CN113980155A - Method for extracting heparin in animal lungs - Google Patents
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- 229920000669 heparin Polymers 0.000 title claims abstract description 72
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 title claims abstract description 69
- 229960002897 heparin Drugs 0.000 title claims abstract description 67
- 210000004072 lung Anatomy 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 52
- 241001465754 Metazoa Species 0.000 title claims abstract description 49
- 238000001914 filtration Methods 0.000 claims abstract description 42
- 239000012528 membrane Substances 0.000 claims abstract description 42
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000011347 resin Substances 0.000 claims abstract description 39
- 229920005989 resin Polymers 0.000 claims abstract description 39
- 150000001875 compounds Chemical class 0.000 claims abstract description 29
- 238000000926 separation method Methods 0.000 claims abstract description 28
- 238000001179 sorption measurement Methods 0.000 claims abstract description 22
- 238000001035 drying Methods 0.000 claims abstract description 20
- 230000003647 oxidation Effects 0.000 claims abstract description 16
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 14
- 238000001556 precipitation Methods 0.000 claims abstract description 14
- 239000000706 filtrate Substances 0.000 claims description 44
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 40
- 238000003756 stirring Methods 0.000 claims description 34
- 239000011780 sodium chloride Substances 0.000 claims description 31
- 102000004190 Enzymes Human genes 0.000 claims description 28
- 108090000790 Enzymes Proteins 0.000 claims description 28
- 239000012530 fluid Substances 0.000 claims description 28
- 108091005658 Basic proteases Proteins 0.000 claims description 18
- 239000003480 eluent Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 101710163270 Nuclease Proteins 0.000 claims description 15
- 102000004142 Trypsin Human genes 0.000 claims description 15
- 108090000631 Trypsin Proteins 0.000 claims description 15
- 239000012588 trypsin Substances 0.000 claims description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 11
- 239000002244 precipitate Substances 0.000 claims description 11
- 239000003957 anion exchange resin Substances 0.000 claims description 10
- 229920002239 polyacrylonitrile Polymers 0.000 claims description 9
- 230000001376 precipitating effect Effects 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 4
- 239000012465 retentate Substances 0.000 claims description 3
- 150000001408 amides Chemical class 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 229920002492 poly(sulfone) Polymers 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 16
- 102000004169 proteins and genes Human genes 0.000 abstract description 5
- 108090000623 proteins and genes Proteins 0.000 abstract description 5
- 239000000284 extract Substances 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000002429 anti-coagulating effect Effects 0.000 abstract description 2
- 210000004379 membrane Anatomy 0.000 description 34
- 229940088598 enzyme Drugs 0.000 description 24
- 230000000052 comparative effect Effects 0.000 description 19
- 239000000243 solution Substances 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 13
- 239000012982 microporous membrane Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 230000001590 oxidative effect Effects 0.000 description 7
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 5
- 229960001008 heparin sodium Drugs 0.000 description 5
- 239000003146 anticoagulant agent Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108090000526 Papain Proteins 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 229940127219 anticoagulant drug Drugs 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229940055729 papain Drugs 0.000 description 3
- 235000019834 papain Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000001631 haemodialysis Methods 0.000 description 2
- 230000000322 hemodialysis Effects 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
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- 244000144972 livestock Species 0.000 description 2
- 239000000463 material Substances 0.000 description 2
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- 230000004048 modification Effects 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000004019 antithrombin Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of heparin production, and particularly relates to a method for extracting heparin from animal lungs. The method adopts the steps of compound enzymolysis, resin adsorption and elution, membrane separation, oxidation filtration, alcohol precipitation and drying to treat the animal lungs and extract the heparin therein, so that other impurities such as protein and the like contained in the animal lungs can be separated from the heparin to the maximum extent, the anticoagulant effect of the heparin is prevented from being influenced by the existence of the impurities, and the titer of the extracted heparin in the animal lungs is obviously improved; in addition, the method is simple to operate and is very suitable for large-scale industrial production of heparin.
Description
Technical Field
The invention belongs to the technical field of heparin production. More particularly, it relates to a method for extracting heparin from animal lungs.
Background
The main utilization parts of the cultivated livestock are meat and fur, the market demands of other visceral organs such as lungs and the like are less, the price is low, the economic benefit is low, most of the livestock are discarded except a small amount of animal lungs which are directly eaten or processed into feed fertilizers, and the waste of biological resources is greatly caused. In fact, the lungs of animals contain very abundant heparin, and can be used as a source of heparin production material. The heparin is a mucopolysaccharide sulfate anticoagulant substance extracted from animal tissues, generates an anticoagulant function through the combination with antithrombin, and is the most effective anticoagulant drug with the largest clinical dosage in the world. Heparin is mainly applied to cardiovascular and cerebrovascular diseases and hemodialysis treatment, and is the only effective specific drug in the hemodialysis treatment; in addition, heparin or derivatives thereof also have multiple biological functions of regulating blood fat, resisting inflammation, resisting allergy, regulating immunity and the like, so far, no product can completely replace the clinical function of heparin, and the heparin or derivatives thereof is still one of the most important anticoagulant drugs at present.
At present, heparin is extracted mainly by taking small intestinal mucosa as a raw material by adopting a salt hydrolysis-resin exchange method, an enzymolysis-resin exchange method, an ultrasonic-assisted salt hydrolysis method, a salt hydrolysis and enzymolysis combined method and the like. However, the above method cannot be directly applied to the extraction of heparin from animal lungs, because heparin in animal lungs can be combined with impurities such as various proteins, heparin analogues, nucleic acid analogues, and small molecular substances existing in lungs, the conventional heparin extraction method for small intestine mucosa cannot separate heparin from impurities well, and the residual impurities can directly affect the anticoagulation effect of heparin, thereby reducing potency. Aiming at the problem of extracting heparin from animal lungs, Chinese patent application provides a preparation process for extracting a crude product of heparin sodium by utilizing pig lungs, which mainly extracts the heparin in the pig lungs through the steps of enzymolysis of special enzyme for the heparin sodium, resin adsorption, precipitation and the like; however, the special enzyme for heparin sodium adopted by the process is mainly papain and alkaline protease, can only remove proteins, but can not remove other impurities such as heparin analogues and small molecular substances existing in lung heparin, and still exist in a heparin sodium crude product, the activity is low, and the titer is only 90 u/mg.
Therefore, it is urgently needed to provide a method for extracting heparin from animal lungs with high efficiency, and the titer of the obtained heparin can be remarkably improved.
Disclosure of Invention
The invention aims to solve the technical problems that the existing method for extracting the heparin from the lungs of the animals is difficult to completely remove impurities and the titer of the obtained heparin is compromised, provides a method for efficiently extracting the heparin from the lungs of the animals, and can obviously improve the titer of the obtained heparin.
The invention aims to provide a method for extracting heparin in animal lungs.
The above purpose of the invention is realized by the following technical scheme:
a method for extracting heparin from animal lungs comprises the following steps:
s1, compound enzymolysis: adding water into the animal lungs for homogenizing, adjusting the pH of the homogenate to 8.0-8.8 and the temperature to 40-45 ℃, adding compound enzyme for complete enzymolysis, filtering, and collecting filtrate;
s2, resin adsorption and elution: adjusting the pH of the filtrate obtained in the step S1 to 8.2-8.5, adding anion exchange resin, stirring until complete adsorption, filtering, collecting the resin, eluting with 12-16 wt% saline, and collecting the eluent;
s3, membrane separation: performing membrane separation on the eluent obtained in the step S2 by using a polyacrylonitrile membrane or a polysulfonamide membrane to obtain trapped fluid;
s4, oxidation and filtration: adding 30-35 wt% of hydrogen peroxide solution into the trapped fluid obtained in the step S3, filtering after complete oxidation, and collecting filtrate;
s5, alcohol precipitation and drying: adjusting the pH of the filtrate obtained in the step S4 to be neutral, adding saturated saline water, uniformly stirring, adding an ethanol solution, uniformly stirring, completely precipitating, collecting precipitate, and drying to obtain the compound I;
wherein the complex enzyme consists of alkaline protease, trypsin and nuclease.
The invention firstly adopts a complex enzyme composed of alkaline protease, trypsin and nuclease to carry out enzymolysis on impurities such as protein, nucleic acid analogues and the like in animal lungs, then sequentially removes impurities such as mineral substances, metal salts, pigments and the like through anion exchange resin, then intercepts heparin through membrane separation, removes impurities with smaller molecular weight, and finally sequentially carries out oxidation and alcohol precipitation to obtain the heparin product with less impurity residual quantity and higher titer.
Further, in step S1, the complex enzyme is prepared from alkaline protease, trypsin and nuclease in a weight ratio of 10: (3-5): (0.3-0.5). Preferably, the compound enzyme is prepared by mixing alkaline protease, trypsin and nuclease according to the weight ratio of 10: (4-5): (0.4-0.5). More preferably, the compound enzyme is prepared by mixing alkaline protease, trypsin and nuclease according to the weight ratio of 10: 5: 0.5.
Furthermore, in the step S1, the addition amount of the compound enzyme is 0.05-0.2% of the mass of the homogenate. Preferably, the addition amount of the complex enzyme is 0.1-0.2% of the mass of the homogenate.
Further, in step S1, the enzymolysis time is 3-4 hours.
Preferably, in step S1, the volume ratio of the animal lungs to the water is 1 (3-4).
Preferably, in step S1, the filtering is performed by using a screen of 80-100 meshes.
Further, in step S2, the elution with 12-16 wt% saline is performed by eluting with 12-14 wt% saline, and then with 15-16 wt% saline. Preferably, the elution is carried out for 2-3 h by using 12-14 wt% saline which is 3-4 times the volume of the resin, then for 1-2 h by using 15-16 wt% saline which is 2 times the volume of the resin, and the two eluates are combined.
Preferably, in step S2, the complete adsorption needs 3-4 h.
Preferably, in step S2, the filtering screen is 80-100 mesh.
Further, in step S3, the polyacrylonitrile membrane or the polysulfonamide membrane has a molecular weight cut-off of 3000-4000 Da.
Further, in step S3, when the volume of the retentate is 15-20% of the total volume of the eluate obtained in step S2, the membrane separation is stopped.
Further, in step S4, the addition amount of the hydrogen peroxide solution is 0.8-1.2% of the volume of the trapped liquid obtained in step S3.
Furthermore, in step S4, the time for oxidation is 16-24 h.
Preferably, in step S4, the filtration is performed by using a 0.22-0.45 μm microporous membrane filter.
Further, in step S5, the ethanol solution has an ethanol volume concentration of 80 to 100%.
Preferably, in step S5, the ethanol solution is added in an amount of 1.5 to 2 times the total volume of the filtrate obtained in step S4 and the brine.
Preferably, in the step S5, the time required for complete precipitation is 8-12 h.
Furthermore, the animal lungs mainly comprise lungs of pigs, cows and sheep.
Through determination, the titer of the heparin prepared by the method is more than 100 u/mg.
The invention has the following beneficial effects:
the invention relates to a method for extracting heparin in animal lungs, which adopts the steps of compound enzymolysis, resin adsorption and elution, membrane separation, oxidation filtration, alcohol precipitation and drying to treat the animal lungs and extract the heparin therein, so that other impurities such as protein and the like in the animal lungs can be separated from the heparin to the maximum extent, the anticoagulant effect of the heparin is prevented from being influenced by the existence of the impurities, and the titer of the extracted heparin in the animal lungs is obviously improved; in addition, the method is simple to operate and is very suitable for large-scale industrial production of heparin.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1A method for extracting heparin from animal lungs
The method for extracting the heparin in the animal lung specifically comprises the following steps:
s1, compound enzymolysis: adding 3 times of water into animal lungs, crushing and homogenizing, adjusting the pH of homogenate to 8.0 and the temperature to 40 ℃, adding complex enzyme (composed of alkaline protease, trypsin and nuclease according to the weight ratio of 10: 3: 0.3) with the mass of 0.05% of the homogenate, carrying out enzymolysis reaction for 3 hours until the reaction is complete, filtering by using a 80-mesh screen, and collecting filtrate;
s2, resin adsorption and elution: adjusting the pH value of the filtrate obtained in the step S1 to 8.2, adding anion exchange resin, stirring for 3 hours until complete adsorption, filtering with a 80-mesh screen, collecting the resin, eluting with 12-14 wt% saline which is 3 times the volume of the resin for 2 hours, eluting with 15-16 wt% saline which is 2 times the volume of the resin for 1 hour, and combining the eluates obtained in the two times;
s3, membrane separation: performing membrane separation on the eluent obtained in the step S2 by using a polyacrylonitrile membrane (the molecular weight cutoff is 3000Da), and stopping the membrane separation when the volume of the trapped fluid is 15% of the total volume of the eluent obtained in the step S2 to obtain trapped fluid;
s4, oxidation and filtration: adding 30 wt% hydrogen peroxide solution with the volume of 0.8% of the trapped fluid into the trapped fluid obtained in the step S3, continuously stirring and filtering by using a 0.45 mu m microporous membrane filter after oxidizing for 16h at normal temperature, and collecting filtrate;
s5, alcohol precipitation and drying: and (4) adjusting the pH value of the filtrate obtained in the step (S4) to 7.0, adding saturated saline, uniformly stirring, adding 95% ethanol solution with the volume being 1.5 times of the total volume of the filtrate and the saline, uniformly stirring, precipitating for 8 hours till the solution is complete, collecting precipitate, and drying under reduced pressure and vacuum to obtain the compound.
Example 2A method for extracting heparin from animal lungs
The method for extracting the heparin in the animal lung specifically comprises the following steps:
s1, compound enzymolysis: adding 3.5 times of water into animal lung, pulverizing, homogenizing, adjusting pH to 8.5, heating to 42 deg.C, adding complex enzyme (composed of alkaline protease, trypsin and nuclease at weight ratio of 10: 4: 0.4) 0.1% of homogenate mass, performing enzymolysis reaction for 3.5 hr, filtering with 80 mesh screen, and collecting filtrate;
s2, resin adsorption and elution: adjusting the pH value of the filtrate obtained in the step S1 to 8.4, adding anion exchange resin, stirring for 3.5h until complete adsorption, filtering with a 80-mesh screen, collecting the resin, eluting with 12-14 wt% saline water which is 3.5 times the volume of the resin for 2.5h, eluting with 15-16 wt% saline water which is 2 times the volume of the resin for 1.5h, and combining the eluates obtained in two times;
s3, membrane separation: performing membrane separation on the eluent obtained in the step S2 by using a polysulfone amide membrane (the molecular weight cutoff is 4000Da), and stopping membrane separation when the volume of the trapped fluid is 18 percent of the total volume of the eluent obtained in the step S2 to obtain trapped fluid;
s4, oxidation and filtration: adding 30 wt% hydrogen peroxide solution with the volume of 1% of the trapped fluid into the trapped fluid obtained in the step S3, oxidizing for 20 hours at normal temperature, continuously stirring and filtering by using a 0.45 mu m microporous membrane filter, and collecting filtrate;
s5, alcohol precipitation and drying: and (4) adjusting the pH value of the filtrate obtained in the step (S4) to 7.0, adding saturated saline, uniformly stirring, adding 95% ethanol solution with the volume being 1.8 times of the total volume of the filtrate and the saline, uniformly stirring, precipitating for 10 hours till the precipitate is complete, collecting the precipitate, and drying under reduced pressure and vacuum to obtain the compound. Example 3A method for extracting heparin from animal lungs
The method for extracting the heparin in the animal lung specifically comprises the following steps:
s1, compound enzymolysis: adding 4 times of water into animal lungs, crushing and homogenizing, adjusting the pH of homogenate to 8.8 and the temperature to 45 ℃, adding complex enzyme (composed of alkaline protease, trypsin and nuclease according to the weight ratio of 10: 5: 0.5) with the mass of 0.2% of the homogenate, carrying out enzymolysis reaction for 4 hours until the reaction is complete, filtering by using a 80-mesh screen, and collecting filtrate;
s2, resin adsorption and elution: adjusting the pH value of the filtrate obtained in the step S1 to 8.5, adding anion exchange resin, stirring for 4 hours until complete adsorption, filtering with a 100-mesh screen, collecting the resin, eluting with 12-14 wt% saline which is 4 times the volume of the resin for 3 hours, eluting with 15-16 wt% saline which is 2 times the volume of the resin for 2 hours, and combining the eluates obtained in the two times;
s3, membrane separation: performing membrane separation on the eluent obtained in the step S2 by using a polyacrylonitrile membrane (the molecular weight cutoff is 3000Da), and stopping the membrane separation when the volume of the trapped fluid is 20% of the total volume of the eluent obtained in the step S2 to obtain trapped fluid;
s4, oxidation and filtration: adding 30 wt% hydrogen peroxide solution with the volume of 1.2% of the trapped fluid into the trapped fluid obtained in the step S3, oxidizing for 24h at normal temperature, continuously stirring and filtering by using a 0.45 mu m microporous membrane filter, and collecting filtrate;
s5, alcohol precipitation and drying: and (4) adjusting the pH value of the filtrate obtained in the step (S4) to 7.0, adding saturated saline, uniformly stirring, adding 95% ethanol solution with the volume being 2.0 times of the total volume of the filtrate and the saline, uniformly stirring, precipitating for 10 hours till the precipitate is complete, collecting the precipitate, and drying under reduced pressure and vacuum to obtain the compound. Comparative example 1A method for extracting heparin from animal Lung
The method for extracting the heparin in the animal lung specifically comprises the following steps:
s1, compound enzymolysis: adding 3 times of water into animal lungs, crushing and homogenizing, adjusting the pH of homogenate to 8.0, adjusting the temperature to 40 ℃, adding complex enzyme (composed of alkaline protease, trypsin and nuclease according to the weight ratio of 8: 3: 0.3) with the mass of 0.05% of the homogenate, carrying out enzymolysis reaction for 3 hours until the reaction is complete, filtering by using a 80-mesh screen, and collecting filtrate;
s2, resin adsorption and elution: adjusting the pH value of the filtrate obtained in the step S1 to 8.2, adding anion exchange resin, stirring for 3 hours until complete adsorption, filtering with a 80-mesh screen, collecting the resin, eluting with 12-14 wt% saline which is 3 times the volume of the resin for 2 hours, eluting with 15-16 wt% saline which is 2 times the volume of the resin for 1 hour, and combining the eluates obtained in the two times;
s3, membrane separation: performing membrane separation on the eluent obtained in the step S2 by using a polyacrylonitrile membrane (the molecular weight cutoff is 3000Da), and stopping the membrane separation when the volume of the trapped fluid is 15% of the total volume of the eluent obtained in the step S2 to obtain trapped fluid;
s4, oxidation and filtration: adding 30 wt% hydrogen peroxide solution with the volume of 0.8% of the trapped fluid into the trapped fluid obtained in the step S3, continuously stirring and filtering by using a 0.45 mu m microporous membrane filter after oxidizing for 16h at normal temperature, and collecting filtrate;
s5, alcohol precipitation and drying: and (4) adjusting the pH value of the filtrate obtained in the step (S4) to 7.0, adding saturated saline, uniformly stirring, adding 95% ethanol solution with the volume being 1.5 times of the total volume of the filtrate and the saline, uniformly stirring, precipitating for 8 hours till the solution is complete, collecting precipitate, and drying under reduced pressure and vacuum to obtain the compound.
The method of the comparative example 1 is different from that of the example 1 in that the composition of the compound enzyme is changed in the comparative example 1, and the compound enzyme is prepared from alkaline protease, trypsin and nuclease according to the weight ratio of 8: 3: 0.3 composition, the rest of the operations and parameters refer to example 1.
Comparative example 2A method for extracting heparin from animal Lung
The method for extracting the heparin in the animal lung specifically comprises the following steps:
s1, compound enzymolysis: adding 3 times of water into animal lung, pulverizing, homogenizing, adjusting pH to 8.0, adjusting temperature to 40 deg.C, adding complex enzyme (composed of alkaline protease and trypsin at weight ratio of 10: 3) 0.05% of homogenate quality, performing enzymolysis reaction for 3 hr to complete reaction, filtering with 80 mesh screen, and collecting filtrate;
s2, resin adsorption and elution: adjusting the pH value of the filtrate obtained in the step S1 to 8.2, adding anion exchange resin, stirring for 3 hours until complete adsorption, filtering with a 80-mesh screen, collecting the resin, eluting with 12-14 wt% saline which is 3 times the volume of the resin for 2 hours, eluting with 15-16 wt% saline which is 2 times the volume of the resin for 1 hour, and combining the eluates obtained in the two times;
s3, membrane separation: performing membrane separation on the eluent obtained in the step S2 by using a polyacrylonitrile membrane (the molecular weight cutoff is 3000Da), and stopping the membrane separation when the volume of the trapped fluid is 15% of the total volume of the eluent obtained in the step S2 to obtain trapped fluid;
s4, oxidation and filtration: adding 30 wt% hydrogen peroxide solution with the volume of 0.8% of the trapped fluid into the trapped fluid obtained in the step S3, continuously stirring and filtering by using a 0.45 mu m microporous membrane filter after oxidizing for 16h at normal temperature, and collecting filtrate;
s5, alcohol precipitation and drying: and (4) adjusting the pH value of the filtrate obtained in the step (S4) to 7.0, adding saturated saline, uniformly stirring, adding 95% ethanol solution with the volume being 1.5 times of the total volume of the filtrate and the saline, uniformly stirring, precipitating for 8 hours till the solution is complete, collecting precipitate, and drying under reduced pressure and vacuum to obtain the compound.
The method of the comparative example 2 is different from the method of the example 1 in that the composition of the compound enzyme is changed in the comparative example 2, and the compound enzyme is prepared by mixing alkaline protease and trypsin according to the weight ratio of 10: 3 composition, rest operations and parameters refer to example 1.
Comparative example 3A method for extracting heparin from animal Lung
The method for extracting the heparin in the animal lung specifically comprises the following steps:
s1, compound enzymolysis: adding 3 times of water into animal lungs, crushing and homogenizing, adjusting the pH of homogenate to 8.0 and the temperature to 40 ℃, adding complex enzyme (composed of alkaline protease, papain and nuclease according to the weight ratio of 10: 3: 0.3) with the mass of 0.05% of the homogenate, carrying out enzymolysis reaction for 3 hours until the reaction is complete, filtering by using a 80-mesh screen, and collecting filtrate;
s2, resin adsorption and elution: adjusting the pH value of the filtrate obtained in the step S1 to 8.2, adding anion exchange resin, stirring for 3 hours until complete adsorption, filtering with a 80-mesh screen, collecting the resin, eluting with 12-14 wt% saline which is 3 times the volume of the resin for 2 hours, eluting with 15-16 wt% saline which is 2 times the volume of the resin for 1 hour, and combining the eluates obtained in the two times;
s3, membrane separation: performing membrane separation on the eluent obtained in the step S2 by using a polyacrylonitrile membrane (the molecular weight cutoff is 3000Da), and stopping the membrane separation when the volume of the trapped fluid is 15% of the total volume of the eluent obtained in the step S2 to obtain trapped fluid;
s4, oxidation and filtration: adding 30 wt% hydrogen peroxide solution with the volume of 0.8% of the trapped fluid into the trapped fluid obtained in the step S3, continuously stirring and filtering by using a 0.45 mu m microporous membrane filter after oxidizing for 16h at normal temperature, and collecting filtrate;
s5, alcohol precipitation and drying: and (4) adjusting the pH value of the filtrate obtained in the step (S4) to 7.0, adding saturated saline, uniformly stirring, adding 95% ethanol solution with the volume being 1.5 times of the total volume of the filtrate and the saline, uniformly stirring, precipitating for 8 hours till the solution is complete, collecting precipitate, and drying under reduced pressure and vacuum to obtain the compound.
The method of the comparative example 3 is different from the method of the example 1 in that the composition of the complex enzyme is changed in the comparative example 3, and the adopted complex enzyme is prepared from alkaline protease, papain and nuclease according to the weight ratio of 10: 3: 0.3 composition, the rest of the operations and parameters refer to example 1.
Comparative example 4A method for extracting heparin from animal Lung
The method for extracting the heparin in the animal lung specifically comprises the following steps:
s1, compound enzymolysis: adding 3 times of water into animal lungs, crushing and homogenizing, adjusting the pH of homogenate to 8.0 and the temperature to 40 ℃, adding complex enzyme (composed of alkaline protease, trypsin and nuclease according to the weight ratio of 10: 3: 0.3) with the mass of 0.05% of the homogenate, carrying out enzymolysis reaction for 3 hours until the reaction is complete, filtering by using a 80-mesh screen, and collecting filtrate;
s2, resin adsorption and elution: adjusting the pH value of the filtrate obtained in the step S1 to 8.2, adding anion exchange resin, stirring for 3 hours until complete adsorption, filtering with a 80-mesh screen, collecting the resin, eluting with 12-14 wt% saline which is 3 times the volume of the resin for 2 hours, eluting with 15-16 wt% saline which is 2 times the volume of the resin for 1 hour, and combining the eluates obtained in the two times;
s3, oxidation and filtration: adding 30 wt% hydrogen peroxide solution with the volume of 0.8% of the eluent into the eluent obtained in the step S2, oxidizing for 16h at normal temperature, continuously stirring and filtering by using a 0.45 mu m microporous membrane filter, and collecting filtrate;
s4, alcohol precipitation and drying: and (4) adjusting the pH value of the filtrate obtained in the step (S3) to 7.0, adding saturated saline, uniformly stirring, adding 95% ethanol solution with the volume being 1.5 times of the total volume of the filtrate and the saline, uniformly stirring, precipitating for 8 hours till the solution is complete, collecting precipitate, and drying under reduced pressure and vacuum to obtain the compound.
Of these, comparative example 4 is different from example 1 in that comparative example 4 is free from the step of membrane separation, and the rest of the operation and parameters are referred to example 1.
Test example heparin potency assay
The titer of heparin obtained in the examples and comparative examples was measured by the following method:
placing frozen and stored goat plasma in a water bath kettle at 37 ℃ for natural thawing, and filtering with absorbent cotton into a clean and dry conical flask for later use; precisely sucking 160 μ L, 170 μ L, 180 μ L, 190 μ L and 200 μ L of refrigerated heparin sodium standard, placing into 10ml test tube with plug, and sequentially adding 1ml of sheep plasma and 0.8ml of 0.25 wt% CaCl along the tube wall2And (3) covering the test tube plug, reversing for 3-4 times, shaking up to wet the inner wall without generating bubbles, vertically putting the test tube plug into a water bath kettle at 37 ℃ for 1 hour, observing and recording the half-freezing point of the standard product. Preparing a solid sample and a liquid sample of the heparin to be detected, diluting the liquid sample of the heparin to be detected step by step, comparing the half-freezing points of the standard sample by a sheep plasma coagulation method, and finally obtaining the half-freezing point of the sample.
The results are shown in Table 1.
TABLE 1 results of heparin titer measurement obtained in examples and comparative examples
| Group of | Potency (u/mg) |
| Example 1 | 110 |
| Example 2 | 115 |
| Example 3 | 120 |
| Comparative example 1 | 95 |
| Comparative example 2 | 90 |
| Comparative example 3 | 85 |
| Comparative example 4 | 75 |
As shown in the table, the potency of the heparin prepared by the embodiment of the invention is over 100u/mg, the contained impurities are less, the influence of the impurities on the potency of the heparin is avoided, and the higher potency can be achieved; and the preparation method is simple and suitable for large-scale industrial production. Compared with the comparative examples 1-4, the composition or preparation method of the complex enzyme is changed respectively, impurities in the obtained heparin cannot be completely removed, the anticoagulant activity of the final product is directly influenced, and the titer is obviously reduced.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for extracting heparin from animal lungs is characterized by comprising the following steps:
s1, compound enzymolysis: adding water into the animal lungs for homogenizing, adjusting the pH of the homogenate to 8.0-8.8 and the temperature to 40-45 ℃, adding compound enzyme for complete enzymolysis, filtering, and collecting filtrate;
s2, resin adsorption and elution: adjusting the pH of the filtrate obtained in the step S1 to 8.2-8.5, adding anion exchange resin, stirring until complete adsorption, filtering, collecting the resin, eluting with 12-16 wt% saline, and collecting the eluent;
s3, membrane separation: performing membrane separation on the eluent obtained in the step S2 by using a polyacrylonitrile membrane or a polysulfonamide membrane to obtain trapped fluid;
s4, oxidation and filtration: adding 30-35 wt% of hydrogen peroxide solution into the trapped fluid obtained in the step S3, filtering after complete oxidation, and collecting filtrate;
s5, alcohol precipitation and drying: adjusting the pH of the filtrate obtained in the step S4 to be neutral, adding saturated saline water, uniformly stirring, adding an ethanol solution, uniformly stirring, completely precipitating, collecting precipitate, and drying to obtain the compound I;
wherein the complex enzyme consists of alkaline protease, trypsin and nuclease.
2. The method according to claim 1, wherein in step S1, the complex enzyme is prepared by mixing alkaline protease, trypsin and nuclease in a weight ratio of 10: (3-5): (0.3-0.5).
3. The method as claimed in claim 1, wherein in step S1, the addition amount of the complex enzyme is 0.05-0.2% of the mass of the homogenate.
4. The method according to claim 1, wherein in step S1, the enzymolysis time is 3-4 h.
5. The method according to claim 1, wherein in step S2, the elution with 12 to 16 wt% saline is performed by eluting with 12 to 14 wt% saline and then with 15 to 16 wt% saline.
6. The method according to claim 1, wherein in step S3, the polyacrylonitrile membrane or polysulfone amide membrane has a molecular weight cut-off of 3000-4000 Da.
7. The method of claim 1, wherein in step S3, when the volume of the retentate is 15-20% of the total volume of the eluate obtained in step S2, the membrane separation is stopped.
8. The method of claim 1, wherein in step S4, the hydrogen peroxide solution is added in an amount of 0.8-1.2% by volume of the retentate obtained in step S3.
9. The method according to claim 1, wherein in step S4, the oxidation time is 16-24 h.
10. The method according to claim 1, wherein in step S5, the ethanol solution has an ethanol volume concentration of 80-100%.
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| CN117327205A (en) * | 2023-08-10 | 2024-01-02 | 浩泰健康济盛(吉林)生物科技有限公司 | Method for extracting heparin from pig lungs |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212149A (en) * | 2011-06-15 | 2011-10-12 | 天津宝迪农业科技股份有限公司 | Preparation process for extracting crude sodium heparin from pig lungs |
| CN104448046A (en) * | 2014-12-24 | 2015-03-25 | 青岛九龙生物医药有限公司 | Production process for extracting crude heparin sodium products from animal lungs |
| CN106432549A (en) * | 2016-10-05 | 2017-02-22 | 陈石良 | Method for extracting sodium heparin from animal lung and sodium heparin |
| CN111560087A (en) * | 2020-06-28 | 2020-08-21 | 揭阳市润达肠衣有限公司 | Purification method of high-quality heparin sodium |
-
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212149A (en) * | 2011-06-15 | 2011-10-12 | 天津宝迪农业科技股份有限公司 | Preparation process for extracting crude sodium heparin from pig lungs |
| CN104448046A (en) * | 2014-12-24 | 2015-03-25 | 青岛九龙生物医药有限公司 | Production process for extracting crude heparin sodium products from animal lungs |
| CN106432549A (en) * | 2016-10-05 | 2017-02-22 | 陈石良 | Method for extracting sodium heparin from animal lung and sodium heparin |
| CN111560087A (en) * | 2020-06-28 | 2020-08-21 | 揭阳市润达肠衣有限公司 | Purification method of high-quality heparin sodium |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117327205A (en) * | 2023-08-10 | 2024-01-02 | 浩泰健康济盛(吉林)生物科技有限公司 | Method for extracting heparin from pig lungs |
| CN117327205B (en) * | 2023-08-10 | 2024-07-02 | 浩泰健康济盛(吉林)生物科技有限公司 | Method for extracting heparin from pig lungs |
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