Genome-wide identification and characterization of the JAZ gene family in Gynostemma pentaphyllum reveals the COI1/JAZ/MYC2 complex potential involved in the regulation of the MeJA-induced gypenoside biosynthesis.

The Jasmonate ZIM domain (JAZ) proteins, functioning as critical suppressors for jasmonic acid (JA) signal transduction in plants, occupy crucial roles in multiple biological processes, particularly in the orchestration of secondary metabolic pathways. However, the mechanism underlying the JA-induced gypenosides accumulation in Gynostemma pentaphyllum remains poorly elucidated. Our research led to the identification of 11 distinct JAZ members in G. pentaphyllum (GpJAZs). According to the classification approach of AtJAZ, we allocated these members into five subgroups that shared similar conserved motif compositions. Subsequently, we identified the presence of various cis-acting elements associated with light stimuli, hormone responses, and stress signals within the promoter regions of the GpJAZ gene family. The expression levels of GpJAZ genes in different tissues were quite different, and the majority of GpJAZ genes exhibited varying degrees of response to methyl jasmonate (MeJA) induction. Yeast two-hybrid (Y2H) assays revealed interactions between GpJAZ1/2/4/5/7/9/10 and GpMYC2, whereas GpCOI1 protein was found to interact with GpJAZ1/2/4/5, thereby forming the COI1/JAZ/MYC2 complex. Furthermore, as an activator of gypenoside metabolic pathway, GpMYC2 could activate the promoter activity of the gypenoside metabolism-related genes to varying degrees by binding to their promoters, indicating that the COI1/JAZ/MYC2 module involved in the MeJA-induced regulation of gypenosides. In summary, our findings present an exhaustive examination of the JAZ gene family, furnishing a significant lead for delving deeper into the molecular mechanisms that drive the MeJA-induced enhancement of gypenosides accumulation in G. pentaphyllum.

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 XIII regulates lipid metabolism in HepG2 hepatocytes and ameliorates nonalcoholic steatohepatitis in mice.

Gypenoside XIII is isolated from Gynostemma pentaphyllum (Thunb.) Makino. In mice, G. pentaphyllum extract and gypenoside LXXV have been shown to improve non-alcoholic steatohepatitis (NASH). This study investigated whether gypenoside XIII can regulate lipid accumulation in fatty liver cells or attenuate NASH in mice. We used HepG2 hepatocytes to establish a fatty liver cell model using 0.5 mM oleic acid. Fatty liver cells were treated with different concentrations of gypenoside XIII to evaluate the molecular mechanisms of lipid metabolism. In addition, a methionine/choline-deficient diet induced NASH in C57BL/6 mice, which were given 10 mg/kg gypenoside XIII by intraperitoneal injection. In fatty liver cells, gypenoside XIII effectively suppressed lipid accumulation and lipid peroxidation. Furthermore, gypenoside XIII significantly increased SIRT1 and AMPK phosphorylation to decrease acetyl-CoA carboxylase phosphorylation, reducing fatty acid synthesis activity. Gypenoside XIII also decreased lipogenesis by suppressing sterol regulatory element-binding protein 1c and fatty acid synthase production. Gypenoside XIII also increased lipolysis and fatty acid ß-oxidation by promoting adipose triglyceride lipase and carnitine palmitoyltransferase 1, respectively. In an animal model of NASH, gypenoside XIII effectively decreased the lipid vacuole size and number and reduced liver fibrosis and inflammation. These findings suggest that gypenoside XIII can regulate lipid metabolism in fatty liver cells and improve liver fibrosis in NASH mice. Therefore, gypenoside XIII has potential as a novel agent for the treatment of NASH.

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.

Gypenosides ameliorate high-fat diet-induced nonalcoholic fatty liver disease in mice by regulating lipid metabolism.

Gypenosides (GP), extracted from the traditional Chinese herb Gynostemma pentaphyllum (Thunb.) Makino, have been used to treat metabolic disorders, including lipid metabolism disorders and diabetes. Although recent studies have confirmed their beneficial effects in nonalcoholic fatty liver disease (NAFLD), the underlying therapeutic mechanism remains unclear. In this study, we explored the protective mechanism of GP against NAFLD in mice and provided new insights into the prevention and treatment of NAFLD. Male C57BL6/J mice were divided into three experimental groups: normal diet, high-fat diet (HFD), and GP groups. The mice were fed an HFD for 16 weeks to establish an NAFLD model and then treated with GP for 22 weeks. The transcriptome and proteome of the mice livers were profiled using RNA sequencing and high-resolution mass spectrometry, respectively. The results showed that GP decreased serum lipid levels, liver index, and liver fat accumulation in mice. Principal component and heatmap analyses indicated that GP significantly modulated the changes in the expression of genes associated with HFD-induced NAFLD. The 164 differentially expressed genes recovered using GP were enriched in fatty acid and steroid metabolism pathways. Further results showed that GP reduced fatty acid synthesis by downregulating the expression of Srebf1, Fasn, Acss2, Acly, Acaca, Fads1, and Elovl6; modulated glycerolipid metabolism by inducing the expression of Mgll; promoted fatty acid transportation and degradation by inducing the expression of Slc27a1, Cpt1a, and Ehhadh; and reduced hepatic cholesterol synthesis by downregulating the expression of Tm7sf2, Ebp, Sc5d, Lss, Fdft1, Cyp51, Nsdhl, Pmvk, Mvd, Fdps, and Dhcr7. The proteomic data further indicated that GP decreased the protein expression levels of ACACA, ACLY, ACSS2, TM7SF2, EBP, FDFT1, NSDHL, PMVK, MVD, FDPS, and DHCR7 and increased those of MGLL, SLC27A1, and EHHADH. In conclusion, GP can regulate the key genes involved in hepatic lipid metabolism in NAFLD mice, providing initial evidence for the mechanisms underlying the therapeutic effect of GP in NAFLD.

Gynostemma pentaphyllum polysaccharides ameliorate non-alcoholic steatohepatitis in mice associated with gut microbiota and the TLR2/NLRP3 pathway.

Recent studies have revealed the pivotal role of gut microbiota in the progress of liver diseases including non-alcoholic steatohepatitis (NASH). Many natural herbs, such as Gynostemma pentaphyllum (GP), have been extensively applied in the prevention of NASH, while the bioactive components and underlying mechanism remain unclear. The aim of this study was to investigate whether the polysaccharides of GP (GPP) have a protective effect on NASH and to explore the potential mechanism underlying these effects. C57BL/6 male mice were fed with a methionine-choline-deficient (MCD) diet for 4 weeks to induce NASH and administered daily oral gavage of sodium carboxymethylcellulose (CMC-Na), low dose of GPP (LGPP), high dose of GPP (HGPP), and polyene phosphatidylcholine capsules (PPC), compared with the methionine-choline-sufficient (MCS) group. Our results showed that the symptoms of hepatic steatosis, hepatocyte ballooning, liver fibrosis, and oxidative stress could be partially recovered through the intervention of GPP with a dose-dependent effect. Furthermore, gut microbiome sequencing revealed that HGPP altered the composition of gut microbiota, mainly characterized by the enrichment of genera including Akkermansia, Lactobacillus, and A2. Moreover, hepatic transcriptome analysis indicated that the anti-inflammatory effect of HGPP might be associated with toll-like receptor (TLR) and nod-like receptor (NLR) signaling pathways. HGPP could inhibit the expression of TLR2 and downregulate the expression of the NLRP3 inflammasome, as well as the pro-inflammatory cytokine tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. In summary, GPP could ameliorate NASH possibly mediated via the modulation of gut microbiota and the TLR2/NLRP3 signaling pathway, indicating that GPP could be tested as a prebiotic agent in the prevention of NASH.

Cardioprotective Effect of Gynostemma pentaphyllum against Streptozotocin Induced Cardiac Toxicity in Rats via Alteration of AMPK/Nrf2/HO-1 Pathway.

Gynostemma pentaphyllum (GP) is a plant commonly used in diabetic therapy in China. GP having potent antioxidant effect against various free radicals. The purpose of the current investigation to identify the cardioprotective effect of GP against streptozotocin (STZ)/ high fat diet (HFD) induced cardiac dysfunction in rats via alteration of AMPK/Nrf2/HO-1 pathway. Wistar rats were used for the current protocol. The rats were received the intraperitoneal injection of STZ and HFD to induce the cardiac remodelling. Blood glucose level, insulin and lipid parameters were estimated. Blood pressure and heart rate were also estimated. Cardiac parameters, antioxidant, cytokines, total protein and inflammatory mediators were analysed. The mRNA expression was detected using the RT-qPCR, respectively. GP significantly (p < 0.001) decreased the BGL and improved the insulin level. GP altered the ratio of heart/BW, liver/BW, and lung/BW. GP treatment significantly (p < 0.001) suppressed the heart rate and blood pressure (diastolic, systolic and mean pressure). GP significantly (p < 0.001) reduced the level of TC, LDL, TG, VLDL and increased the level of HDL. DCM induced rats received the GP administration exhibited reduction in the level of CK and LDH. GP significantly (p < 0.001) reduced the levels of MDA, hydrogen peroxide, peroxynitrite, ROS and increased the level of GSH, SOD, CAT and GPx. GP significantly (p < 0.001) reduced the levels of cytokines (TNF-α, IL-6, IL-1ß) and inflammatory parameters (COX-2 and NFκB). GP significantly (p < 0.001) suppressed the NLRP3 and NF-κB expression. GP also boosted mitochondrial biogenesis by boosting the PGC-1α, HO-1 and Nrf2 expression in cardiac tissue. GP treatment showed the cardioprotective effects against STZ induced diabetic cardiac dysfunction via alteration of AMPK/Nrf2/HO-1 pathway.

Gynostemma pentaphyllum and Gypenoside-IV Ameliorate Metabolic Disorder and Gut Microbiota in Diet-Induced-Obese Mice.

Gynostemma pentaphyllum (G. pentaphyllum) is a perennial liana herb of the Cucurbitaceae family which has both nutraceutical and pharmacological functions. The objective of the current study was to investigate the preventative effects of G. pentaphyllum and Gypenoside-IV (GP-IV, a saponin monomer in G. pentaphyllum) on metabolic symptoms in high fat diet induced obese (DIO) mice with gut microbiota dysbiosis. G. pentaphyllum water extract (GPWE, 150 mg/kg•d- 1) and GP-IV (50 mg/kg•d- 1) were orally administered to DIO mice by gavage for 10 weeks. The results showed that both GPWE and GP-IV prevented obesity development by decreasing body weight gain, reducing fat mass/body weight ratio and inhibiting adipocyte hypertrophy. GPWE and GP-IV also improved lipid profile and glucose tolerance effectively. Moreover, GPWE and GP-IV treatments partly restored gut microbiota in DIO mice. Typically, GPWE and GP-IV reduced Firmicutes to Bacteroidetes ratio, increased the abundance of certain health-promoting bacteria and reduced the abundance of microbiota that were associated with metabolic disorders. We conclude that GPWE and GP-IV can ameliorate metabolic symptoms possibly via modulating gut microbiota in DIO mice.

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.

Gypenosides regulate autophagy through Sirt1 pathway and the anti-inflammatory mechanism of mitochondrial autophagy in systemic lupus erythematosus.

To study the mechanism of gynostemma pentaphyllum saponins (GpS) regulating mitochondrial autophagy and anti-inflammatory through Sirtuin 1 (Sirt1) pathway in systemic lupus erythematosus (SLE). JURKAT cells were cultured in vitro, RT-PCR and western blotting (WB) were utilized to identify the expression of related-proteins in Sirt1 pathway and global autophagy and mitochondrial autophagy markers in JURKAT before and after GpS treatment induced by ultraviolet B (UVB), and the related-mechanism of GpS regulation of autophagy was analyzed. The SLE model was established to analyze the alleviating effects of GpS on various symptoms of lupus mice. Sirt1/AMPK/mTOR pathway was activated in UVB induced JURKAT cells. After the addition of GpS, WB revealed that the phosphorylation of AMPK decreased, the phosphorylation of mTOR increased, the expression of Sirt1 protein decreased, and the activation of the pathway was inhibited. Moreover, autophagy of JURKAT cells wasinhibited. In order to further verify the role of Sirt1 pathway, we activated Sirt1 expression in cells by constructing lentiviral vectors, and the therapeutic effect of GpS was significantly reduced. These results indicate GpS can exert autophagy regulation by inhibiting the activity of Sirt1 pathway. To treat SLE. GpS can significantly reduce the level of autoantibodies, kidney inflammation, immune complex deposition and urinary protein excretion, improve kidney function in lupus-prone mice. GpS can regulate autophagy and mitochondrial autophagy through Sirt1 pathway, which may be a potential mechanism for GpS to reduce the level of autoantibodies, kidney inflammation, immune complex deposition and urinary protein excretion, improve kidney function in lupus-prone mice.

Gene excavation and expression analysis of CYP and UGT related to the post modifying stage of gypenoside biosynthesis in Gynostemma pentaphyllum (Thunb.) Makino by comprehensive analysis of RNA and proteome sequencing.

Previous studies have revealed that gypenosides produced from Gynostemma pentaphyllum (Thunb.) Makino are mainly dammarane-type triterpenoid saponins with diverse structures and important biological activities, but the mechanism of diversity for gypenoside biosynthesis is still unclear. In this study, a combination of isobaric tags for relative and absolute quantification (iTRAQ) proteome analysis and RNA sequencing transcriptome analysis was performed to identify the proteins and genes related to gypenoside biosynthesis. A total of 3925 proteins were identified by proteomic sequencing, of which 2537 were quantified. Seventeen cytochrome P450 (CYP) and 11 uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) candidate genes involved in the side chain synthesis and modification of gypenosides were found. Seven putative CYPs (CYP71B19, CYP77A3, CYP86A7, CYP86A8, CYP89A2, CYP90A1, CYP94A1) and five putative UGTs (UGT73B4, UGT76B1, UGT74F2, UGT91C1 and UGT91A1) were selected as candidate structural modifiers of triterpenoid saponins, which were cloned for gene expression analysis. Comprehensive analysis of RNA sequencing and proteome sequencing showed that some CYPs and UGTs were found at both the transcription and translation levels. In this study, an expression analysis of 7 CYPs and 5 UGTs that contributed to gypenoside biosynthesis and distribution in G. pentaphyllum was performed, providing consistent results that will inspire more future research on vital genes/proteins involved in gypenoside biosynthesis.

Identification of three groups of ginsenoside biosynthetic UDP-glycosyltransferases from Gynostemma pentaphyllum.

Ginsenosides are glycosylated dammarene-type triterpenes that have been identified in distantly related Panax ginseng and Gynostemma pentaphyllum. The phylogenetic relatedness of the ginsenoside biosynthetic genes in the two species was previously unknown. The final steps of ginsenoside biosynthesis are the glycosylations of hydroxylated triterpenes, protopanaxadiol (PPD) and protopanaxatriol (PPT), and their glycosylated forms by UDP-glycosyltransferases (UGTs). Ginsenoside biosynthetic UGTs have been identified in Panax but not in Gynostemma. Through a biochemical screening of Gynostemma UGTs (GpUGTs), we herein identified three groups of ginsenoside biosynthetic GpUGTs. These groups comprise: two GpUGTs that belong to the UGT71 family and glucosylate the C20-OH positions of PPD- and PPT-type ginsenosides; one GpUGT that belongs to the UGT74 family and glucosylates the C3-OH position of PPD-type ginsenosides; and two GpUGTs that belong to the UGT94 family and add a glucose to the C3-O-glucosides of PPD-type ginsenosides. These GpUGTs belong to the same UGT families as the ginsenoside biosynthetic Panax UGTs (PgUGTs). However, GpUGTs and PgUGTs belong to different subfamilies. Furthermore, cucumber UGTs orthologous to GpUGTs do not glucosylate ginsenosides. These results collectively suggest that, during evolution, P. ginseng and G. pentaphyllum independently opted to use the same UGT families to synthesize ginsenosides.

The triterpenoid sapogenin (2α-OH-Protopanoxadiol) ameliorates metabolic syndrome via the intestinal FXR/GLP-1 axis through gut microbiota remodelling.

Gypenosides, extracts of Gynostemma yixingense, have been traditionally prescribed to improve metabolic syndrome in Asian folk and local traditional medicine hospitals. However, the mechanism of its action remains unclarified. In this work, our results indicated that chronic administration of 2α-OH-protopanoxadiol (GP2), a metabolite of gypenosides in vivo, protected mice from high-fat diet-induced obesity and improved glucose tolerance by improving intestinal L-cell function. Mechanistically, GP2 treatment inhibited the enzymatic activity of bile salt hydrolase and modulated the proportions of the gut microbiota, which led to an increase in the accumulation of tauro-ß-muricholic acid (TßMCA) in the intestine. TßMCA induced GLP-1 production and secretion by reducing the transcriptional activity of nuclear receptor farnesoid X receptor (FXR). Transplantation of GP2-remodelled fecal microbiota into antibiotic-treated mice also increased the intestinal TßMCA content and improved intestinal L-cell function. These findings demonstrate that GP2 ameliorates metabolic syndrome at least partly through the intestinal FXR/GLP-1 axis via gut microbiota remodelling and also suggest that GP2 may serve as a promising oral therapeutic agent for metabolic syndrome.

Integrated transcriptome and miRNA analysis uncovers molecular regulators of aerial stem-to-rhizome transition in the medical herb Gynostemma pentaphyllum.

BACKGROUND: Gynostemma pentaphyllum is an important perennial medicinal herb belonging to the family Cucurbitaceae. Aerial stem-to-rhizome transition before entering the winter is an adaptive regenerative strategy in G. pentaphyllum that enables it to survive during winter. However, the molecular regulation of aerial stem-to-rhizome transition is unknown in plants. Here, integrated transcriptome and miRNA analysis was conducted to investigate the regulatory network of stem-to-rhizome transition. RESULTS: Nine transcriptome libraries prepared from stem/rhizome samples collected at three stages of developmental stem-to-rhizome transition were sequenced and a total of 5428 differentially expressed genes (DEGs) were identified. DEGs associated with gravitropism, cell wall biosynthesis, photoperiod, hormone signaling, and carbohydrate metabolism were found to regulate stem-to-rhizome transition. Nine small RNA libraries were parallelly sequenced, and seven significantly differentially expressed miRNAs (DEMs) were identified, including four known and three novel miRNAs. The seven DEMs targeted 123 mRNAs, and six pairs of miRNA-target showed significantly opposite expression trends. The GpmiR166b-GpECH2 module involved in stem-to-rhizome transition probably promotes cell expansion by IBA-to-IAA conversion, and the GpmiR166e-GpSGT-like module probably protects IAA from degradation, thereby promoting rhizome formation. GpmiR156a was found to be involved in stem-to-rhizome transition by inhibiting the expression of GpSPL13A/GpSPL6, which are believed to negatively regulate vegetative phase transition. GpmiR156a and a novel miRNA Co.47071 co-repressed the expression of growth inhibitor GpRAV-like during stem-to-rhizome transition. These miRNAs and their targets were first reported to be involved in the formation of rhizomes. In this study, the expression patterns of DEGs, DEMs and their targets were further validated by quantitative real-time PCR, supporting the reliability of sequencing data. CONCLUSIONS: Our study revealed a comprehensive molecular network regulating the transition of aerial stem to rhizome in G. pentaphyllum. These results broaden our understanding of developmental phase transitions in plants.

Hybrid sequencing of the Gynostemma pentaphyllum transcriptome provides new insights into gypenoside biosynthesis.

BACKGROUND: Gypenosides are a group of triterpene saponins from Gynostemma pentaphyllum that are the same as or very similar to ginsenosides from the Panax species. Several enzymes involved in ginsenoside biosynthesis have been characterized, which provide important clues for elucidating the gypenoside biosynthetic pathway. We suppose that gypenosides and ginsenosides may have a similar biosynthetic mechanism and that the corresponding enzymes in the two pathways may have considerable similarity in their sequences. To further understand gypenoside biosynthesis, we sequenced the G. pentaphyllum transcriptome with a hybrid sequencing-based strategy and then determined the candidate genes involved in this pathway using phylogenetic tree construction and gene expression analysis. RESULTS: Following the PacBio standard analysis pipeline, 66,046 polished consensus sequences were obtained, while Illumina data were assembled into 140,601 unigenes with Trinity software. Then, these output sequences from the two analytical routes were merged. After removing redundant data with CD-HIT software, a total of 140,157 final unigenes were obtained. After functional annotation, five 2,3-oxidosqualene cyclase genes, 145 cytochrome P450 genes and 254 UDP-glycosyltransferase genes were selected for the screening of genes involved in gypenoside biosynthesis. Using phylogenetic analysis, several genes were divided into the same subfamilies or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis. Using real-time PCR technology, their expression patterns were investigated in different tissues and at different times after methyl jasmonate induction. Since the genes in the same biosynthetic pathway are generally coexpressed, we speculated that GpOSC1, GpCYP89, and GpUGT35 were the leading candidates for gypenoside biosynthesis. In addition, six GpWRKYs and one GpbHLH might play a possible role in regulating gypenoside biosynthesis. CONCLUSIONS: We developed a hybrid sequencing strategy to obtain longer length transcriptomes with increased accuracy, which will greatly contribute to downstream gene screening and characterization, thus improving our ability to elucidate secondary metabolite biosynthetic pathways. With this strategy, we found several candidate genes that may be involved in gypenoside biosynthesis, which laid an important foundation for the elucidation of this biosynthetic pathway, thus greatly contributing to further research in metabolic regulation, synthetic biology and molecular breeding in this species.

Research on the Potential Mechanism of Gypenosides on Treating Thyroid-Associated Ophthalmopathy Based on Network Pharmacology.

Thyroid-associated ophthalmopathy is the commonest orbital disease in adults. However, shortcomings still exist in treatments. The aim of this study was to identify the efficacy and potential mechanism of gypenosides in the treatment of thyroid-associated ophthalmopathy. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform was screened for active compounds of gypenosides, and targets were predicted using Swiss Target Prediction. The targets of thyroid-associated ophthalmopathy were obtained from Online Mendelian Inheritance in Man, Comparative Toxicogenomic Database and GeneCards Human gene database. Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome Pathways were determined based on the common targets. Protein-protein interaction (PPI) network was constructed to further understand of relationship among target genes, compounds and proteins. Molecular docking was performed to investigate the binding ability between gypenosides and hub genes. A total of 70 targets for gypenosides and 804 targets for thyroid-associated ophthalmopathy were obtained with 8 common targets identified. GO analysis and KEGG pathway analysis revealed that the hub genes were enriched in JAK-STAT, while Reactome pathways analysis indicated genes enriched in interleukin pathways. PPI network showed STAT1, STAT3, and STAT4 were at the center. Additionally, molecular docking indicated that STAT1 and STAT3 display good binding forces with gypenosides. This study indicates that target genes mainly enriched in JAK-STAT signaling pathway, particularly in STATs, which can be combined with gypenosides. This may suggest that gypenosides have curative effect on thyroid-associated ophthalmopathy via the JAK-STAT pathway.

Metabolomic mechanisms of gypenoside against liver fibrosis in rats: An integrative analysis of proteomics and metabolomics data.

AIMS: To investigate mechanisms and altered pathways of gypenoside against carbon tetrachloride (CCl4)-induced liver fibrosis based on integrative analysis of proteomics and metabolomics data. METHODS: CCl4-induced liver fibrosis rats were administrated gypenoside. The anti-fibrosis effects were evaluated by histomorphology and liver hydroxyproline (Hyp) content. Protein profiling and metabolite profiling of rats liver tissues were examined by isobaric tags for relative and absolute quantitation (iTRAQ) approach and gas chromatography-mass spectrometer (GC-MS) technology. Altered pathways and pivotal proteins and metabolites were searched by integrative analysis of proteomics and metabolomics data. The levels of some key proteins in altered pathways were determined by western blot. RESULTS: Histopathological changes and Hyp content in gypenoside group had significant improvements (P<0.05). Compared to liver fibrosis model group, we found 301 up-regulated and 296 down-regulated proteins, and 9 up-regulated and 8 down-regulated metabolites in gypenoside group. According to integrative analysis, some important pathways were found, including glycolysis or gluconeogenesis, fructose and mannose metabolism, glycine, serine and threonine metabolism, lysine degradation, arginine and proline metabolism, glutathione metabolism, and sulfur metabolism. Furthermore, the levels of ALDH1B1, ALDH2 and ALDH7A1 were found increased and restored to normal levels after gypenoside treated (P<0.05). CONCLUSIONS: Gypenoside inhibited CCl4-induced liver fibrosis, which may be involved in the alteration of glycolysis metabolism and the protection against the damage of aldehydes and lipid peroxidation by up-regulating ALDH.

Biotransformation of gypenoside XVII to compound K by a recombinant ß-glucosidase.

OBJECTIVE: To study the ß-glucosidase gene (bgy1) from Lactobacillus brevis that was cloned and expressed in Escherichia coli BL21 (DE3) and then using it for the biotransformation of gypenoside XVII. RESULTS: The bgy1 gene consists of 2283 bp encoding 761 amino acids, with homology to the glycosyl hydrolase family-3 protein domain. The enzyme (Bgy1) hydrolyzed the glucose moieties at the C-3 position and the outer glucose moieties at the C-20 position of gypenoside XVII. Using 0.1 mg enzyme ml(-1) in 20 mM sodium phosphate buffer at 30 °C and pH 6.0, 1 mg gypenoside XVII ml(-1) was transformed into 0.58 mg compound K ml(-1) within 6 h, with a corresponding molar conversion yield of 89 %. CONCLUSION: The recombinant Bgy1 is considered potentially useful for the practical preparation of compound K.

Simultaneous determination of gypenoside LVI, gypenoside XLVI, 2α-OH-protopanaxadiol and their two metabolites in rat plasma by LC-MS/MS and its application to pharmacokinetic studies.

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