Chemical Constituents From Gynostemma Pentaphyllum And Discriminant Analysis Of Gynostemma Pentaphyllum Sapoins From Different Regions

ObjectiveGynostemma pentaphyllum(Thunb.)Makino belongs to Cucurbitaceae family.It is a traditional Chinese herbal medicine in China and has a long history of medicinal use.It is one of plants with ginsenosides besides of Araliaceae family and has many functions such as clearing away heat and toxin,stopping coughing,anti-cancer,anti-aging,and improving lipid metabolism.It is famous as "Southern ginseng".The objective of this study is to expect to discover new compounds and enrich the chemical constituents of G.pentaphyllum by targeting isolation and structure identification;set the oxidative damage model of human neuroblastoma cancer SH-SY5Y cells induced by hydrogen peroxide(H2O2)and evaluate the neuroprotective activity of the new saponins;identify the main chromatographic peaks of G.pentaphyllum by LC-MS and distinguish the G.pentaphyllum in different region by multi variable analysis and pattern recognition.Methods1.The new compounds from G.pentaphyllum were discovered combined of literature comparison and LC-MS analysis.The constituents of G.pentaphyllum were taget isolated by different chromatography such as silica gel,macroporous resin,middle pressure chromatography,semi-preparative and analytical HPLC.They were identified using physical and chemical properties,UV,MS,NMR and CD.2.The inhibitory activity of novel saponins on human non-small cell lung cancer A549 cells,hepatocarcinoma HepG2 cells and neuroblastoma SH-SY5Y cells were investigated by MTT assay.The oxidative damage model of neuroblastoma SH-SY5Y cells induced by H2O2 was established and the neuroprotective effect new saponins of different concentrations were evaluated.3.Inertsil ODS-SP(4.6 ×250 mm,5 μm)C18 column was eluted with acetonitrile-0.1%formic acid as mobile phase gradient.The detection wavelength was 203 nm.The mass spectrometry equipped with electrospray ionization(ESI)was used as detector and operated under the negative ion mode.Twenty one batches of G.pentaphyllum produced in Guangxi,Fujian and Shaanxi were analyzed by cluster analysis,principal component analysis and corresponding analysis.Results1.Ten compounds(compounds 1-10)were isolated from G.pentaphyllum produced in Pingli,Shaanxi province.From them,five compounds(compound 1-5)were new compounds and compounds 2,3 and 7 were isomers.The 10 compounds were identified as gypenoside S1(1),gypenoside S3(2),gypenoside S2(3),gypenoside S4(4)and gypenoside S5(5),gypenoside XLIX(6),gypenosides VN2(7),ombuin(8),ombuin-3-O-β-D-glucopyranoside(9),ombuoside(10),respectively.2.The new saponins showed weak cytotoxicity to A549 cells,HepG2 cells and SH-SY5Y cells with less than 10%inhibition at 100 μM.However,they showed neuroprotective activities on the oxidative damage of SH-SY5Y cells induced by H2O2.The survival of SH-SY5Y cells induced by H2O2 at 250 μM was 65.3%and the new saponins treatment group increased the survival rate by 2.5-19.7%with concentration dependent.3.The fingerprints of G.pentaphyllum produced in Guangxi,Fujian and Shanxi were established by LC-MS,and 9 common peaks and 10 characteristic peaks were calibrated.According to the mass spectrometryinformation and literature comparison,16 chromatographic peaks were qualitatively analyzed.Combined with cluster analysis,principal component analysis and correspondence analysis,G.pentaphyllum from different region was distinguished using the common peaks and characteristic peaks.ConclusionTen components were isolated and elucidated from G.pentaphyllum.Among them,five gypenosides were new saponins,which has somewhat enriched chemical structures of gypenosides.The anti-cancer activity of novel gypenosides was much weak,but all showed neuroprotection,which provided drug candidates for neuroprotection in clinic.The LC-MS and pattern recognition analysis can be used to distinguish G.pentaphyllum from different regions and the targeting compounds of interest would be separated.