CN104761635A - Hirudin extraction separation technique - Google Patents
Hirudin extraction separation technique Download PDFInfo
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- CN104761635A CN104761635A CN201510131284.2A CN201510131284A CN104761635A CN 104761635 A CN104761635 A CN 104761635A CN 201510131284 A CN201510131284 A CN 201510131284A CN 104761635 A CN104761635 A CN 104761635A
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Abstract
The invention discloses a hirudin extraction separation technique which comprises the following steps: smashing and homogenizing hirudo to obtain an extracting solution, filtering through a ceramic filter membrane, and cleaning the ceramic filter membrane with distilled water; merging the ceramic filter membrane filtrate and cleaning solution, filtering through an organic filter membrane, and cleaning the organic filter membrane with distilled water; eluting the organic filter membrane filtrate and cleaning solution through a gel chromatographic column, and detecting the eluate by a high pressure liquid chromatograph; collecting the fraction with the hirudin chromatographical purity of 80% above, concentrating the collected eluate through a nanofiltration membrane, purifying the concentrated solution through a reverse-phase c18 semipreparative column, and collecting the target peak value; and carrying out freeze drying to obtain the freeze-dried hirudin pure product. By adopting the filtration and separation mode of combining the ceramic filter membrane and organic film, most impurities in the hirudo homogenate can be removed, thereby enhancing the extraction efficiency. The technique is convenient to operate, avoids the use of the organic solvent, and greatly lowers the extraction preparation cost.
Description
Technical field
The present invention relates to a kind of filtration extracting method of anticoagulant compounds, be specifically related to a kind of extraction and separation process of r-hirudin.
Background technology
R-hirudin is a kind of anticoagulative substance extracted from hirudinaria manillensis (being commonly called as leech).Leech, head is loaded in China's Shennong's Herbal, after all record in the monographs such as Compendium of Material Medica and " Chinese animal drugs ".1884, Haycraft found that Hirudo extract has blood coagulation resisting function, nineteen fifty-five, MarkwMdt points out that this anticoagulative substance is protein, name as r-hirudin, after 1986, gene recombination r-hirudin comes out, for the further investigation of r-hirudin and clinical application open bright prospects.Root it is documented, the low molecular polypeptide that natural hirudin is made up of 65 amino acid, and its effect has the following aspects:
1, have blood coagulation resisting function: the r-hirudin of different sources and structure, its anticlotting mechanism is not quite similar.Therefore natural hirudin can affect blood coagulation and thrombosis from multiple link.
2, antithrombin effect: the antithrombin effect of r-hirudin sees European Hementaria officianalis (hiNmedic5nalis) L1j, Asia buffalo leech (Hirudinaria manillensis) and India leech etc.By the r-hirudin extracted in these leech containing 6 similar cysteine residues that distribute, there is similar three-dimensional structure: its N holds by disulfide formation imporosity, and is combined with thrombin activity site; Its C end is rich in acidic amino acid residue, be then combined with the Fibrinogen binding site of zymoplasm; The amino-acid residue coordinative role of intermediate zone.This structure can reach r-hirudin and zymoplasm be 1: 1 ratio form stable mixture, thus have the restraining effect of high special to zymoplasm, play the blood coagulation resisting function of Trombin inhibiting.
3, antiplatelet effects: 1992, Como etc. extract one and are called Hirudinaria manillensis antiplatelet protein from Mexico leech, there is the effect that specificity suppresses collagen-induced platelet to be assembled, and to other inductors (as blood, zymoplasm etc.) induced platelet aggregation unrestraint effect.1994, Kmel etc. extract a kind of polypeptide containing 39 amino-acid residues from the leech of North America, its three-dimensional conformation and r-hirudin are very similar, and the RGD sequence of C end identifies with platelet membrane GPIIb/IIIa phase, thus the platelet aggregation that inhibit Fibrinogen to mediate.
4, fibrin degradation (former) effect: 1984, Malin-conlco etc. extracted a kind of material being called hementin from the huge leech of Amazon.This material can the particular rib chain of fibrin degradation (former), has antithrombin activity and the platelet aggregation that can significantly suppress ADP induce, has obvious effect for what prevent thrombotic diseases.
Chinese patent ZL2010105266817 disclosed " a kind of method extracting high anticoagulating active r-hirudin from natural leech ", the method is combined by two or more materials in sodium-chlor, a water acetyl-cysteine salt, Methionin, neutral protease, papoid, be dissolved in deionized water and be mixed with stimulation fluid, in stimulation fluid, the concentration expressed in percentage by weight of each material is 0.01 ~ 5%, and wherein enzyme concn is at 100U ~ 300U/ml.Being weighed by living leech is placed in container, be that 1:1 ~ 5 times add described stimulation fluid by leech weight ratio stimulation fluid volume, stimulate and within 3 hours, make leech secrete juice, juice obtains r-hirudin crude product through 16000 ~ 20000r/min centrifugation, through 0.2 micron of stainless steel membrane micro-filtration, then carrying out reverse osmosis nanofiltration and concentrate, is then that 1:6 ~ 10 add medical starch allotment by solid than the weightmeasurement ratio of liquid, spray-dried, obtain r-hirudin finished product.This extracting method is complicated, and operation easier is high, and magazine is removed thorough not, and extraction efficiency is low, and organic solvent uses many, and environmental pollution is large.
Summary of the invention
The object of the invention is to solve prior art Problems existing, the extraction and separation process of the r-hirudin that a kind of easy handling, cost are low, efficiency is high, purity is good is provided.
The technical scheme realizing the object of the invention is a kind of technique of r-hirudin extraction and isolation, comprises the following steps:
Step one: filter leech extracting solution: the extracting solution after leech is smashed homogenate filters through ceramic filtration membrane, collects filtered liquid, then carries out ceramic filtration membrane cleaning with distilled water; Merge ceramic filtration membrane filtered liquid and scavenging solution, then through organic membrane filtration, collect filtered liquid, then carry out organic filter membrane cleaning with distilled water;
Step 2: wash-out: organic membrane filtration liquid step one obtained and scavenging solution by gel chromatographic columns wash-out, then detect elutriant by high pressure liquid chromatograph;
Step 3: purifying: collected by the flow point that r-hirudin chromatographic purity reaches more than 80%, concentrated by the elutriant collected through nanofiltration membrane again, after obtaining nanofiltration concentrated solution, carries out purifying by concentrated solution through anti-phase c18 semipreparative column, collects target peak;
Step 4: carry out lyophilize to the liquid that step 3 obtains, obtains freeze-drying r-hirudin sterling.
In described step one, the volume carrying out ceramic filtration membrane cleaning with distilled water is 25 ~ 30% of ceramic filtration membrane filtered liquid; The volume carrying out organic filter membrane cleaning with distilled water is 10 ~ 15% of organic membrane filtration liquid.
The working temperature of described step one is 25 DEG C ~ 35 DEG C, and pressure is 0.05Mpa ~ 0.3Mpa.
Ceramic filtration membrane specification in described step one is 30KDa ~ 300KDa molecular weight cut-off; And the molecular weight cut-off of rear nonrecoverable ceramic filtration membrane is less than the molecular weight cut-off of a front ceramic filtration membrane.
Organic filter membrane specification in described step one is 0.8 ~ 1KDa molecular weight cut-off.
In described step one, before first time and second time distilled water clean, add the rinse that the distilled water of working as time cleaning distilled water 5% volume carries out container and film.
Gel chromatographic columns in described step 2 is SephadexG-25.
Nanofiltration membrane in described step 3 is 0.5 ~ 0.8KDa; Anti-phase c18 semipreparative column is Zorbax, 300SB-C18; The elutriant of anti-phase c18 semipreparative column is: 0.1 ~ 1M acetonitrile.
Freeze temperature in described step 4 is :-30 DEG C ~-10 DEG C.
Have employed technique scheme, the present invention has following beneficial effect: the mode that (1) the present invention adopts ceramic filtration membrane and organic membrane to carry out combined filtering to be separated, and ceramic filtration membrane is filtered at least one times, most of impurity in leech homogenate can be removed, improve the efficiency of extraction, and easy to operate, avoid the use of organic solvent, extract preparation cost and greatly reduce.
(2) the present invention adopts gel chromatographic columns wash-out, adopts nanofiltration membrane to concentrate, takes full advantage of the high efficiency feature that membrane filtration is concentrated, effectively reduce product cost and operation easier.
(3) the present invention adopts reverse phase preparative column to carry out purifying, and freeze-drying processes r-hirudin, decreases the use of organic solvent, optimizes extraction process, reduce cost.
Embodiment
Prepare leech and extract stoste: fresh living leech is cleaned up, and is twisted into leech mud.The physiological saline and the leech mud that add 0.5 times of volume 0.9% mix.20 DEG C are cooled to, collected by centrifugation mother liquor after being warmed to 50 DEG C of insulation 10min.By the solid matter physiological saline lixiviate twice of 0.9% after centrifugal, finally merge mother liquor.Extraction stoste used in the present invention is not limited to above-mentioned stoste, also can use the stoste that other modes obtain.Prepare 1L leech and extract stoste.
(example 1)
1L leech is extracted stoste filter through ceramic filtration membrane, ceramic filtration membrane specification is 100KDa molecular weight cut-off, cleans with the distilled water of ceramic filtration membrane filtered liquid 30% volume, obtains filtrate and scavenging solution amounts to 1.03L; Again filtrate and scavenging solution are passed through 30KDa ceramic filtration membrane, clean with the distilled water of filtered liquid 30% volume, obtain filtrate and scavenging solution total 0.87L.Again filtrate and scavenging solution are passed through organic membrane filter, organic membrane specification is 1KDa molecular weight cut-off, cleans with the distilled water of organic membrane filtration liquid 15% volume, obtains filtrate and scavenging solution total 0.75L.By the merging filtrate that obtains by SephadexG-25 gel chromatographic columns wash-out, elutriant is deionized water.HPLC detects elutriant, collect the flow point of r-hirudin chromatographic purity more than 80%, obtain elutriant 0.52L, again elutriant is concentrated by nanofiltration membrane, nanofiltration membrane specification is 500 molecular weight cut-offs, obtain 0.42L concentrated solution, concentrated solution is carried out purifying by anti-phase c18 post, and elutriant is: 0.5M acetonitrile.Collect target peak, and by the elutriant collected as-20 DEG C of lyophilizes, obtain 18.6mg r-hirudin lyophilized products.
(example 2)
1L leech is extracted stoste to add and filter through ceramic filtration membrane, ceramic filtration membrane specification is 150KDa molecular weight cut-off, cleans with the distilled water of ceramic filtration membrane filtered liquid 30% volume, obtains filtrate and scavenging solution amounts to 0.96L; Again filtrate and scavenging solution are passed through 30KDa ceramic filtration membrane, clean with the distilled water of ceramic filtration membrane filtered liquid 30% volume, obtain filtrate and scavenging solution total 0.8L; Again filtrate and scavenging solution are passed through organic membrane filter, organic membrane specification is 1KDa molecular weight cut-off, cleans with the distilled water of organic membrane filtration liquid 15% volume, obtains filtrate and scavenging solution total 0.68L.By the merging filtrate that obtains by SephadexG-25 gel chromatographic columns wash-out, elutriant is deionized water.HPLC detects elutriant, collect the flow point of r-hirudin chromatographic purity more than 80%, obtain elutriant 0.47L, again elutriant is concentrated by nanofiltration membrane, nanofiltration membrane specification is 500 molecular weight cut-offs, obtain 0.38L concentrated solution, concentrated solution is carried out purifying by anti-phase c18 post, and elutriant is: 0.5M acetonitrile.Collect target peak, and by the elutriant collected as-20 DEG C of lyophilizes, obtain 18.1mg r-hirudin lyophilized products.
(example 3)
1L leech is extracted stoste to add and filter through ceramic filtration membrane, ceramic filtration membrane specification is 300KDa molecular weight cut-off, cleans with the distilled water of ceramic filtration membrane filtered liquid 30% volume, obtains filtrate and scavenging solution amounts to 0.97L; Again filtrate and scavenging solution are passed through 30KDa ceramic filtration membrane, clean with the distilled water of ceramic filtration membrane filtered liquid 30% volume, obtain filtrate and scavenging solution total 0.82L; Again filtrate and scavenging solution are passed through organic membrane filter, clean with the distilled water of organic membrane filtration liquid 15% volume, organic membrane specification is 1KDa molecular weight cut-off, obtains filtrate and scavenging solution total 0.7L.By the merging filtrate that obtains by SephadexG-25 gel chromatographic columns wash-out, elutriant is deionized water.HPLC detects elutriant, collect the flow point of r-hirudin chromatographic purity more than 80%, obtain elutriant 0.51L, again elutriant is concentrated by nanofiltration membrane, nanofiltration membrane specification is 500 molecular weight cut-offs, obtain 0.37L concentrated solution, concentrated solution is carried out purifying by anti-phase c18 post, and elutriant is: 0.5M acetonitrile.Collect target peak, and by the elutriant collected as-20 DEG C of lyophilizes, obtain 17.5mg r-hirudin lyophilized products.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; be understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any amendment made, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. a technique for r-hirudin extraction and isolation, is characterized in that comprising the following steps:
Step one: leech extracting solution is filtered: the extracting solution after leech is smashed homogenate, through ceramic filtration membrane filtration at least one times, is collected filtered liquid, then carried out ceramic filtration membrane cleaning with distilled water; Merge ceramic filtration membrane filtered liquid and scavenging solution, then through organic membrane filtration, collect filtered liquid, then carry out organic filter membrane cleaning with distilled water;
Step 2: wash-out: organic membrane filtration liquid step one obtained and scavenging solution by gel chromatographic columns wash-out, then detect elutriant by high pressure liquid chromatograph;
Step 3: purifying: collected by the flow point that r-hirudin chromatographic purity reaches more than 80%, concentrated by the elutriant collected through nanofiltration membrane again, after obtaining nanofiltration concentrated solution, carries out purifying by concentrated solution through anti-phase c18 semipreparative column, collects target peak;
Step 4: carry out lyophilize to the liquid that step 3 obtains, obtains freeze-drying r-hirudin sterling.
2. the technique of a kind of r-hirudin extraction and isolation according to claim 1, is characterized in that: in described step one, and the volume carrying out ceramic filtration membrane cleaning with distilled water is 25 ~ 30% of ceramic filtration membrane filtered liquid; The volume carrying out organic filter membrane cleaning with distilled water is 10 ~ 15% of organic membrane filtration liquid.
3. the technique of a kind of r-hirudin extraction and isolation according to claim 2, is characterized in that: the working temperature of described step one is 25 DEG C ~ 35 DEG C, and pressure is 0.05Mpa ~ 0.3Mpa.
4. the technique of a kind of r-hirudin extraction and isolation according to claim 3, is characterized in that: the ceramic filtration membrane specification in described step one is 30KDa ~ 300KDa molecular weight cut-off; And the molecular weight cut-off of rear nonrecoverable ceramic filtration membrane is less than the molecular weight cut-off of a front ceramic filtration membrane.
5. the technique of a kind of r-hirudin extraction and isolation according to claim 4, is characterized in that: the organic filter membrane specification in described step one is 0.8 ~ 1KDa molecular weight cut-off.
6. according to the technique of a kind of r-hirudin extraction and isolation one of claim 1 to 5 Suo Shu, it is characterized in that: in described step one, before first time and second time distilled water clean, add the rinse that the distilled water of working as time cleaning distilled water 5% volume carries out container and film.
7. the technique of a kind of r-hirudin extraction and isolation according to claim 6, is characterized in that: the gel chromatographic columns in described step 2 is SephadexG-25.
8. the technique of a kind of r-hirudin extraction and isolation according to claim 7, is characterized in that: the nanofiltration membrane in described step 3 is 0.5 ~ 0.8KDa; Anti-phase c18 semipreparative column is Zorbax, 300SB-C18; The elutriant of anti-phase c18 semipreparative column is: 0.1 ~ 1M acetonitrile.
9. the technique of a kind of r-hirudin extraction and isolation according to claim 8, is characterized in that: the freeze temperature in described step 4 is :-30 DEG C ~-10 DEG C.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106928347A (en) * | 2017-03-13 | 2017-07-07 | 佛山市爱的生物科技有限公司 | A kind of separation method of hirudin |
| CN107353338A (en) * | 2017-07-27 | 2017-11-17 | 宁波博睿修存生物科技有限公司 | A kind of method for separating pigment molecular in hirudin zymotic fluid |
| CN109957008A (en) * | 2019-04-16 | 2019-07-02 | 广东双骏生物科技有限公司 | The method for extraction and purification of hirudin mutant and its application |
| CN112480243A (en) * | 2021-01-06 | 2021-03-12 | 广西科康科技集团有限公司 | Large-scale hirudin separation and purification production process method and equipment |
| US20210317162A1 (en) * | 2019-04-16 | 2021-10-14 | Sungen Bioscience Co., Ltd. | Method for extraction and purification of hirudin mutant and use thereof |
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| CN104031059A (en) * | 2014-05-21 | 2014-09-10 | 虞龙 | Membrane extraction separation technique of epothilone |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106928347A (en) * | 2017-03-13 | 2017-07-07 | 佛山市爱的生物科技有限公司 | A kind of separation method of hirudin |
| CN107353338A (en) * | 2017-07-27 | 2017-11-17 | 宁波博睿修存生物科技有限公司 | A kind of method for separating pigment molecular in hirudin zymotic fluid |
| CN107353338B (en) * | 2017-07-27 | 2020-11-17 | 宁波博睿修存生物科技有限公司 | Method for separating pigment molecules in hirudin fermentation liquor |
| CN109957008A (en) * | 2019-04-16 | 2019-07-02 | 广东双骏生物科技有限公司 | The method for extraction and purification of hirudin mutant and its application |
| US20210317162A1 (en) * | 2019-04-16 | 2021-10-14 | Sungen Bioscience Co., Ltd. | Method for extraction and purification of hirudin mutant and use thereof |
| CN109957008B (en) * | 2019-04-16 | 2021-12-24 | 广东双骏生物科技有限公司 | Extraction and purification method of hirudin mutant and application thereof |
| CN112480243A (en) * | 2021-01-06 | 2021-03-12 | 广西科康科技集团有限公司 | Large-scale hirudin separation and purification production process method and equipment |
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