CN100439402C - Method for extracting heparin using lecithin liquid membrane separating method - Google Patents
Method for extracting heparin using lecithin liquid membrane separating method Download PDFInfo
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- CN100439402C CN100439402C CNB2006100693335A CN200610069333A CN100439402C CN 100439402 C CN100439402 C CN 100439402C CN B2006100693335 A CNB2006100693335 A CN B2006100693335A CN 200610069333 A CN200610069333 A CN 200610069333A CN 100439402 C CN100439402 C CN 100439402C
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- heparin
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- low molecular
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
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Abstract
The invention relates to a method used to extract low molecule heparin from sea slug wastes and cactus. It includes the following steps: beche-de-mer intestine emulsifying latex emulsion breaking heparin extracting solution is added resin to absorb, wash, and elute; using alcohol precipitation heparin crude product to refine heparin. The invention has the advantages of original at home and aboard, high yield, high efficiency, low energy consumption, low cost, environmental protection, huge market competition potential etc.
Description
One, technical field
The present invention relates to a kind of method of extracting Low molecular heparin, especially from sea slug wastes, extract the method for Low molecular heparin, belong to heparin extracting method technical field.
Two, background technology
Because the special affinity of stichopus japonicus heparin and reticuloendothelial cell makes heparin-protein complex be difficult to stripping, add that the antiplasmin activity in the stichopus japonicus intestines is strong, heparin is destroyed seriously, therefore, utilize general heparin extractive technique can only obtain the heparin of constant.
At present, preparing Low molecular heparin both at home and abroad, is raw material with the commodity heparin mostly, adopts acid, alkali control depolymerization, obtain the Low molecular heparin fragment by the finished product depolymerized heparin, or classified method, through mediums of exchange such as macroporous resins, obtain the Low molecular heparin component, the cost height, the product biological activity is low.
Adopting the nitrous acid depolymerization to prepare Low molecular heparin from LAAH (low-anticoagulant heparin) is that recovery rate is low, and the nitrous acid that adopts can make the biological activity of heparin reduce.
Heparin preparation relates to many-sides such as separation in the modern biological specialty, extraction, and many-sided knowledge such as pharmacology, experiment, and is interdisciplinary and professional very strong, and needs suitable working experience.
Three, summary of the invention
The object of the present invention is to provide and a kind ofly adopt lecithin liquid membrane separating method, be the method for the extraction Low molecular heparin that improves greatly of raw material, recovery rate with the stichopus japonicus waste.
The present invention adopts liquid Membrane Separation Technique, makes heparin when forming oil-in-water emulsion, is come out by tensio-active agent " washing " from endothelial tissue, and is sealed by fine, effectively isolates with enzyme.Meanwhile, make enzyme form micelle by surfactant and help and proteinic thorough hydrolysis, thereby effectively improve recovery rate, the leaching process heparin structure does not destroy, the heparin distribution coefficient that obtains is low, almost is efficient, the stable Low molecular heparin of simple substance level.
Gordian technique of the present invention is analyzed as follows:
(1) feasibility of extraction Low molecular heparin from the stichopus japonicus waste
China's traditional medical theory is thought that the sea cucumber nature and flavor are sweet, salty, warm, is gone into the heart, lung, spleen, kidney channel.Modern nutriology studies show that sea cucumber includes 50 multiple nutritional components, is the nutritive food of high protein, lower fat, low sugar, no cholesterol, and its content of mineral substances is also very high.But, still be not enough to explain the tonic effect of sea cucumber with these nutritive ingredients, so present research has been carried out deep analysis to sea cucumber, found that to exist a lot of special composition in the sea cucumber that these special composition have been achieved tonic effect and the pharmaceutical use of sea cucumber.
The very high collagen protein of content is arranged in the stichopus japonicus body wall, and the stichopus japonicus collagen protein can compare favourably on nutritive ingredient with donkey-hide gelatin, Colla Plastri Testudinis, Soft-shelled Turtle shell glue, the Deerhorn Glue of traditional Chinese medicine, and its function is supplying vital essence and marrow, passage, nourishing blood, in addition, delay body aging, can make skin keep smooth and elasticity.
The similar ginsenoside of the chemical structure of stichopus japonicus saponin, its biological medical action comprise anti-experimental tumor cell, suppress pathogenic bacterium, anti-oxidant, regulate immunologic function, promote hematopoiesis, antifatigue etc.
The sea cucumber drug action, successively there is the expert to propose in the past 30 years with sea cucumber treatment tumour, aplastic anemia and diabetes, and obtain good effect, and when assisting a ruler in governing a country such as the treatment of common diseases such as insane crazy, gastric and duodenal ulcer, chronic viral hepatitis B, chronic nephritis, hypertension, pulmonary tuberculosis, neurasthenia, phlebothrombosis, hemorrhoid, prolapse of the anus and constipation and for general after being ill or in the various herbal cuisine or diet therapy formulation worked out in the postpartum recovery process, sea cucumber has also obtained admitting widely and using.
20th century middle and late, adopt modern means of science and technology such as chemistry and pharmacology each constituents of sea cucumber to be carried out the result of biological activity determination, the drug action of the effective component of sea cucumber, confirmed the past three, four tcm clinical practice experience sea cucumber health care medical functions of being summed up over 100 years, and be provided with under the positive reference substance contrast, carry out organ in integral body, also found the new activity of sea cucumber composition in the test of pesticide effectiveness on cell or the molecular level, from sea cucumber itself, all kinds of crude extractive, the pharmacologically active of component or single component is very extensive, almost contain current main illness, such as tumour to the harm of human health formation, cardiovascular disorder, viral hepatitis, acquired immune deficiency syndrome (AIDS), immunity illness and senile disease etc.
The stichopus japonicus mucopolysaccharide is a class important component of sea cucumber body wall, the structure of now determining two kinds of sea cucumber polysaccharide HG and HF is special, all peculiar by sea cucumber, sea cucumber polysaccharide degree with polysaccharide more than its content in tissue is big very rare in animal based food resource.Over nearly 20 years both at home and abroad to sea cucumber polysaccharide, particularly the pharmacological research of HG confirms that it has multiple drug effect: antagonism kinds of experiments animal tumor, the artificial lung of antagonism MA-73 mammary cancer shifts, improve the body cell immunologic function, improve and strengthen animal body immunologic hypofunction situation because of using cancer therapy drug to cause, have the serum cholesterol of reduction and triglyceride levels, multiple efficacies such as anti-inflammatory action.
(2) extraction of Low molecular heparin
Influence the consumption that heparin cracked factor mainly contains temperature, time, pH value and oxygenant.Fixing under the condition of other influence factor, analyzing the influence of temperature and time heparin cracking degree.At a certain temperature, the molecular weight of Low molecular heparin reduces with the prolongation in reaction times.Under certain hour, the molecular weight of Low molecular heparin reduces with the rising of temperature.Therefore, controlled temperature and time just can obtain the Low molecular heparin of certain molecular weight.
(3) liquid Membrane Separation Technique
With liquid Membrane Separation Technique separation and Extraction biologically active substance from the marine organisms tissue, have that device is simple, processing ease, advantage such as easily manufactured, and separation efficiency height, energy consumption is low, that the extract biological activity is preserved is complete, cost is low, the height of tiring, toxic side effect are little, has opened up the frontier of traditional Chinese medicine to modernized Chinese medicine development.
Purpose of the present invention is achieved through the following technical solutions.
The present invention utilizes liquid Membrane Separation Technique, and the method for extracting Low molecular heparin from the stichopus japonicus waste can be divided into following hierarchical structure:
1, emulsification, breakdown of emulsion layer: the utilization emulsifying technology, add tensio-active agent in the emulsion process, from the stichopus japonicus waste, extract the heparin extracting solution; The utilization liquid Membrane Separation Technique makes heparin when forming oil-in-water emulsion, is come out by tensio-active agent " washing " from endothelial tissue, and is sealed by fine, effectively isolates with enzyme;
2, extract phase: make enzyme form micelle by surfactant and help proteinic thorough hydrolysis, as, molecular weight is 5800 Low molecular heparin crude product through the refining molecular weight that obtains is 5300 elaboration, product is a white powder, is 82.1% by crude product to the recovery rate of elaboration, the leaching process heparin structure does not destroy, and the heparin distribution coefficient that obtains is low, almost is efficient, the stable Low molecular heparin of simple substance level.
The present invention adopts lecithin liquid membrane separating method to extract the method for heparin, and its extraction process flow process is as follows
The present invention adopts lecithin liquid membrane separating method to extract the method for heparin, and its raw material adopts the stichopus japonicus intestines;
The tensio-active agent of selecting is a Yelkin TTS, or nonyl phenol phosphoric acid fat.
Refining step selects ion exchange resin to make with extra care.
The present invention adopts the lecithin liquid membrane isolation technique, and the gordian technique of extracting Low molecular heparin from sea slug wastes is
1. solve the special avidity of stichopus japonicus heparin and reticuloendothelial system and endotheliocyte, select high effect nontoxic not influence the tensio-active agent Yelkin TTS that heparin is tired, or nonyl phenol phosphoric acid fat, the extraction that improves heparin-protein complex;
2. solve the decomposition of heparin-protein complex, selection can conveniently be eliminated, and can make enzyme form micelle again, has both protected heparin to exempt from destruction, helps the lyase and the surfactant of proteinic thorough hydrolysis;
3. during the liquid film breakdown of emulsion,, adopt corresponding millipore filtration and breaking method according to the molecular weight of extract Low molecular heparin;
4. select ion exchange resin refining, exchange capacity is strong, and balance is fast, and is with short production cycle, solves the recovery rate of heparin in the extracting solution emphatically.
The present invention is that raw material extracts Low molecular heparin with the stichopus japonicus waste, be domestic and international initiative, the elaboration recovery rate that Low molecular heparin of the present invention extracts is more than 82%, improves greatly than the recovery rate 50% that adopts the nitrous acid depolymerization to prepare from LAAH (low-anticoagulant heparin).
Four, specific embodiments:
Below provide the specific embodiment of the present invention, be used for that the invention will be further described.
Embodiment adopts the method for following lecithin liquid membrane separating method extraction heparin, but the present invention is not limited to the following stated embodiment.
Adopt lecithin liquid membrane separating method to extract the method for heparin, its extraction process flow process is as follows
Raw material adopts the stichopus japonicus intestines, and tensio-active agent is yellow Yelkin TTS of dawn or nonyl phenol phosphoric acid fat, and refining step selects ion exchange resin to make with extra care.
External low molecule heparin product is many to be that raw material makes with the commodity heparin, product of the present invention is that to have made molecular weight be the Low molecular heparin of 5000-5600d to raw material with the stichopus japonicus intestinal mucosa, wherein the LMWH of 5300d has strong FXa effect and more weak anti-FII effect, check through t, the OT value of Low molecular heparin all has tangible prolongation than LAAH, heparin, physiological saline, illustrate its to thrombotic restraining effect obviously greater than LAAH and heparin.Compare with heparin, the anti-FXa/FIIa value of this Low molecular heparin is big, and subcutaneous injection bioavailability height is eliminated slow in vivo, long half time, oral easy absorption, little to the side effect of thrombocyte and steatolysis aspect, bleeding tendency is also little, and is safe and convenient to use, thereby the clinical application potentiality are big.
Anti-FXa activity is an important prerequisite of Low molecular heparin antithrombotic potentiality.Generally with the antithrombotic potentiality than value representation Low molecular heparin of anti-FXa activity with anticoagulating active (APTT or anticoagulant efficiency), its value is big more, and anti thrombotic action is strong more, and bleeding tendency is more little.The anti-FXa/APTT value of heparin is about 1, and Low molecular heparin is generally 1.5-4, its international standard substance is 2.51, the anti-FXa/APTT value of the Low molecular heparin of the 5300d of the present invention's preparation is 3.62, antithrombotic acitivity is the strongest, the anti-Fax/APTT value for preparing from LAAH (low-anticoagulant heparin) than domestic employing nitrous acid depolymerization is high, is higher than the international standard substance prescribed value, locates international most advanced level.
Therefore be the feedstock production Low molecular heparin with the stichopus japonicus intestinal mucosa, both can make full use of the stichopus japonicus waste, increase economic benefit, eliminate of the pollution of stichopus japonicus waste environment, clinical good effect, the high antithrombotic acitivity medicine that toxic side effect is little of providing is provided again, certain theory and practical significance are arranged.
Innovative point of the present invention is
(1) adopts liquid Membrane Separation Technique from stichopus japonicus waste, Root and stem of Cholla, to extract the preparation biological activity and preserve complete Low molecular heparin, belong to initiative both at home and abroad;
(2) technology advanced person of the present invention, the recovery rate height; Employing ion exchange resin is refining, solves the recovery rate of heparin in the extracting solution emphatically;
(3) take the liquid Membrane Separation Technique in the modern biotechnology to extract the preparation Low molecular heparin, the efficient height, energy consumption is low;
(4) adopting the stichopus japonicus waste is main raw material, has environment protection significance widely, and the Low molecular heparin of development is with low cost, and at home and abroad competition potential is huge on the market;
(5) be widely used in clinical, progressively replace unfractionated heparin at antithrombotic, antitumor, antiphlogistic activity, improve aspect the body immunity.
The main products technical indicator that reaches is as follows
The color and luster white powder
Stability is to thermally-stabilised
Water-soluble soluble in water, stronger water absorbability is arranged in air
Molecular weight ranges 1200-15000d, peak value is at 4000-6000d
Distribution coefficient≤1.5
Anti-Fxa/APTT value 1.5-4
Protein content does not contain protein substantially
The key technical indexes contrast table
The present invention of contrast content prior art
Molecular weight ranges 3000-80000 1200-15000d, peak value is at 4000-6000d
(molecular weight is concentrated)
(distribution coefficient is few, molecular weight is concentrated in distribution coefficient＞2≤1.5
, similarity is good, purity is high)
The anti-Fxa 70iu/mg 76iu/mg (improve 10%) that tires
Protein content does not contain protein (it is few to contain protein impurities) on a small quantity substantially
The anti-FIIa 1.5 1.6 of anti-Fxa/ (biological activity is preserved complete)
Transformation period (min) 111 300 (long half time, good stability)
Recovery rate 50% 82% (height)
Claims (2)
1, adopt lecithin liquid membrane separating method to extract the method for heparin, it is characterized in that its extraction process flow process is as follows:
The tensio-active agent of selecting in the above-mentioned emulsion process is an Ovum Gallus domesticus Flavus lecithin.
2, extract the method for heparin according to the described employing lecithin liquid membrane separating method of claim 1, it is characterized in that refining step selects ion exchange resin to make with extra care.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100693335A CN100439402C (en) | 2006-10-12 | 2006-10-12 | Method for extracting heparin using lecithin liquid membrane separating method |
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| CNB2006100693335A CN100439402C (en) | 2006-10-12 | 2006-10-12 | Method for extracting heparin using lecithin liquid membrane separating method |
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| Publication Number | Publication Date |
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| CN1935850A CN1935850A (en) | 2007-03-28 |
| CN100439402C true CN100439402C (en) | 2008-12-03 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1264391A (en) * | 1997-07-16 | 2000-08-23 | 阿克佐诺贝尔公司 | Process for the production of heparin |
| CN1283636A (en) * | 2000-09-07 | 2001-02-14 | 上海惠海生化制品厂 | Heparin and its preparing process |
| CN1749284A (en) * | 2005-09-19 | 2006-03-22 | 南京健友生物化学制药有限公司 | Purifying method for low molecule heparin |
-
2006
- 2006-10-12 CN CNB2006100693335A patent/CN100439402C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1264391A (en) * | 1997-07-16 | 2000-08-23 | 阿克佐诺贝尔公司 | Process for the production of heparin |
| CN1283636A (en) * | 2000-09-07 | 2001-02-14 | 上海惠海生化制品厂 | Heparin and its preparing process |
| CN1749284A (en) * | 2005-09-19 | 2006-03-22 | 南京健友生物化学制药有限公司 | Purifying method for low molecule heparin |
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| CN1935850A (en) | 2007-03-28 |
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Granted publication date: 20081203 Termination date: 20121012 |