Genome-wide characterization of the bHLH gene family in Gynostemma pentaphyllum reveals its potential role in the regulation of gypenoside biosynthesis.

BACKGROUND: Gynostemma pentaphyllum, an ancient Chinese herbal medicine, serves as a natural source of gypenosides with significant medicinal properties. Basic helix-loop-helix (bHLH) transcription factors play pivotal roles in numerous biological processes, especially in the regulation of secondary metabolism in plants. However, the characteristics and functions of the bHLH genes in G. pentaphyllum remain unexplored, and their regulatory role in gypenoside biosynthesis remains poorly elucidated. RESULTS: This study identified a total of 111 bHLH members in G. pentaphyllum (GpbHLHs), categorizing them into 26 subgroups based on shared conserved motif compositions and gene structures. Collinearity analysis illustrated that segmental duplications predominately lead to the evolution of GpbHLHs, with most duplicated GpbHLH gene pairs undergoing purifying selection. Among the nine gypenoside-related GpbHLH genes, two GpbHLHs (GpbHLH15 and GpbHLH58) were selected for further investigation based on co-expression analysis and functional prediction. The expression of these two selected GpbHLHs was dramatically induced by methyl jasmonate, and their nuclear localization was confirmed. Furthermore, yeast one-hybrid and dual-luciferase assays demonstrated that GpbHLH15 and GpbHLH58 could bind to the promoters of the gypenoside biosynthesis pathway genes, such as GpFPS1, GpSS1, and GpOSC1, and activate their promoter activity to varying degrees. CONCLUSIONS: In conclusion, our findings provide a detailed analysis of the bHLH family and valuable insights into the potential use of GpbHLHs to enhance the accumulation of gypenosides in G. pentaphyllum.

Gypenoside L inhibits hepatocellular carcinoma by targeting the SREBP2-HMGCS1 axis and enhancing immune response.

Hepatocellular carcinoma (HCC) is a malignant tumor that occurs in the liver, with a high degree of malignancy and relatively poor prognosis. Gypenoside L has inhibitory effects on liver cancer cells. However, its mechanism of action is still unclear. This study aims to investigate the inhibitory effects of gypenoside L on HCC in vitro and in vivo, and explore its potential mechanisms. The results showed that gypenoside L reduced the cholesterol and triglyceride content in HepG2 and Huh-7 cells, inhibited cell proliferation, invasion and metastasis, arrested cell cycle at G0/G1 phase, promoted cell apoptosis. Mechanistically, it targeted the transcription factor SREPB2 to inhibit the expression of HMGCS1 protein and inhibited the downstream proteins HMGCR and MVK, thereby regulating the mevalonate (MVA) pathway. Overexpression HMGCS1 led to significant alterations in the cholesterol metabolism pathway of HCC, which mediated HCC cell proliferation and conferred resistance to the therapeutic effect of gypenoside L. In vivo, gypenoside L effectively suppressed HCC growth in tumor-bearing mice by reducing cholesterol production, exhibiting favorable safety profiles and minimal toxic side effects. Gypenoside L modulated cholesterol homeostasis, enhanced expression of inflammatory factors by regulating MHC I pathway-related proteins to augment anticancer immune responses. Clinical samples from HCC patients also exhibited high expression levels of MVA pathway-related genes in tumor tissues. These findings highlight gypenoside L as a promising agent for targeting cholesterol metabolism in HCC while emphasizing the effectiveness of regulating the SREBP2-HMGCS1 axis as a therapeutic strategy.

Gypenoside A Protects Human Myocardial Cells from Ischemia/Reperfusion Injury via the circ_0010729/miR-370-3p/RUNX1 Axis.

Ischemia/reperfusion (I/R) injury is one of the major causes of cardiovascular disease. Gypenoside A (GP), the main active component of Gynostemma pentaphyllum, alleviates myocardial I/R injury. Circular RNAs (circRNAs) and microRNAs (miRNAs) are involved in the I/R injury. We explored the protective effect of GP on human cardiomyocytes (HCMs) via the circ_0010729/miR-370-3p/RUNX1 axis. Overexpression of circ_0010729 abolished the effects of GP on HMC, such as suppression of apoptosis and increase in cell viability and proliferation. Overexpression of miR-370-3p reversed the effect of circ_0010729 overexpression, resulting in the stimulation of HMC viability and proliferation and inhibition of apoptosis. The knockdown of miR-370-3p suppressed the effects of GP in HCMs. RUNX1 silencing counteracted the effect of miR-370-3p knockdown and maintained GP-induced suppression of apoptosis and stimulation of HMC viability and proliferation. The levels of RUNX1 mRNA and protein were reduced in cells expressing miR-370-3p. In conclusion, this study confirmed that GP alleviated the I/R injury of myocardial cell via the circ_0010729/miR-370-3p/RUNX1 axis.

New natural protein tyrosine phosphatase 1B inhibitors from Gynostemma pentaphyllum.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease mainly caused by insulin resistance, which can lead to a series of complications such as cardiovascular disease, retinopathy, and its typical clinical symptom is hyperglycaemia. Glucosidase inhibitors, including Acarbose, Miglitol, are commonly used in the clinical treatment of hypoglycaemia. In addition, Protein tyrosine phosphatase 1B (PTP1B) is also an important promising target for the treatment of T2DM. Gynostemma pentaphyllum is a well-known oriental traditional medicinal herbal plant, and has many beneficial effects on glucose and lipid metabolism. In the present study, three new and nine known dammarane triterpenoids isolated from G. pentaphyllum, and their structures were elucidated by spectroscopic methods including HR-ESI-MS,1H and 13C NMR and X-ray crystallography. All these compounds were evaluated for inhibitory activity against α-glucosidase, α-amylase and PTP1B. The results suggested that compounds 7∼10 were potential antidiabetic agents with significantly inhibition activity against PTP1B in a dose-dependent manner.

New dammarane-type triterpenoids from hydrolyzate of total Gynostemma pentaphyllum saponins with protein tyrosine phosphatase 1B inhibitory activity.

Protein tyrosine phosphatase 1B (PTP1B) is a key factor and regulator of glucose, lipid metabolism throughout the body, and a promising target for treatment of type 2 diabetes mellitus (T2DM). Gynostemma pentaphyllum is a famous oriental traditional medicinal herbal plant and functional food, which has shown many beneficial effects on glucose and lipid metabolism. The aim of the present study is to assess the inhibitory activity of five new and four known dammarane triterpenoids isolated from the hydrolysate product of total G. pentaphyllum saponins. The bioassay data showed that all the compounds exhibited significant inhibitory activity against PTP1B. The structure-activity relationship showed that the strength of PTP1B inhibitory activity was mainly related to the electron-donating group on its side chain. Molecular docking analysis suggested that its mechanism may be due to the formation of competitive hydrogen bonding between the electron-donating moiety and the Asp48 amino acid residues on the PTP1B protein.

Effects of gypenoside L-containing Gynostemma pentaphyllum extract on fatigue and physical performance: A double-blind, placebo-controlled, randomized trial.

This study was conducted to investigate the effect of Gynostemma pentaphyllum extract containing gypenoside L (GPE) on improving the cognitive aspects of fatigue and performance of the motor system. One hundred healthy Korean adults aged 19-60 years were randomized to the treatment (GPE for 12 weeks) and control groups, and efficacy and safety-related parameters were compared between the two groups. Maximal oxygen consumption (VO2 max) and O2 pulse were significantly higher in the treatment group than in the control group (p = 0.007 and p = 0.047, respectively). After 12 weeks, the treatment group showed significant changes such as decreases in the levels of free fatty acids (p = 0.042). In addition, there were significant differences in the rating of perceived exertion (RPE) (p < 0.05) and value of temporal fatigue between the treatment and control groups on the multidimensional fatigue scale (p < 0.05). Moreover, the level of endothelial nitric oxide synthase (eNOS) in the blood was significantly higher in the treatment group than in the control group (p = 0.047). In summary, oral administration of GPE has a positive effect on resistance to exercise-induced physical and mental fatigue.

The application of mixed stabilizing materials promotes the feasibility of the intercropping system of Gynostemma pentaphyllum/Helianthus annuus L. on arsenic contaminated soil.

Intercropping technology and stabilizing materials are common remediation techniques for soils contaminated with heavy metals. This study investigated the feasibility of the Gynostemma pentaphyllum (G. pentaphyllum)/Helianthus annuus L. (H. annuus) intercropping system on arsenic (As) contaminated farmland through field and pot experiments and the regulation of plant As absorption by the application of mixed stabilizing materials in this intercropping system. Field experiments demonstrated that intercropping with H. annuus increased the As concentration in G. pentaphyllum leaves to 1.79 mg kg-1 but still met the requirements of the national food standard of China (2 mg kg-1) (GB2762-2017). Meanwhile, G. pentaphyllum yield in the intercropping system decreased by 15.09%, but the difference was insignificant (P > 0.05). Additionally, the As bioconcentration (BCA) per H. annuus plant in the intercropping system was significantly higher than that in the monoculture system, increasing by 76.37% (P < 0.05). The pot experiment demonstrated that when granite powder, iron sulfate mineral, and "Weidikang" soil conditioner were applied to the soil collectively, G. pentaphyllum leaf As concentration in the intercropping system could be significantly reduced by 42.17%. Rhizosphere pH is the most crucial factor affecting As absorption by G. pentaphyllum in intercropping systems. When these three stabilizing materials were applied simultaneously, the As bioaccumulation (BCA) per H. annuus plant was significantly higher than that of normal intercropping treatment, which increased by 71.12% (P < 0.05), indicating that the application of these stabilizing materials significantly improved the As removal efficiency of the intercropping system. Dissolved organic carbon (DOC) concentration in the rhizosphere soil is the most pivotal factor affecting As absorption by H. annuus. In summary, the G. pentaphyllum-H. annuus intercropping model is worthy of being promoted in moderately As polluted farmland. The application of granite powder, iron sulfate mineral, and "Weidikang" soil conditioner collectively to the soil can effectively enhance the potential of this intercropping model to achieve "production while repairing" in the As polluted farmland.

The Content and Principle of the Rare Ginsenosides Produced from Gynostemma pentaphyllum after Heat Treatment.

Ginsenoside Rg3, Rk1, and Rg5, rare ginsenosides from Panax ginseng, have many pharmacological effects, which have attracted extensive attention. They can be obtained through the heat treatment of Gynostemma pentaphyllum. In this study, scanning electron microscopy (SEM) and thermal gravity-differential thermal gravity (TG-DTG) were employed to investigate this process and the content change in ginsenosides was analyzed using liquid chromatography-mass spectrometry (LC-MS). SEM and TG-DTG were used to compare the changes in the ginsenosides before and after treatment. In SEM, the presence of hydrogen bond rearrangement was indicated by the observed deformation of vascular bundles and ducts. The before-and-after changes in the peak patterns and peaks values in TG-DTG indicated that the content of different kinds of compounds produced changes, which all revealed that the formation of new saponins before and after the heat treatment was due to the breakage or rearrangement of chemical bonds. Additionally, the deformation of vascular bundles and vessels indicated the presence of hydrogen bond rearrangement. The glycosidic bond at the 20 positions could be cleaved by ginsenoside Rb3 to form ginsenoside Rd, which, in turn, gave rise to ginsenoside Rg3(S) and Rg3(R). They were further dehydrated to form ginsenoside Rk1 and Rg5. This transformation process occurs in a weak acidic environment provided by G. pentaphyllum itself, without the involvement of endogenous enzymes. In addition, the LC-MS analysis results showed that the content of ginsenoside Rb3 decreased from 2.25 mg/g to 1.80 mg/g, while the contents of ginsenoside Rk1 and Rg5 increased from 0.08 and 0.01 mg/g to 3.36 and 3.35 mg/g, respectively. Ginsenoside Rg3(S) and Rg3(R) were almost not detected in G. pentaphyllum, and the contents of them increased to 0.035 and 0.23 mg/g after heat treatment. Therefore, the rare ginsenosides Rg3(S), Rg3(R), Rk1, and Rg5 can be obtained from G. pentaphyllum via heat treatment.

Gypenosides Synergistically Reduce the Extracellular Matrix of Hepatic Stellate Cells and Ameliorate Hepatic Fibrosis in Mice.

Liver fibrosis resulting from chronic liver damage is becoming one of the major threats to health worldwide. Active saponin constituents isolated from Gynostemma pentaphyllum were found to possess a protective effect in liver diseases. Here, we obtained a naturally abundant gypenoside, XLVI, and evaluated its liver protection activity in both animal and cellular models. The results showed that it ameliorated acute and chronic liver injuries and lightened the process of fibrogenesis in vivo. XLVI can inhibit TGF-ß-induced activation of hepatic stellate cells and ECM deposition in vitro. The underlying mechanism study verified that it upregulated the protein expression of protein phosphatase 2C alpha and strengthened the vitality of the phosphatase together with a PP2Cα agonist gypenoside NPLC0393. These results shed new light on the molecular mechanisms and the potential therapeutic function of the traditional herb Gynostemma pentaphyllum in the treatment of liver fibrosis.

[Identification of terpene synthase gene family in Gynostemma pentaphyllum and expression pattern analysis under abiotic stresses].

The present study aimed to investigate the composition of the terpene synthase(TPS) gene family in Gynostemma pentaphyllum and its role in abiotic stresses. The G. pentaphyllum TPS gene family was identified and analyzed at the genome-wide level using bioinformatics analysis, and the expression patterns of these family members were analyzed in different tissues of G. pentaphyllum as well as under various abiotic stresses. The results showed that there were 24 TPS gene family members in G. pentaphyllum with protein lengths ranging from 294 to 842 aa. All of them were localized in the cytoplasm or chloroplasts and unevenly distributed on the 11 chromosomes of G. pentaphyllum. The results of the phylogenetic tree showed that the G. pentaphyllum TPS gene family members could be divided into five subfamilies. As revealed by the analysis of promoter cis-acting elements, TPS gene family members in G. pentaphyllum were predicted to respond to a variety of abiotic stresses such as salt, low temperature, and dark stress. The analysis of gene expression patterns in different tissues of G. pentaphyllum revealed that nine TPS genes were tissue-specific in expression. The qPCR results showed that GpTPS16, GpTPS17, and GpTPS21 responded to a variety of abiotic stresses. This study is expected to provide references in guiding the further exploration of the biological functions of G. pentaphyllum TPS genes under abiotic stresses.

[Comparison on anti-inflammatory activity of Gynostemma pentaphyllum processed with different methods].

The aim of this study is to investigate the effects of Gynostemma pentaphyllum dried with two different methods(air drying and heating) on inflammation in acute lung injury(ALI) mice in vivo and in vitro. Lipopolysaccharide(LPS) was sprayed into the airway of wild type C57BL/6J male mice to establish the model, and the drug was injected into the tail vein 24 h after modeling. Lung function, lung tissue wet/dry weight(W/D) ratio, the total protein concentration, interleukin 6(IL-6), IL-1ß, and tumor necrosis factor-α(TNF-α) in the bronchoalveolar lavage fluid(BALF), and pathological changes of the lung tissue were used to evaluate the effects of different gypenosides on ALI mice. The results showed that total gypenosides(YGGPs) and the gypenosides substituted with one or two glycosyl(GPs_(1-2)) in the air-dried sample improved the lung function, significantly lowered the levels of IL-1ß and TNF-α in BALF, and alleviated the lung inflammation of ALI mice. Moreover, GPs_(1-2) had a more significant effect on inhibiting NO release in RAW264.7 cells. This study showed that different drying methods affected the anti-inflammatory activity of G. pentaphyllum, and the rare saponins in the air-dried sample without heating had better anti-inflammatory activity.

Mechanisms of Gynostemma pentaphyllum against non-alcoholic fibre liver disease based on network pharmacology and molecular docking.

As a progressive chronic disease, the effective treatment for non-alcoholic fibre liver disease (NAFLD) has not yet been thoroughly explored at the moment. The widespread use of Gynostemma pentaphyllum (Thunb) for its anti-insulin resistance effect indicates that potential therapeutic value may be found in Thunb for NAFLD. Hence, this research aims to discover the latent mechanism of Thunb for NAFLD treatment. To achieve the goal of discovering the latent mechanism of Thunb for NAFLD treatment, molecular docking strategy integrated a network phamacology was adopted in the exploration. We acquire Thunb compounds with activeness from TCMSP database. We collect the putative targets of Thunb and NAFLD to generate the network. Key targets and mechanism are screened by PPI analysis, GO and KEGG pathway enrichment analyses. Molecular docking simulation is introduced into the study as assessment method. Through network analysis and virtual screening based on molecular docking, 2 targets (AKT 1 and GSK3B) are identified as key therapeutic targets with satisfying binding affinity. Main mechanism is believed to be the biological process and pathway related to insulin resistance according to the enrichment analyses outcomes. Particularly, the P13K-AKT signalling pathway is recognized as a key pathway of the mechanism. In conclusion, the study shows that Thunb could be a potential treatment against NAFLD and may suppress insulin resistance through the P13K-AKT signalling pathway. The result of the exploration provides a novel perspective for approaching experimental exploration.

GP-14 protects against severe hypoxia-induced neuronal injury through the AKT and ERK pathways and its induced transcriptome profiling alteration.

Gypenosides are major bioactive ingredients of G. pentaphyllum. In our previous study, we found that gypenosides had neuroprotective effects against hypoxia-induced injury. In the current study, we focused on the protective effects of gypenoside-14 (GP-14), which is one of the newly identified bioactive components, on neuronal injury caused by severe hypoxia (0.3% O2). The results showed that GP-14 pretreatment alleviated the cell viability damage and apoptosis induced by hypoxia in PC12 cells. Moreover, GP-14 pretreatment also attenuated primary neuron injuries under hypoxic conditions. Additionally, GP-14 pretreatment significantly ameliorated neuronal damage in the hippocampal region induced by high-altitude cerebral edema (HACE). At the molecular level, GP-14 pretreatment reversed the decreased activities of the AKT and ERK signaling pathways caused by hypoxia in PC12 cells and primary neurons. To comprehensively explore the possible mechanisms, transcriptome sequencing was conducted, and these results indicated that GP-14 could alter the transcriptional profiles of primary neuron. Taken together, our results suggest that GP-14 acts as a neuroprotective agent to protect against neuronal damage induced by severe hypoxia and it is a promising compound for the development of neuroprotective drugs.

Gypenoside A attenuates dysfunction of pancreatic ß cells by activating PDX1 signal transduction via the inhibition of miR-150-3p both in vivo and in vitro.

Saponin gypenoside A (GP) has shown its potential to handle diabetes mellitus. MicroRNA-150-3p (miR-150-3p) is closely related to the dysfunction of pancreatic ß cells by targeting PDX1. Given the function of GP is related to its regulation on different miRs, the current study assessed the role of miR-150-3p as a therapeutic target for the hypoglycemic effects of GP. Pancreatic ß cell dysfunction was induced in mice using the high-fatty diet (HFD) method and then handled with GP. Changes in insulin release and resistance and the activity of the miR-150-3p/PDX1 axis were detected. The expression of miR-150-3p was induced to confirm its central in the effects of GP. The results of in vivo tests were then validated with in vitro assays. HFD administration suppressed glucose tolerance, delayed insulin release, and induced insulin resistance and pancreas apoptosis in mice, which was indicative of the dysfunction of ß pancreatic cells. Changes in pancreatic ß function were associated with the increased expression of miR-150-3p and suppressed expression of PDX1. After the administration of GP, the impairments of the pancreas were alleviated and the expression of miR-150-3p was inhibited, contributing to the restored level of PDX1. The injection of miR-150-3p agomir counteracted the protective effects of GP. In in vitro assays, the pretransfection of miR-150-3p mimetics also counteracted the protective effects of GP on pancreatic ß cells against palmitic acid. Collectively, miR-150-3p played a key role in the protective effects of GP against pancreatic ß cell dysfunction by inhibiting PDX1 expression.

The effect of an orally-dosed Gynostemma pentaphyllum extract (ActivAMP®) on body composition in overweight, adult men and women: A double-blind, randomised, placebo-controlled study.

BACKGROUND: The present study examined the effect of a herbal supplement containing a Gynostemma pentaphyllum (Gpp) extract (ActivAMP®) with respect to improving body composition in overweight males and females. METHODS: One-hundred and seventeen men and women aged over 18 years completed 16 weeks of daily supplementation with either Gpp or a placebo. Participants underwent dual-energy X-rays to assess body composition (fat mass, lean mass and mass distribution), as well as anthropometric measures (weight, height, hip and waist circumference), in addition to blood tests to assess inflammatory and safety markers. RESULTS: Following 16 weeks of treatment, the Gpp group had a significant reduction in total body weight, body mass index, total fat mass and gynoid fat mass compared to the placebo group. Blood measures showed plasma triglyceride, alanine aminotransferase and tumour necrosis factor-α to be statistically different between groups at week 16. Subgroup analysis of gender for fat distribution showed males in the Gpp group had a significant reduction in visceral fat compared to males in the placebo group and females in the Gpp group had a significant reduction in gynoid fat compared to the placebo group. CONCLUSIONS: Gpp was capable of altering fat mass and fat distribution in overweight and obese males and females compared to a placebo.

Effects of Curcuma longa L., Eucommia ulmoides Oliv. and Gynostemma pentaphyllum (Thunb.) Makino on Cytokine Production in Stimulated Peripheral Blood Mononuclear Cells in Patients with Tuberculosis.

BACKGROUND: Tuberculosis (TB) infection triggers the innate and adaptive immune responses. Eucommia ulmoides Oliv., Gynostemma pentaphyllum (Thunb.) Makino, and Curcuma longa L. extracts exhibit various immunomodulatory effects. OBJECTIVE: This study aimed to determine the effects of 3 extracts used in Traditional Chinese Medicine (TCM) on cytokine production in peripheral blood mononuclear cells (PBMCs) obtained from patients with TB. DESIGN: The research team performed an in vitro study with self controls. SETTING: The study took place at the Department of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Taiwan. PARTICIPANTS: 18 patients diagnosed with pulmonary TB were enrolled in the study. INTERVENTION: Purified protein derivative (PPD)-stimulated PBMCs were cultured for 48 h in the presence and absence of 0.05 or 0.1 mg/mL of herbal extracts. OUTCOME MEASURES: Cytokine levels of interferon (IFN)-γ, interleukin (IL)-10, IL-12, tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-ß1 in the culture supernatant were measured. RESULTS: C longa L., E ulmoides Oliv. and G pentaphyllum (Thunb.) Makino extracts decreased IFN-γ production in PPD-stimulated PBMCs. C longa L. extract did not exhibit a marked and consistent effect on the production of IL-10, IL-12, TNF-α and TGF-ß1. E ulmoides Oliv. extract increased the production of IL-10, TNF-α and TGF-ß1. G pentaphyllum (Thunb.) Makino extract increased the production of IL-10, IL-12, TNF-α and TGF-ß1. CONCLUSION: These results show that G pentaphyllum (Thunb.) Makino might enhance cell immunity since it increased the production of IL-12 and TNF-α with dose effect.

A novel natural PPARγ agonist, Gypenoside LXXV, ameliorates cognitive deficits by enhancing brain glucose uptake via the activation of Akt/GLUT4 signaling in db/db mice.

Targeting the PPARγ might be a potential therapeutic strategy for diabetes-associated cognitive decline (DACD). In this study, Gypenoside LXXV (GP-75), a dammarane-type triterpene compound isolated from Gynostemma pentaphyllum, was found to be a novel PPARγ agonist using a dual-luciferase reporter assay system. However, whether GP-75 has protective effects against DACD remains unknown. Interestingly, intragastric administration of GP-75 (40 mg/kg/day) for 12 weeks significantly attenuated the cognitive deficit in db/db mice. GP-75 treatment significantly improved the glucose tolerance and lipid metabolism, and suppressed neuroinflammation. Notably, GP-75 treatment dramatically increased the uptake of glucose by the brain, as detected by 18 F-FDG PET. Incubation of primary cortical neurons with GP-75 significantly increased 2-deoxyglucose uptake. In addition, GP-75 treatment markedly increased the p-Akt (Ser 473)/total Akt levels and the expression levels of PPARγ and GLUT4, while decreasing the levels of p-IRS-1 (Ser 616)/total IRS-1. Importantly, all of these protective effects mediated by GP-75 were abolished by cotreatment with the PPARγ antagonist, GW9662. However, GP-75-mediated PPARγ upregulation was not affected by coincubation with the phosphatidylinositol 3-kinase inhibitor, LY294002. Collectively, GP-75 might be a novel PPARγ agonist that ameliorates cognitive deficit by enhancing brain glucose uptake via the activation of Akt/GLUT4 signaling in db/db mice.

Gypenoside A from Gynostemma pentaphyllum Attenuates Airway Inflammation and Th2 Cell Activities in a Murine Asthma Model.

Our previous study found that oral administration of Gynostemma pentaphyllum extract can attenuate airway hyperresponsiveness (AHR) and reduce eosinophil infiltration in the lungs of asthmatic mice. Gypenoside A is isolated from G. pentaphyllum. In this study, we investigated whether gypenoside A can effectively reduce asthma in mice. Asthma was induced in BALB/c mice by ovalbumin injection. Asthmatic mice were treated with gypenoside A via intraperitoneal injection to assess airway inflammation, AHR, and immunomodulatory effects. In vitro, gypenoside A reduced inflammatory and oxidative responses in inflammatory tracheal epithelial cells. Experimental results showed that gypenoside A treatment can suppress eosinophil infiltration in the lungs, reduce tracheal goblet cell hyperplasia, and attenuate AHR. Gypenoside A significantly reduced Th2 cytokine expression and also inhibited the expression of inflammatory genes and proteins in the lung and bronchoalveolar lavage fluid. In addition, gypenoside A also significantly inhibited the secretion of inflammatory cytokines and chemokines and reduced oxidative expression in inflammatory tracheal epithelial cells. The experimental results suggested that gypenoside A is a natural compound that can effectively reduce airway inflammation and AHR in asthma, mainly by reducing Th2 cell activation.

Effects of Plant Elicitors on Growth and Gypenosides Biosynthesis in Cell Culture of Giao co lam (Gynostemma pentaphyllum).

Giao co lam (Gynostemma pentaphyllum (Thunb.) Makino) is used in Northeast and Southeast Asia countries for the treatment of various diseases, including hepatitis, diabetes, and cardiovascular disease. G. pentaphyllum saponins (gypenosides) are the major components responsible for the pharmacological activities. In this study, different concentrations of abiotic (25-200 µM methyl jasmonate-MeJA and salicylic acid-SA) or biotic elicitors (1-5 g/L yeast extract-YE and Fusarium biomass) were used as plant elicitors, in order to investigate their influences on cell growth and gypenosides accumulation in G. pentaphyllum suspension cells. Suspension cells were grown on a MS medium containing 2.0 mg/L KIN and 0.5 mg/L IBA, with initial inoculum sizes of 3 g and shaking speeds of 120 rpm for 18 days. Gypenoside and Rb1 contents were measured by colorimetric and HPLC methods. Among three elicitors, SA was suitable for gypenosides accumulation in individual treatment. The cell biomass had the same values in elicitated and control suspension cells. Gypenosides content in cells treated with 100 µM salicylic acid after 6 days of culture reached a maximum value of 79.721 mg gypenoside/g dry biomass (including 0.093 mg ginsenoside Rb1/mg dry weight), which was 2.18-folds higher than that of the natural product. The elicitation promises an efficiency strategy for the production gypenosides in Gynostemma pentaphyllum suspension cells.

Enzymatic Biotransformation of Gypenoside XLIX into Gylongiposide I and Their Antiviral Roles against Enterovirus 71 In Vitro.

Biotransformation of specific saponins in the valuable medical plants to increase their bioavailability and pharmaceutical activities has attracted more and more attention. A gene encoding a thermophilic glycoside hydrolase from Fervidobaterium pennivorans DSM9078 was cloned and expressed in Escherichia coli. The purified recombinant enzyme, exhibiting endoglucanase cellulase activity, was used to transform gypenoside XLIX into gylongiposide I via highly selective and efficient hydrolysis of the glucose moiety linked to the C21 position in gypenoside XLIX. Under the optimal reaction conditions for large scale production of gylongiposide I, 35 g gypenoside XLIX was transformed by using 20 g crude enzyme at pH 6.0 and 80 °C for 4 h with a molar yield of 100%. Finally, 11.51 g of gylongiposide I was purified using a silica gel column with 91.84% chromatographic purity. Furthermore, inhibitory activities of gypenoside XLIX and gylongiposide I against Enterovirus 71 (EV71) were investigated. Importantly, the EC50 of gypenoside XLIX and gylongiposide I calculated from viral titers in supernatants was 3.53 µM and 1.53 µM, respectively. Moreover, the transformed product gylongiposide I has better anti-EV71 activity than the glycosylated precursor. In conclusion, this enzymatic method would be useful in the large-scale production of gylongiposide I, which would be a novel potent anti-EV71 candidate.