Studies On Characterization And Anticoagulant Activity Analysis Of The Degradation Products From Laminaria Japonica Polysaccharide LJP4

Laminaria japonica is a kind of famous traditional medicine and food plants. Its nutritional value and health effects is all outstanding. Sulfated polysaccharide, a family of water-soluble heteropolysaccharides, are found in brown algae and marine invertebrates. Polysaccharides isolated from Laminaria japonica exhibit various biological activities, such as anticoagulant, antithrombotic and anticancer activities as well as the protective function of neural tissues. Anticoagulant and antithrombotic effects are the most notable functions of Laminaria japonica-derived polysaccharides. Related research reports found that the activity of kelp are related to its polysaccharide sulfate. Due to the big molecular weight and complicated structure of the Laminaria japonica, less the studies on its structure and activity and structure-activity relationship could be expored.In this study, we systematically investigated the physicochemical properties of the four product, and degraded the LJP4 which it is rich in fucose and sulfate group, then the more systematic study which the degradation products of structure and anticoagulant activity was carried out, specific research results are as follows:1?Extraction, isolation, purification and determination of the physical and chemical properties of Laminaria japonicaLaminaria japonica powder was subjected to hot water extraction and ethanol precipitation to obtain preliminary polysaccharides LJP. Then LJP was separated by anion column (DEAE- cellulose column), dialyzed and lyophilized, to afford four product, LJP1, LJP2, LJP3 and LJP4. The physicochemical properties of these products were measured. The results are as follows:LJP1:the total sugar content was 80.74%, the protein content was 2.4%, and the sulfate content was 9.28%, the average molecular weight (MW) was estimated to be 540kDa; the LJP1 was made up of fucose and mannose in the molar ratio of 1.0:0.79;LJP2:the total sugar content was 61.72%, the protein content was 0.1%, and the sulfate content was 32.36%, the average molecular weight (MW) was estimated to be 515kDa; the LJPlwas made up of fucose, xylose, mannose and galactose in the molar ratio of 1.0: 0.09:0.25:0.15;LJP3:the total sugar content was 55.62%, the protein content was 2.4%, and the sulfate content was 11.29%, the average molecular weight (MW) was estimated to be 551kDa; the LJPlwas made up of fucose, mannose and galactose in the molar ratio of 1.0:0.14:0.31;LJP4:the total sugar content was 50.93%, the protein content was 2.1%, and the sulfate content was 35.23%, the average molecular weight (MW) was estimated to be 544kDa; the LJPlwas made up of fucose and galactose in the molar ratio of 1.0:0.19.Studies have shown that the content of sulfate groups and its sites have a significant effect on the anticoagulant activity of the polysaccharide, and sulfate content of LJP4 are the highest in these four components, so in the follow-up study, we will be in-depth and systematic study of the LJP4.2?Preliminary analysis degradation and anticoagulant activity of LJP4LJP4 be used degraded by oxidation degradation by using the orthogonal experiment to optimize such hydrolysis conditionsto get three degradation products. The optimized conditions were:500mM H2O2, 1mM Fe2+,50?,2h,4h,6h. The degradation products obtained the successively molecular weight of about 170,26 and 19 kDa. Then we have measured the anticoagulant activity in vitro of three degradation products in terms of the APTT (Activated partial thromboplastin time), PT (Prothrombin time) and TT (Thrombin time) three indicators. The results showed that:LJP4 was the most active on anticoagulant activity in the four products. However, LJP4-2, with the molecular weight of about 26kDa, possessed higher anticoagulant activity over other three degradation products, but the activity of the low molecular weight of LJP4-2 is weaker than LJP4.3?Structure analysis of LJP4-2Using the new method of study sulfate polysaccharides established by our lab and a series of methods such as gas chromatography, mass spectrometry and gas-mass spectrometry, we finally deduce the structure of LJP4-2, which is connected to the main chain of 1?6 galactose to fucose branched of fucosegalactan repeating units, branched to connect 1?3 Fuc majority, a small amount of 1?4 containing fragment. A small amount of sulfated fucose place in C-3 position, and all the ends of the C-3 position are sulfated.