Solation Of The Functional Components Of Akebiae Fructus And Its Mechanism Of Inducing Apoptosis Of Gastric Cancer Cells

Objective:The purpose of this study was to explore the separation of the effective substances of Pseudosin,one of the components of cancer-eliminating and detoxifying prescription,and the mechanism of inducing apoptosis of gastric cancer BGC-823 cells.By extracting and analyzing the active components of Akebiae fructus,and screening its anti-tumor active sites,the effects of Akebiae fructus on the proliferation and apoptosis of gastric adenocarcinoma cell BGC-823 were observed,and the anti-tumor mechanism of Akebiae fructus active sites was discussed.The results of this study will provide experimental basis for the discovery of the components of the functional substances and the mechanism of antitumor action of Antitumor,and provide clues for the study of the mechanism of Xiaoaijiedu Formula in the prevention and treatment of gastric cancer.Methods:1.Extraction and separation process of Akebiae fructus and screening of active partsThe seeds were soaked in anhydrous ethanol,filtered and concentrated by rotary evaporator to obtain crude extract.The crude extract was dispersed in 5L pure water,extracted with different polarity solvents,petroleum ether and ethyl acetate respectively,and then concentrated under pressure to obtain the ethyl acetate layer.The ethyl acetate extract was mixed with reversed-phase silica gel(C18),separated by low pressure column chromatography(C18),detected by TLC,and the same fraction was combined.The active site B14 was identified by cell screening in vitro,and the active part of Akebiae fructus was analyzed by liquid phase and mass spectrometry.2.Effect of the active site of Akebiae fructus on the proliferation of gastric cancer cellsGastric adenocarcinoma cell line BGC-823 was selected to determine the IC50 of B14 by MMT.MTT,EDU and flow cytometry were used.To observe the effects of active fraction B14 on the morphology,survival,proliferation and apoptosis of BGC-823 cells.3.Study on the action mechanism of the active fraction of Akebiae fructus on gastric cancer cellsBGC-823 cells in each group were collected,and the changes of mitochondrial membrane potential after B14 intervention were detected by flow cytometry.Total cell protein,cytoplasmic protein and mitochondrial protein were isolated and extracted from BGC-823 cells in each group.The protein expression levels of ERK,Bad,Bcl-2,BAK,Caspase-3,cytochrome C and Aif were detected by Western Blot,so as to discover and clarify the mechanism of action of B14 active part of Fructase in inducing human gastric cancer cell line BGC-823.Results:1.The ethyl acetate extract of Akebiae fructus was mixed by reversed-phase silica gel(C18)dry method,separated by low pressure column chromatography(C18),and detected by TLC.The same fractions were combined to obtain 25 fractions,which were recorded as B1-B25.B14 group was divided into active parts by cell experiment in vitro.2.Using α-ivy saponin as the standard,the liquid phase analysis showed that the active fraction of B14 mainly contained two compounds,of which α-ivy saponin accounted for about 37%,and the other component was not clear yet.Although we scanned the hydrogen and carbon spectra of B14 by liquid mass spectrometry,we still could not identify another component due to the purity of B14 and the comparison with the compound library.3.After the stimulation of B14 from the active part of Akebiae fructus,the gastric cancer cells BGC-823 had significant morphological changes,and the cell viability and proliferation were significantly inhibited,and apoptosis occurred,and the above inhibitory effects were positively correlated with the concentration of B14.In previous transcriptome sequencing experiments,we found that B14 could up-regulate the mRNA expression of Aif,which was verified by biological repetition.Therefore,we believe that the mechanism of B14 is likely to promote the apoptosis of tumor cells by regulating Aif and its related upstream signaling pathways to mediate the mitochondrial apoptosis pathway.4.B14 induced depolarization of mitochondrial membrane potential in BGC-823 cells.The occurrence of this depolarization may be related to the up-regulation of pro-apoptotic gene BAK\Bax\Bad and down-regulation of anti-apoptotic gene BCL-2.Among them,the upregulation of BAD may be due to the inhibition of ERK signaling pathway leading to the phosphorylation and transfer of BAD to mitochondria.The change of mitochondrial membrane permeability promotes the release of pro-apoptotic factors in mitochondria:the expression of cytochrome C is up-regulated,which in turn activates Caspase 3;The expression of Aif,an apoptosis inducer independent of the Caspase family,increased at the same time,which induced apoptosis of BGC-823 cells under the combined action of the two factors.These results suggest that B14 can regulate the ERK signaling pathway and promote the up-regulation of pro-apoptotic genes in the Bcl-2 family,leading to mitochondrial apoptosis,thereby inhibiting the proliferation of gastric cancer cells.Our study found that this will provide clues for the further study of the functional components of Akebiae fructus,and provide a theoretical basis for the anti-tumor application research of the cancer-eliminating and detoxifying prescription.Conclusion:The active part of Akebiae fructus B14 mainly contains α-ivy saponin and another unknown component.The mechanism of its action may be through inhibiting ERK pathway,affecting the key targets in the downstream mitochondrial apoptosis pathway,resulting in the apoptosis of tumor cells,so as to achieve the effect of tumor inhibition.