The Characteristic Spectrum Of Saponins Of Schizocapsa Plantaginea Hance And The Antitumor Activity Or Screening Of Effective Components In Vitro

Objectives:1.To detect ingredient of saponins of Schizocapsa plantaginea Hance by comparing High Performance Liquid Chromatography with Ultraviolet and Evaporative Light Scattering Detector,and to establish the characteristic spectra of total saponins of Schizocapsa plantaginea Hance.Acetonitrile-water mobile phase gradient Elution,finding the main anti-tumor active ingredients in total saponins,and investigated the stability of saponins of Schizocapsa plantaginea Hance ethanol aqueous solution.2.To establish the characteristic spectra of saponins of Schizocapsa plantaginea Hance(SFSP_60%)were established by High Performance Liquid Chromatography with Ultraviolet and Evaporative Light Scattering Detector,to investigate the interaction of various effective components in saponins and its effect on the anti-hepatoma activity in vitro.3.The use of total saponins of Schizocapsa plantaginea Hance,compounds SPHSI and SPHSII screened tumor cells that could be inhibited proliferation activity and their preliminary study on apoptosis of human non-small cell lung cancer H460 and A549.Methods:1.80%ethanol was used to extract Schizocapsa plantaginea Hance tubers under normal pressure,The ethanol extracts were extracted with petroleum ether,ethyl acetate and n-butanol in turn.The n-butanol group was enriched the saponins with D101 macroporous resin,according to 50%⁹5.Ethanol gradient elution to collect the saponins;The 60%ethanol fraction was separated and purified by silica gel column and Sephadex LH-20 gel column to obtainsaponinsofSchizocapsaplantagineaHancecompound.2.High-performance liquid chromatography（UV）coupled with ELSD was used to establish the ELSD characteristic chromatogram of steroidal saponin in Schizocapsa plantaginea Hance,and the stability of the aqueous solution of saponins of Schizocapsa plantaginea Hance ethanol in refrigerator at 4°C for half a year was observed.At the same time,the MTT assay was used to observe effect of SFSP_60%on the proliferation of human hepatocellular BEL-7404 cells and the the inhibitory effect of SFSP_60%,SPHSI and SPHSII on the proliferation of human hepatoma SMMC-7721 cells.3.MTT assay was used to observe the inhibitory effects of SFSP_Total,SPHSⅠand SPHSⅡon the proliferation of non-small cell lung cancer H460 and A549,human ovarian cancer cell SKOV3and human nasopharyngeal carcinoma cell CNE-1.The effects of SFSP_Total,SPHSⅠand SPHSⅡon the apoptotic morphology of human non-small cell lung cancer H460 and A549 were observed by AO-EB staining.Results:1.After 22.3kg of roots of the Schizocapsa plantaginea Hance were extracted,extracted,and eluted with D101 drilling resin to obtain 42.79 g of total saponins（The content is 98.56%）.The 60%ethanol components were repeatedly separated and purified by silica gel column and Sephadex LH-20 gel column to obtain 11915.7mg of SPHSⅠand 1180.7mg SPHSⅡ.In the chromatogram of the total saponin of Schizocapsa plantaginea Hance evaporative light scattering detector,there are 10 distinct characteristic peaks appearing between 0 and 30 min,of which two compounds are SPHSI and SPHSII.Compounds 1-8 may be similar saponin compounds to the SPHSI and SPHSII cores.Based on the area normalization method,the relative percentages of SPHSⅠand SPHSⅡin the SFSP_总were calculated to be 42.52%and1.61%,compound 1 0.64%,compound 2 1.06%,compound 3 4.46%,and compound 4 14.61%.Compound 5 is 3.73%,Compound 6 is 2.00%,Compound7 is 6.49%,and Compound 8 is 21.44%.In the chromatogram,the relative ratio of SPHSI to SPHSII is 26.355:1;SFSP_60%evaporative light scattering detector of cleavable saponin shows five distinct characteristic peaks at 0 to 30 min,indicating that at least five compounds of the SFSP_60%are present,two of which are SPHSI and SPHSII,compound 1-3 may also be similar to the SPHSI and SPHSII parent saponins.Based on the area normalization method,the relative percentages of SPHSI and SPHSII in SFSP_60%were 84.28%and 3.02%,compound 1 was 2.21%,compound 2 was 6.31%,compound 3 was 2.73%,and the relative ratio between SPHSI and SPHS II was 27.937:1.The SFSP60%ethanol solution can be stored for at least 2 months at 4°C in a refrigerator.However,in the solution within 2 to 6 months,there is a possibility that the inside of the solution may undergo degradation,sugar chain shedding,and other reactions that affect the solution Stability;The monomer SPHSI and SPHSII ethanol aqueous solution,stored at 4°C in a refrigerator,can last for at least 6months without significant degradation or change inside the solution.2.MTT assay was used to detect the proliferation inhibition rate of SMMC-7721 cells in order to finally calculate its half inhibitory concentration(IC₅₀).The IC₅₀0 values of SFSP _60%（content:98.55%）at 24,48,and 72 h were3.75,1.56,and 0.95μg/mL,respectively,and the IC₅₀0 values of SPHSI（purity:96.91%）at 24,48,and 72 h were 2.048,1.193,and 0.632μg/mL,SPHSII（purity:98.07%）at 24,48,and 72 h were 1.941,1.054,and 0.499μg/mL,respectively.SPHSI+II was detected at 24,48,and 72 hours,IC₅₀0 values were1.728,0.799,and 0.470μg/mL,respectively.The results showed that the inhibitory effect of the administration group on the proliferation of SMMC-7721cells was in a time-and dose-dependent manner（all P<0.05）,and the viability size was SFSP_60%<SPHSⅠ,SPHSⅡ<SPHSⅠ+Ⅱ.3.SFSP_Total,SPHSI and SPHSII significantly inhibited the proliferation of human non-small cell lung cancer H460 and A549,human ovarian cancer cell SKOV3,and human nasopharyngeal carcinoma cell CNE-1 in a dose-and time-dependent manner（all P<0.05）.The anti-tumor effect of SFSP_Totalotal on A549and SKOV3 cells was relatively good;SPHSI had better anti-tumor effect on H460 and SKOV3 cells;SPHSII had better anti-tumor effect on H460 cells.The results of AO-EB staining showed that the nucleus of the control group was clear and complete with green fluorescence;the morphology of cells in the administration group changed significantly,such as the early apoptotic nuclear chromatin stained green with a condensation or round bead,and the late apoptotic.The nuclear chromatin of cells is orange-red and it is in the form of a condensation or round bead.Conclusions:1.Preliminary establishment of a high performance liquid chromatography（ELSD）chromatogram of SFSP_Totalotal and SFSP_60%,and High-performance liquid evaporative light scattering detection method is superior to ultraviolet detection method;SFSP_60%and SFSP_70%is the main anti-tumor active ingredient in total saponins,and SFSP_60%,SPHSI,SPHSII aqueous ethanol solution can be stored in a refrigerator at 4°C for 2 months without significant change,SPHSI and SPHSII can be stored for a least 6months.2.SPHSI and SPHSII play a major anti-tumor effect,and the anti-tumor effect is the result of the joint action of various components in the SFSP_Totalotal and SFSP _60%,in addition,the combined effect of various components may be to reduce the overall anti-tumor activity of the drug.3.SFSP_Total,SPHSI and SPHSII have inhibitory effects on human non-small cell lung cancer H460 and A549,human ovarian cancer cell SKOV3,human nasopharyngeal carcinoma cell CNE-1,which have a broad-spectrum antibacterial effect,and have apoptosis-inducing effect on H460 and A549 cells.