| Hypomesus olidus is a kind of small freshwater fish, which has a high nutritional value and economic value, and the process of it is very convenient, that is why so popular to domestic and foreign consumers.Current research on Hypomesus olidus are still in their farming,fishing techniques,resource distribution,food processing and preservation technology, and studies on its extractive and its biological activity is extremely rare, only found in my laboratory study that the extractive from Hypomesus olidus having anticoagulant function.In this paper,extraction,physicochemical properties and anticoagulant mechanism of Hypomesus olidus are studied.The specific contents and results are as follows:1. Ultrasonic assisted alkaline hydrolysis of polysaccharides extracted from the head of Hypomesus olidus,and the optimal ultrasonic-assisted extraction conditions by response surface methodology were obtained as follows: ultrasonic power 275 W, ultrasonic treatment time 50 min, and liquid-solid ratio 5 mL/g,Under this condition, Hypomesus olidus crude polysaccharide yield was 7.73 %, compared with the traditional method was increased by 20.44 %.2. Using Sevag method,Trypsin combined with Sevage method and papain combined with Sevage method are used to remove polysaccharide protein from Hypomesus olidus crude polysaccharide.Compareing the three methods of protein clearance rate and loss rate of polysaccharide found that the method of trypsin combined with sevage has the highest protein clearance and minimum loss rate of polysaccharide, respectively 85.48% and 28.78%.3. Y-1,Y-2 and Y-3,were preliminary separated and purified by DEAE-D22 cellulose,and the polysaccharide yield of them were 536.8 mg/g,392.7 mg/g and 276.2 mg/g.4. Y-1,Y-2 and Y-3 were further separated and purified by Sephadex G-100 gel column chromatograph to obtain purified polysaccharide GYT-1,GYT-2 and GYT-3.The content of total sugar of GYT-1,GYT-2 and GYT-3 were 81.54%,80.13% and 77.67%;the protein of them were 0.24%,0.15% and 1.32%;the sulfate ester of then were 3.58%,1.04% and 7.65%;the uronic acid of them were 2.38%,6.87% and 8.59%.5. The results of UV and IR spectrum analysis showed that all of GYT-1,GYT-2 and GYT-3 has the characteristic absorption peaks of polysaccharides,and found that GYT-3 is a kind of Glycosaminoglycan.6. In the vitro anticoagulant assay indicated that all of Hypomesus olidus crude polysaccharide GYT,preliminary purified polysaccharide Y-1 and Y-3,refined polysaccharide GYT-1 and GYT-3 could prolong both the activated partial thromboplastin time(APTT) and thrombin time(TT)(P<0.05),And the influence of prothrombin time(PT) is not significant.The result indicated that they inhibited both the intrinsic and common pathways of coagulation so as to achieve the effect of anticoagulant.And when the polysaccharides were in the same concentration, the anticoagulant activity of GYT-3 was the maximum.7. GYT-3 was mainly composed of Ara,Fru,Glc and Lac by high performance liquid chromatography(HPLC) comparing with the monosaccharide standard peak time.And the monosaccharide composition molar ratio of them was 0.595:1:2.026:0.273 calculated with internal standard method.8. The results of mechanism of anticoagulation research showed that both the effects of the prolongation of APTT and TT,and inhibition of factor â…¡a and â…¡a were positive correlation of AT-â…¢ concentration in plasma.While plasma was lack of AT-â…¢,both of the anticoagulant activity of GYT-3 and its inhibitory of factors â…¡a and â…¡a also disappeared.All these results suggest that the anti-clotting activity of Hypomesus olidus polysaccharide GYT-3 requires participation or regulation of AT-â…¢ in plasma. |
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