Activity-Oriented Study On The Isolation,Identification And Anti-Colorectal Cancer Mechanism Of The Chemical Components Of Solanum Nigrum L.

Objective:The ability of active ingredients of Solanum nigrum L.to inhibit the viability of a variety of tumor cells,but few studies have been reported on the activity of active components of Solanum nigrum L.to inhibit colorectal cancer,and the mechanism of action is still unclear,which hinders the application and development of active ingredients.In this project,we extracted and isolated the 60%ethanol extract of Solanum nigrum L.and screened out the components with good anti-colorectal cancer activity.Solasonine（SS）was used as the target of the study to elucidate its mechanism of action against colorectal cancer cell proliferation in vitro and in vivo,and to investigate the effect of SS on histone deacetylase（HDAC）.Methods:1 Using D-101 macroporous adsorbent resin column chromatography,the total saponins fraction of the ethanolic extract of Solanum nigrum L.was initially separated,after which the fraction with the best anti-colorectal cancer activity was screened in combination with MTT method.Then the compounds were further separated and purified using silica gel column chromatography,ODS column chromatography,and semi-preparative HPLC.¹H-NMR and ¹³C-NMR techniques were used for the structural identification of the compounds,and MTT method was used to excavate the fraction of Lobelia with out best anti-colorectal cancer activity.2 Screening of potentially acting targets for SS based on Pharm Mapper,Swiss Target Prediction and BATMAN-TCM databases.Screening of colorectal cancer disease-related targets based on Gene Cards,Online Mendelian Inheritance Database（OMIM）,Drug Bank and Therapeutic Target Database（TTD）databases.The common targets of drug targets and disease targets were taken for protein-protein（PPI）interaction analysis,and GO analysis and KEGG analysis of the common targets were performed using the DVAID database.3 SW620 colorectal cancer cells were cultured.The Ed U method with clone formation assay was used to evaluate the effect of SS on the proliferation of SW620 cells.Flow cytometry was used to detect cell cycle and apoptosis（Annexin V/PI double staining）,JC-1to detect mitochondrial membrane potential,and Western blot for expression of mitochondrial apoptosis-related and cycle-related proteins.4 SW620 cells were treated with class I HDAC inhibitor Entinostat.Clone formation assay was used to detect the proliferation of SW620 cells,flow cytometry to detect cell cycle and apoptosis（Annexin V/PI double staining）,and Western blot was used to detect changes in the expression of mitochondrial apoptosis pathway-related proteins,cell cycle-related proteins,class I HDAC and histones.The binding ability of SS to the target protein class I HDAC was assessed by molecular docking technique.5 SW620 colorectal cancer cells were cultured and transplanted subcutaneously on the right dorsal surface of nude mice to construct a subcutaneous transplantation tumor model for human colorectal cancer mice（35 mice）.After the mice formed subcutaneous tumors,they were randomly divided into five groups,control group,5-fluorouracil group（5-FU,25mg/kg）,SS group（50 mg/kg）,entinostat group（Entinostat,Ent,15 mg/kg）and SS（50mg/kg）+Ent（15 mg/kg）,which were administered intraperitoneally.The mice were executed by cervical dislocation at the end of the experiment,and the mice were stripped of subcutaneous transplanted tumors for immunohistochemistry,HE staining and Western blot.Results:1 Seven compounds were isolated and purified from the components of Solanum nigrum L.with good antitumor activity,namely solamargine（1）,diosgenin（2）,solasodine（3）,daucosterol（4）,solasonine（5）,quercetin（6）andβ-sitosterol（7）.MTT results showed that compound 5（solasonine）had the best anti-colorectal cancer activity.2 A total of 382 SS potential target genes and 11347 colorectal cancer disease-related genes were identified in the network pharmacological study,among which 299 were common targets of drugs and diseases.GO enrichment analysis showed that the potential biological processes of SS treatment of colorectal cancer are mainly signal transduction,apoptosis and cell proliferation,and the potential therapeutic targets are mainly concentrated in the"pathway in cancer"signaling pathway.3 The study of SS on SW620 cell proliferation and apoptosis showed that SS inhibited SW620 cell activity and proliferation in a dose-dependent manner.SS can block SW620 cell cycle in S phase by up-regulating the expression of p21 and inhibiting the expression of CDK2.SS can induce SW620 cell apoptosis by promoting the expression of Bax,Cyt C,Caspase-3 and p53,inhibiting the expression of Bcl2,reducing mitochondrial membrane potential,and activating mitochondrial apoptosis pathway.4 The SS study on the regulation of SW620 cell proliferation and apoptosis by Class I DAC showed that entinote inhibited SW620 cell viability in a dose-dependent manner.Entinote and solanine synergically blocked SW620 cell cycle in S phase,and up-regulated the expression of p21 and inhibited the expression of CDK2.Entinote and solanline synergically inhibited the expression of deacetylated histone HDAC1/3/8,promoted the expression of non-histone p53,and then up-regulated the expression of Bax,Cyt C and Caspase-3,inhibited the expression of Bcl2,activated the mitochondrial apoptotic pathway,and finally induced the apoptosis of SW620 cells.In addition,solanine and entinote synergistically promote acetylation of histones H3k27,H3k18,and H3k9.Molecular docking results further indicated that the regulation of Solanasine on the proliferation and apoptosis of SW620 cells may be related to the regulation of Class I HDAC activity.5 In vivo studies showed that SW620 cells could form subcutaneous graft tumors in nude mice.After drug treatment,the proliferation rate of subcutaneous graft tumors in mice in the treatment group was relatively slower than that in the control group,and the tumor weight in the treatment group was significantly lower than that in the control group.Western blot results showed that compared with the control group,the expression of cycle-related protein p21 was increased in the treatment group,while the expression of CDK2 was decreased.The expressions of apoptosis-related proteins Bax,Cyt C,Caspase-3 and p53were increased,while the expression of Bcl2 was decreased.The expression of deacetylase HDAC1/2/3/8 decreased,while the expression of histones H3k27ac,H3k18ac and H3k9ac increased.HE staining showed that the cell distribution in the treatment group was looser than that in the control group.Immunohistochemical results showed that the expressions of Ki-67,Caspase-3 and p53 were significantly increased in the treatment group.Conclusion:In this study,7 compounds were obtained from Solanum nigrum L.with anti-colorectal cancer activity,among which solanine was the best.Studies on the mechanism of action have shown that solanine can inhibit the proliferation of colorectal cancer cells by blocking cell cycle and inducing cell apoptosis,and its mechanism is related to the inhibition of Class I HDAC activity.