CN101974085B - Method for extracting high-anticoagulation-activity hirudin from natural leeches - Google Patents
Method for extracting high-anticoagulation-activity hirudin from natural leeches Download PDFInfo
- Publication number
- CN101974085B CN101974085B CN 201010526681 CN201010526681A CN101974085B CN 101974085 B CN101974085 B CN 101974085B CN 201010526681 CN201010526681 CN 201010526681 CN 201010526681 A CN201010526681 A CN 201010526681A CN 101974085 B CN101974085 B CN 101974085B
- Authority
- CN
- China
- Prior art keywords
- hirudin
- leech
- stimulation
- crude product
- natural
- 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 - Fee Related
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a method for extracting high-anticoagulation-activity hirudin from natural leeches, which comprises the steps that two kinds or a plurality of kinds of substances in sodium chloride, acetyl cysteine hydrochloride monohydrate, lysine, neutral protease and papain are combined and dissolved in deionized water to be prepared into stimulation liquid; the weight percentage concentration of each substance in the stimulation liquid is 0.01 to 5%, wherein the enzyme concentration is 100U to 300U/ml; the living leeches are weighed and placed into a container; the stimulation liquid is added according to the weight and volume ratio of the leeches to the stimulation liquid of 1 to (1-5); the leeches secrete exudate after the stimulation is carried out for 3 hours; the exudateis separated to obtain the hirudin crude product through 16000 to 20000r/min centrifugation; the hirudin crude product is micro-filtered through a stainless steel membrane with the thickness of 0.2 micrometer; the micro-filtration is carried out through an organic membrane with the molecular weight of 10000 to 3000 Dalton; the reverse osmosis, the nano-filtration and the concentration are carriedout; starch for drugs is added according to the weight and volume ratio of the solid to the liquid of 1 to (6-10) to allocate the mixture; and the spray drying is carried out to obtain the leech finished product. Finally, the extraction yield of the hirudin is above 30%, and the activity unit of the product is stabilized between 600AT-U/g to 1000AT-U/g.
Description
Technical field
The present invention relates to a kind of method of from natural leech, extracting the high anticoagulating active r-hirudin.
Background technology
The single chain polypeptide that r-hirudin is made up of 65 amino-acid residues, relative molecular weight are 7000, iso-electric point is 3.9, water soluble, physiological saline and pyridine, are insoluble to ethanol, ether, acetone and benzene.Therefore, the extraction of r-hirudin, separation and purifying can be undertaken by the ultimate principle of its physics and chemical property and general protein purification.According to molecular size, solvability, adsorptivity, institute is electrically charged and feature such as the special avidity size of aglucon, adopt normal saline extraction, ethanol precipitation and dialysis method are extracted crude product, are carried out the separation and purification of elaboration with gel chromatography, ion exchange chromatography, high performance liquid chromatography, isoelectrofocusing and affinity chromatography etc.
Natural hirudin at first is found in the saliva of leech, is secreted out when leech is sucked blood.Viewpoint was thought in the past, and the sialisterium of leech is hirudin, and in fact, r-hirudin spreads all over leech whole body each several part, and just r-hirudin content how much.
Domestic prior art adopt when gathering raw material cuts to fall leech head or artificial external force extruding leech more, and these two kinds of methods all only lay particular emphasis on the salivary gland secretion of leech head, and have ignored other position juices.Mainly adopt normal saline extraction when extracting anticoagulative substance, ethanol precipitation and dialysis method are extracted refining.Generally below 20%, anticoagulating active maintains about 100AT-U/g the yield of products obtained therefrom, and the activity index instability, also can't form to produce in batches to extract, can only be for Clinical Application and laboratory provide some datas on a small quantity.The backwardness of extractive technique art has seriously restricted the widespread use of r-hirudin on field of medicaments.
Summary of the invention
The object of the present invention is to provide a kind of method of from natural leech, extracting the high anticoagulating active r-hirudin.This method does not need the manual compression leech, has both simplified the collection operation, but the repeated multiple times effect of gathering live body leech juice again.Final r-hirudin yield is more than 30%, and activity unit is at 600AT-U/g to 1000AT-U/g.
The present invention is achieved through the following technical solutions above-mentioned purpose: a kind of method of extracting the anticoagulating active r-hirudin from natural leech, this method comprises the steps:
1.1 two or more materials in sodium-chlor, a water acetyl-cysteine salt hydrochlorate, Methionin, neutral protease, the papoid are made up, be dissolved in and be mixed with stimulation fluid in the deionized water, the concentration expressed in percentage by weight of each material is 0.01～5% in the stimulation fluid, and wherein enzyme concn is at 100U～300U/ml.The live body leech weighed to be placed in the container, is 1: 1～5 times by leech weight ratio stimulation fluid volume and adds described stimulation fluids, stimulates to make leech secrete juice in 3 hours.Juice obtains the r-hirudin crude product through 16000～20000r/min centrifugation.
1.2 the r-hirudin crude product is through 0.2 micron stainless steel membrane micro-filtration, again through the organic membrane ultrafiltration of 10000～3000 Dalton molecular weights, carry out the reverse osmosis nanofiltration again and concentrate, be 1: 6～10 to add the medical starches allotment by solid than the weightmeasurement ratio of liquid then, spray-dried, get the r-hirudin finished product.
Outstanding advantage of the present invention is:
1, adopt described stimulation fluid, can stimulate the leech whole body to secrete a large amount of juices, and the live body leech can repeatedly stimulate the product juice, recycle.This method is than traditional external force extruding leech or directly cut to fall leech head method, raw material availability height.
2, adopt the ultrafiltration post of 100,000 Dalton molecular weights to carry out the ultrafiltration process extraction, make the hirudin anticoagulant activity unit that obtains at 600AT-U/g to 1000AT-U/g.
Embodiment
Embodiment 1
Neutral protease, the 150U enzyme papoid of measuring alive with 5g sodium-chlor, 0.3g one water acetyl-cysteine salt hydrochlorate, 5g Methionin, 100U enzyme are lived and measured fully is dissolved in the 1000mL deionized water, is mixed with stimulation fluid.The live body leech weighed to be placed in the container, adds described stimulation fluids by 1.25 times of leech weight, and placing stimulated leech to secrete juice in 1 hour.Pulling the live body leech out continuation after stimulation finishes raises in order to reuse.Juice concentrates through organic membrane ultrafiltration, the reverse osmosis nanofiltration of 20000r/min centrifugation, 10000 Dalton molecular weights successively, be 1: 6～10 to add the medical starches allotment by solid than the weightmeasurement ratio of liquid at last, through spraying drying, get the r-hirudin finished product.
Embodiment 2
Papoid with 15g sodium-chlor, 0.2g one water acetyl-cysteine salt hydrochlorate, 300U enzyme are lived and measured fully is dissolved in the 1000mL deionized water, is mixed with stimulation fluid.The live body leech weighed to be placed in the container, adds described stimulation fluids by 1.25 times of leech weight, and placing stimulated leech to secrete juice in 1 hour.Pulling the live body leech out continuation after stimulation finishes raises in order to reuse.Juice successively through the organic membrane ultrafiltration of 18000r/min centrifugation, 7000 Dalton molecular weights, carry out the reverse osmosis nanofiltration and concentrate, be 1: 6～10 to add the medical starches allotment by solid than the weightmeasurement ratio of liquid at last, through spraying drying, get the r-hirudin finished product.
Embodiment 3
Neutral protease with 25g sodium-chlor, 0.1g one water acetyl-cysteine salt hydrochlorate, 0.5g Methionin, 250U enzyme are lived and measured fully is dissolved in the 1000mL deionized water, is mixed with stimulation fluid.The live body leech weighed to be placed in the container, adds described stimulation fluids by 1.25 times of leech weight, and placing stimulated leech to secrete juice in 1 hour.Pulling the live body leech out continuation after stimulation finishes raises in order to reuse.Juice concentrates through organic membrane ultrafiltration, the reverse osmosis nanofiltration of 17000r/min centrifugation, 4000 Dalton molecular weights successively, be 1: 6～10 to add the medical starches allotment by solid than the weightmeasurement ratio of liquid at last, through spraying drying, get the r-hirudin finished product.
Claims (4)
1. a method of extracting the high anticoagulating active r-hirudin from natural leech is characterized in that this method comprises the steps:
1.1 with 5g sodium-chlor, a 0.3g water acetyl-cysteine salt hydrochlorate, 5g Methionin, the neutral protease that the 100U enzyme is lived and measured, the papoid that the 150U enzyme is lived and measured, fully be dissolved in the 1000mL deionized water, be mixed with stimulation fluid, the concentration expressed in percentage by weight of each material is 0.01～5% in the stimulation fluid, wherein enzyme concn is at 100U～250U/1000ml, the live body leech weighed be placed in the container, be that 1:1～5 times add described stimulation fluids by leech weight ratio stimulation fluid volume, stimulate and made leech secretion juice in 3 hours, juice is through 16000～20000r/min centrifugation, obtain the r-hirudin crude product
1.2 the r-hirudin crude product is through 0.2 micron stainless steel membrane micro-filtration, again through the organic membrane ultrafiltration of 10000～3000 Dalton molecular weights, carry out the reverse osmosis nanofiltration again and concentrate, be that 1:6～10 add the medical starches allotment by solid than the weightmeasurement ratio of liquid then, spray-dried, get the r-hirudin finished product.
2. a method of extracting the high anticoagulating active r-hirudin from natural leech is characterized in that this method comprises the steps:
1.1 with 15g sodium-chlor, a 0.2g water acetyl-cysteine salt hydrochlorate, the papoid that the 300U enzyme is lived and measured, fully be dissolved in the 1000mL deionized water, be mixed with stimulation fluid, the concentration expressed in percentage by weight of each material is 0.01～5% in the stimulation fluid, wherein enzyme concn is at 300U/1000ml, the live body leech weighed be placed in the container, be that 1:1～5 times add described stimulation fluids by leech weight ratio stimulation fluid volume, stimulate and made leech secretion juice in 3 hours, juice is through 16000～20000r/min centrifugation, obtain the r-hirudin crude product
1.2 the r-hirudin crude product is through 0.2 micron stainless steel membrane micro-filtration, again through the organic membrane ultrafiltration of 10000～3000 Dalton molecular weights, carry out the reverse osmosis nanofiltration again and concentrate, be that 1:6～10 add the medical starches allotment by solid than the weightmeasurement ratio of liquid then, spray-dried, get the r-hirudin finished product.
3. a method of extracting the high anticoagulating active r-hirudin from natural leech is characterized in that this method comprises the steps:
1.1 with 25g sodium-chlor, a 0.1g water acetyl-cysteine salt hydrochlorate, 0.5g Methionin, the neutral protease that the 250U enzyme is lived and measured, fully be dissolved in the 1000mL deionized water, be mixed with stimulation fluid, the concentration expressed in percentage by weight of each material is 0.01～5% in the stimulation fluid, wherein enzyme concn is at 250U/1000ml, the live body leech weighed be placed in the container, be that 1:1～5 times add described stimulation fluids by leech weight ratio stimulation fluid volume, stimulate and made leech secretion juice in 3 hours, juice is through 16000～20000r/min centrifugation, obtain the r-hirudin crude product
1.2 the r-hirudin crude product is through 0.2 micron stainless steel membrane micro-filtration, again through the organic membrane ultrafiltration of 10000～3000 Dalton molecular weights, carry out the reverse osmosis nanofiltration again and concentrate, be that 1:6～10 add the medical starches allotment by solid than the weightmeasurement ratio of liquid then, spray-dried, get the r-hirudin finished product.
4. the method for extracting the high anticoagulating active r-hirudin from natural leech according to claim 1 is characterized in that described natural leech is the natural leech of live body.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010526681 CN101974085B (en) | 2010-11-01 | 2010-11-01 | Method for extracting high-anticoagulation-activity hirudin from natural leeches |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010526681 CN101974085B (en) | 2010-11-01 | 2010-11-01 | Method for extracting high-anticoagulation-activity hirudin from natural leeches |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101974085A CN101974085A (en) | 2011-02-16 |
| CN101974085B true CN101974085B (en) | 2013-09-25 |
Family
ID=43574002
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010526681 Expired - Fee Related CN101974085B (en) | 2010-11-01 | 2010-11-01 | Method for extracting high-anticoagulation-activity hirudin from natural leeches |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101974085B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102964446B (en) * | 2012-11-28 | 2014-08-13 | 周维官 | Method for repeatedly extracting natural hirudin from live vampire leech |
| CN104774259A (en) * | 2015-04-21 | 2015-07-15 | 虞龙 | Method for extracting hirudin from leeches based on membrane |
| CN107661357A (en) * | 2017-11-16 | 2018-02-06 | 广西远程水蛭养殖有限公司 | Leech medicinal liquor preparation method |
| CN107875175A (en) * | 2017-11-16 | 2018-04-06 | 广西远程水蛭养殖有限公司 | Leech capsule |
| CN112480243B (en) * | 2021-01-06 | 2023-05-05 | 广西科康科技集团有限公司 | Process and equipment for large-scale hirudin separation and purification production |
| CN113813291B (en) * | 2021-11-10 | 2023-05-26 | 鲁南制药集团股份有限公司 | Preparation method of animal medicinal material freeze-dried powder |
| CN114699432B (en) * | 2022-05-19 | 2024-02-23 | 广西蛭郎中生物科技有限公司 | Method for extracting anti-coagulation crude product of Poecilobdella manillensis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1423929A (en) * | 2003-01-15 | 2003-06-18 | 周维海 | Culture of hiruto and extraction of hirutin |
| CN1883510A (en) * | 2006-06-29 | 2006-12-27 | 周维海 | A method for preparing hirudin by making leech bloodsucker as raw material and application thereof |
| CN101108879A (en) * | 2007-08-03 | 2008-01-23 | 广西博泰恩生物科技有限公司 | Method for extracting active ingredient of natural leech essence |
-
2010
- 2010-11-01 CN CN 201010526681 patent/CN101974085B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1423929A (en) * | 2003-01-15 | 2003-06-18 | 周维海 | Culture of hiruto and extraction of hirutin |
| CN1883510A (en) * | 2006-06-29 | 2006-12-27 | 周维海 | A method for preparing hirudin by making leech bloodsucker as raw material and application thereof |
| CN101108879A (en) * | 2007-08-03 | 2008-01-23 | 广西博泰恩生物科技有限公司 | Method for extracting active ingredient of natural leech essence |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101974085A (en) | 2011-02-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101974085B (en) | Method for extracting high-anticoagulation-activity hirudin from natural leeches | |
| CN109400678A (en) | A kind of anti-oxidant and DPP-IV inhibitory activity peptide in stichopus japonicus source | |
| CN104042548B (en) | A kind of snail mucus extract and preparation method thereof and the application in cosmetics | |
| CN105339007A (en) | Treatment of peripheral vascular disease using protein solutions | |
| CN106434803B (en) | A kind of Goat Placenta antioxidation polypeptide and its enzymolysis preparation and application | |
| CN102925522A (en) | Method for preparing pilose antler peptide dry powder by enzymolysis method | |
| CN104263788A (en) | Preparation method of sea cucumber polypeptide | |
| CN101294186A (en) | Method for preparing donkey-hide gelatin oligopeptide | |
| CN102453106A (en) | Preparation method of bletilla striata polysaccharide | |
| CN101020715B (en) | Process of extracting and preparing deer nerve growth factor (DEER NGF) | |
| CN104761635A (en) | Hirudin extraction separation technique | |
| CN101991038B (en) | Giant salamander oligosaccharide peptide health food | |
| CN104974239A (en) | Method for separating and purifying cobra venom neurotoxin with combination of ion exchange and reverse phase chromatography | |
| CN105950693B (en) | The extracting method of Cow placeta biologically active polypeptide | |
| CN101054414B (en) | Method of extracting and preparing deer DGF | |
| KR100328877B1 (en) | A composition for external application containing β-1, 6-branched-β-1, 3-glucan having anti-inflammatory and anti-irritant effect | |
| CN103804506A (en) | Method for extracting heparin and dermatan sulfate from small intestine lixivium | |
| CA2455655A1 (en) | A glycoprotein with antidiabetic, antihypertensive, antiobesity and antihyperlipidemic effects from grifola frondosa, and a method for preparing same | |
| CN103468765A (en) | Biological extracting and purifying method for beer yeast soluble 1,3-beta-D-glucan | |
| CN101081865B (en) | Abstraction of pilose antler releasing somatomedin (DEER GHRF) and preparation method thereof | |
| CN1062741C (en) | Animal blood hirudo hydrolytic method and hirudo hydrolytic blood | |
| JP6198867B2 (en) | Wound treatment | |
| CN105646658B (en) | Ray porosa chondroprotein antioxidant peptide and application thereof | |
| CN101684151B (en) | Protein matter with coagulation activity | |
| CN101711791B (en) | Method for preparing cat's-foot water extract and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20180710 Address after: 530007, 3288, room 3, Kechuang building, 25-1 Keyuan Avenue, Nanning, the Guangxi Zhuang Autonomous Region. Patentee after: Nanning and Lanji Food Technology Co., Ltd. Address before: 530007 biological industrial incubator 60, Keyuan Avenue, Nanning, the Guangxi Zhuang Autonomous Region 4, 3 unit 302 Patentee before: Nanning Jinbei Biological Technology Co., Ltd. |
|
| TR01 | Transfer of patent right | ||
| CP01 | Change in the name or title of a patent holder |
Address after: Room 3288, 3rd floor, Kechuang Building, 25-1 Keyuan Avenue, Nanning City, Guangxi Zhuang Autonomous Region Patentee after: Nanning He Lan Ji Biotechnology Co., Ltd. Address before: Room 3288, 3rd floor, Kechuang Building, 25-1 Keyuan Avenue, Nanning City, Guangxi Zhuang Autonomous Region Patentee before: Nanning and Lanji Food Technology Co., Ltd. |
|
| CP01 | Change in the name or title of a patent holder | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130925 Termination date: 20201101 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |