Extraction, Purification Of Heparin And Preparation Of Low Molecular Weight Heparin By Nitrous Acid

Heparin is a polysaccharide produced by mast cells in animal connect-ive tissue, and mainly exists in the pig small intestine mucosa and liver and lung tissue cells. At present, the heparin was mainly extracted from t-he pig small intestine mucosa, which is the byproduct in meat products en-terprises. Because the technology and equipment are not new and the pro-fessional and technical personnel are few in most of the small enterprises, the production of the good and bad is intermingled, and the products are mainly exported as raw heparin API. To improve the product quality and processing precision, extending industrial chain for raising quality and effic-iency are extremely urgent. Heparin has good anticoagulant effects, it is t-he first choice when preventing and treatmenting the deep vein thrombosi-s (DVT) thromboembolic disorders. As an anticoagulant, heparin has been used in the clinic for 70 years, but there is still bleeding in the clinical a-pplications, which limits its clinical application. Whereas, low molecular weight heparin has many advantages, such as producing good and lasting antithrombotic effect when at a low level of anticoagulation, little bleeding, long plasma half-life and so on. But now, our country’s research on low molecular weight heparin is still in the primary stage, and the use of low molecular weight heparin mainly relys on imports. So in this study, the pr-eparation technology of heparin was investigated to improve the tradition-al production technology and the quality of heparin products. Meanwhile, t-he nitrite on preparing low molecular weight heparin was studied to provi-de certain theoretical basis in producing low molecular weight heparin. T- he main results were as follows:1. The study on detecting the heparin potency. Two manufacturers’he-parin potency was detected, and the results of sheep plasma, chromogenic substrate, azure A method were 148 U/mg and 164 U/mg,145.0 U/mg a-nd 156.0 U/mg,145.5 U/mg and 161.4 U/mg, respectively. Three method-s could be used for determination of heparin sodium potency, and the dete-rmination results were reliable and basically identical. In the application, t-he actual conditions and the advantages and disadvantages of each metho-d should be considered. The azure A method was used for rapid detection in the experiment, but the other two methods based on detection of antic-oagulant activity were mainly used for the detection of pesticide effect of the final products.2. The study on preparing raw heparin sodium. In the preparation of raw heparin sodium, three main steps were disscussed:enzymolysis, resin adsorption and desorption. The influence of the factors (temperature, pH, salt concentration, time) on heparin potency, the adsorption rate and hepari-n yield was studied. And according to the results of the orthogonal tests, the optimized conditions of preparing raw heparin were as follows:a. en-zymolysis:material liquid ratio of 1:8, salt concentration 0.5 mol/L, the ad-ding quantity of trypsin 0.1%, enzymolysis liquid pH value of 8.8, enzy-molysis temperature 60 ℃, enzymolysis time 3.5 h. b. adsorption:the add-ing quantity of D254 resin 2.8%, adsorption temperature 55 ℃, salt conce-ntration 0.2 mol/L, adsorption liquid pH value of 8.5, adsorption time 7 h. c. elution:eluent salt concentration 3.0 mol/L, eluent pH8.5, elution temp-erature 45 ℃, elution time 3.5 h, elution twice. The raw heparin potency was 83 U/mg, yield was 2.12%, protein content of 19.26 mg/g, products were in pale yellow. Finally, the requirement of refined heparin sodium fo-r raw materials was achieved, and the production was stable and efficient, and the effect was satisfactory.3. Study on refining raw heparin. In refining technology, the effects o-f the once oxidation, twice oxidation with the enzymolysis and enzymolys-is combining oxidation on refining crude heparin were analyzed, and the protein content, potency, impurity UV absorption OD260, OD280, and produc -t color and other indicators were analyzed. Finally, the enzymolysis(the q-uantity of trypsin 0.1%, enzymolysis temperature 40 ℃, enzymolysis liquid pH value of 7-9, enzymolysis time 5 h) combining oxidation(the quantit-y of H2O21.5%, oxidation pH8.5-9.5, oxidation temperature 25 ℃, oxidat-ion time 15 h) was defined as the best way for refining raw heparin, the protein content of the final product was 6.13mg/g, anticoagulant activity was 139.5U/mg, the OD260 and OD280 reached the provisions of the Chine-se Pharmacopoeia, and the product was analyzed by Fourier transform inf rared spectrometer, the spectrum was consistent with standard heparin pro-duct peak shape, the product was white, method was stable, and the result-s were good.4. Study on preparing low molecular weight heparin with nitrous acid. Four influencing factors, namely nitrite concentration, degradation temperat-ure, solution pH and degradation time were selected, and then the impact of the four factors on the quality of LMWH degradated were analyzed. A-nalysis was done by polyacrylamide gel electrophoresis (PAGE), and the optimum parameters of preparation of low molecular weight heparin were 0.6% of nitrite concentration, liquid pH2.6, the degradation temperature 25 ℃ and degradation time 4.0 h, and the LMWH yield was 45.4% unde-r the condition.5. Research on the quality of the low molecular weight heparin. The sheep plasma method and the chromogenic substrate method were used for detecting the potency of low molecular weight heparin, the resuslts were 185 U/mg, anti-FXa potency= 122.5 U/mg, and anti-FⅡa potency= 43.1 U/mg, respectively.And anti-FXa/anti-FⅡa=2.8>1.5, which was conform to the requirment of the 2015 edition of China Pharmacopoeia. Viscosity me-thod was developed for the determination of low molecular weight hepari-n and the average molecular weight was 3292.5 Da. The gel permeation chromatography and capillary electrophoresis were preliminaryly used in an-alyzing low molecular weight heparin, the separation conditions were dete-rmined and the results showed that both methods could effectively analyz-e the molecular weight of low molecular weight heparin.The analysis method of heparin product was established, and the basi- c processes of extracting heparin from small intestine mucosa and purificat-ing heparin as well as preparing LMWH with nitrite were achieved, whic-h had guiding significance for preparing new heparin product with intesti-ne mucosa.