Heparin sodium is a kind of mucopolysaccharide sulfate anticoagulant. It is sodium salt of CSSO3 extracted from the intestinal mucosa of pigs or cattle, which belongs to mucopolysaccharide. At present, it is the most widely used anticoagulant in the world. This paper introduces a process of extracting heparin sodium from pig intestinal mucosa by enzymolysis.
The extraction process of heparin sodium includes the following steps:
1.Enzymolysis: Put porcine small intestinal mucosa into a blender and add 0.5% bovine amylase (35,000 U/g) by weight of raw material. After mixing evenly, adjust ph to 8.0-9.0. After heating to 45-50℃, the enzymatic hydrolysis was carried out for 2.8 hours by constant temperature stirring. The pH value of the reaction system should be kept stable during the enzymatic hydrolysis process.
2.Enzyme inactivation: The enzymolysis solution was heated to 90℃ for 10 minutes, and then cooled immediately.
3. Filtration: The quench liquid is filtered twice by a filter, first by a coarse filter with a pore diameter of 2 microns, and then by a filter with a pore diameter of 0.22 microns. After removing impurities, the filtrate is obtained.
4. Enzymatic hydrolysis of protein: Adjust the pH of the filtrate to 7.5-9.0, then heat it to 45-55℃, add flavor protease of 0.5% weight of raw material (38000 U/g), stir the filtrate at 45-55℃ for 4-6 hours. In order to control the stability of the ph of the reaction system, the enzymolysis solution was heated to 90℃ for 10 minutes and then cooled immediately.
5. Protein removal: Remove protein with a 5 micron filter and concentrate the quenched solution.
6. Precipitation: In a liquid concentrator, 95wt% ethanol was added at equal density, stirred and precipitated for 10 hours, then the supernatant was removed. The crude heparin sodium was obtained by adding anhydrous ethanol for dehydration and freeze drying.
7. Refining: adjust pH to 1.5 with pure water of 9 times the weight of crude heparin sodium, then dissolve the crude product, filter to remove impurities, adjust pH of filtrate to 11, record the volume of the solution at this time, then add 30wt% hydrogen peroxide to 4% of the volume, keep the oxidation at 25℃, pay attention to control the pH of the reaction system to be stable at 11. After 30 hours of oxidation, adjust pH to 6.5, then add 95wt% ethanol with the same density as the solution, stir and stand for 10 hours to remove the supernatant, add anhydrous ethanol with the same density to the precipitate for dehydration, filter out the ethanol precipitate, ventilate, evaporate the precipitate to remove the residual ethanol, vacuum drying, and obtain the purification of heparin sodium.