CN1273012C - Culture of hiruto and extraction of hirutin - Google Patents
Culture of hiruto and extraction of hirutin Download PDFInfo
- Publication number
- CN1273012C CN1273012C CN 03113566 CN03113566A CN1273012C CN 1273012 C CN1273012 C CN 1273012C CN 03113566 CN03113566 CN 03113566 CN 03113566 A CN03113566 A CN 03113566A CN 1273012 C CN1273012 C CN 1273012C
- Authority
- CN
- China
- Prior art keywords
- hirudin
- hiruto
- hirudinaria manillensis
- vigor
- leech
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000000605 extraction Methods 0.000 title abstract description 5
- 102000007625 Hirudins Human genes 0.000 claims abstract description 68
- 108010007267 Hirudins Proteins 0.000 claims abstract description 68
- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 claims abstract description 68
- 229940006607 hirudin Drugs 0.000 claims abstract description 67
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 19
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- 239000012043 crude product Substances 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 11
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 10
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
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- 239000007853 buffer solution Substances 0.000 claims description 6
- 235000011187 glycerol Nutrition 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 6
- 235000002639 sodium chloride Nutrition 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Substances [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
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- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 3
- 239000004472 Lysine Substances 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 3
- 229960003121 arginine Drugs 0.000 claims description 3
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- 235000010323 ascorbic acid Nutrition 0.000 claims description 3
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- 229960004838 phosphoric acid Drugs 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
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- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 2
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- OTQCKZUSUGYWBD-BRHMIFOHSA-N lepirudin Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)C(C)C)[C@@H](C)O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)[C@@H](C)O)C1=CC=C(O)C=C1 OTQCKZUSUGYWBD-BRHMIFOHSA-N 0.000 description 1
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Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a culture method for hirudinaria manillensis and an extraction method of hirudin. Excellent seed sources are introduced, or strong hirudinaria manillensis are trapped in the field, and artificial taming and feeding of grading and segment and artificial propagation are carried out; after the hirudinaria manillensis are fed to a certain extent, chemical substance is adopted to stimulate the fresh and live hirudinaria manillensis to secrete saliva out; afterwards, the saliva is further separated and filtered to obtain coarse products of hirudin, and then, deep-machining is carried out to the coarse products of hirudin to obtain the hirudin according with medical use. The content of anti-coagulative substance of the hirudinaria manillensis is higher than that of other breeds of hirudinaria manillensis, the effect of medical use is good, and the method for extracting the hirudin is simple; after the hirudin is extracted, the hirudinaria manillensis is put back to a water pool to be cultivated, and can be extracted again after one week, or ten days; each fresh hirudinaria manillensis can be repeatedly extracted for 6 to 7 times, and about 6 to 7 ml of the coarse products of hirudin can be extracted for each time; thus, the present invention has the advantages of good quality of the products, resource saving and improved utilization value of the hirudinaria manillensis.
Description
Technical field
The present invention relates to the extracting method of Hementaria officianalis hirudin, particularly from the hiruto live body, extract the method for natural hirudin.
Background technology
Hirudin is a kind of anticoagulative substance that extracts from hirudinaria manillensis (leech).Leech, head is stated from China's Shennong's Herbal, and the back is all recorded in Compendium of Material Medica and monographs such as " 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 a protein, name and be hirudin, the genetic recombination hirudin comes out after 1986, for bright prospects have been opened up in the further investigation and the clinical practice of hirudin.According to the document record, the low molecular polypeptide that natural hirudin is made up of 65 amino acid, its effect has the following aspects: 1, have blood coagulation resisting function.The hirudin of separate sources and structure, its anticlotting mechanism is not quite similar.Therefore natural hirudin can influence blood coagulation and thrombosis from a plurality of links.2, antithrombase effect.The antithrombase effect of hirudin sees European Hementaria officianalis (hiNmedic5nalis) L1j, Asia buffalo leech (hiruto) and India leech etc.Contain 6 similar cysteine residues that distribute by the hirudin that extracts in these leech, have similar three-dimensional structure: its N end forms tight structure by disulfide bond, and combines with the thrombin activity site; Its C end is rich in acidic amino acid residue, then combines with the fibrinogen binding site of fibrin ferment; The amino acid residue coordinative role of mesozone.This structure can reach the ratio that hirudin and fibrin ferment be 1: 1 and form stabilized complex, thereby fibrin ferment is had the inhibitory action of high special, plays the blood coagulation resisting function of Trombin inhibiting.3, antiplatelet effects.1992, Como etc. extracted a kind of hiruto antiplatelet protein that is called from the Mexico leech, had specificity and suppressed the effect that collagen-induced platelet is assembled, and other derivants (as blood, fibrin ferment etc.) induced platelet is assembled the unrestraint effect.1994, Kmel etc. extract a kind of polypeptide that contains 39 amino acid residues from the leech of North America, its three-dimensional conformation is very similar to hirudin, and the RGD sequence of C end is discerned mutually with platelet membrane GPIIb/IIIa, thereby has suppressed the platelet aggregation effect of fibrinogen mediation.4, fibrin degradation (former) effect.1984, Malin-conlco etc. extracted a kind of material that is called hementin from the huge leech of Amazon.This material can fibrin degradation (former) the particular rib chain, have antithrombin activity and can significantly suppress the platelet aggregation that ADP induces, for the tangible effect that has of prevention thrombotic diseases.
The clinical practice of hirudin, at present, because the appearance of lepirudin 023 ludon, clinically be used for the treatment of unstable angina (USA), acute myocardial infarction AMI (AMI), angioplasty, postoperative thrombosis, haemodialysis, extracorporal circulatory system, disseminated intravascular coagulation (DIC) more, and be used for antitumor etc.
But more stable under the hirudin drying regime. under the room temperature in water stable existence 6 months, 80 ℃ down heating be not destroyed in 15 minutes.The pH value then stability decreases that raises can be stablized 15min in 0.1moL HCl or 0.1mol NaOH.Health protease sodium and protease do not destroy the activity of hirudin, and papain pepsin and subtilopeptidase A then can make it to lose activity.
Aspect hirudiniculture, at present in order to obtain hirudin, leech is all being cultured in various places, the species of leech have multiple, as eurysome golden thread leech, Hirudo medicinalis, Hirudo japonica, hiruto, the huge leech of Amazon etc., the species that China cultures mostly are eurysome golden thread leech greatly, mainly be to raise easily because the eurysome golden thread leech bodily form is big, recognize have many places to organize the hirudiniculture field in China at present, as Beijing through Indexing of Scien. and Tech. Literature, Wujiang, Jiangsu, the Zhengzhou City, Cao County, Shandong etc., but the species that they culture mostly are eurysome golden thread leech greatly, raising eurysome golden thread leech is based on the snail alive of throwing something and feeding, and is aided with earthworm, the larva of insect etc.
Hiruto (golden-rimmed leech) is especially area and the distinctive leech species of some countries in Southeast Asia such as Guangxi, Guangdong, Hainan of China south China, best in quality, it is comparatively extensive to distribute in Guangxi, it is much higher that the contained hirudin anticoagulant blood of these species material particularly contains anticoagulative substance content than the eurysome golden thread leech of present bibliographical information than other kind leech, medicinal effects is good, and eurysome golden thread leech is hirudin not.Because hiruto is not easy growth in the area of cold weather, raising also is not easy very much, and not seeing at present has the document record of culturing hiruto.
Aspect the extraction of hirudin, leech have many methods at present, and domestic hirudin extracts, and main the employing extracted with chemical agent its rubbing or dry back hirudiniculture after to a certain degree with it, this method waste resource, and extraction process complexity.
" aquatile journal " the 2nd phase in 1997 report, the Markwardt of Germany took up a job as a doctor first and isolated purer hirudin in the leech nineteen fifty-five, and after this purifying of hirudin and standardized technique are gradually improved.Existing many so far relevant research report and patents, most work are to utilize the head of Hirudo medicinalis even whole health to do raw material, through ethanol dehydration, add series of steps such as acetone precipitation then and obtain the hirudin semifinished product.Markwardt adopted steps such as ethanol precipitation, cation exchange, gel filtration and anion exchange to obtain the pure product of hirudin in 1970.WdsmaM in 1985 and Marhyardt have improved purification process, adopt ion exchange to combine with affinity chromatography and have obtained highly purified hirudin.People such as Johmnes had reported a kind of five step method of purification in 1986, had wherein used the high performance liquid chromatography new technology.
Chinese patent also discloses the extracting method of some hirudins, Chinese patent＜application number〉92107076＜denomination of invention〉extracting method of medical natural hirudiu, be after just leech is cleaned impurity such as removing earth, integral body is smashed into muddy to pieces, the leech slurry is heated to 60-80 ℃ again, and kept 2-3 minute, the glacial acetic acid of adding 10% is adjusted pH value at 4.4-4.6 in the leech slurry after heating, be cooled to 25-30 ℃ immediately, add 10%Na again
2CO
3Adjust pH value at 6-7, the leech slurry is through centrifugal collection mother liquor, the physiological saline lixiviate successively of residue adding 0.9% 2-3 time, collect whole leaching liquors, then mother liquor dialysis 36 hours, changed water once every 12 hours, dislysate is with 95% above precipitation with alcohol, and adding 95% ethanol earlier, to make its content be 61%, placed 12 hours, supernatant inclines, it is 85% that the ethanol that adds same concentration again makes its content, places 24 hours, obtains the hirudin precipitation, (7) will precipitate low temperature drying and get semifinished product, (8) semifinished product dissolves with an amount of redistilled water.
＜application number〉95105650＜denomination of invention〉animal watery blood leech Hydrolyze method and Hirudohydrolysis blood thereof, it is the new method of utilizing about animal blood, make blood-eating hirudo fully draw animal blood, with physics or chemical method blood is flowed out in the leech body after several minutes to a few hours, collect the orange red blood body that flows out, from blood, extract hirudin again.The Hirudohydrolysis blood that this method obtains have contain in the animal blood of small molecular weight protein or peptide, also contain the plasmase (former), hyaluronidase of hirudin, digestion blood and other and be difficult to the useful leech secretion measured at present.Can effectively degrade animal blood and make it to contain the leech secretion such as hirudin, hyaluronidase of medical value of this law, leech also can repeatedly be used, and improves its availability.
Summary of the invention
The inventor is through in a few years research and exploration, recognize that the contained hirudin anticoagulant blood material of hiruto (golden-rimmed leech) particularly contains anticoagulative substance content height than the eurysome golden thread leech of present bibliographical information than other species leech, after the good advantage of medicinal effects, after the experience test of many times, finally find out the method for from hiruto, extracting hirudin, obtained favorable economic benefit.
Technical scheme of the present invention is as follows:
Capture healthy and strong hiruto, be placed on and carry out domestication, nursing in water body clean water pond or the pond by introduction fine provenance or field, and keep suitable temperature, carry out artificial propagation, wait to raise to a certain degree, can extract hirudin.
The method of extraction hirudin adopts the whole making beating of leech mostly or dries and use solvent extraction then at present, though having introduced, document make blood-eating hirudo fully draw animal blood, with physics or chemical method blood is flowed out in the leech body after several minutes to a few hours, collect the orange red blood body that flows out, from blood, extract the water quality element again, this method is still complicated, and cost is also higher.
The method that the present invention extracts hirudin comprises that the hiruto raw material is selected and cleaning, adopts chemical substance to stimulate fresh and alive hiruto to make it secrete saliva, then its saliva is filtered, and the hirudin crude product is carried out deep processing again and obtains meeting medical hirudin.
Above-described hiruto raw material is selected and cleaned is that the hiruto of pulling maturation, health in raising the pond out is put into the pond that fills clear water, changes water every 2 days and cleans once, and feeding did not so keep 10-30 days, used distilled water immersion 10-30 minute at last.
The method of above-described hiruto fed to appetite inducing substance and extruding is the hiruto that will clean up with the abundant fed to appetite hiruto of inducing substance, then hiruto being pushed makes it secrete saliva (hirudin crude product), the inducing substance that is adopted comprises mineral salt, the inorganic acid that organic acid and concentration are lower, select acetate for use, hydrochloric acid, phosphoric acid, citric acid, arginine, lysine, ascorbic acid, cystine, sodium chloride, potassium chloride, sodium carbonate, one or both of sodium phosphate or potassium dihydrogen phosphate, two or more mixtures, its concentration expressed in percentage by weight is 0.1-3%, and the PH of the aqueous solution is between 4.5-6.8.Concrete grammar is to configure pure mixed aqueous solution earlier, allows hiruto fully have enough, and then it is pushed, and makes it secrete saliva, and every fresh and alive hiruto can extract 6-7 time repeatedly, can extract about hirudin crude product 6-7 milliliter at every turn.
The hirudin crude product is carried out deep processing comprise separation and purifying and anion exchange chromatography, gel filtration chromatography;
Separate and the process of purifying is: add 4 times of acetone of moisture 15% of putting precooling in the refrigerator the salivary gland secretion extract that will in above-mentioned hiruto body, extract, stir rearmounted refrigerator overnight, sucking-off supernatant acetone is also centrifugal, the trichloroacetic acid that adds precooling in precipitation makes its dissolving, the centrifugal residue of removing treats that sample carries out column chromatography.
The process of anion exchange chromatography is: get an amount of DEAE-C adding distil water and make its expansion, respectively soak with NaOH-NaCl mixed liquor, HCl and NaOH-NaCl mixed liquor successively according to a conventional method and be washed with distilled water to neutrality after 30 minutes.The dress post is to desired height, with citric acid one sodium citrate buffer solution balance, sample carries out gradient elution and draws elution curve with recorder on salivary gland secretion concentrate or the above-mentioned trichloroacetic acid solution, the Fractional Collections eluent, after testing, to there be vigor partly to concentrate in the bag filter and concentrate, survey total protein content with two cheek methods that contract again, continue following column chromatography with glycerine.
The process of gel filtration chromatography is: get an amount of adding distil water and soak 2-4h, use NaoH-NaCl mixed liquid dipping 0.5h according to a conventional method, suction filtration is removed alkali lye and is washed with distilled water to neutrality, and heating is boiled and removed bubble.The dress post is to desired height, with TRIS-HCl-NaCl buffer solution balance, draw elution curve, the Fractional Collections eluent with sample on the above-mentioned concentrate that the vigor eluent arranged, wash-out and with recorder, detect vigor respectively, will have vigor partly to concentrate in the bag filter and concentrate with glycerine.Behind the survey vigor, stand-by spectrophotometer is in the interscan of 200nm to 400nm wave-length coverage again.
Compared with prior art, outstanding substantive distinguishing features of the present invention and obvious improvement are:
1, hiruto (golden-rimmed leech) contains anticoagulative substance and particularly contains anticoagulative substance content height than the eurysome golden thread leech of present bibliographical information than other kind leech, and also the medicinal effects than Hirudo japonica is good.
2, the very important point is that the present invention adopts the distinctive hiruto live body in Guangxi to extract the method for hirudin, every fresh and alive hiruto can be extracted 6-7 time repeatedly, can extract about the 6-7 milliliter, the hirudin or the drying that obtain liquid through measures such as centrifugation, ion exchanges obtain the solid hirudin at every turn.Can extract weekly 1 time, the so not only simple operation easily of method, good product quality economizes on resources again, improves the hiruto value.Hiruto is cultured and can be extracted in 1 year, and the life cycle of hiruto is about 5 years.
Embodiment
Embodiment 1, taking-up maturation, healthy hiruto are put into the pond that fills clear water in raising the pond, changed water cleaned once every 2 days, feeding not, so kept 10-30 days, use distilled water immersion 10-30 minute at last, with the hiruto that cleans up the aqueous solution with acetate, citric acid, sodium chloride, sodium carbonate mixture, its concentration is 0.1%, PH is 4.5, be dissolved in behind the distilled water fully fed to appetite hiruto, hiruto pushed make it secrete saliva (hirudin crude product) then, and the concentration that adds acid makes the PH of the aqueous solution between 5-6.5.Concrete grammar is to configure pure acid solution earlier, allows hiruto fully have enough, and then it is pushed, make it secrete saliva and obtain the hirudin crude product, then to its saliva through centrifugal removal of impurities, separate again and purifying.
Separate and the preparation of purified salivary glandular secretion thing is to add 4 times of acetone of moisture 15% of putting precooling in the refrigerator the salivary gland secretion extract that 250-300ml is extracted in above-mentioned hiruto body, stir rearmounted refrigerator overnight, sucking-off supernatant acetone is also centrifugal, the trichloroacetic acid (about 25ml) that adds precooling in precipitation makes its dissolving, the centrifugal residue of removing treats that sample carries out column chromatography.
Anion exchange chromatography is got an amount of DEAE adding distil water and is made its expansion, respectively soaks with 0.5MOL/L NaOH-NaCl mixed liquor, 0.5mol/L HCl and 0.5mol/L NaOH-NaCl mixed liquor successively according to a conventional method to be washed with distilled water to neutrality after 30 minutes.The dress post (2cm * 60cm) to desired height, 0.1mol/L citric acid-sodium citrate buffer solution balance with pH4.6, sample carries out gradient elution and draws elution curve with recorder, the Fractional Collections eluent on the salivary gland secretion concentrate of 5ml or the above-mentioned trichloroacetic acid solution.After testing, will there be vigor partly to concentrate in the bag filter and concentrate, survey total protein content with two cheek methods that contract again, continue following column chromatography with glycerine.
Anion exchange chromatography is got an amount of DEAE-C adding distil water and is made its expansion, respectively soaks with 0.5MOL/LNaOH-NaCl mixed liquor, 0.5mol/L HCl and 0.5mol/L NaOH-NaCl mixed liquor successively according to a conventional method to be washed with distilled water to neutrality after 30 minutes.The dress post (2cm * 60cm) to desired height, 0.1mol/L citric acid-sodium citrate buffer solution balance with pH4.6, sample carries out gradient elution and draws elution curve with recorder, the Fractional Collections eluent on the salivary gland secretion concentrate of 5ml or the above-mentioned trichloroacetic acid solution.After testing, will there be vigor partly to concentrate in the bag filter and concentrate, survey total protein content again, continue following column chromatography with glycerine.
Embodiment 2, take out maturation in the pond from raising, healthy hiruto is put in the pond that fills clear water, changed water cleaned once every 2 days, feeding not, so kept 10-30 days, use distilled water immersion 10-30 minute at last, with the hiruto arginine that cleans up, cystine, the aqueous solution of sodium phosphate mixture, its concentration is 0.5%, PH is between 4.5-6.8, be dissolved in behind the distilled water fully fed to appetite hiruto, then to hiruto push make its secrete saliva (hirudin crude product) then to its saliva through centrifugal removal of impurities, separate and purifying by the method for embodiment 1 again.
Embodiment 3, taking-up maturation, healthy hiruto are put into the pond that fills clear water in raising the pond, changed water cleaned once every 2 days, feeding not, so kept 10-30 days, use distilled water immersion 10-30 minute at last, with the hiruto that cleans up the aqueous solution with hydrochloric acid, sodium carbonate, potassium dihydrogen phosphate mixture, its concentration is 3%, PH is 6, be dissolved in behind the distilled water fully fed to appetite hiruto, hiruto pushed make it secrete saliva (hirudin crude product) then, these hirudin crude products are purified according to a conventional method again.
Embodiment 4, take out maturation in the pond from raising, healthy hiruto is put in the pond that fills clear water, changed water cleaned once every 2 days, feeding not, so kept 10-30 days, use distilled water immersion 10-30 minute at last, with the hiruto ascorbic acid that cleans up, cystine, the aqueous solution of sodium chloride mixture, its concentration is 0.5%, PH is dissolved in behind the distilled water fully fed to appetite hiruto between 5.5, hiruto is pushed to make it secrete saliva (hirudin crude product) then, then to its saliva through centrifugal removal of impurities, separate and purifying by the method for embodiment 1 again.
Embodiment 5, take out maturation in the pond from raising, healthy hiruto is put in the pond that fills clear water, changed water cleaned once every 2 days, feeding not, so kept 10-30 days, use distilled water immersion 10-30 minute at last, with the hiruto phosphoric acid that cleans up, lysine, sodium carbonate, sodium chloride, the aqueous solution of potassium chloride mixture, its concentration is 1.5%, PH is dissolved in behind the distilled water fully fed to appetite hiruto between 5.0, hiruto is pushed to make it secrete saliva (hirudin crude product) then, then to its saliva through centrifugal removal of impurities, separate and purifying by the method for embodiment 1 again.
Claims (5)
1, a kind of method of from the hiruto live body, extracting natural hirudin, it is characterized in that: hiruto is raised to a certain degree, earlier hiruto is selected and cleaning, adopt inducing substance to stimulate hiruto to salivate, then these salivas are obtained natural hirudin through purifies and separates, again the hirudin crude product is carried out deep processing and obtain meeting medical hirudin;
The fresh and alive hiruto of described stimulation makes its chemical substance of secreting saliva be: acetate, hydrochloric acid, phosphoric acid, citric acid, arginine, lysine, ascorbic acid, cystine, sodium chloride, potassium chloride, sodium carbonate, sodium phosphate or potassium dihydrogen phosphate wherein one or both, two or more mixture, its aqueous solution concentration expressed in percentage by weight is 0.1-3%, and PH is between 4.5-6.8;
Described the hirudin crude product is carried out the process that deep processing comprises separation, purifying, anion exchange chromatography and gel filtration chromatography.
2, the method for from the hiruto live body, extracting natural hirudin according to claim 1, it is characterized in that: non-ox leech is selected and cleans is to take out healthy hiruto to be put into the pond that fills clear water in raising the pond, changed water cleaned once every 2 days, feeding not, so kept 10-30 days, use distilled water immersion 10-30 minute at last.
3, the method for from the hiruto live body, extracting natural hirudin according to claim 1, it is characterized in that: the process of purifies and separates is: add 4 times of acetone of moisture 15% of putting precooling in the refrigerator the salivary gland secretion extract that will extract in above-mentioned hiruto body, stir rearmounted refrigerator overnight, sucking-off supernatant acetone is also centrifugal, the trichloroacetic acid that adds precooling in precipitation makes its dissolving, the centrifugal residue of removing treats that sample carries out column chromatography.
4, the method of from the hiruto live body, extracting natural hirudin according to claim 1, it is characterized in that: the process of anion exchange chromatography is: get an amount of DEAE adding distil water and make its expansion, use the NaOH-NaCl mixed liquor according to a conventional method successively, HCl and NaoH-NaCl mixed liquor respectively soak after 30 minutes and are washed with distilled water to neutrality, the dress post is to desired height, with citric acid-sodium citrate buffer solution balance, sample carries out gradient elution and draws elution curve with recorder on salivary gland secretion concentrate or the above-mentioned trichloroacetic acid solution, the Fractional Collections eluent, after testing, to have vigor partly to concentrate in the bag filter concentrates with glycerine, survey total protein content again, continue following column chromatography.
5, the method of from the hiruto live body, extracting natural hirudin according to claim 1, it is characterized in that: the process of gel filtration chromatography is: get an amount of adding distil water and soak 2-4h, use NaOH-NaCl mixed liquid dipping 0.5h according to a conventional method, suction filtration is removed alkali lye and is washed with distilled water to neutrality, heating is boiled and is removed bubble, the dress post is to desired height, with TRIS-HCl-NaCl buffer solution balance, with sample on the above-mentioned concentrate that the vigor eluent arranged, wash-out is also drawn elution curve with recorder, the Fractional Collections eluent, detect vigor respectively, to have vigor partly to concentrate in the bag filter concentrates with glycerine, behind the survey vigor, use spectrophotometer again in the wave-length coverage interscan.
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| CN102146136A (en) * | 2010-12-24 | 2011-08-10 | 南宁乙翔生物科技有限公司 | Method for extracting hirudin by stimulating living leech with electricity |
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2003
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| CN102146136A (en) * | 2010-12-24 | 2011-08-10 | 南宁乙翔生物科技有限公司 | Method for extracting hirudin by stimulating living leech with electricity |
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