CN103772530A - Method for extraction separation of heparin sodium through enzymolysis - Google Patents
Method for extraction separation of heparin sodium through enzymolysis Download PDFInfo
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- CN103772530A CN103772530A CN201410023683.2A CN201410023683A CN103772530A CN 103772530 A CN103772530 A CN 103772530A CN 201410023683 A CN201410023683 A CN 201410023683A CN 103772530 A CN103772530 A CN 103772530A
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- heparin sodium
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Abstract
The invention discloses a method for extraction separation of heparin sodium through enzymolysis. The method is characterized by comprising the steps of cleaning and mincing small intestine of pigs, adding water of same volume to obtain a small intestine stock solution; activating a bio-enzyme to obtain a bio-enzyme solution; mixing 30mL of the bio-enzyme solution and 10-15L of the small intestine stock solution, regulating pH value to 8.0-9.0, heating to 45-55 DEG C, adding 10-20mL of collagenase, preserving heat for 2-4 hours, continuing heating to 85-90 DEGC, preserving heat for 4 hours, filtering and collecting a reaction solution; and drying to obtain a crude heparin sodium product, wherein the collagenase is added in enzymolysis process and enzymolysis temperature is improved step by step, so as to sufficiently guarantee complete breakage of a connecting bond between heparin and hydrolyzing collagen. The method disclosed by the invention can increase product yield and achieve environmental protection and conservation in production process, and the method is suitable for industrial production.
Description
Technical field
The present invention relates to the preparation method of organic high molecular compound, particularly a kind of enzymolysis process extracts the method that separates heparin sodium.
Background technology
Heparin sodium is the sodium salt of the CSSO3 extracted in the intestinal mucosa by pig or ox, belongs to mucopolysaccharide material, is white or off-white powder, and odorless, tasteless, has water absorbability, soluble in water, is insoluble to the organic solvents such as ethanol, ether, acetone and benzene.
At present heparin is in the world the most effectively and the anticoagulation medicine of quantity maximum, is mainly used in cardiovascular and cerebrovascular diseases and hemodialysis, in hemodialysis, is wherein unique effective specific medicament.Clinical application and studies show that, heparin, except having blood coagulation resisting function, also has other multiple biological activity and clinical applications, comprise reducing blood lipid, anti-in film smooth muscle cell proliferation, promote the effects such as fibrinolysis.
Existing traditional heparin sodium production technique is enzymolysis and extraction, saltouts refining.By pancreatin gastric enzyme heparin in enzymolysis and extraction raw material chitterlings under suitable pH and temperature condition, obtain heparin crude product stoste.Heparin crude product stoste at a certain temperature, due to adding of salts solution, constantly open by the connecting key of foreign protein and heparin, and sodium ion and heparin reaction bonded generate heparin sodium simultaneously, and product is refined purifying.This technique is simple, and convenient, cost is low.But its main drawback is that the heparin rate of recovery is low, the salt solution liquid recovery utilization rate that contains a large amount of heparin is low, has produced a large amount of waste materials.
Summary of the invention
The object of the present invention is to provide a kind of rate of recovery high, the enzymolysis process that waste material is few extracts the method that separates heparin sodium.
The technical solution that realizes the object of the invention is:
Enzymolysis process extracts a method that separates heparin sodium, comprises the following steps:
Step 1, raw material processing; Chitterlings are cleaned with clear water, rubbed into rotten shape, stir and add isopyknic water, make small intestine stoste;
Step 2, activation biological enzyme; Get biological enzyme stoste, with NaOH solution adjust pH to 8.0～9.0, leave standstill 1～2 hour, collect and obtain biological enzyme liquid, for subsequent use;
Step 3, enzymolysis; 30mL biological enzyme liquid and 10～15L small intestine stoste are poured in reactor, with NaOH solution adjusting pH value to 8.0～9.0, be heated to 45～55 ℃, add again Collagenase 10～20mL, be incubated 2～4 hours, continue to be heated to 85～90 ℃, be incubated 4 hours, filter and collect reaction solution, for subsequent use;
Step 4, dry; By the dry reaction solution heparin sodium crude that makes.
Wherein, described in step 1, in raw material treating processes, can add concentration is sanitas 1～2ml of 0.1wt%.
Wherein, described in step 1, in raw material treating processes, chitterlings derive from fresh or freezing chitterlings.
Wherein, biological enzyme stoste is directly taken from the Pancreas Sus domestica that pulverizes or with commercially available gastric enzyme and pancreatin, according to the mass ratio preparation of 1:2 described in step 2.
Wherein, when enzymolysis process is heated to 45～55 ℃ described in step 3, preferably add Collagenase 15mL, be incubated 3 hours.
Wherein, step 4 can adopt lyophilize or vacuum drying method to make heparin sodium crude.
The present invention compared with prior art, its remarkable advantage: add Collagenase in enzymolysis process, adopt the method for heating stage by stage, guarantee the abundant cracking of heparin and foreign protein connecting key, the raising heparin rate of recovery; In reaction process, do not use the conventional solvents of traditional technology such as ethanol, salt, resin elution liquid, the equal recoverable of gained intermediate reaction liquid, the thought of " Green Chemistry " is applied to whole extraction process, as far as possible less with an organic solvent, make the extraction process of heparin sodium succinctly distinct, be easy to industrialization.
Embodiment
Embodiment mono-
Step 1: raw material processing.8kg fresh pig small intestine is cleaned with clear water, rubbed into rotten shape, and under fully stirring, add isopyknic water to mix, then to add 1～2ml concentration be that the sanitas of 0.1wt% mixes, make small intestine stoste.
Step 2: activation biological enzyme.Get the pancreas of pig, more fresh better, pulverize, add NaOH solution, adjust pH to 8.0～9.0, leave standstill 1～2 hour.
Step 3: enzymolysis.30mL biological enzyme liquid and about 15L small intestine stoste are poured in reactor and heated, add NaOH solution, regulate pH value to 8.5, be heated to 50 ℃, then add Collagenase 15mL, be incubated 3 hours, continue to be heated to 85～90 ℃, be incubated 4 hours, filter and collect reaction solution.
Step 4: dry.Reaction solution lyophilize is made to heparin sodium crude 5.6kg.
Measure heparin sodium according to " Chinese Pharmacopoeia " method and tire, can obtain the heparin sodium crude rate of recovery and reach 70.2%.
Embodiment bis-
Step 1: raw material processing.8kg fresh pig small intestine is cleaned with clear water, rubbed into rotten shape, and under fully stirring, add isopyknic water to mix, make small intestine stoste.
Step 2: activation biological enzyme.With commercially available gastric enzyme and pancreatin, prepare according to the mass ratio of 1:2, make 30mL biological enzyme stoste.Add NaOH solution, adjust pH to 8.0～9.0, leave standstill 1～2 hour, collect and obtain biological enzyme liquid.
Step 3: enzymolysis.30mL biological enzyme liquid and about 12L small intestine stoste are poured in reactor and heated, add NaOH solution, regulating pH value is 8.5, be heated to 50 ℃, then add Collagenase 15mL, be incubated 2 hours, continue to be heated to 85～90 ℃, be incubated 4 hours, filter and collect reaction solution.
Step 4: dry.Reaction solution vacuum-drying is made to heparin sodium crude 4.9kg.
Measure heparin sodium according to " Chinese Pharmacopoeia " method and tire, can obtain the heparin sodium crude rate of recovery and reach 66.9%.
Claims (6)
1. enzymolysis process extracts a method that separates heparin sodium, it is characterized in that, comprises the following steps:
Step 1, raw material processing; Chitterlings are cleaned with clear water, rubbed into rotten shape, stir and add isopyknic water, make small intestine stoste;
Step 2, activation biological enzyme; Get biological enzyme stoste, use NaOH solution, adjust pH to 8.0～9.0, leave standstill 1～2 hour, collect and obtain biological enzyme liquid, for subsequent use;
Step 3, enzymolysis; 30mL biological enzyme liquid and 10～15L small intestine stoste are poured in reactor, used NaOH solution, regulate pH value to 8.0～9.0, be heated to 45～55 ℃, add again Collagenase 10～20mL, be incubated 2～4 hours, continue to be heated to 85～90 ℃, be incubated 4 hours, filter and collect reaction solution, for subsequent use;
Step 4, dry; By the dry reaction solution heparin sodium crude that makes.
2. enzymolysis process extracts the method that separates heparin sodium according to claim 1, it is characterized in that, it is sanitas 1～2ml of 0.1wt% that step 1 raw material treating processes adds concentration.
3. enzymolysis process extracts the method that separates heparin sodium according to claim 1, it is characterized in that, the chitterlings in step 1 raw material treating processes derive from fresh or freezing chitterlings.
4. enzymolysis process extracts and separates the method for heparin sodium according to claim 1, it is characterized in that, step 2 biological enzyme stoste is directly taken from the Pancreas Sus domestica that pulverizes or with commercially available gastric enzyme and pancreatin, prepared according to the mass ratio of 1:2.
5. enzymolysis process extracts the method that separates heparin sodium according to claim 1, it is characterized in that, when step 3 enzymolysis process is heated to 45～55 ℃, adds Collagenase 15mL, is incubated 3 hours.
6. enzymolysis process extracts the method that separates heparin sodium according to claim 1, it is characterized in that, step 4 adopts lyophilize or vacuum drying method to make heparin sodium crude.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410023683.2A CN103772530A (en) | 2014-01-20 | 2014-01-20 | Method for extraction separation of heparin sodium through enzymolysis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410023683.2A CN103772530A (en) | 2014-01-20 | 2014-01-20 | Method for extraction separation of heparin sodium through enzymolysis |
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| CN103772530A true CN103772530A (en) | 2014-05-07 |
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| CN201410023683.2A Withdrawn CN103772530A (en) | 2014-01-20 | 2014-01-20 | Method for extraction separation of heparin sodium through enzymolysis |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0386053A1 (en) * | 1987-11-06 | 1990-09-12 | Opocrin Spa | Non-anticoagulant heparan sulfate, process for extraction from organs, and pharmaceutical compositions thereof. |
| CN101463095A (en) * | 2007-12-20 | 2009-06-24 | 大英县茂森生物化工厂 | Novel technique for producing heparin |
| CN102924626A (en) * | 2012-09-19 | 2013-02-13 | 杭州龙扬生物科技有限公司 | Process for extracting heparin sodium efficiently from small intestines of pigs |
-
2014
- 2014-01-20 CN CN201410023683.2A patent/CN103772530A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0386053A1 (en) * | 1987-11-06 | 1990-09-12 | Opocrin Spa | Non-anticoagulant heparan sulfate, process for extraction from organs, and pharmaceutical compositions thereof. |
| CN101463095A (en) * | 2007-12-20 | 2009-06-24 | 大英县茂森生物化工厂 | Novel technique for producing heparin |
| CN102924626A (en) * | 2012-09-19 | 2013-02-13 | 杭州龙扬生物科技有限公司 | Process for extracting heparin sodium efficiently from small intestines of pigs |
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Application publication date: 20140507 |