Preparation And Antithrombotic Activity Of Low Molecular Weight Fucoidan Extracted From Laminaria Japonica Areschi

The number of people dies from cardiovascular disease comes up to 40.7% of the total deaths due to illness every year in China. This proportion is much higher than any other causes of death, including cancer. Thrombosis is largely the result of cardiovascular disease. Therefore, the development of natural antithrombotic drugs with high efficient but no side effects becomes the research emphases in the world today.Fucoidan is a characteristic water-soluble heteroglycan, existing in brown alga, which contains sulfates. The chemical composition and structure of fucoidan change with different algae species, growth seasons and extraction methods. At present, more studies have been done on the preparation of fucoidan from Sargassum fusiforme, Undaria pinnatifida, Ascophyllum nodosum and Fucus vesiculosus, but not Laminaria japonica. What's more, the structure and action mechanism of high molecular weight fucoidan from Laminaria japonica are still not clear. So it is necessary to intensively study the structure and structure-activity relationship with antithrombotic activity of low molecular weight fucoidan from Laminaria japonica in our waters. (One)Raw sugar, extracted from Laminaria Japonica Areschi in the Yellow Sea, was used as raw matetial, which was purified to get medium molecular weight fucoidan by Q-Sepharose FF anion-exchange and degraded to get low molecular weight fucoidan in different molecular weight by the free radical oxidation method. During the process of preparation and chemical analysis of fucoidan, different preparation conditions, product yield, molecular weight, sulfate content, neutral sugar composition and functional groups were analyzed. Finally, we got the best 4 from all of the samples: F1,F2,LMWF4a,LMWF4b.(Two) Medium molecular weight fucoidan and low molecular weight fucoidan containing high sulfate and high-sulfate were used to study the antithrombotic activity, and the carotid artery thrombosis model was induced by the DC stimulation(Indicators:the whole blood vessels blocked time, TFPI,TXB2 and 6-keto-PGF1α). The effective dose of inhibiting thrombus were used on the normal experimental animals in coagulation system(Indicators:APTT, PT, TT, ATⅢand FIB), fibrinolysis system (Indicators:the clot lysis time , the euglobulin lysis time , t-PA, U-PA and PAI)and platelet systems(Indicators:platelet aggregation induced by the ADP, AA and thrombin), combined with the human platelet experiments in vitro (Indicators:platelet aggregation induced by the sample, ADP, AA and thrombin) to explore the mechanism of medium molecular weight fucoidan and low molecular weight fucoidan containing high sulfate and high-sulfate The main conclusions are as follow:1. Sulfate connects of medium molecular weight fucoidan is C2 or C3 bit in the equatorial key position and mainly contains fucose and galactose.2. In the radical degradation of raw sugar, the higher the concentration of H2O2, the lower the molecular weight of the product and the sulfate and fucose content of product will decrease.It mainly contains fucose and galactose, disulfide replacement could be exist between C2 and C4 bit.the low molecular weight fucoidan between 300-2500Da comes from 9% H2O2 and 4.5% H2O2, has no significant difference in the composition, the molecular weights are 1.5kDa. The sulfate content of low molecular weight fucoidan degraded by 9% H2O2 of raw sugar was significantly lower than that degraded by 4.5% H2O2. Products are mainly concentrated between 2500-6000Da. In the whole products, the highest sulfate content of the component is LMWF4, 30.28%.3. A heated solvent and degassing method were used in the traditional barium chloride-gelatin method, and the costs of laboratory reagents and equipment are lower, so it is generally suitable for determination of sulfate content in the laboratory. Fewer samples were used by ion chromatography than modified barium chloride-gelatin method in the determination of sulfate content, and the interference of other ions and impurities were avioded. So ion chromatography is more suitable for the detection of sulfate content in the Laminaria Japonica Areschi.4. The higher molecular weight of fucoidan, the stronger the antithrombotic capacity is.5. The antithrombotic effect of medium molecular weight fucoidan mainly increase extrinsic coagulation pathway ,inhibit platelet aggregation by platelet metabolism of arachidonic acid , but it is not obvious in extend the endogenous pathway of coagulation and fibrinolytic system, and they can promote platelet aggregation induced by ADP and thrombin in vitro on human .6. The antithrombotic effect of low molecular weight fucoidan mainly increase extrinsic and endogenous coagulation pathway, active fibrinolytic system, inhibit platelet aggregation by platelet and vascular metabolism pathway of arachidonic acid.