomal lipid per-oxidation showed that Gypenosides ( GP ) , the total saponin of Gy-nostemma pentaphyllum Makino, produced a marked inhibitory effect on the lipid peroxidation either spontaneously or induced by Fe2+-cystein, Vit C-NADPH and CCl4. The inhibitory effect was found to be stronger than that of Ginsenosides. GP also resisted the decrease of fluidity of liver microsomal and mitochondrial membrane induced by Fe2+-cystein.

MEMORY FACILITATION INDUCED BY GYNOSTEMMA PENTAPHYLLUM AND GYPENOSIDE Ⅲ ( GINSENOSIDER b_1 ) IN MICE / 中国药理学通报

Aquatic, 20% alcoholic & 95% alcoholic extractions of the aeri -al parts of Gynostemma pentaphyllum from Guangdong Provin -ce contain several gypenosides as same as some ginsenosides. The effects of the extractions and gypenoside Ⅲ (ginsenoside Rb 1 ) on memory in mice were studied using one trial passive avoidance responses. Results indicated that Gynostemma pentaphyllum improved impairment of acquisition of memory produced by anisodine, and disruption of consolidation of memory caused by cycloheximide and chlo-ramphenicol. Gynostemma was capable of antagonizing alcoh cl-induc- ed deficit of retrieval. Similar effects were observed with Rb1 in the impairments of acquisition of memory caused by alcohol and pen-tobarbitone sodium. It is clear that Gynostemma pentaphyllum may be an effective memory-enhancing agent.