Study On The Purification,Structure And Vitro Antioxidant Activity Of Sulfated Polysaccharide From Undaria Pinnitifida

In this dissertation, we conducted a series of study on sulfated polysaccharide (S1 and S2) which were isolated and purified from Undaria pinnitafida. Phsico-chemical characteristics and chemical structure of S1 and S2 were determined. The antioxidant activity in vitro was examined. The following results were achieved.1. Dried algae were extracted in HC1 (pH 2) and the residues were extracted with HCl at 60℃for six hour, for two times. The extract were precipitated with threefold of the volume of 90% ethanol (v/v) and vacuum-dried to get the preliminary polysaccharide products. The yield of the crude polysaccharide under these conditions was 2.4%. Proteins from the crude polysaccharide were removed by pronase and Sevag method. The protein content of the crude polysaccharide was only 1.51% after removing 88.5% of the protein. Then it was purified by a DEAE-Sephadex A-25 column eluting with a step gradient of 0-2 M NaCl. Total sugar content of the elution was determined by the phenol-sulfuric acid method to get a suitable concentration of NaCl. S1 and S2 were obtained by stage eluting. The fractions were further applied to Superdex-200 and Sephacryl S-300 column chromatography. S1 and S2, respectively, appeared as only a single and symmetrically sharp peak indicating the two fractions were homogenous polysaccharides.2. Phsico-chemical characteristics was analyzed and determined by chemical methods. The total sugar content of S1 and S2 was estimated as 40.54%,33.50%. Protein in S1 and S2 were 1.39%,1.41% by coomassie Brilliant blue method and uronic acid were 12.97% and 9.97% measured by a modified carbazole method. The molecular weight of S1 and S2 were determined by gel filtration and found to be 150000,94620. The sulfate content in S1 and S2 were 34.29%,33.99% with sodium rhodizonate reagents. The results demonstrated that the polysaccharides (S1 and S2) were high sulfated polysaccharides. Solvolytic desulfation of S1 and S2 removed 82% of the sulfate giving risen to the partially desulfated fractions (DS-1,DS-2)3. The structure of S1 and S2 were analyzed and determined by GC,FI-IR and GC-MS techniques. The results showed that the two sulfated polysaccharides both contained rhamnose,xylose,galctose and glucuronic acid. But the molar ratio was different for S1 and S2. Rhamnose was the main sugar unit, accounting for 51.44 mol%,40.30 mol%, additional to rhamnose, xylose and galactose were also seen in the samples. These results demonstrated that the polysaccharide isolated from U. pinnitafida, was high rhamnose-containing sulfated polysaccharide. IR spectrum demonstrated several bands corresponding to sulfate and carboxy group, which was consistent with the results of chemical methods. The results of GC-MS showed S1 contained a linear backbone built up of 1→2 Rha,1→4 Xyl, 1→3 Gal residues. This backbone had braches at position 4-O of Rha,2-O of Xyl,6-O of Gal. The terminal residues were Rha, Xyl, Glc.4. Antioxidant activities of the polysaccharide fractions were evaluated by assays of various antioxidants in vitro systems, including superoxide anion, DPPH and hydroxyl radical-scavenging activity and metal chelating ability. The results showed sulfated polysaccharide fractions possessed good antioxidant properties and had stronger antioxidant abilities than de-sulfated polysaccharides. Available data obtained by in vitro models suggested that the correlation between the sulfate content and antioxidant activity was positive.