Effects Of Saponins From Schizocapsa Plantaginea Hance On Apoptosis And Cell Cycle Regulation In Hepatocellular Carcinoma Cells And Purification Of The Saponins

Objective:To investigate the effects of saponins from Schizocapsa plantaginea Hance(SSPH)on apoptosis and cell cycle regulation in hepatocellular carcinoma SMMC-7721 cells and its mechanism.To study the separation and purification of SSPH and establish the preparation method of saponin compounds from Schizocapsa plantaginea Hance,investigate the effect of saponin compounds on inhibition in hepatocellular carcinoma cells,validate the anti-hepatocarcinoma compounds from Schizocapsa plantaginea Hance.Method:After incubation of human hepatocellular carcinoma SMMC-7721 cells with varying concentrations of SSPH for 24h,apoptosis and cell cycle regulation were assessed by flow cytometry with Annexin V-PE/7-AAD double staining and PI staining,the activity of caspase-3,caspase-8,caspase-9 were measured by spectrophotometry.Schizocapsa plantaginea Hance tubers were extracted by reflux in 80%ethanol and the ethanol extract was extracted with petroleum ether,ethyl acetate and n-BuOH.Then n-BuOH fraction was adsorbed with D101 macroporous resin by gradient elution of ethanol.The effects of different fractions on inhibition in hepatocellular carcinoma BEL-7404 cells were measured by MTT assay,during the extraction and separation process of Schizocapsa plantaginea Hance.Ethanol fractions with high activity were separated by silica gel column chromatography,then purified by Sephadex LH-20,silica gel with repeatedly many times to obtain saponin compounds.The purity of compound was identified by HPLC and the structure was identified by NMR.The effect of saponin compound on inhibition in hepatocellular carcinoma SMMC-7721 cells was measured by MTT assay.Results:FCM indicated that apoptosis in SSPH groups were significantly higher than that in control group and early apoptosis was mainly induced by low concentration of SSPH in SMMC-7721 cells.The late apoptosis was gradually increased with the increase of SSPH and the effect of SSPH on apoptosis in SMMC-7721 cells was increased with the increase of concentration.The percentage of late apoptosis were(0.85±0.21)%and(31.67±3.34)%in control group and 15?g/mL SSPH group,the difference was significant(p<0.01).The percentage of G1/G0 phase in SSPH groups were significantly lower than that in control group,while the percentage of S phase and G2/M phase were significantly higher and the effect on cell cycle in SMMC-7721 cells was in a dose-dependent manner regulated by SSPH.The percentage of S phase and G2/M phase were(4.73±0.41)%,(16.03d±1.53)%in control group,respectively,and they were increased to(9.33±1.07)%,(32.58±2.49)%with 15?g/mL SSPH treatment,the difference was significant(p<0.01).The acti-vity of caspase-3,caspase-8,caspase-9 in SMMC-7721 cells were in a dose-dependent manner increased by the treatment of SSPH,compare with the control group,the difference was significant(p<0.01).The ethanol extract from Schizocapsa plantaginea Hance exhibited proliferation inhibitory activity in BEL-7404 cells and the IC50 was 95.4?g/mL.The effective components of ethanol extract were concentrated in n-BuOH fraction by extraction and the IC50 was 58.7?g/mL in BEL-7404 cells which was lower than ethanol extract.The n-BuOH fraction was adsorbed with D101 macroporous resin and the effective components were concentrated in 70%ethanol and 100%ethanol fractions.The IC50 were 4.5?g/mL,4.9?g/mL in BEL-7404 cells,respectively,which were significantly lower than n-BuOH fraction.70%ethanol fraction(2.0g)was purified with repeatedly many times to obtain taccaoside I(313.3mg)from Schizocapsa plantaginea Hance and the purity was 95.68%.Its structure was established as(25S)-spirost-5-en-3?-yl-O-?-L-rhamnopyranosyl-(1?2)-O-[O-?-D-glucopyran osyl-(1?4)-?-L-rhamnopyranosyl-(1?3)]-?-D-glucopyranoside by analysis of its 1H NMR and 13C NMR spectra.Taccaoside I exhibited obvious proliferation inhibitory activity in SMMC-7721 cells and the IC50 was 2.502?g/mL.Conclusion:1.SSPH can induce apoptosis in human hepatocellular carcinoma SMMC-7721 cells and arrest of cell cycle at S phase and G2/M phase nonspecifically,and its apoptosis underlying mechanism is maybe due to its ability to promote the expression of caspase.2.The separation of taccaoside I from Schizocapsa plantaginea Hance belongs to the first time and its preparation method is preliminary established.3.Taccaoside I can obviously inhibit the growth of human hepatocellular carcinoma cells and the effect is stronger than total saponins,and it is maybe the main effective component of Schizocapsa plantaginea Hance to anti-hepatic carcinoma.