| Grifola frondosa is well known as a highly nutritious and officinal edible fungus. Polysaccharide, one of the main active components extraced from Grifola frondosa, has various functions such as stimulating immune system, anticancer, antivirus and so on. However, a part of these active polysaccharides were extracted alkalinely from the mycelia of submerged fermentation of Grifola frondosa are water-insoluble, which limits their applications in the medicine field. The chemical modification can improve the solubility of natural polysaccharides to enhance their activities, and enlarge their application range. The sulfation of polysaccharides is the most interesting field of its chemical modification. Lot attention has been paid to sulfated polysaccharides because of their anticoagulant and antivirus activity. And the sites of sulfation, sulfate content, molecular weight, physical and chemical characterizations are the key factors for these activities.This thesis mainly focuses on the isolation of water-insoluble polysaccharide GF4A from Grifola frondosa, the optimized conditions for the sulfation of GF4A, the characterization, structure analysis and bioactivities of the derivative SGF4A. Systemically study is carried out to find out the optimum preparative conditions using Me3N·SO3-DMF. The activity results showes that SGF4A has anticoagulant and immunomodulating activities. The characterizations of SGF4A are studied on the basis of the research above. The main results are shown as follows:1. Water-insoluble polysaccharide GF4A is extracted in cool alkaline solution from the mycelia of submerged fermentation of Grifola frondosa, and purified with gel-permeation chromatography. A pure water-insoluble polysaccharide GF4A with sugar content of 99.4% is obtained. While it could be dissolved in 0.5mol/L NaOH solution. The primary structure of GF4A is elucidated by chemical method, such as acid hydrolysis, periodate oxidation, Smith degradation, and spectroscopic techniques IR, 13C-NMR etc.. The results show that GF4A contains glucose, mannose and xylose, with the molar ratio of 9:2:1. Every 12 sugar residues consist a repeating unit, in which the backbone is composed of sixα-1,3-D-Glc and twoα-1,3-D-Man. Each unit also has two branches, one is consisted of threeβ-1,4-Glc, and the other is anα-1,4-Xyl.2. The reaction conditions are studied to prepare sulfated GF4A .The degree of sulfation, yield, Mw and capability of sulfate reagents are used as the guideline to compare three different sulfation system. According to the results, sulfation using Me3N·SO3-DMF is the optimal method. In this reaction, a high yield of sulfation (153%) was achieved with the following optimized conditions: mass ratio of Me3N·SO3 to GF4A, 5:1; temperature, 80oC; reaction time, 6 hours. The degree of sulfation is 1.6 and the relative molecular mass 88 KD. The activity results in vivo showed that sulfated polysaccharide had a good antitumor activity. 3. The physical and chemical characters of three bathes of SGF4A are studied according to the appendix of The Pharmacopoeia of China. The results indicate that SGF4A is water-soluble in room temperature with the highest concentration of 50%, Mw is between 85 kD ~ 88 kD, degree of sulfation is 1.5~1.6. The UV and IR spectra of SGF4A showed that characteristic absorptions of sulfated eater bond at 265nm and 1240cm-1, 810cm-1 respectively, which demonstrate that the modification of sulfation is successful. Relative signal in 13C-NMR spectrum indicate that the C6-OH has been sulfated completely, and the sulfation also occurs at some C2-OH and C4-OH position. The activity results in vivo showed that sulfated polysaccharide had a good anticoagulant activity and immunomodulating activity.The series of optimal conditions studied in this thesis is suitable for preparing the water-soluble sulfated polysaccharide SGF4A with high content of sulfation and high yield. This study will lay a foundation for more application of Grifola frondosa in medicine fields and accelerate the development of officinal fungus in our country.
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