Isolation And Identification Of Polyphenols Of Trapa Bispinosa Roxb Shell And The Mechanism Of Anti-Tumor In Vitro

The purpose of this research was to isolate the active components from TbR shell and clarify the mechanism of its anti-tumor.In this paper,the fractional extraction,silica gel,Sephadex LH-20 gel column chromatography and liquid phase separation techniques were used to separate the polyphenols from the TbR shell and the anti-tumor active components were tracked and screened.The structures were identified by HPLC-MS and NMR techniques and their anti-tumor mechanisms were studied in vitro.1.Three Monomer Compounds were separated from the ethanol extract of the TbR shell.1,2,6-trigalloyl-β-D-glucose,1,2,3,6-tetragalloyl-β-D-glucose and 1,2,3,4,6-penta-galloyl-β-D-Glucose were identifiedand the contents of the three compounds in ethanol extract were 38 mg/g,120 mg/g,and 8 mg/g,respectively.1,2,3,4,6-penta-galloyl-β-D-glucose had significant inhibitory effects on the proliferation of human gastric cancer SGC7901 and human hepatoma HepG2 cells.The IC50 of SGC7901 cells at 24h was 40.85μg/mL,which was better than the positive drug 5-FU.The inhibitory effect of 1,2,3,6-tetragalloyl-β-D-glucose on SGC7901 cells was better,and 1,2,6-Trigalloyl-β-D-glucose has a better inhibitory effect on HepG2 cells.2.1,2,3,6-tetragalloyl-β-D-glucose can affect the cell cycle of SGC7901.The dose of 200μg/mL for 48h could arrest cells in G1 phase and induce apoptosis in SGC7901 cells.The intracellular calcium ion concentration increased and the mitochondrial membrane potential decreased.Transcriptome sequencing analysis showed that the differentially expressed genes were mainly enriched in the P53 signaling pathway associated with apoptosis.The results of RT-qPCR and Western blot showed that 1,2,3,6-tetra-galloyl-β-D-glucose could induction the apoptosis of SGC7901 cells by up-regulate the expression of P21,PUMA,PERP and IGF-BP3 gene mRNA,down-regulate CyclinD gene mRNA,increase the expression of Cytochrome c,Caspase-3 and Caspase-9 protein,decreased the expression of Bcl-2 protein.3.1,2,3,4,6-penta-galloyl-β-D-glucose can affect the cell cycle of tumor cells.12.5～50μg/mL could keep HepG2 and SGC7901 cells in S phase,100～200μg/mL could block HepG2 in G1 phase,SGC7901 block in G2 phase.It could induce apoptosis in HepG2 and SGC7901 cells.The apoptosis rate was 51.02%at drug concentration 200μg/mL.The 1,2,3,4,6-penta-galloyl-β-D-glucose can decrease the mitochondrial membrane potential and increase the intracellular calcium ion concentration in the two tumor cells.The results of RT-qPCR and Western blot showed that 1,2,3,4,6-penta-galloyl-β-D-glucose could induce the apoptosis of SGC7901 and HepG2 cells by up-regulate the expression of PERP and IGF-BP3 gene mRNA,increase the expression of P53,P21,PUMA,Bax,cytochrome C,Caspase-3 and Caspase-9 protein,decreased the expression of Bcl-2 protein.