Isolation And Purification Of Isoflavone From Chickpea And Transcriptomics Study Of Its Inhibition Effects On Human Breast Cancer Cells

Breast cancer is one of the most common malignant tumors among women.The search on novel natural chemopreventive agents and new natural chemical enhancers in the treatment process is currently hot topics in the prevention and treatment of breast cancer.Epidemiological studies have suggested that higher consumption of legumes containing large amounts of phytoestrogen might contribute to the lower incidence in Asia of breast cancer.The main reason is the consumption of legumes containing a large amount of phytoestrogen.Isoflavonoids are a type of bioactive substances widely found in leguminous plants such as soybeans and chickpeas.They have a wide range of physiological activities,and their molecular structures are similar to those of animal female hormones.They are called phytoestrogens.Isoflavones are a class of phytoestrogens that are structurally similar to mammalian estrogens.Isoflavones can bind to estrogen receptors,induce weak estrogen-like effects,inhibit cancer cell proliferation and induce apoptosis effectively.It is a potential cancer chemopreventive agent.Studies have shown that germination increases the phenolic content of seeds,and particularly in chickpea,the isoflavone content is increased by over 100 fold.Germination can be used as chickpea enrichment of total isoflavones.Chickpea sprouts contain a higher content of total isoflavones,but other chemical components are complex.The simple extraction process cannot meet the higher quality requirements,and a certain purification method is needed to remove a large amount of protein,polysaccharides,fatty acids,tannins and other impurities contained in the crude extract.In this study,chickpea sprouts were used as raw materials to determine the optimal extraction process of isoflavones at 70℃,1.5 h,liquid-solid ratio of 25 : 1,the optimal extraction process of isoflavones at 70℃,1.5 h,liquid-solid ratio of 25 : 1,and 60% ethanol.Macroporous resin HPD-300 as a filler,an aqueous solution of isoflavone content of 3.05 mg / m L,adsorbing at a rate of 2 times the volume per hour,removing impurities by eluting with 5 times the volume of 20% ethanol,and finally eluting with 6 times the volume with 95% ethanol at a rate of 2 times the volume per hour were determined for the optimal purification process conditions.It was found that 29 chemical components and inferred 14 possible chemical components in chickpea isoflavones.After comparing with the standard products,Ononin,Sissotrin,Formononetin and Biochanin A with higher content were identified.This research provides technical parameters and material basis for the study of isoflavones in chickpeas.Chickpea isoflavones could not only significantly inhibit the survival,proliferation,and migration of MCF-7 cells but could also induce their apoptosis.In this study,isoflavones extracted from chickpea sprouts were added to the MCF-7 cells for 48 hours.Using RNA-sequencing,we analyze the m RNA and Lnc RNA expression changes.The protein-protein interaction(PPI)network was provided by stringdb.Gene and Lnc RNA co-expression were analyzed by Pearson correlation analysis,survival analysis results were provided by human protein atlas website,and gene expression was verified by q RT-PCR.Transcriptome results showed differential expression of a total of 1094 m RNA and 378 Lnc RNA.KEGG pathways analysis results revealed that inhibition of cell proliferation mainly came from up-regulation of apoptosis in the pathway and down-regulation of m RNA splicing in the pathway.The down-regulation of top10 co-expression genes in Lnc RNA were mainly HNRNP family genes,which interacted with apoptosis-related genes through UBC.Abnormal expression of hub genes(degree>10)on PPI network were beneficial to improve the survival of breast cancer patients.The inhibitory effect of chickpea isoflavones on the proliferation of human breast cancer MCF-7 cells may be achieved by inducing tumor cell apoptosis and attenuating RNA synthesis.At the same time,Lnc RNAs may be involved in the above-mentioned processes to coordinate the regulation.Survivalanalysis was consistent with quantitative q RT-RNA results,however,not every hub gene can be used as a high risk factor for breast cancer prediction.Up-regulated NME2,FOS and down-regulated DDX5,TOP2 B,CCT2 of hub genes can significantly improve the survival time of breast cancer patients.Chickpea isoflavones inhibit the proliferation of human breast cancer cells by regulating the expression of hub genes NME2,FOS,DDX5,TOP2 B,and CCT2 in the PPI network,which suggested that four genes can be used as targets or clinical treatment reference indicators and could provide theoretical support for the prevention and treatment of human breast cancer.Through the integrated analysis of wet and dry experiments,we found that isoflavones in chickpeas may promote the apoptosis of MCF-7 cells by inhibiting the expression of HNRNP family genes mediated by Lnc RNAs.The up-regulation and down-regulation of hub genes in the PPI network triggered by chickpea isoflavones have positive effects on improving the survival time of breast cancer patients.Our results reveal a potential mechanism for chickpea isoflavones to inhibit MCF-7 breast cancer cell proliferation and provide a reference for the development of new anti-cancer drugs used in breast cancer.