Extraction, Purification And Structure Of Fucoidan From Hizikia Fusiforme

Hizikia fusiforme belongs to sargasaceae of heophyta, which has good nutrition and can be used as food and medicine. Hizikia fusiforme is a characteristic algae of China and the resources are abundant. Fucoidan isolated from Hizikia fusiforme is a kind of sulfated-polysaccharide containing fucose, which is a very important material for its various biological activities. But till now there are only few simple studies on fucoidan of Hizikia fusiforme in China. The objective of this dissertation is to study the methods of extraction and purification, structure and anticoagulant activity of fucoidan of Hizikia fusiforme harvested in Dongtou, Zhejiang Province. The main results are as follows.The chemical compositions of powder of Hizikia fusiforme were moisture 12.2%, ash 15.4%, protein 12.3%, crude fat 1.6% and crude fiber 40.4%. The compositions of polysaccharides in Hizikia fusiforme were algin 28.4%, fucoidan 8.1% and laminaran 0.5%.The effects of acid extraction and aqueous extraction on the extraction efficiency and macromolecule structure of fucoidan were studied. The results showed that acid method was better than aqueous method according to the extraction efficiency. However, fucoidan was hydrolyzed during the acid extraction while the intact macromocule structure of fucoidan was kept during the aqueous extraction. For crude polysaccharides of acid extraction it was suitable to be purified by fractional precipitation with alcohol, and for crude polysaccharides of aqueous extraction to be purified by calcium chloride method.The chemical components of fucoidan of Hizikia fusiforme were fucose 18-20%, uronic acid 12-19%, sulfate groups 11-13% and ash 24-34%. The neutral sugars in fucoidan contained fucose, galactose and mannose residues which were major, glucose, xylose, arabinose and rhamnose residues which were minor. The metal elements in fucoidan were mainly K, Ca and Na.The fractionation and physical chemistry properties of fucoidan from aqueous extraction were studied. Fucoidan was graded into three fractions: F1, F2 and F3 by chromatography on a DEAE Sepharose CL-6B column. F3 was further graded into three fractions: F31, F32 and F33 by chromatography on a Sepharose CL-6B column, which were shown to be homogeneous by gel filtration chromatography and agarose gel electrophoresis.The five fractions (Fl, F2, F31, F32, F33) all contained fucose (18-33%), mannose and galactose predominantly, also contained sulfate groups (3-24%), uronic acids (13-32%) and little protein (1-2%), and Mw from 26 kDa to 959 kDa. The uronic acids in F32 and fucoidanfrom aqueous extraction were mainly GlcA and little ManA, but in F33 ManA was primary. F33 contained 2% N element, mostly of which probably came from nucleic acid according to amino acid analysis, UV and nuclease treatment. IR spectra suggested that F31, F32 and F33 had mainly a-glycosidic bonds and little (3-glycosidic bonds. The optical rotation suggested that fucose constituents in F32 and F33 had mainly a-L anomeric configuration.The structure of F32 was studied by periodate oxidation, Smith degradation, partial hydrolysis, desulfation, carboxyreduction, GC, methylation analysis, GC/MS and NMR. The results showed the main chain of F32 was composed of —>2)-a-D-Man(l—* and —>4)-P-D-GlcA(l-? alternately while little —?4)-p-D-Gal(l—> was mixed in the main chain. The branch points were at C-3 of->2)Man(l->, C-2 of->4)Gal(l-> and C-2 of->6)Gal(l->. About 2/3 of fucose were at the nonreducing ends, and left of them were 1,4, 1,3 and 1,2 glycosidic linkage. About 2/3 of xylose were at the nonreducing ends, and left of them were 1,4 glycosidic linkage. Most of mannose was 1,2 glycosidic linkage, and 2/3 of them had branch in C-3. Galactose was mainly 1,6 glycosidic linkage. The configuration of sugar constituents was a-D-Manp, a-L-Fucp, a-D-Xylp, 0-D-Galp and P-D-GlcAp.Sulfate groups in F32 were at C-6 of ->2,3)Man(l->, C-4 and C-6 of->2)Man(l->, C-3 of —>6)Gal(l—>, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate group in GlcA and xylose. F32 contained 1.2% protein, which linked with glycan through O-glycosidic bonds. The reducing ends of sugars linked maily with Thr, and little with Ser.Some problems on structural analysis of polysaccharide were deeply studied. 1) Determination of the position of sulfate group in sulfated polysaccharides. Because besides the C-O-S vibration in 820-850 cm'1 region, there was also C-H bending vibration of sugar reducing end, which affected the judgment on the position of sulfate group, so error conclusion would be obtained if the position of sulfate group was determined only by the infrared. It was necessary to synthesize the results of IR, stability of sulfate esters to alkali, and methylation analysis if the correct conclusion wanted to be acquired. 2) Methylation of polysaccharides undissolved in DMSO but dissolved in water. Polysaccharide sample was firstly dissolved in little water, followed by adding DMSO. Then the 3 A molecular sieves was added to remove the water left in DMSO. Finally the sample was methylated by Needs method. The methylation degree of the product was high. 3) Determination of the uronic acids in polysaccharide by GC. The uronic acids in the polysaccharide were firstly reduced to their neutral sugars through carboxyreduction. Then the neutral sugars in the polysaccharide were determined by GC after derivatization of aldononitrile acetates. Comparing with the change of the neutral sugars before and after carboxyreduction, the composition of the uronic acids canbe calculated. 4) Structural analysis of small molecule production obtained by partial acid hydrolysis of polysaccharide. Their structure could be determined by LC/MS and methylation. The analysis results could be as the supplement and verifier of polysaccharide structure. For the methylation analysis of mixture composed of monosaccharides and oligosaccharides, the monosaccharides in mixture should be removed firstly, and the left oligosaccharides were purified furthest. The purified oligosaccharides were reduced to translate the sugar residue of reducing end into alditol, and then methylated. Only so the credible results could be acquired.The relationship between the structure of fucoidan separated from Hizikia fusiforme and its anticoagulant activity was studied. The results showed that fucoidan could prolong APTT distinctly, but delay TT little, which indicated that anticoagulant activity mainly came from the endogenesis coagulant approach restrained by fucoidan. The coagulation time and the bleeding time of mice could be prolonged after them were fed with fucoidan of Hizikia fusiforme. Sulfate group was necessary to anticoagulant activity of fucoidan. Uronic acid was not necessary but it could strengthen the anticoagulant activity of fucoidan. The anticoagulant activity of fucoidan was also related to the position of sulfate group. The anticoagulant activity of F32 was mainly decided by sulfate groups of fucose.