Study On Modification And Anticoagulant Activity Of Chitosan

Chitosan was a unique basic polysaccharide in nature, which was a bundant resources, low prices, excellent biological activity and of widely used in many aspects such as the pharmaceutical, food, agriculture and chemicals. At present, the heparin was one of the most commonly used anti-clotting drugs in clinical application. Although it had a high anticoagulant activity, it could lead side effects such as internal hemorrhage. Therefore, in this paper we modified the chitosan and hoped to get products which were similar to the heparin in structure-activity, to provide a scientific basis for the clinical research. The main studies are as follows:1. The physical and chemical properties of chitosanA low-molecular-weight chitosan was prepared by H2O2oxidation, and its physical and chemical properties were investigated. The results implied that degree of deacetylation of a low-molecular-weight chitosan and chitosan both are85.2%. The molecular weight of the sample dropped from6.3×105to1.3×104, and the sample was colored from white to yellow.2. Study on sulfation of a low-molecular-weight chitosanSulfonation reaction tempreture, the amounts of chlorosulfonic acid and sulfonation reaction time influencing the S-content were investigated, respectively. By orthogonal experiments, the optimum sulfonation reaction conditions as follows:sulfonation reaction temperature70℃, the amounts of chlorosulfonic acid3mL and sulfonation reaction time4h, the highest S-content of sulfated chitosans is8.94%.3. Anticoagulant activity in vitro tests of sulfated chitosans The anticoagulant activities of low-molecular-weight sulfated chitosans were investigated by the classical coagulation assays APTT, PT and TT, and meanwhile the saline solution and heparin were used as negative and positive control, respectively. Anticoagulant activity in vitro tests showed that low-molecular-weight sulfated chitosans could prolong APTT, but according to the PT assay low-molecular-weight sulfated chitosans were no significant differences in modulating prothrombin time. The results implied that a low-molecular-weight sulfated chitosan initiates via the intrinsic pathway in the coagulation of blood. The sulfated chitosans prolonged APTT compared to saline control a dose-dependent manner.Ranging from4.61%to8.94%, the higher the S-content, the better the anticoagulant activity.4. Antimicrobial activity of sulfated chitosansAntimicrobial activity of sulfated chitosans was measured by filted paper inhibition zone method. The results implied that chitosan and its derivatives had antimicrobial activity compared to the saline solution. Low-molecular-weight sulfated chitosans showed good antimicrobial activity.The higher the S-content of sulfated chitosans, the better the antimicrobial activity. The sample2#with S%of7.96%showed best antimicrobial activity among Staphylococcus anreu and Bacillus subtilis, and The sample1#with S%of8.94%showed best antimicrobial activity among Escherichia coli.5. Anticoagulant activities of sulfated carboxylchitosansO-carboxymethyl chitosan was prepared firstly. Alkalization time, Carboxylation reaction tempreture, the amounts of chloroethanoic acid and carboxylation reaction time influencing the degree of substitution of carboxylchitosans were investigated, respectively. Preparation and anticoagulant activities of sulfated carboxylchitosans were studied. The results implied that anticoagulant activity of sulfated carboxylchitosans depended on not only DS of carboxymethyl groups but also the S-content. The sample with DS of0.668and S%of8.316%showed best anticoagulant activity. Sulfated carboxylchitosans could prolong APTT, but according to the PT assay sulfated carboxylchitosans were no significant differences in modulating prothrombin time. The results implied that sulfated carboxylchitosans initiated via the intrinsic pathway in the coagulation of blood.