Study On A Novel Extraction And Purification Technology Of Heparin And Bioactivity Of Degraded Heparin

Heparin sodium is known as a kind of polysaccharide, which is widely distributed in mammalian lung, liver, and small intestinal mucosa. Being an anticoagulant, some other biology functions like antithrombotic, blood fat regulation, anti-inflammatory and anti-anaphylaxis are also shown for heparin. By chemical, enzymatic or physical degradation, the GAG fragments of 3000 ~8000Da molecular weight isolated from the degradation heparin sodium is called low molecular weight heparin (LMWH). LMWH shows stronger biological functions than crude heparin. At present, few heparin factories are there very in China, however, most of the producers are very small scale workshops, and based on primary processing technology and equipment, which leads to the lower yield of heparin, and lower profit. Accordingly, by using the porcine small intestine mucous membrane as raw materials, new technologies of heparin extraction, separation and purification, preparation of LMWH, and sulfate modification were developed in this study, and the biological activity of LMWH was researched too.1. Research on the technologies parameters of ultrasound assisted enzyme hydrolysis extraction porcine small intestine heparin. Single factor experiment and quadratic rotary combinational design was applied to optimize the conditions of ultrasound assisted and enzyme hydrolysis extraction heparin. As the results indicated that the optimized parameters were: ultrasonic power 300W, ultrasonic time 40 min, ratio of material to solution 15 mL/mg, ultrasonic temperature 40℃, enzyme addition quantity 1.85 %,pH value 8.7, enzyme hydrolysis temperature 58℃,enzyme hydrolysis time 2.6 h; the addition quantity of precipitation agent 0.8%, treatment temperature 50℃, treatment time 1h. The yield of heparin was 40% higher than traditional salt fraction, and the extraction time was shortened 2 h.2. Research on technology parameters Simulated Moving Bed (SMB) purification heparin sodium. Strong alkalinous anion ion exchange resin was applied to separate heparin from the crude extraction solution, single factor experiment and orthogonal experiment were applied to study the effect of factors on heparin separation, the results were: feed flow rate 1.5 mL/min, 2mol/LNaCl elution flow rate 1.5mL/min, pH 8.0, elution temperature 45℃, the heparin recovery was 95.8%. Based on single column experiment data, the process conditions of SMB were established, and the results showed: feed flow rate at 20 mL/min, eluent concentration 2 mol/LNaCl, elution flow rate 20 mL/min, temperature 45℃, pH 8.0, switching time 20 min, the purity of heparin sodium was 98.2%, and the heparin sodium potency was 150 U/mg, which was corresponded with the standards of the 2005 Chinese Pharmacopoeia.3. Research on the technology parameters of nitrous acid degradation method preparation LMWH and conditions of LMWH sulfation modification. By using the extracted porcine small intestine heparin as the raw material, quadratic rotary combinational design was applied to optimize the preparation technology parameters of nitrous acid degradation method preparation LMWH. The results were: reaction time 4h, reaction temperature 25℃, nitrous acid concentration 0.6%, pH 2.6. As a result, the average molecular weight of LMWH was 4600±95Da, The content of molecular weight of less than 8000Da was 89.95±1.2%, and the recovery of LMWH was 80±2.3%. With replace degree as the index, by using single factor experiment, modification conditions of the Wolfrom method, DMF method, concentrated sulfuric acid method, and sodium acid sulfate method were compared. Wolfrom method was chosen as the optimum condition for LMWH sulfation modification. The optimized conditions of Wolfrom method sulfation modification LMWH were as following: reaction temperature 10℃, reaction time 3h, and pH3.2, the replace degree was 0.48. Determined with the method of 2005 Chinese Pharmacopoeia, all the indexes were consistent with the standards of it.4. Research on the biological activity tests of LMWH and multi-sulfated heparin. With LMWH as experimental materials to study the growth inhibition for four kinds of human cancer in vitro, and with LMWH and multi-sulfated heparin as experimental materials to study the blood lipid lowering effect for hyperlipidemia mouse. The results of MTT, flow cytometry tests showed: LMWH had obviously inhibitory action on human gastric cancer cells (SGC-7901), human breast cancer cells (MCF-7), hepatoma cells (HCCLM3) proliferation (P <0.01), and could reduce the activity of human lung cancer cells (A549) (P <0.05), furthermore, the inhibitory action of multi-sulfated heparin was more obvious. Flow cytometry tests also showed that LMWH and multi-sulfated heparin could block cell cycle progression, and cause cell morphologic change, and showed a significant dose - effect and time - dependent manner, with lower rate of cancer cell apoptosis (48%). The results of blood lipid lowering test for hyperlipidemia mouse showed that different doses of LMWH and multi-sulfated heparin both could reduce cholesterol in the blood of hyperlipidemic mice, the middle-dose group and high-dose group were extremely significant, and the cholesterol-lowering effect of multi-sulfated heparin was better than LMWH. Moreover, the high-dose group of LMWH could reduce riglycerides in hyperlipidemia mouse blood, with an obvious weight loss, on the other hand, the MDA content of middle-dose group and high-dose group was significantly reduced, which indicated that LMWH could enhance the antioxidant capacity of organism.Conclusion: Conclusion: with porcine small intestine as the raw material, by means of quaternionic orthogonally rotational combination design and SAS, SPSS software for statistical analysis, the critical technology parameters were optimized, and the corresponding mathematic model was built. Moreover, the application of ultrasound assisted extraction and SMB separation and purification technologies changed the conventional technology, shorten the processing time, reduced the running cost, and improved the quality of the product. Combination of nitrous acid degradation method and Wolfrom method sulfation modification increased the biological activity of LMWH. The results of confirmatory experiments showed that all the indexes of heparin and LMWH. were consistent with the standards of 2005 Chinese Pharmacopoeia. Therefore, studies on new technologies of heparin extraction and preparation of LMWH possess excellent practical application importance and great promotion value.