Studies On Isolation, Purification, Antitumor Activity And Its Molecular Mechanism Of Saponin From Solanum Nigruml

In this study, saponins were isolated from Solanum nigrum L.(SNL), and screened for anticancer activity by MTT method with human hepatocellular cancer SMMC-7721cells, human gastric cancer MGC-803cells, and human lung carcinoma cell line NCI-H157as tumor model. We examined the antitumor activity of saponins on human gastric cancer MGC-803cells in vitro and to investigate its possible mechanisms. There are four parts in this dissertation.The first part:Isolation, purification of saponins from SNL.Coarsely powdered of dried SNL was refluxed twice with70%ethanol for2.5h. The extraction was condensed and then extracted with n-butanol. The n-butanol extract was dissolved in methanol, and precipitated with acetone-ether (1:1) to obtain different saponin fractions.which were. used as samples screened for antitumor new drugs.The active sites were isolation on silica gel column chromatographyand eluted with stepwise elution with methanol-chloroform (85:15,80:20,70:30), repeatedly many times to obtain saponin monomer.The second part:The screening for anti-tumor saponins from SNL. We studied the inhibitory effects of saponin from SNL on SMMC-7721, MGC-803cells, and NCI-H157carcinoma cell lines by MTT assay.The results showed that the saponins from SNL (30%section, sub-section solanigroside P, β1-solasonine) and solamargine could significantly inhibit the growth of the cells in a dose and time dependent manner.The third part:Apoptosis induced by saponins from Solanum nigrum L. in human gastric cancer MGC-803cells apoptosis.The morphological analysis for apoptosis was conducted using hematoxylin/giemsa/eosin/hoechst33258staining. After treatment with20μg/mL SNL saponin, the morphological changes of MGC-803cells were observed by light or fluorescence microscope. The results showed that the cells shrank and became smaller and rounder, and cell density reduced dramatically. After treatment with the saponins,typical apotosis morphological changes such as chromatin condensation, plasma membrane blebs, DNA fragmentation and apoptotic body formation were observed by fluorescence miscrope, which suggested that the cells treated undergo typical apoptosis.The cell cycle distribution and the apopototic ratio were analyzed by PI staining and flow cytometry. A significant increase of sub-G1DNA contents (%) in human gastric cancer MGC-803cells was observed following treatment with the saponins in a dose and time dependent manner.The results showed that the cell population of the G2/M phase was reduced concurrently with the increase of the S phase population, after treatment with the saponins.The fourth part:The mechanism of saponins from Solanum nigrum L. induce apoptosis in human gastric cancer MGC-803cells.The expression of p53, Bcl-2and Bax was determined using immunocytochemical method. The results showed that saponins (sub-section solanigroside P, β1-solasonine) from Solanum nigrum L. and solamargine suppressed mutant p53and Bcl-2expression and augmented Bax expression.In conclusion, saponins (sub-section solanigroside P, β1-solasonine) from SNL and solamargine exhibited antitumor activity on SMMC-7721, MGC-803and NCI-H157cells. Their antitumor activity on MGC-803was implemented via apoptosis, which might be related to the cell cycle arrest in S phase, the increase of the ratio of Bax to Bcl-2and the decrease of the expression of mutant p53.