Study On The Chitosan Derivstives Of Amino Acid In Anticoagulations

Heparin is one of the best anticoagulant in clinical anticoagulant effect. It could form stable compounds with a lot of clotting factors, such as blood coagulation factor IX, prothrombin and thrombin. The compounds could inhibit the activity of the clotting factors to reach a good anticoagulant effect. But the specific identification of long chain heparin molecular is low. Long chain heparin molecular could combine with protein except clotting factors. Long-term use, it may cause high blood potassium or hemorrhage syndrome. Meanwhile, heparin could not be synthesized artificially and the price of heparin is very expensive.Study found that chitosan had the analogous structure of heparin. Carboxyl was an important anticoagulant group in the structure. It had excellent biocompatibility and degradability. So use chitosan to develop anticoagulant drugs would have the features of low-cost, high safety and convenient production. It could be used for the prevention and treatment of cardiovascular disease, blood preservation, blood anticoagulant in uremic dialysis or extracorporeal circulation, blood anticoagulant of the surface of the medicinal materials in checking or treating patients and implants surgery, et al. In consequence, using chitosan to develop anticoagulant drugs has a good development prospect.Chitosan of high deacetylation degree would be used to react with glycine-epoxy chloropropane, aspartic acid-epoxy chloropropane and arginine-epoxy chloropropane in aqueous medium to compound chitosan glycine derivative, chitosan aspartic acid derivative and chitosan arginine derivative. With rate of charge, reaction temperature, reaction time and concentration of NaOH solution as study factors, four factors three levels orthogonal experiment was designed to determine the optimum condition of synthesis of three amino acid derivatives. The influence of different factors on the degree of substitution was investigated by single factor experiment. The structures of three chitosan derivatives were analyzed by infrared spectrum and the molecular structures of the derivatives were confirmed preliminary. Anticoagulation property of chitosan derivatives were qualitative and quantitative analysis through testing the substitution degree of derivatives, chelating properties, whole blood coagulation time (CT), activated partial thrombin time (APTT) and prothrombin time (PT). The reason and mechanism of anticoagulant function were preliminary discussion in this dissertation. Finally, external hemolysis ratio of derivatives was tested to discuss the blood compatibility preliminary. The results showed that:1. Piecewise washing method was employed, chitosan was treated with40%NaOH for6h. Chitosan of high deacetylation degree was obtained as deacetylation degree was94.25%and molecular weight was100300.2. Substitution degree of carboxyl in the chitosan derivative served as a measured standard. The optimum condition for synthesis of chitosan glycine derivative by four factors three levels orthogonal experiment as follows:rate of charge of intermediates of chitosan react with glycine-epoxy chloropropane (mole ratio)1:4, reaction temperature40℃, reaction time4h, the concentration of NaOH solution5mol/L. Among of study factors, rate of charge of intermediates of chitosan react with glycine-epoxy chloropropane had a great influence on substitution degree of derivatives. Then temperature, reaction time and the concentration of NaOH solution had an influence on substitution degree of derivative. By single factor experiment analysis, the optimum condition for synthesis of chitosan glycine derivative as follows:rate of charge (mole ratio)1:4, reaction temperature50℃, reaction time6h, the concentration of NaOH solution10mol/L; the optimum condition for synthesis of chitosan arginine derivative as follows:rate of charge (mole ratio)1:4, reaction temperature40℃, reaction time4h, the concentration of NaOH solution10mol/L; the optimum condition for synthesis of chitosan aspartic acid derivative as follows:rate of charge (mole ratio)1:6, reaction temperature50℃, reaction time8h, the concentration of NaOH solution10mol/L.3. Calcium ion chelating performance of products was tested. Chitosan itself almost didn’t have calcium ion chelating performance. But with the increase of the substitution degree, chelating properties of the products gradually enhanced. Under the same substitution degree, chelating properties of the products as follows:chitosan aspartic acid derivative> chitosan glycine derivative> chitosan arginine derivative. 4. The structure of products was tested by the infrared spectrum. The products were obtained by hydroxyl of Chitosan reacting etherification reaction with amino acid. And carboxyl content in the product molecules as follows:chitosan aspartic acid derivative> chitosan glycine derivative> chitosan arginine derivative. The sort agreed with chelating properties.5. Whole blood coagulation time (CT), activated partial thrombin time (APTT) and prothrombin time (PT) showed that:chitosan itself didn’t have the anticoagulant performance, after grafting amino acids, most of the products had anti-coagulation activity. Meanwhile, with the increase of the substitution degree, the anticoagulant effect of the products enhanced significantly. Anti-coagulation activity of chitosan arginine derivative was the highest, reach35u/mg, which was equal to1/3anti-coagulation activity of heparin. The anticoagulant reason of chitosan amino acid derivatives mainly had two points:firstly, carboxyl had calcium ion chelating performance; secondly, the special molecular structure of the product itself. Chitosan amino acid derivatives could both have influence on endogenous and exogenous coagulation pathway to achieve the effect of anticoagulant.6. Chitosan amino acid derivatives didn’t have external hemolysis reaction and had good blood compatibility.