Studies On The Isolation,Purification And Immunomodulatory Activity Of Extracellular Polysaccharide From Grifola Frondosa

The aim of this thesis is to obtain an extracellular polysaccharide by submerged culture of Grifola Frondosa, and investigated its chemical composition and structure by isolation and purification. In addition, several immunological tests were performed to investigate the immunomodulatory activity and the action mechanism. The research will provide a theoretical basis for the development of new polysaccharide immunomodulatory products.In this thesis, a crude extracellular polysaccharide named CGFP was extracted from Grifola frondosa by alcohol precipitation method after submerged fermentation culture. The final purified fractions were obtained by enzymes-sevag for deproteinization, macroporous absorption resin AB-8for decoloration, ultrafiltration and Sephadex G-100gel colume for further isolation and purification, and named GFP-A,GFP-B. Of the fractions, because of its higher immunological active, GFP-A was selected by the proliferation activity of RAW264.7cells by MTT method. The investigations on Sephadex G-75gel colume and HPLC indicated that GFP-A was the macroscopic homogeneous component and had a molecular weight of37kDa. The physicochemical properties of GFP-A incated that GFP-A was a non-starch neutral polysaccharide, excluding polyphenols, proteins and nucleic acids, contained a-D-glucoside bond and pyranose ring. In addition, GC analysis revealed that GFP-A was mainly composed of rhamnose, arabinose, xylose, mannose, glucose, galactose, by the molar ratio of0.28:031:0.30:0.06:7.98:0.61.The proliferation activity of GFP-A towards RAW264.7cells with different concentrations and treatment time was studied. The results of MTT assay showed that GFP-A were able to promote the" proliferation of RAW264.7cells in significant dose-dependent and time-dependent manners, respectively. When the GFP-A was at a concentration of80ng/mL and treatment time of48h, the cell proliferation index reached a maximum of137.5%. The analysis of scanning electron microscope showed that the activation of RAW264.7cells could be also promoted by adding GFP-A, and the results of AO staining indicated that GFP-A could activate RAW264.7cells and improve the level of intracellular nucleic acid metabolism. According to neutral red phagocytosis experiment, the phagocytic activity of RAW264.7cells was significantly enhanced by GFP-A. In addition, in a certain range of concentration, GFP-A was able to increase the release of NO in RAW264.7cells, and upregulate the mRNA expression of mmunological factor TNF-a, IL-1β, IL-6, IL-12, IFN-γ and iNOS of RAW264.7cells.