Anti-cancer Effects Of Polysaccharides From The Grifola Frondosa On HT-29 Cells And Its Mechanisms

Grifola frondosa is a medicinal and edible fungus of which the polysaccharides has a unique biological activity,non-toxic and safely edible. In recent years, domestic and foreign scholars have also been increasingly concerned about the study of the fungus' biological activity. In this paper, the fruiting bodies of Grifola frondosa were isolated and purified to obtain polysaccharides. Their chemical compositions and structures were also further studied. At the same time, the anticancer activity and mechanism of polysaccharides were explored by using cell experiments,which provided a theoretical basis for the development of new functional polysaccharides products.In this thesis, the crude polysaccharides were obtained by water extraction and alcohol precipitation. The crude polysaccharides was deproteinized by using the Sevag method, destained by concentrated ammonia and impurities were removed by ultrafiltration to obtain crude polysaccharide. By gel chromatography obtained the homogeneous component from crude polysaccharide. It was designated as GFD. GFD is a macroscopic homogeneous component by high performance liquid chromatograp-hy (HPLC) and its molecular weight is about 54kDa. The results of color test show that the polysaccharide is a non-starch polysaccharide without reducing carbonyl and phenolic hydroxyl groups. Gas chromatography test shows that GFD is composed of galactose, rhamnose, xylose and glucose, with a molar ratio of 1.00: 1.09: 1.57: 43.90.And mass fraction is 4.9%,7.9%,8.16%,79.04%. Infrared spectroscopy used to analyze and characterize the structure of GFD and were confirmed an ?-type pyranpolyaccharide.The MTT test detected that GFD can inhibit the proliferation of human colon cancer cell HT-29. The result showed that GFD of inhibiting effect were time-depend-ent and concentration-dependent. With a polysaccharide concentration of 160?g/mL,at a time of 48h,the cell inhibition rate of maximum was 54.9%. The results of scanning electron microscopy showed that the apoptotic body gradually increased with the prolongation of time, which indicated that GFD could induce the apoptosis of HT-29 cells. The results of AO/EB staining and DAPI staining indicated that GFD inducing HT-29 cells apoptosis based on the typical apoptotic morphological changes.The results of Western blot showed that GFD could cause the decrease of PI3K in the cytoplasm of HT-29 cells and the increase of p-JNK. The expression of Bcl-2 on mitochondria was decrease and the expression of Bax was increase. The release of Cytochrome C from mitochondria which arose the Caspases cascade enhanced by the decrease of Bcl-2-to-Bax expression ratio and intensified the expression of Caspase-8.GFD ultimately lead to the apoptosis of HT-29 cells by the increase of Caspase-8.