Research On Chemical Composition And Antiinflammatory Activity Of Gypenosides From Tetraploid Gynostemma Pentaphyllum

Gynostemma pentaphyllum（Thunb.Makino）is a natural plant with abundant biological activity and is widely distributed in China and many countries in Southeast Asian.As a medicinal and edible plant material,gynostemma pentaphyllum is taken worldwide as food or medicine to prevent or treat some diseases such as hyperlipidemia,hyperglycemia,atherosclerosis and even cancer,all of which are associated with inflammation.Gypenosides is the main active ingredient of gynostemma pentaphyllum.In recent years,research on activities,especially the anti-inflammatory activity of gypenosides has received extensive attention.In this study,the leaves of tetraploid and diploid gynostemma pentaphyllum（origin:Pingli County,Shanxi Province）were used as raw materials to extract and isolate total saponins,and the chemical composition of these saponions were analyzed.Then the LPS-stimulated RAW264.7 macrophage cells were used as an inflammatory model to investigate the anti-inflammatory activity of gypenosides of tetraploid gynostemma pentaphyllum in vitro,and the mechanism of its anti-inflammatory activity was explored.After the four steps of extraction and separation including ethanol extraction,extraction,macroporous resin separation and semi-preparative HPLC separation,the gypenosides of tetraploid and diploid gynostemma pentaphyllum accounted for 7.43 and2.05 mg/g dry leaves,respectively.In addition,UPLC/Q-TOF-MS analysis showed that the gypenosides of tetraploid gynostemma pentaphyllum were composed of four major saponins including isomers of gypenoside（GP）1 and GP 2(C₄₇H₇₆O₁₈),GP3(C₄₇H₇₆O₁₇),and GP4(C₄₆H₇₄O₁₇),of which the peak areas were 14.95%,15.28%,20.02%and 44.84%,respectively.And the gypenosides of diploid gynostemma pentaphyllum were composed of five major saponins including isomers of gypenoside（GP）1 and GP2(C₄₇H₇₆O₁₈),GP3(C₄₇H₇₆O₁₇),GP4(C₄₆H₇₄O₁₇)and GP(C₄₆H₇₄O₁₆),of which the peak areas were 7.82%,16.13%,15.47%,28.79%and 16.87%,respectively.In vitro experiments showed that,in LPS-induced RAW264.7 macrophages cells,tetraploid gypenosides significantly inhibited NO release and pro-inflammatory factor TNF-αmRNA expression at concentrations of 50,100,150 and 200μg/mL（p<0.01）,inhibited the protein secretion of the pro-inflammatory factor TNF-αat concentrations of 100,150 and 200μg/mL（p<0.01）,and inhibited the pro-inflammatory factors IL-6,IL-1β,and COX-2 mRNA expression and IL-6 protein secretion levels at concentrations of 150 and 200μg/m L（p<0.01）.And the inhibition above all performed in a dose-dependent manner.Besides,gypenosides（100μg/mL）also inhibited mRNA expression of MCP-1 with no significant dose dependence.Moreover,studies on the mechanism of anti-inflammatory activity indicate that tetraploid gypenosides（200μg/mL）might down-regulate the activation and phosphorylation of IκB kinase（IKK）and MAPKs signaling proteins（JNK and ERK1/2）,thereby significantly inhibiting the activation and nuclear translocation of NF-κB p65and AP-1（c-Fos and c-Jun）downstream of them in LPS-induced RAW264.7macrophages cells,and thus relieving inflammation.These results suggested that the gypenosides from tetraploid gynostemma pentaphyllum might have potential anti-inflammatory effect and use for improving human health,providing a basis for the development of food and medicine related to Gynostemma pentaphyllum.