Study On The Preparation Of Natural Lmwh And The Modification Condition Of Sulfation

As an important anticoagulant drugs, low molecular weight heparin is a fragment of glycosaminoglycan isolated from heparin of molecular weight 3000 ~ 8000Da ,which is treated by chemical, enzymatic or physical degradation methods. Compared with traditional heparin, low molecular weight heparin has a strong antithrombotic effect and a lower bleeding side effect, higher bioavailability, longer plasma half-life, etc., and it seems that it can replace heparin in the international market. In order to further enhance the characteristics of low molecular weight heparin on the anti-FXa and anti-FⅡa activity ratio, less 8000Da content, high yield, etc., heparin was taken as the raw material, by comparison the advantages and disadvantages between different preparation merhods, and optimization the preparation technology parameters, a reference can be provided for chinese livestock and poultry by-product utilization technology .1. Comparison and analysis of different low molecular weight heparin preparative technique. Physical and chemical methods for preparation low molecular weight heparin such as organic solvent method, dialysis filtration method, hydrogen peroxide method, nitrite method,β-elimination degradation of was compared in this study. Single factor experiment and orthogonal experiment was applied, by aggregate analysis of the average molecular weight, yield, content of molecular weight less than 8000Da, and the difference with standard preparation, nitrous acid degradation method was chosen to prepare low molecular weight heparin.2. Optimization and determination of the technology parameters of nitrous acid degradation method preparation low molecular weight heparin. By using Single-factor Experiment , Second Rotation Orthogonal Combination of Experiment, and response surface methods analysis, the preparation technology parameters of nitrous acid degradation method preparation low molecular weight heparin were researched, and the optimized parameters were: reaction time was 4h, reaction temperature 25℃, nitrite concentration 0.6%, pH2.6. As a result, the average molecular weight of low molecular weight heparin was 4600Da±95Da, the content of molecular weight of less than 8000Da was 89.95%±1.2%, the different of molecular weight with standard sample was 2.1%,recovery was 80%±2.3%; the content small molecular weight 8000Da was higher than the national and international standard. Nevertheless, the activity of low molecular weight heparin was reduction because of sulfate groups were degradation in the processing course of nitrous acid degradation.3. Study on low molecular weight heparin sulfated group modification conditions. Combined with the structure and nature of low molecular weight heparin, Wolfrom method, DMF method, concentrated sulfuric acid method, and sodium acid sulfate method were chosen, by using single-factor experiment and orthogonal experiment, took the replace degree as a indicator, compared with the modification conditions of the above four methods, the anti-FXa to the FⅡa activity and infrared structure of the fours kinds of modified low molecular weight heparins were determined, finally, the Wolfrom method was decided as the conditions for low molecular weight heparin sulfate group modification. 4. The determination of physical and chemical properties of Low molecular weight heparin. In accordance with the specify of the Chinese Pharmacopoeia, the physical and chemical properties of low molecular weight heparin were identified, the results showed that all the indicators were consistent with the requirements of Chinese Pharmacopoeia.