CN105693886A - Preparation method of heparin sodium - Google Patents
Preparation method of heparin sodium Download PDFInfo
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- CN105693886A CN105693886A CN201610242697.2A CN201610242697A CN105693886A CN 105693886 A CN105693886 A CN 105693886A CN 201610242697 A CN201610242697 A CN 201610242697A CN 105693886 A CN105693886 A CN 105693886A
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- 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
- C08B37/0078—Degradation products
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- 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
- C08B37/0081—Reaction with amino acids, peptides, or proteins
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- Polymers & Plastics (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to a preparation method of heparin sodium, belongs to the field of biological pharmacy and aims to solve the problem that existing preparation of heparin sodium is high in production hardware facility input and high in production cost and heparin sodium loss is high in the extraction process. The preparation method includes: subjecting bovine liver as a raw material to enzymatic hydrolysis and ultrasonic-assisted extraction, decoloring with clay and removing purities via ammonium sulfate, preparing crude heparin sodium by ethanol deposition, further treating the crude heparin sodium with enzymes in bovine blood such as superoxide dismutase, purifying, and carrying to resin adsorption, desorption and drying to obtain the heparin sodium. The heparin sodium prepared herein is low in cost, high in purity and high in extraction efficiency.
Description
Technical field
The preparation method that the present invention relates to a kind of heparin sodium, belongs to field of biological pharmacy。
Background technology
Heparin sodium is the sulfuric acid ester material of a kind of acid mucopolysaccharide produced by the mastocyte of animal connective tissue, because it has strong anticoagulation, it it is the choice drug of the thrombotic diseases such as preventing and treating deep-vein thrombosis formation, along with going deep into of research, it is found that heparin sodium does not only have the effect of anticoagulant, antithrombus formation and adjustment blood fat, also have antiinflammatory, antiallergic, antiviral, the various biological function such as anticancer。Though heparin sodium is for the history in clinic existing more than 60 years, but so far but without a kind of product that can replace it completely, so it remains most important anticoagulation and antithrombotic biochemical drug, and can only extract from the portion of tissue of animal at present, it is impossible to synthetic。
The preparation of heparin sodium, controls requirement to material and ambient temperature higher, and hardware facility puts into bigger；This method adopts strong acidic condition operation (pH1.5) at Deproteinization process procedure, to heparin activity loss, (heparin solution is very unstable under low pH value environment relatively greatly, very easily degrade inactivation), affect final activity yield and titer, operability is not strong；This method adopts and isolation technics is concentrated by ultrafiltration, and ultrafiltration apparatus is expensive, and hardware puts into bigger；The ionic equilibrium exchanged form that this method adopts, is realize the heparin sodium conversion to heparin sodium in the way of sodium at high concentration ion is constantly got involved, it is impossible to ensure adequacy and the continuous-stable of Dynamic ion balance conversion。Abroad prepare heparin sodium and all adopt ion exchange, it is raw material that this method needs to select refined heparin sodium, cost of material is high, and pH can be caused in ion exchange process sharply to decline, have a strong impact on yield and the titer of product, and exchange process needs to carry out under low temperature puies forward condition, causing that heparin sodium produces hardware facility and puts into relatively big, production cost is higher。
Summary of the invention
The technical problem to be solved: at present producing hardware facility and put into bigger preparing heparin sodium, production cost is higher, the problem that heparin sodium loss is big in extraction process simultaneously, the present invention is by with Hepar Bovis seu Bubali for raw material, pass through enzymolysis, ultrasound assisted extraction, re-use hargil decolouring and ammonium sulfate remove impurity, thick heparin sodium is prepared in alcohol analysis, then pass through the enzyme such as superoxide dismutase in Sanguis Bovis seu Bubali thick heparin sodium is processed further, it is purified, finally by resin absorption, De contamination and dry, prepare heparin sodium, heparin sodium cost prepared by the present invention is low, purity is high, extraction ratio is high。
For solving above-mentioned technical problem, the present invention adopts the technical scheme as described below to be:
(1) weigh fresh beef liver to put into pulper is ground into slurry, again by slurry and compound protease 7:1 in mass ratio, mix homogeneously, put in container, adding the ellagic acid solution that mass fraction is 1.5% in container, addition is the 70～75% of mixture quality, and using mass fraction is that the salt acid for adjusting pH of 10% is to 6.0～6.5, being moved to by container in ultrasonator, vibrate 2～3h under 23～25KHz；
(2) after above-mentioned vibration terminates, mixture in container is put in agitator, filters after 6000r/min stirs 30～40min, collect filtrate, again by solid-to-liquid ratio 1:9,100 whitish eye soil and filtrate are stirred, being added thereto to ammonium sulfate stirring subsequently until filtering after producing without precipitation, collecting filtrate, dehydrated alcohol is added again until producing without precipitation in filtrate, collect by filtration precipitate, obtain thick heparin sodium, standby；
(3) Sanguis Bovis seu Bubali is collected, by the hydrogen peroxide 4:1 by volume that itself and mass fraction are 20%, mix homogeneously, the protease of Sanguis Bovis seu Bubali quality 5～7% and the phospholipase of Sanguis Bovis seu Bubali quality 8～9% is added again in mixture, at 35 DEG C, stir enzymolysis 3～5h with 150r/min, subsequently enzymolysis mixture is put in centrifuge, with 9000～12000r/min centrifugation 5～10min, collect supernatant, obtain treatment fluid；
(4) by solid-to-liquid ratio 1:3, standby thick heparin sodium is mixed homogeneously with the treatment fluid of above-mentioned gained, stir 5～8h with 120r/min, be added thereto to the D208 resin of the quality such as thick heparin sodium subsequently again, after stirring 30min, collect by filtration filtrate, subsequently it is filtered by after solid-to-liquid ratio 1:2 and sodium chloride solution stirring De contamination 1～2h that mass fraction is 8%, collect filtrate to be positioned at 4 DEG C and stand 30min, refilter, filtrate is carried out spray drying, collect dried object, heparin sodium can be obtained。
The heparin sodium of gained of the present invention obtains after testing: titer of heparin sodium is 138～148IU/mg, and the titer response rate is 96～99.2%, and heparin sodium is white。
The present invention compared with additive method, Advantageous Effects:
(1) the heparin sodium cost that prepared by the present invention is low, and purity is high, and extraction ratio is high；
(2) present invention process is simple, and discharging of waste liquid amount is few, process conditions environmental protection, and steady quality。
Detailed description of the invention
Weigh fresh beef liver to put into pulper is ground into slurry, again by slurry and compound protease 7:1 in mass ratio, mix homogeneously, put in container, adding the ellagic acid solution that mass fraction is 1.5% in container, addition is the 70～75% of mixture quality, and using mass fraction is that the salt acid for adjusting pH of 10% is to 6.0～6.5, being moved to by container in ultrasonator, vibrate 2～3h under 23～25KHz；After above-mentioned vibration terminates, mixture in container is put in agitator, filters after 6000r/min stirs 30～40min, collect filtrate, again by solid-to-liquid ratio 1:9,100 whitish eye soil and filtrate are stirred, being added thereto to ammonium sulfate stirring subsequently until filtering after producing without precipitation, collecting filtrate, dehydrated alcohol is added again until producing without precipitation in filtrate, collect by filtration precipitate, obtain thick heparin sodium, standby；Collect Sanguis Bovis seu Bubali, by the hydrogen peroxide 4:1 by volume that itself and mass fraction are 20%, mix homogeneously, the protease of Sanguis Bovis seu Bubali quality 5～7% and the phospholipase of Sanguis Bovis seu Bubali quality 8～9% is added again in mixture, at 35 DEG C, stir enzymolysis 3～5h with 150r/min, subsequently enzymolysis mixture is put in centrifuge, with 9000～12000r/min centrifugation 5～10min, collect supernatant, obtain treatment fluid；By solid-to-liquid ratio 1:3, standby thick heparin sodium is mixed homogeneously with the treatment fluid of above-mentioned gained, stir 5～8h with 120r/min, be added thereto to the D208 resin of the quality such as thick heparin sodium subsequently again, after stirring 30min, collect by filtration filtrate, subsequently it is filtered by after solid-to-liquid ratio 1:2 and sodium chloride solution stirring De contamination 1～2h that mass fraction is 8%, collect filtrate to be positioned at 4 DEG C and stand 30min, refilter, filtrate is carried out spray drying, collect dried object, heparin sodium can be obtained。
Example 1
Weigh fresh beef liver to put into pulper is ground into slurry, again by slurry and compound protease 7:1 in mass ratio, mix homogeneously, put in container, adding the ellagic acid solution that mass fraction is 1.5% in container, addition is the 72% of mixture quality, and using mass fraction is that the salt acid for adjusting pH of 10% is to 6.0, being moved to by container in ultrasonator, vibrate 2h under 24KHz；After above-mentioned vibration terminates, mixture in container is put in agitator, filters after 6000r/min stirs 35min, collect filtrate, again by solid-to-liquid ratio 1:9,100 whitish eye soil and filtrate are stirred, being added thereto to ammonium sulfate stirring subsequently until filtering after producing without precipitation, collecting filtrate, dehydrated alcohol is added again until producing without precipitation in filtrate, collect by filtration precipitate, obtain thick heparin sodium, standby；Collect Sanguis Bovis seu Bubali, by the hydrogen peroxide 4:1 by volume that itself and mass fraction are 20%, mix homogeneously, the protease of Sanguis Bovis seu Bubali quality 6% and the phospholipase of Sanguis Bovis seu Bubali quality 8% is added again in mixture, at 35 DEG C, stir enzymolysis 4h with 150r/min, subsequently enzymolysis mixture is put in centrifuge, with 10000r/min centrifugation 8min, collect supernatant, obtain treatment fluid；By solid-to-liquid ratio 1:3, standby thick heparin sodium is mixed homogeneously with the treatment fluid of above-mentioned gained, stir 7h with 120r/min, be added thereto to the D208 resin of the quality such as thick heparin sodium subsequently again, after stirring 30min, collect by filtration filtrate, subsequently it is filtered by after solid-to-liquid ratio 1:2 and the sodium chloride solution stirring De contamination 2h that mass fraction is 8%, collect filtrate to be positioned at 4 DEG C and stand 30min, refilter, filtrate is carried out spray drying, collect dried object, heparin sodium can be obtained。
The heparin sodium of gained of the present invention obtains after testing: titer of heparin sodium is 142IU/mg, and the titer response rate is 98.6%, and heparin sodium is white。
Example 2
Weigh fresh beef liver to put into pulper is ground into slurry, again by slurry and compound protease 7:1 in mass ratio, mix homogeneously, put in container, adding the ellagic acid solution that mass fraction is 1.5% in container, addition is the 75% of mixture quality, and using mass fraction is that the salt acid for adjusting pH of 10% is to 6.5, being moved to by container in ultrasonator, vibrate 3h under 25KHz；After above-mentioned vibration terminates, mixture in container is put in agitator, filters after 6000r/min stirs 40min, collect filtrate, again by solid-to-liquid ratio 1:9,100 whitish eye soil and filtrate are stirred, being added thereto to ammonium sulfate stirring subsequently until filtering after producing without precipitation, collecting filtrate, dehydrated alcohol is added again until producing without precipitation in filtrate, collect by filtration precipitate, obtain thick heparin sodium, standby；Collect Sanguis Bovis seu Bubali, by the hydrogen peroxide 4:1 by volume that itself and mass fraction are 20%, mix homogeneously, the protease of Sanguis Bovis seu Bubali quality 7% and the phospholipase of Sanguis Bovis seu Bubali quality 9% is added again in mixture, at 35 DEG C, stir enzymolysis 5h with 150r/min, subsequently enzymolysis mixture is put in centrifuge, with 12000r/min centrifugation 10min, collect supernatant, obtain treatment fluid；By solid-to-liquid ratio 1:3, standby thick heparin sodium is mixed homogeneously with the treatment fluid of above-mentioned gained, stir 8h with 120r/min, be added thereto to the D208 resin of the quality such as thick heparin sodium subsequently again, after stirring 30min, collect by filtration filtrate, subsequently it is filtered by after solid-to-liquid ratio 1:2 and the sodium chloride solution stirring De contamination 2h that mass fraction is 8%, collect filtrate to be positioned at 4 DEG C and stand 30min, refilter, filtrate is carried out spray drying, collect dried object, heparin sodium can be obtained。
The heparin sodium of gained of the present invention obtains after testing: titer of heparin sodium is 148IU/mg, and the titer response rate is 99.2%, and heparin sodium is white。
Example 3
Weigh fresh beef liver to put into pulper is ground into slurry, again by slurry and compound protease 7:1 in mass ratio, mix homogeneously, put in container, adding the ellagic acid solution that mass fraction is 1.5% in container, addition is the 70% of mixture quality, and using mass fraction is that the salt acid for adjusting pH of 10% is to 6.0, being moved to by container in ultrasonator, vibrate 2h under 23KHz；After above-mentioned vibration terminates, mixture in container is put in agitator, filters after 6000r/min stirs 30min, collect filtrate, again by solid-to-liquid ratio 1:9,100 whitish eye soil and filtrate are stirred, being added thereto to ammonium sulfate stirring subsequently until filtering after producing without precipitation, collecting filtrate, dehydrated alcohol is added again until producing without precipitation in filtrate, collect by filtration precipitate, obtain thick heparin sodium, standby；Collect Sanguis Bovis seu Bubali, by the hydrogen peroxide 4:1 by volume that itself and mass fraction are 20%, mix homogeneously, the protease of Sanguis Bovis seu Bubali quality 5% and the phospholipase of Sanguis Bovis seu Bubali quality 8% is added again in mixture, at 35 DEG C, stir enzymolysis 3h with 150r/min, subsequently enzymolysis mixture is put in centrifuge, with 9000r/min centrifugation 5min, collect supernatant, obtain treatment fluid；By solid-to-liquid ratio 1:3, standby thick heparin sodium is mixed homogeneously with the treatment fluid of above-mentioned gained, stir 5h with 120r/min, be added thereto to the D208 resin of the quality such as thick heparin sodium subsequently again, after stirring 30min, collect by filtration filtrate, subsequently it is filtered by after solid-to-liquid ratio 1:2 and the sodium chloride solution stirring De contamination 1h that mass fraction is 8%, collect filtrate to be positioned at 4 DEG C and stand 30min, refilter, filtrate is carried out spray drying, collect dried object, heparin sodium can be obtained。
The heparin sodium of gained of the present invention obtains after testing: titer of heparin sodium is 138IU/mg, and the titer response rate is 96%, and heparin sodium is white。
Claims (1)
1. the preparation method of a heparin sodium, it is characterised in that concrete preparation process is:
(1) weigh fresh beef liver to put into pulper is ground into slurry, again by slurry and compound protease 7:1 in mass ratio, mix homogeneously, put in container, adding the ellagic acid solution that mass fraction is 1.5% in container, addition is the 70～75% of mixture quality, and using mass fraction is that the salt acid for adjusting pH of 10% is to 6.0～6.5, being moved to by container in ultrasonator, vibrate 2～3h under 23～25KHz；
(2) after above-mentioned vibration terminates, mixture in container is put in agitator, filters after 6000r/min stirs 30～40min, collect filtrate, again by solid-to-liquid ratio 1:9,100 whitish eye soil and filtrate are stirred, being added thereto to ammonium sulfate stirring subsequently until filtering after producing without precipitation, collecting filtrate, dehydrated alcohol is added again until producing without precipitation in filtrate, collect by filtration precipitate, obtain thick heparin sodium, standby；
(3) Sanguis Bovis seu Bubali is collected, by the hydrogen peroxide 4:1 by volume that itself and mass fraction are 20%, mix homogeneously, the protease of Sanguis Bovis seu Bubali quality 5～7% and the phospholipase of Sanguis Bovis seu Bubali quality 8～9% is added again in mixture, at 35 DEG C, stir enzymolysis 3～5h with 150r/min, subsequently enzymolysis mixture is put in centrifuge, with 9000～12000r/min centrifugation 5～10min, collect supernatant, obtain treatment fluid；
(4) by solid-to-liquid ratio 1:3, standby thick heparin sodium is mixed homogeneously with the treatment fluid of above-mentioned gained, 5～8h is stirred with 120r/min, it is added thereto to the D208 resin of the quality such as thick heparin sodium subsequently again, filtrate is collected by filtration after stirring 30min, subsequently filtrate is filtered by after solid-to-liquid ratio 1:2 and sodium chloride solution stirring De contamination 1～2h that mass fraction is 8%, collect filtrate to be positioned at 4 DEG C and stand 30min, refilter, filtrate is carried out spray drying, collect dried object, heparin sodium can be obtained。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610242697.2A CN105693886A (en) | 2016-04-19 | 2016-04-19 | Preparation method of heparin sodium |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610242697.2A CN105693886A (en) | 2016-04-19 | 2016-04-19 | Preparation method of heparin sodium |
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| CN105693886A true CN105693886A (en) | 2016-06-22 |
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| CN201610242697.2A Withdrawn CN105693886A (en) | 2016-04-19 | 2016-04-19 | Preparation method of heparin sodium |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107383242A (en) * | 2017-08-10 | 2017-11-24 | 盐城盛大肠衣食品有限公司 | A kind of ultrasonic enzymolysis and extraction liquaemin technology |
| CN110092848A (en) * | 2019-05-14 | 2019-08-06 | 山东辰龙药业有限公司 | A kind of preparation method of Bemiparin sodium |
-
2016
- 2016-04-19 CN CN201610242697.2A patent/CN105693886A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107383242A (en) * | 2017-08-10 | 2017-11-24 | 盐城盛大肠衣食品有限公司 | A kind of ultrasonic enzymolysis and extraction liquaemin technology |
| CN110092848A (en) * | 2019-05-14 | 2019-08-06 | 山东辰龙药业有限公司 | A kind of preparation method of Bemiparin sodium |
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Application publication date: 20160622 |