Chemical Constituents From Gynostemma Pentaphyllum And Their Inhibitory Effect On SGC-7901 Cells

ObjectiveGynostemma pentaphyllum(Thunb.)Makino as a health product,has the effects of anti-tumor,anti-oxidation and anti-tumor.the main active components are saponins and flavonoids.Especially,there are saponins with strong anti-tumor activity in heat-treated G.pentaphyllum,but some isomers are difficult to separate.The aim of this study was to screen the effective components of heat-treated G.pentaphyllum with inhibitory activity on human gastric cancer SGC-7901 cells,and to develop its isolation method.Methods1.Compounds were separated by HP-20 macroporous adsorption resin,silica gel column chromatography,Sephadex LH-20 gel column,semi preparative high performance liquid chromatography and analytical high performance liquid chromatography,and compounds were identified by MS,~1D NMR and ~2D NMR.2.MTT method was used to detect the inhibitory effect of isolated monomer compounds on SGC-7901 cells;the effect of effective components on the clone formation ability of SGC-7901 cells was detected by cell clone formation experiment;Hoechst was used The morphological changes of SGC-7901 cells were observed by 33258 experiment;the effects of effective components on SGC-7901 cells were observed by JC-1 experiment;the migration of SGC-7901 cells was studied by scratch test and Transwell experiment.3.A new and efficient method for separating isomers in natural products was developed by acetylation modification and NaOH reduction.The optimum conditions were optimized by single factor test.Results1.Eleven compounds(1-11)were isolated from heat-processed G.pentaphyllum,which compound 1-8 were saponins and 9-11 were flavonoids.They were identified as 20(S)-ginsenoside Rg3(1),20(R)-ginsenoside Rg3(2),ginsenoside RK1(3),ginsenoside Rg5(4),gypenoside L(5),gypenoside LI(6),damulin B(7),damulin A(8),neocoplanoside(9),kaempferol(10),and lemono-3-o-βd-glucoside(11),respectively.Among them,compounds 3,4 and 9 were first isolated from G.pentaphyllum.2.MTT test indicated that the 8 saponins isolated from G.pentaphyllum showed inhibitory activities on SGC-7901 cells.The inhibition effect of gypenoside LI on SGC-7901 cells was the strongest with the IC50 value of 33.12 μM.The isomers,gypenoside L and gypenoside LI inhibited the ability of SGC-7901 cell cloning significantly,induced apoptosis of SGC-7901 cells through mitochondrial pathway,and inhibitd the migration of SGC-7901 cells.3.The two acetylation products with high substitution were obtained by acetylation of gypenoside L and gypenoside LI in the system of anhydrous pyridine acetic anhydride.The results showed that the physical and chemical properties of the two acetylation products were significantly different.After separation and purification with silica gel column,the two acetylation products were reduced with NaOH and gypeneposite was successfully gypenoside L and gypenoside LI achieve efficient separation.Through single factor test,the factors affecting the acetylation reaction were optimized,including the material ratio,reaction temperature and reaction time.The optimum reaction conditions were obtained:in the anhydrous pyridine solvent,the ratio of gypenoside L to acetic anhydride was 1:315,and the reaction time was 4 hours at 30℃.ConclusionEleven compounds were isolated from G.pentaphyllum,including ginsenoside Rk1,ginsenoside Rg5 and neocoplanoside for the first time.gypenoside L and gypenoside LI can significantly inhibit the formation of SGC-7901 cells,and induce apoptosis and cell migration of SGC-7901 cells through mitochondrial pathway.The physical and chemical properties of gypenoside L and gypenoside LI were modified in the anhydrous pyridine acetic anhydride system.After the separation and purification with silica gel column,the two acetylation products were reduced with NaOH,and gypenoside L and gypenoside LI were successfully obtained,and the high efficiency separation was achieved.