A kind of production method extracting heparin from pig lung
Technical field
The present invention relates to biomedicine field, particularly relate to a kind of production method extracting heparin from pig lung.
Background technology
Heparin is acidic mucopolysaccharide sulfuric acid ester anticoagulant.Recent study proves that heparin also has reducing blood lipid, and for many years, world market is always very vigorous to the demand of heparin.Obtain because heparin extracts processing from pluck, output is subject to a definite limitation, often can not meet the market requirement.From the later stage nineties 20th century, world market heparin demand significantly increases, and China's heparin is produced and exported year after year all to rise by a relatively large margin, and export volume and export amount of money are quick growing trend.China has become premier heparin bulk drug big export country in the world.Calendar year 2001, national heparin product export amount of money is 4,460 ten thousand dollars.2002, export amount of money reached 5,894 ten thousand dollars, increases 32.17 % than last year.Before heparin product ranks among in China's pharmaceutical products export total money sequence 5, be only second to vitamins C, citric acid, vitamin-E and sulfanilamide (SN).
The heparin product that China produces, except meeting domestic market, also exports in a large number.For a long time, China's products export is smooth and easy, as long as quality reaches requirement, usually has how many just outlet how many.In contrast thereto, due to the Economic Development Status of China and the restriction of living standards of the people, to this costliness (injection fine work 8 ~ 10 dollars/mega, for oral use is 7 ~ 8 dollars/mega) anticoagulant current home consumption amount minimum, mainly earn foreign exchange, sell to the developed countries and regions such as Japan, the U.S., Europe.
At present, the method of domestic production heparin, mainly salt solution and acid hydrolyzation, such as publication number is CN101735340A, CN101649008A, the Chinese invention application of CN101519459A etc., these two kinds of methods are all adopt pig intestinal mucosa to be raw material, in the basic conditions by salt solution, enzymolysis, go out after partial protein, by Trimethylamine 99 quaternary ammonium strong basicity cationic exchange resin adsorption agent wash-out, in elutriant in addition alcohol settling both heparin sodium primary raw materials is sold, its shortcoming reaches 48h at the production cycle, temperature of reaction is 95-100 DEG C, energy consumption is large, hydrolysis not exclusively, yield is low, produce 100,000,000 international unit (IU) heparin crude product and need 2800-3000 root pig intestinal mucosa, products obtained therefrom purity is low, crude product is at most 80 containing heparin and tires/milligram, and produce a large amount of large intestines slags and waste water, contaminate environment, and resin regeneration difficulty in production process, this preparation method is long due to the production cycle, is hydrolyzed not exclusively, not thoroughly, obtained heparin sodium purity is low, yield is low, it is low to tire, and is difficult to carry out Produce on a large scale, and can not meets the heavy demand in market.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, there is provided that a kind of life cycle of the product is short, purity is high, height of tiring, impurity are few, the pig lung that utilizes of environmental protection extracts the production method of heparin crude product, this technique adopts biologic enzymolysis method to extract heparin crude product from the pig lung that starting material are easy to get.
The technical scheme that the present invention realizes object is as follows:
A kind of production method extracting heparin from pig lung, it is characterized in that: step is as follows: (1) pre-treatment: pig lung is cleaned, after removing inside and outside dirt and fat, rub into rotten shape, and under fully stirring, add water stirring and obtain pig lung mixed solution, wherein the mass ratio of pig lung and water is 1:3-4, (2) enzymolysis: adjust pig lung mixed solution pH value to be 6.5 ~ 8.0, add the chondroitin sulfate prozyme of 1.5% ~ 2.5% of pig lung weight, stir, be warming up to 30 ~ 45 DEG C, after insulation reaction 30 ~ 60min, the pH value keeping reaction solution is 7.0 ~ 7.5, add 0.25% ~ 0.5% trypsinase of pig lung weight, stir, insulation 2 ~ 4h, continue to be warmed up to 90 ~ 100 DEG C, keep 5 ~ 10min, cooling, obtain pig lung enzymolysis solution (3) removing protein: filter pig lung enzymolysis solution, remove surface oil lipid layer, filtrate pH value is adjusted to be 8.5 ~ 10.5, stir the finings adding 0.1% ~ 0.4% of pig lung weight when being warmed up to 55 ~ 65 DEG C, continue to be warming up to 90 ~ 95 DEG C, insulation reaction 2 ~ 4h, be cooled to normal temperature, (4) ultrafiltration: aforesaid liquid 0.2 ~ 0.4 μm of filter membrane is filtered, obtains filtered liquid, (5) oxidation precipitation: add hydrogen peroxide, make the content of hydrogen peroxide in filtered liquid be 10 ~ 30g/kg, normal-temperature reaction 8 ~ 12h filters, obtain filtrate, filtrate is adjusted pH to 4.5 ~ 5.5, after adding isopyknic dehydrated alcohol precipitates overnight, removing supernatant liquid, throw out is crude product heparin, (6) lyophilize: crude product heparin is put into vacuum freezing drying oven, design temperature-20 ~ 10 DEG C, vacuum tightness 0.05 ~ 0.08Mpa, time of drying 2 ~ 4h.
Described a kind of production method extracting heparin from pig lung, is characterized in that: described step (2) chondroitin sulfate prozyme is the combination of in chondrosulphatase A, B and C two kinds or more.
Described a kind of production method extracting heparin from pig lung, is characterized in that: described step (3) finings is natural 1+1 finings, is made into the solution of 1% before use with Glacial acetic acid.
Described a kind of production method extracting heparin from pig lung, is characterized in that: the acid used of described step (5) adjust pH is one in Glacial acetic acid, oxalic acid, citric acid or two kinds of mixing.
Advantage of the present invention and beneficial effect are: the enzyme solution that 1, the present invention adopts more thoroughly can dissolve pig lung slurries all sidedly, enzymolysis is stopped with trypsinase, Vacuum Freezing & Drying Technology stabilizes the quality of product and improves yield, and the crude product impurity simultaneously obtained is few, height of tiring; 2, adopt in production method of the present invention finings come alternative ethanol repeatedly alcohol precipitation remove the traditional technology of protein, there is clearance high, saved the usage quantity of a large amount of ethanol, reduced production cost; 3, present invention process is reasonable in design, produces nontoxic, harmless, biological green environmental protection, and pig lung raw material sources are easy to get, and operation cost is low, is applicable to large-scale industrial production.
Embodiment
Embodiment 1: a kind of production method extracting heparin from pig lung, it is characterized in that: step is as follows: (1) pre-treatment: pig lung 5kg is cleaned, after removing inside and outside dirt and fat, rub into rotten shape, and under fully stirring, add the stirring of 15kg water and obtain pig lung mixed solution, (2) enzymolysis: adjust pig lung mixed solution pH value to be 6.5 with sodium hydroxide, add chondroitin sulfate A (CSA) and the C of 75g, stir, be warming up to 30 DEG C, after insulation reaction 30min, the pH value keeping reaction solution is 7.0, add 12.5g trypsinase, stir, insulation 2h, continue to be warmed up to 90 DEG C, keep 5min, cooling, obtain pig lung enzymolysis solution (3) removing protein: filter pig lung enzymolysis solution, remove surface oil lipid layer, filtrate pH value is adjusted to be 8.5 with sodium hydroxide, stir the finings adding 5g when being warmed up to 55 DEG C, continue to be warming up to 90 DEG C, insulation reaction 2h, be cooled to normal temperature, (4) ultrafiltration: aforesaid liquid 0.2 μm of filter membrane is filtered, obtains filtered liquid, (5) oxidation precipitation: add hydrogen peroxide, makes the content of hydrogen peroxide in filtered liquid be that 10/kg, normal-temperature reaction 8h filter, obtain filtrate, filtrate is adjusted pH to 4.5, after adding the dehydrated alcohol precipitates overnight of 20kg with Glacial acetic acid, removing supernatant liquid, throw out is crude product heparin, (6) lyophilize: crude product heparin is put into vacuum freezing drying oven, design temperature-20 DEG C, vacuum tightness 0.05Mpa, time of drying 2h, both heparin crude product 20 g, it is tired as every milligram of 100 units.
Embodiment 2: a kind of production method extracting heparin from pig lung, it is characterized in that: step is as follows: (1) pre-treatment: 100kg pig lung is cleaned, after removing inside and outside dirt and fat, rub into rotten shape, and under fully stirring, add the stirring of 350kg water and obtain pig lung mixed solution (2) enzymolysis: adjust pig lung mixed solution pH value to be 7.0 with sodium hydroxide, add chondroitin sulfate A (CSA) and the B of the 2kg of pig lung weight, stir, be warming up to 40 DEG C, after insulation reaction 45min, the pH value keeping reaction solution is 7.0, add 0.3kg trypsinase, stir, insulation 3h, continue to be warmed up to 95 DEG C, keep 7min, cooling, obtain pig lung enzymolysis solution (3) removing protein: filter pig lung enzymolysis solution, remove surface oil lipid layer, filtrate pH value is adjusted to be 9.0 with sodium hydroxide, stir the finings adding 0.2kg when being warmed up to 60 DEG C, continue to be warming up to 93 DEG C, insulation reaction 3h, be cooled to normal temperature, (4) ultrafiltration: aforesaid liquid 0.2 μm of filter membrane is filtered, obtains filtered liquid, (5) oxidation precipitation: add hydrogen peroxide, makes the content of hydrogen peroxide in filtered liquid be that 20g/kg, normal-temperature reaction 10h filter, obtain filtrate, filtrate is adjusted pH to 5.0, after adding 350kg dehydrated alcohol precipitates overnight with oxalic acid, removing supernatant liquid, throw out is crude product heparin, (6) lyophilize: crude product heparin is put into vacuum freezing drying oven, design temperature-10 DEG C, vacuum tightness 0.07Mpa, time of drying 3h, both heparin crude product 0.5kg, it is tired as every milligram of 90 units.
Embodiment 3: a kind of production method extracting heparin from pig lung, it is characterized in that: step is as follows: (1) pre-treatment: 1000kg pig lung is cleaned, after removing inside and outside dirt and fat, rub into rotten shape, and under fully stirring, add the stirring of 4000kg water and obtain pig lung mixed solution (2) enzymolysis: adjust pig lung mixed solution pH value to be 8.0, add chondroitin sulfate B and the C of the 25kg of pig lung weight, stir, be warming up to 45 DEG C, after insulation reaction 60min, the pH value keeping reaction solution is 7.5, add 5kg trypsinase, stir, insulation 4h, continue to be warmed up to 100 DEG C, keep 10min, cooling, obtain pig lung enzymolysis solution (3) removing protein: filter pig lung enzymolysis solution, remove surface oil lipid layer, filtrate pH value is adjusted to be 10.5 with sodium hydroxide, stir the finings adding 4kg when being warmed up to 65 DEG C, continue to be warming up to 95 DEG C, insulation reaction 4h, be cooled to normal temperature, (4) ultrafiltration: aforesaid liquid 0.4 μm of filter membrane is filtered, obtains filtered liquid, (5) oxidation precipitation: add hydrogen peroxide, makes the content of hydrogen peroxide in filtered liquid be that 30g/kg, normal-temperature reaction 12h filter, obtain filtrate, filtrate is adjusted pH to 5.5, after adding the dehydrated alcohol precipitates overnight of 4000kg with citric acid, removing supernatant liquid, throw out is crude product heparin, (6) lyophilize: crude product heparin is put into vacuum freezing drying oven, design temperature 0 DEG C, vacuum tightness 0.08Mpa, time of drying 4h, both heparin crude product 4.8kg, it is tired as every milligram of 100 units.