Steroidal Sapoins Isolated From Schizocapsa Plantaginea Hance And Its Effect On Apoptosis Of Human Hepatocellular Carcinoma Cells And Mechanism

Objective:1.To establish a method for separating and purifying steroidal saponin compounds from Schizocapsa plantaginea Hance?SPH?.To analysis the against human hepatocellular carcinoma cells effect of saponin compounds from SPH and confirm the specific components which are effective.2.To study the apoptosis induction effect and mechanism of human hepatocellular carcinoma Hep G2 and SMMC-7721 cells of a steroidal saponin compound??SPHS??and a steroidal saponin Compound??SPHS??from SPH.Methods:1.The tubers of SPH were extracted by 80%ethanol under atmospheric conditions and ethanol extract was extracted with petroleum ether,ethyl acetate and n-BuOH in turn.Then n-BuOH fraction was adsorbed with D101 macroporous resin by gradient elution of 50%-95%ethanol.75%ethanol fraction were separated and purified by Silica gel and Sephadex LH-20 column to obtain saponin compounds.The structures were identified by NMR technique.The effects of different fractions on inhibition in SMMC-7721 cells were measured by MTT assay.2.MTT assay and colony formation method were used to observe the inhibitory effect of SPHS?and SPHS?on proliferation of HepG2 and SMMC-7721 cells.Hoechst 33258 staining was used to observe the effect of SPHS?and SPHS?on the apoptotic morphology of Hep G2 and SMMC-7721 cells.The ultrastructure of HepG2 and SMMC-7721 cells treated with SPHS?and SPHS?was observed by transmission electron microscopy?TSEM?.3.The apoptosis,cell cycle,intracellular reactive oxygen species?ROS?and mitochondrial membrane potential?MMP?in HepG2 and SMMC-7721 cells treated with SPHS?and SPHS?were detected by flow cytometry?FCM?,and detected the above indicators after N-acetylcysteine?NAC?treatment.The expression of apoptosis-related proteins and MAPK signal pathway of HepG2and SMMC-7721 cells treated with SPHS?and SPHS?was detected by Western blot?WB?method.Results:1.The ethanol extract from SPH,n-BuOH site,75%ethanol and95%ethanol fractions inhibited the proliferation of SMMC-7721 cells,and the IC₅₀0 were 92.35,56.26,5.69,5.49?g/mL,respectively.75%ethanol fraction was purified by repeated separation to obtain SPHS?123.4 mg,SPHS?203.8mg and stigmasterol 546.8 mg.Its structure was determined as follows?25S?-spirost-5-en-3?-yl-O-?-L-rhamnopyranosyl-?1?2?-O-[O-?-D-glucopyran osyl-?1?4?-?-L-rhamnopyranosyl-?1?3?]-?-D-glucopyranoside,Yamogenin3-0-[?-L-rhamnopyranosyl-?1?3?]-[?-L-rhamnopyranosyl-?1?2?]-?-D-glucopyranoside and stigmasterol 3-O-?-D-gIucopyranoside by analysis of its ¹H NMR and ¹³C NMR spectra.2.SPHS?significantly inhibited the proliferation of HepG2 and SMMC-7721 cells,and the IC₅₀0 of 24 h were 0.91 and 1.00?M,respectively.SPHS?significantly inhibited the proliferation of HepG2 and SMMC-7721cells,and the IC₅₀0 of 24 h were 0.57 and 0.56?M,respectively.The colony formation assay showed that SPHS?and SPHS?significantly inhibited the proliferation of HepG2 and SMMC-7721 cells in a dose-dependent manner?P<0.001?.Hoechst 33258 staining showed that the nucleus of the control group was clear and complete,showing light blue fluorescence.In the administration group,significant morphological changes were observed,such as apoptotic bodies.TSEM results showed that the cell membrane and organellar structure of the control group were intact.While the cells of the administration group were shrunk,the chromatin and chromosome was condensed and concentrated in the cell membrane,resulting in a large number of apoptotic bodies and other apoptosis characteristics.The results of FCM showed that SPHS?and SPHS?induced apoptosis of HepG2 and SMMC-7721 cells in a dose-dependent manner?P<0.001?.And SPHS?and SPHSII blocked HepG2 and SMMC-7721 cells in G2 phase,in a dose-dependent manner?P<0.001?.While NAC partially blocked the apoptosis induction of HepG2 and SMMC-7721 cells by SPHS?and SPHS?,there was significant difference between NAC+SPHS??or SPHS??group and SPHS??or SPHS??group?P<0.001?.And NAC partially blocked the cycle arrest of HepG2 and SMMC-7721 cells by SPHS?and SPHS?,there was significant difference between NAC+SPHS??or SPHS??group and SPHS??or SPHS??group?P<0.001?.3.The results of FCM showed that SPHS?and SPHS?increased the ROS of HepG2 and SMMC-7721 cells in a dose-dependent manner?P<0.001?.And SPHS?and SPHS II decreased the MMP of Hep G2 and SMMC-7721cells in a dose-dependent manner?P<0.001?.The results of FCM showed that NAC partially blocked the increase of ROS in HepG2 and SMMC-7721 cells by SPHS?,NAC+SPHS?group and SPHS?group were significantly different?P<0.001?.And NAC partially blocked the decrease of MMP in HepG2 and SMMC-7721 cells by SPHS?and SPHS?,and there was significant difference between NAC+SPHS??or SPHS??group and SPHS??or SPHS??group?P<0.01 or P<0.05?.The results of WB showed that the expression in HepG2 and SMMC-7721 cells of cleaved caspase-3,-8,-9,Cyto-C and Bax protein was up-regulated by SPHS?and SPHS?,and the expression of Bcl-2and PARP protein was down-regulated.There was no significant effect in the expression of MAPK-related protein.But it significantly increased the phosphorylation level of Erk,P38 and JNK.Conclusions:1.Separating the steroidal saponin compound I,steroidal saponin compound?and the stigmasterol compound from SPH.Establishing a method for separating and purifying steroidal saponin compound I,steroidal saponin compound?from SPH.2.SPHS?and SPHS?can inhibite proliferation and induce apoptosis of human hepatocellular carcinoma Hep G2and SMMC-7721 cells,which may be mediated by ROS-mediated mitochondrial pathway and by the regulation of MAPK signaling pathway.