Structural Identification & Bioactivity Study Of Polysaccharides From Grifola Frondosa And Their Sulfated Derivatives

Grifola frondosa(GF) is a kind of precious edible and medical fungi. Its main active ingredient is polysaccharide.Studies have shown that polysaccharides from GF have activities on anti-tumor,immuno-modulation and hypoglycemia.In order to discover the substance bases of these activities,homogenous fractions have to be isolated.Because of the complicated structure of polysaccharides,it is so hard to purify them that most pharmacological studies on those from GF stay at the level of crude polysaccharides,which has become an obstacle of the development and use of GF resources.Only a few previous reports on isolation of polysaccharides of GF have prepared homogeneous fraction and most of their targets are alkaline-extracted constituents of GF polysaccharides.This dissertation is focused on isolation,purification and preliminary characterization of polysaccharides fractions of GF highly-condensed powder.In order to further develop the medical value,this study also sulfated the homogeneous fractions and preliminarily analyzed their structures.Simultaneously,the biological activities of these homogeneous fractions and their sulfated derivatives were determined. Polysaccharides from Grifols frondosa(GFP) were extracted by hot water and precipitated by 95%EtOH.GFP1-F,GFP1-M and GFP1-L were further purified by DEAE-cellulose and Sephadex G-100 subsequently from GFP. GFP1-F,GFP1-M and GFP1-L were glucan with molecular weight 1.09×10⁵Da,1.93×10⁴Da and 2.76×10³Da.The result of methylation analysis showed that all the three fractions had the same linkage types:1,4-linked Glcp, 1,6-linked Glcp and 1,3,6-linked Glcp.GFP1-FS,GFP1-MS and GFP1-LS were polysaccharide sulfates obtained from GFP1-F,GFP1-M and GFP1-L with chlorosulfonic acid-pyridine.The IR spectrum of GFP1-FS,GFP1-MS and GFP1-LS showed the characteristic absorptions of sulfate ester bond at 1230cm^-1 and 810cm^-1.The ¹³C NMR results indicated the modification mainly occurred at C-6 of the polysaccharide sulfates.GFP1-MS had the greatest sulfated degree with DS 1.07,while DS of GFP1-FS and GFP1-LS were 0.66 and 0.61 respectively.The results of MTT assay indicated that GFP1-MS showed strong inhibitory effect on the growth of A549 cells.The combination of GFP1-FS,GFP1-MS and GFP1-LS with 0.2μg/mL Adr showed good inhibitory effect on the growth of A549 cells,especially GFP1-MS. Scratching Assay suggested that all the three sulfated derivatives had inhibitory effects on HMEC migration,especially GFP1-MS had the strongest activity which may be related with complicated branch structure of GFP1-MS but no direct relationship with DS.