CN110183550A - A kind of preparation process of refined heparin sodium - Google Patents
A kind of preparation process of refined heparin sodium Download PDFInfo
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- CN110183550A CN110183550A CN201910555214.8A CN201910555214A CN110183550A CN 110183550 A CN110183550 A CN 110183550A CN 201910555214 A CN201910555214 A CN 201910555214A CN 110183550 A CN110183550 A CN 110183550A
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- heparin sodium
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- refined
- crude
- preparation process
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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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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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
Abstract
The invention discloses a kind of preparation processes of refined heparin sodium, the following steps are included: crude heparin sodium is dissolved in water obtained crude heparin sodium aqueous solution, composite protein precipitator is added in crude heparin sodium aqueous solution and carries out albumen precipitation, isolates sediment after standing 20-40 minutes；It is filtered, cools down to the crude heparin sodium aqueous solution after sediment has been separated, ethyl alcohol is added in crude heparin sodium aqueous solution until centrifuge dripping is precipitated in sediment, obtains heparin sodium wet product；Heparin sodium wet product is dried, heparin sodium time fine work is obtained；Heparin sodium time fine work is dissolved in water, protein complex enzyme formulation is added in heparin sodium time fine work solution, centrifugation, filtering obtain heparin sodium time fine work clear liquid；By heparin sodium time fine work clear liquid Dynamic Adsorption resin circulation absorption, resin is eluted, ethyl alcohol is added in eluent, filtering, cooling and drying obtain refined heparin sodium.The present invention improves the yield of refined heparin sodium.
Description
Technical field
The present invention relates to a kind of heparin sodium preparation processes, and in particular to a kind of preparation process of refined heparin sodium.
Background technique
Heparin sodium is a kind of mucopolysaccharide sulfate anticoagulant, and the various cardiovascular and cerebrovasculars of prevention and treatment are widely used in clinical use
Disease is always most effective anticoagulation medicine, especially has unique curative effect to arteriovenous thrombus, thromboembolism.Heparin sodium is extensive
It is present in the intestinal mucosa, lung, liver of mammal, content highest especially in pig intestinal mucosa, and from healthy live pig small intestine
The heparin extracted in mucous membrane and the intracorporal heparin molecule structure of people are almost the same.The producer of heparin sodium in the prior art
Method mainly uses fresh pig intestinal mucosa for raw material, passes through cleaning treatment, enzymolysis and extraction, resin adsorption, elution, alcohol precipitation and baking
It is dry to obtain heparin sodium crude.
Traditional manufacturing technique method is that crude heparin sodium is carried out to oxidative decoloration once or twice and ethanol precipitation purification,
Its hydrogen peroxide use is big (3-5%), damages greatly to titer of heparin sodium, yield is low.
Summary of the invention
The technical problem to be solved by the present invention is to be refined by oxidative decoloration to heparin sodium, yield is low, and purpose exists
In providing a kind of preparation process of refined heparin sodium, solves traditional handicraft and heparin sodium is refined by oxidative decoloration, yield
Low problem.
The present invention is achieved through the following technical solutions:
A kind of preparation process of refined heparin sodium, comprising the following steps:
S1, be dissolved in water crude heparin sodium obtained crude heparin sodium aqueous solution, is added in crude heparin sodium aqueous solution
Composite protein precipitator carries out albumen precipitation, isolates sediment after standing 20-40 minutes；
S2, it is filtered, cools down to having separated the crude heparin sodium aqueous solution after sediment in step S1, be reduced to 10-
15 DEG C, the ethyl alcohol of 85-95% is added in 10-15 DEG C of crude heparin sodium aqueous solution until sediment is precipitated, standing 12-24 is small
When, centrifuge dripping obtains heparin sodium wet product；
S3, heparin sodium wet product is dried at 30-35 DEG C, obtains heparin sodium time fine work；
S4, heparin sodium time fine work is dissolved in water, adjusts pH value of solution to 8-9, egg is added in heparin sodium time fine work solution
White matter complex enzyme formulation, centrifugation, filtering obtain heparin sodium time fine work clear liquid；
S5, by heparin sodium time fine work clear liquid with after Dynamic Adsorption resin circulation absorption 3-5 times, resin is eluted,
The ethyl alcohol that temperature is 10 DEG C is added in eluent, until sediment is precipitated, after sediment is precipitated 4-8 hours, solution is at 50-60 DEG C
Lower standing filters after 30-60 minutes, cooling and drying obtains refined heparin sodium.
The present invention is utilized respectively the precipitation method and enzymatic isolation method purifies crude heparin sodium, easy to operate, does not need to liver
Plain sodium is aoxidized, and the structure of heparin sodium will not be destroyed, and purification efficiency is high；
The present invention precipitates heparin sodium using ethyl alcohol, and after ethyl alcohol is added in heparin sodium aqua, heparin sodium is precipitated, extra
Protein precipitant and protein complex enzyme formulation will not influence the precipitation of heparin sodium, extra egg can be extracted after ethanol evaporation
White precipitating reagent and protein complex enzyme formulation, it is energy saving.The present invention when using ethanol precipitation heparin sodium, reduce ethyl alcohol and
The temperature of heparin sodium aqua, on the one hand can prevent ethyl alcohol from volatilizing, and on the other hand effectively reduce heparin sodium and form solid particle mistake
To " package " of the organic solvents such as ethyl alcohol and " co-precipitation " in journey；Enzymatic hydrolysis condition of the present invention is mild, miscellaneous in cracking nucleic acid and albumen
While matter, it can effectively protect the less destruction by protease of heparin sodium.The yield of refined heparin sodium of the present invention is up to 90%, essence
The purity of product heparin sodium is up to 95%.The present invention realizes the enzymatic hydrolysis and adsorption process of heparin sodium under salt-free conditions, to make to arrange
Salinity reduces in the waste water put, and reduces cost for wastewater treatment, improves environmental benefit.
The composite protein precipitator includes the component of following parts by weight: 10-15 parts of 5-sulphosalicylic acids, 3-8 part three
Monoxone, 2-6 part acetonitrile, 5-10 parts of ethyl acetate, 10-20 parts of polyacrylic acid, 0.8-5 parts of sodium-acetate buffers.
The present invention by it is a variety of can precipitating proteins and the good component of compatibility carry out compound, albumen precipitation effect is good,
The acidity of protein precipitant can also be reduced；The preferred ratio range of each component of the present invention, composite protein precipitator pH control
System will not cause to damage in 4-5 to chromatographic column etc..
The composite protein precipitator includes the component of following parts by weight: 13-15 parts of 5-sulphosalicylic acids, 3-6 part three
Monoxone, 4-6 part acetonitrile, 5-7 parts of ethyl acetate, 15-20 parts of polyacrylic acid, 0.8-1.2 parts of sodium-acetate buffers.The present invention is excellent
The ratio range of each component is selected.
The composite protein precipitator includes the component of following parts by weight: 14 parts of 5-sulphosalicylic acids, 4 part of three chloroethene
Acid, 5 parts of acetonitriles, 7 parts of ethyl acetate, 20 parts of polyacrylic acid, 1 part of sodium-acetate buffer.The present invention preferred the matching of each component
Than the sedimentation effect of protein is good, acid small.
The protein complex enzyme formulation includes the component of following parts by weight: 10-20 parts of protease, 5-10 portions of pawpaws
Enzyme, 0.2-1 part lipase, 4-16 parts of nucleases, 8-12 parts of expanded perlites.
The protein complex enzyme formulation includes the component of following parts by weight: 15 parts of protease, 10 parts of papains, 0.8
Part lipase, 12 parts of nucleases, 10 parts of expanded perlites.The present invention prepares complex enzyme formulation using expanded perlite as carrier,
The generation of by-product can be effectively avoided, in addition, hydrolysis result of the present invention is good, the usage amount of enzymatic preparations can be effectively reduced,
Energy saving.
The mass ratio of crude heparin sodium and composite protein precipitator dosage are as follows: 1:0.5-1.The preferred crude product heparin of the present invention
The dosage range of sodium and composite protein precipitator guarantees that protein is deposited completely as far as possible.
The mass ratio of heparin sodium time fine work and protein complex enzyme formulation dosage are as follows: 1:1-1.2.The preferred crude product of the present invention
The dosage range of heparin sodium and protein complex enzyme formulation guarantees that protein is digested completely as far as possible.
Filtering in step S4 and step S5 is all made of ultrafiltration membrance filter.
Step S1 measures the content of heparin sodium in crude heparin sodium and preferably goes out content before preparing crude heparin sodium solution
Crude heparin sodium greater than 60%.The present invention selects the higher crude heparin sodium of heparin sodium content to refine, the efficiency of purification
Height, being capable of energy saving.
Compared with prior art, the present invention having the following advantages and benefits:
1, a kind of preparation process of refined heparin sodium of the present invention be utilized respectively the precipitation method and enzymatic isolation method to crude heparin sodium into
Row purification, it is easy to operate, it does not need to aoxidize heparin sodium, the structure of heparin sodium will not be destroyed, purification efficiency is high；
2, a kind of preparation process of refined heparin sodium of the present invention realizes the enzymatic hydrolysis of heparin sodium and was adsorbed under salt-free conditions
Journey reduces cost for wastewater treatment so that salinity in the waste water of discharge be made to reduce, and improves environmental benefit；
3, a kind of preparation process of refined heparin sodium of the present invention reduces ethyl alcohol and liver when using ethanol precipitation heparin sodium
The temperature of plain sodium solution, on the one hand can prevent ethyl alcohol from volatilizing, and on the other hand effectively reduce heparin sodium and form solid particle process
In to " package " and " co-precipitation " of the organic solvents such as ethyl alcohol.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment, the present invention is made
Further to be described in detail, exemplary embodiment of the invention and its explanation for explaining only the invention, are not intended as to this
The restriction of invention.
Embodiment 1
A kind of preparation process of refined heparin sodium of the present invention, comprising the following steps:
S1, be dissolved in water crude heparin sodium obtained crude heparin sodium aqueous solution, is added in crude heparin sodium aqueous solution
Composite protein precipitator carries out albumen precipitation, isolates sediment after standing 20-40 minutes；
S2, it is filtered, cools down to having separated the crude heparin sodium aqueous solution after sediment in step S1, be reduced to 10-
15 DEG C, the ethyl alcohol of 85-95% is added in 10-15 DEG C of crude heparin sodium aqueous solution until sediment is precipitated, standing 12-24 is small
When, centrifuge dripping obtains heparin sodium wet product；
S3, heparin sodium wet product is dried at 30-35 DEG C, obtains heparin sodium time fine work；
S4, heparin sodium time fine work is dissolved in water, adjusts pH value of solution to 8-9, egg is added in heparin sodium time fine work solution
White matter complex enzyme formulation, centrifugation, filtering obtain heparin sodium time fine work clear liquid；
S5, by heparin sodium time fine work clear liquid with after Dynamic Adsorption resin circulation absorption 3-5 times, resin is eluted,
The ethyl alcohol that temperature is 10 DEG C is added in eluent, until sediment is precipitated, after sediment is precipitated 4-8 hours, solution is at 50-60 DEG C
Lower standing filters after 30-60 minutes, cooling and drying obtains refined heparin sodium.
The mass ratio of crude heparin sodium and composite protein precipitator dosage are as follows: 1:0.5-1.Heparin sodium time fine work and protein
The mass ratio of complex enzyme formulation dosage are as follows: 1:1-1.2.Filtering in step S4 and step S5 is all made of ultrafiltration membrance filter.Step
S1 measures the content of heparin sodium in crude heparin sodium and preferably content is thick greater than 60% out before preparing crude heparin sodium solution
Product heparin sodium.The present invention select the higher crude heparin sodium of heparin sodium content refine, purification it is high-efficient, energy can be saved
Consumption.
The present invention is utilized respectively the precipitation method and enzymatic isolation method purifies crude heparin sodium, easy to operate, does not need to liver
Plain sodium is aoxidized, and the structure of heparin sodium will not be destroyed, and purification efficiency is high；
The present invention precipitates heparin sodium using ethyl alcohol, and after ethyl alcohol is added in heparin sodium aqua, heparin sodium is precipitated, extra
Protein precipitant and protein complex enzyme formulation will not influence the precipitation of heparin sodium, extra egg can be extracted after ethanol evaporation
White precipitating reagent and protein complex enzyme formulation, it is energy saving.The present invention when using ethanol precipitation heparin sodium, reduce ethyl alcohol and
The temperature of heparin sodium aqua, on the one hand can prevent ethyl alcohol from volatilizing, and on the other hand effectively reduce heparin sodium and form solid particle mistake
To " package " of the organic solvents such as ethyl alcohol and " co-precipitation " in journey；Enzymatic hydrolysis condition of the present invention is mild, miscellaneous in cracking nucleic acid and albumen
While matter, it can effectively protect the less destruction by protease of heparin sodium.The yield of refined heparin sodium of the present invention is up to 90%, essence
The purity of product heparin sodium is up to 95%.The present invention realizes the enzymatic hydrolysis and adsorption process of heparin sodium under salt-free conditions, to make to arrange
Salinity reduces in the waste water put, and reduces cost for wastewater treatment, improves environmental benefit.
Embodiment 2
Based on embodiment 1, the composite protein precipitator includes the component of following parts by weight: 10 parts of 5- sulfosalisylics
Acid, 3 parts of trichloroacetic acids, 2 parts of acetonitriles, 5 parts of ethyl acetate, 10 parts of polyacrylic acid, 0.8 part of sodium-acetate buffer.
The present invention by it is a variety of can precipitating proteins and the good component of compatibility carry out compound, albumen precipitation effect is good,
The acidity of protein precipitant can also be reduced；The preferred ratio range of each component of the present invention, composite protein precipitator pH is extremely
Control will not cause to damage in 4-5 to chromatographic column etc..
Embodiment 3
Based on embodiment 1, the composite protein precipitator includes the component of following parts by weight: 15 parts of 5- sulfosalisylics
Acid, 8 parts of trichloroacetic acids, 6 parts of acetonitriles, 10 parts of ethyl acetate, 20 parts of polyacrylic acid, 5 parts of sodium-acetate buffers.
The present invention by it is a variety of can precipitating proteins and the good component of compatibility carry out compound, albumen precipitation effect is good,
The acidity of protein precipitant can also be reduced；The preferred ratio range of each component of the present invention, composite protein precipitator pH is extremely
Control will not cause to damage in 4-5 to chromatographic column etc..
Embodiment 4
Based on embodiment 1, the composite protein precipitator includes the component of following parts by weight: 14 parts of 5- sulfosalisylics
Acid, 4 parts of trichloroacetic acids, 5 parts of acetonitriles, 7 parts of ethyl acetate, 20 parts of polyacrylic acid, 1 part of sodium-acetate buffer.The present invention is preferred
The sedimentation effect of the proportion of each component, protein is good, acid small.The present invention by it is a variety of being capable of precipitating proteins and compatibility
Good component progress is compound, and albumen precipitation effect is good, additionally it is possible to reduce the acidity of protein precipitant.
Embodiment 5
Based on embodiment 1, the protein complex enzyme formulation includes the component of following parts by weight: 10 parts of protease, 5 parts
Papain, 0.2 part of lipase, 4 parts of nucleases, 8 parts of expanded perlites.For the present invention using expanded perlite as carrier, preparation is multiple
Synthase preparation can effectively avoid the generation of by-product, in addition, hydrolysis result of the present invention is good, can effectively reduce enzymatic preparations
Usage amount, energy saving.
Embodiment 6
Based on embodiment 1, the protein complex enzyme formulation includes the component of following parts by weight: 20 parts of protease, 10
Part papain, 1 part of lipase, 16 parts of nucleases, 12 parts of expanded perlites.The present invention is using expanded perlite as carrier, preparation
Complex enzyme formulation can effectively avoid the generation of by-product, in addition, hydrolysis result of the present invention is good, can effectively reduce enzymatic hydrolysis system
The usage amount of agent, energy saving.
Embodiment 7
Based on embodiment 1, the protein complex enzyme formulation includes the component of following parts by weight: 15 parts of protease, 10
Part papain, 0.8 part of lipase, 12 parts of nucleases, 10 parts of expanded perlites.The present invention is using expanded perlite as carrier, system
Standby complex enzyme formulation, can effectively avoid the generation of by-product, in addition, hydrolysis result of the present invention is good, can effectively reduce enzymatic hydrolysis
The usage amount of preparation, energy saving.
Embodiment 8
Based on the above embodiment, the present embodiment determines the pH of protein precipitant in embodiment 2-4 and uses protein
The refined heparin sodium yield of precipitating reagent, purity, the results are shown in Table 1.Refined heparin sodium yield, purity are made with crude heparin sodium
For raw material calculating.
1 refined heparin sodium yield of table and purity detecting
The preferred optimum proportioning range of composite protein precipitator of the present invention, guarantees its pH between 4-5.
Embodiment 9
Based on the above embodiment, the present embodiment determines the fine work liver using protein complex enzyme formulation in embodiment 5-7
Plain sodium yield and purity.The results are shown in Table 2.Refined heparin sodium yield, purity are calculated with crude heparin sodium as raw material.
2 refined heparin sodium yield of table and purity
In conjunction with the usage amount and cost of material of raw material, the preferred embodiment of the present invention 7 as protein complex enzyme formulation most
Good proportion.
Above-described specific embodiment has carried out further the purpose of the present invention, technical scheme and beneficial effects
It is described in detail, it should be understood that being not intended to limit the present invention the foregoing is merely a specific embodiment of the invention
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of preparation process of refined heparin sodium, which comprises the following steps:
S1, be dissolved in water crude heparin sodium obtained crude heparin sodium aqueous solution, is added in crude heparin sodium aqueous solution compound
Protein precipitant carries out albumen precipitation, isolates sediment after standing 20-40 minutes；
S2, it is filtered, cools down to having separated the crude heparin sodium aqueous solution after sediment in step S1, be reduced to 10-15 DEG C,
The ethyl alcohol of 85-95% is added in 10-15 DEG C of crude heparin sodium aqueous solution up to sediment precipitation, stands 12-24 hours, from
Heart drying, obtains heparin sodium wet product；
S3, heparin sodium wet product is dried at 30-35 DEG C, obtains heparin sodium time fine work；
S4, heparin sodium time fine work is dissolved in water, adjusts pH value of solution to 8-9, protein is added in heparin sodium time fine work solution
Complex enzyme formulation, centrifugation, filtering obtain heparin sodium time fine work clear liquid；
S5, by heparin sodium time fine work clear liquid with after Dynamic Adsorption resin circulation absorption 3-5 times, resin is eluted, is being eluted
The ethyl alcohol that temperature is 10 DEG C is added in liquid, until sediment is precipitated, after sediment is precipitated 4-8 hours, solution is quiet at 50-60 DEG C
Filtering, cooling and drying obtain refined heparin sodium after setting 30-60 minutes.
2. a kind of preparation process of refined heparin sodium according to claim 1, which is characterized in that the compound protein precipitating
Agent includes the component of following parts by weight: 10-15 parts of 5-sulphosalicylic acids, 3-8 parts of trichloroacetic acids, 2-6 parts of acetonitriles, 5-10 parts of second
Acetoacetic ester, 10-20 part polyacrylic acid, 0.8-5 parts of sodium-acetate buffers.
3. a kind of preparation process of refined heparin sodium according to claim 2, which is characterized in that the compound protein precipitating
Agent includes the component of following parts by weight: 13-15 parts of 5-sulphosalicylic acids, 3-6 parts of trichloroacetic acids, 4-6 parts of acetonitriles, 5-7 parts of second
Acetoacetic ester, 15-20 part polyacrylic acid, 0.8-1.2 parts of sodium-acetate buffers.
4. a kind of preparation process of refined heparin sodium according to claim 3, which is characterized in that the compound protein precipitating
Agent includes the component of following parts by weight: 14 parts of 5-sulphosalicylic acids, 4 parts of trichloroacetic acids, 5 parts of acetonitriles, 7 parts of ethyl acetate, 20
Part polyacrylic acid, 1 part of sodium-acetate buffer.
5. a kind of preparation process of refined heparin sodium according to claim 1, which is characterized in that the protein complex enzyme
Preparation includes the component of following parts by weight: 10-20 parts of protease, 5-10 parts of papains, 0.2-1 parts of lipase, 4-16 parts of cores
Sour enzyme, 8-12 part expanded perlite.
6. a kind of preparation process of refined heparin sodium according to claim 5, which is characterized in that the protein complex enzyme
Preparation includes the component of following parts by weight: 15 parts of protease, 10 parts of papains, 0.8 part of lipase, 12 parts of nucleases, 10 parts
Expanded perlite.
7. a kind of preparation process of refined heparin sodium according to claim 1, which is characterized in that crude heparin sodium with it is compound
The mass ratio of protein precipitant dosage are as follows: 1:0.5-1.
8. a kind of preparation process of refined heparin sodium according to claim 1, which is characterized in that heparin sodium time fine work and egg
The mass ratio of white matter complex enzyme formulation dosage are as follows: 1:1-1.2.
9. a kind of preparation process of refined heparin sodium according to claim 1, which is characterized in that in step S4 and step S5
Filtering be all made of ultrafiltration membrance filter.
10. a kind of preparation process of refined heparin sodium according to claim 1, which is characterized in that step S1 is thick in preparation
Before product heparin sodium aqua, measures the content of heparin sodium in crude heparin sodium and preferably go out the crude heparin sodium that content is greater than 60%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111138564A (en) * | 2020-01-13 | 2020-05-12 | 苏州健飞肠衣有限公司 | Method for separating, purifying and improving purity of heparin sodium crude product |
| CN114209059A (en) * | 2021-12-20 | 2022-03-22 | 黑龙江乌苏里江制药有限公司 | Acanthopanax senticosus polysaccharide calcium and preparation method and application thereof |
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| CN111138564A (en) * | 2020-01-13 | 2020-05-12 | 苏州健飞肠衣有限公司 | Method for separating, purifying and improving purity of heparin sodium crude product |
| CN114209059A (en) * | 2021-12-20 | 2022-03-22 | 黑龙江乌苏里江制药有限公司 | Acanthopanax senticosus polysaccharide calcium and preparation method and application thereof |
| CN114209059B (en) * | 2021-12-20 | 2024-01-19 | 黑龙江乌苏里江制药有限公司 | Acanthopanax polysaccharide calcium and preparation method and application thereof |
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