CN1143893C - Prepn of active haparin - Google Patents
Prepn of active haparin Download PDFInfo
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- CN1143893C CN1143893C CNB011443170A CN01144317A CN1143893C CN 1143893 C CN1143893 C CN 1143893C CN B011443170 A CNB011443170 A CN B011443170A CN 01144317 A CN01144317 A CN 01144317A CN 1143893 C CN1143893 C CN 1143893C
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- Prior art keywords
- arginine
- damping fluid
- haparin
- separation
- active
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- 102100038356 Kallikrein-2 Human genes 0.000 claims abstract description 25
- 108010035158 arginine esterase Proteins 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000002435 venom Substances 0.000 claims abstract description 12
- 231100000611 venom Toxicity 0.000 claims abstract description 12
- 210000001048 venom Anatomy 0.000 claims abstract description 12
- 238000000926 separation method Methods 0.000 claims abstract description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 241000271039 Agkistrodon Species 0.000 claims description 10
- 241001474977 Palla Species 0.000 claims description 10
- 238000013016 damping Methods 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 10
- 238000004440 column chromatography Methods 0.000 claims description 8
- 239000012141 concentrate Substances 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 238000011033 desalting Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- 239000007974 sodium acetate buffer Substances 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004475 Arginine Substances 0.000 claims description 3
- 229920002684 Sepharose Polymers 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
- 229920002678 cellulose Polymers 0.000 claims description 3
- 239000001913 cellulose Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000008176 lyophilized powder Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- -1 arginine ester Chemical class 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000003292 glue Substances 0.000 claims description 2
- 229960004249 sodium acetate Drugs 0.000 claims description 2
- 235000017281 sodium acetate Nutrition 0.000 claims description 2
- 239000001632 sodium acetate Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 10
- 229940079593 drug Drugs 0.000 abstract description 5
- 208000006454 hepatitis Diseases 0.000 abstract description 4
- 239000003053 toxin Substances 0.000 abstract description 4
- 231100000765 toxin Toxicity 0.000 abstract description 4
- 241001465754 Metazoa Species 0.000 abstract description 2
- 101710138657 Neurotoxin Proteins 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 239000002581 neurotoxin Substances 0.000 abstract description 2
- 231100000618 neurotoxin Toxicity 0.000 abstract description 2
- 231100000614 poison Toxicity 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 239000003440 toxic substance Substances 0.000 abstract description 2
- 241000271897 Viperidae Species 0.000 abstract 2
- 239000008280 blood Substances 0.000 abstract 1
- 210000004369 blood Anatomy 0.000 abstract 1
- 230000006870 function Effects 0.000 abstract 1
- 230000002008 hemorrhagic effect Effects 0.000 abstract 1
- 231100000283 hepatitis Toxicity 0.000 abstract 1
- 239000004615 ingredient Substances 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 230000003908 liver function Effects 0.000 abstract 1
- 239000002075 main ingredient Substances 0.000 abstract 1
- 230000004089 microcirculation Effects 0.000 abstract 1
- 230000000144 pharmacologic effect Effects 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 238000011084 recovery Methods 0.000 abstract 1
- 231100000419 toxicity Toxicity 0.000 abstract 1
- 230000001988 toxicity Effects 0.000 abstract 1
- 230000009385 viral infection Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 5
- 208000019423 liver disease Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 239000003998 snake venom Substances 0.000 description 4
- 108700012359 toxins Proteins 0.000 description 4
- 206010008909 Chronic Hepatitis Diseases 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 208000019425 cirrhosis of liver Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 108010039551 hemorrhagic proteinase IV Proteins 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 240000007164 Salvia officinalis Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229940019336 antithrombotic enzyme Drugs 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 206010019692 hepatic necrosis Diseases 0.000 description 1
- 230000007866 hepatic necrosis Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000005412 red sage Nutrition 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to a method for extracting active haparin from the venom of pit vipers in Jiangsu province and Zhejiang province. The active haparin uses arginine esterase as a main ingredient, and has the pharmacological functions for reducing blood viscosity, improving the microcirculation, inhibiting the development of hepatitis virus infection cells and promoting the recovery of liver functions. Because the venom of pit vipers in Jiangsu province and Zhejiang province contains neurotoxin, hemorrhagic toxin and other harmful ingredients, the method can eliminate the toxic substances through three-column chromatographic plate separation and purification, and the content of the arginine esterase reaches over 90%. Animal experiments show that the active haparin has little toxicity, and the test dose is tens of times more than that used in clinic; therefore, the security of clinical medication can be guaranteed.
Description
Technical field
The present invention relates to a kind of preparation method of biologics, refer to a kind of method of arginine esterase of from the agkistrodon halyx pallas venom of Jiangsu and Zhejiang Provinces, extracting especially, utilize this method can get highly purified arginine esterase, make to be more suitable for being applied in the Remed for hepatopathy as Remed for hepatopathy.
Background technology
Contain arginine esterase in the known agkistrodon halyx pallas venom, once had refining Ahylysantinfarctase to obtain Liaoning Province's pharmaceutical control and administration authentication code in the past, be used for the treatment of the heart, brain, vascular conditions, use the effective of the medicine red sage root always with pharmacologically active.But this medicine is not high in view of the purity of China's snake venom antithrombotic enzyme because of the Ministry of Health, produces disorder, rectifies in 1994, stops original production permission code, and replaces with product innovation " fiber eliminating enzyme " in 1997.Through further discovering, the arginine esterase in the agkistrodon halyx pallas venom is the higher enzyme of specificity, only acts on the arginine ester bond of minority in the peptide chain, to not infringement of tissue, so its side effect is less.Effective in cure to viral hepatitis, liver cirrhosis, hepatic necrosis etc. is a new discovery, and clinical observation result is efficient to be reached more than 80%.Chinese Medical Sciences University senile disease research centre is once used medicine name " soothing the liver special happy " instead with refining Ahylysantinfarctase and is used for the clinical treatment hepatopathy, proof truly has significant curative effect to acute and chronic hepatitis treatment, be specially adapted to the treatment of chronic hepatitis, liver cirrhosis, satisfactory effect, for having clinical commonly used drug now can't be obtained, clinical application is in treatment hepatopathy examples up to a hundred.Treating in the medicine of hepatopathy at present, belong to curative effect newtype drug preferably, has the good clinical application prospect, but the purity of its arginine esterase does not improve, and does not have new pharmaceutical control and administration lot number, and contains more than 20 kind of enzyme and toxalbumin in the snake venom, outstanding neurotoxin and the hemorrhagin toxin that belongs to wherein is the most harmful to human body, think the security that fundamentally guarantees as medicine, must be from these objectionable impuritiess of thorough removal, arginine esterase highly purified be key point of the present invention.
Summary of the invention
Not high for solving with having the resulting arginine esterase purity of method of from agkistrodon halyx pallas venom, extracting arginine esterase now, the technical problem that also contains a large amount of toxic substances, the invention provides a kind of method for preparing liver plain (arginine esterase just) alive of from the agkistrodon halyx pallas venom of Jiangsu and Zhejiang Provinces, extracting, purity can reach more than 90%, thereby guarantees the drug safety degree.
Method of the present invention comprises the steps:
Step a: get Jiangsu and Zhejiang Provinces agkistrodon halyx pallas venom lyophilized powder and be dissolved in 0.05M Tris-HCl, in the pH7.8 damping fluid, 4 ℃ of following centrifugal insolubless of removing are got supernatant liquor;
Step b: supernatant liquor is earlier through DEAE Mierocrystalline cellulose DE-52 column chromatography, and chromatography column with after the Tris-HCl damping fluid balance, carries out gradient elution with the damping fluid that contains NaCl from 0 to 0.5M earlier, collection contains the active component of arginine esterase, concentrate, after desalting, carry out next step separation;
Step c: use the CM-Sepharose column chromatography for separation, balance liquid is a 0.05M sodium-acetate pH5.0 damping fluid, and elutriant contains the sodium-acetate buffer gradient elution of 0 to 0.5M NaCl pH5.0, collects to contain the arginine ester enzyme active component, concentrate, do next step separation after desalting again;
Steps d: doubt the glue filtering separation with the Sephadex-75 column chromatography, elutriant is 0.05M NaHCO
3Contain 0.1M NaCl solution, collect the arginine esterase component, concentrate, desalt, after freezing ten product.
Said desalting is to adopt dialysis method, concentrates the ultrafiltration and concentration method that adopts.
Utilize the resulting product of aforesaid method to show and do not contain toxin and objectionable constituent through animal experiment, purity 90% with (detection of HPLC method).
Contain more than 20 kind of enzyme and toxalbumin in the agkistrodon halyx pallas venom, the difficulty of highly purified wherein a kind of composition very big.The three step chromatographic separation methods that the present invention adopts can be highly purified arginine esterase, removes toxic component, guarantees the security of medication.At first through after tentatively removing insolubles, promptly carry out three step chromatographic separation in the method for the present invention, the isolating purpose of the first step is preliminary purification, and arginine esterase other compositions with snake venom are separated, and is suitable for large-scale processing, each available snake venom raw material 20 grams.It is to use anionite that second step separated, different with the cationite of the first step, once more basic protein is separated, and the purity of arginine esterase is further improved.It is that difference by molecular weight eliminates remaining toxin protein that the 3rd step separated.Because of the molecular weight of agkistrodotoxin is 15000, hemorrhagin toxin is about 50000 to 60000, and the molecular weight of arginine esterase is about 30000, and the difference between molecular weight is big, with gel-filtration (being molecular sieve) method they are separately reached highly purified purpose easily, adopt 0.05M NaHCO
3The solution that contains NaCl can increase the solubleness of arginine esterase as elutriant, thereby obtains maximum yield.Can obtain about 3 milligrams of product in order to last method from agkistrodon halyx pallas venom 1 gram, can adorn about 30 of injection, every arginine esterase is 100 units.
Embodiment
Get Jiangsu and Zhejiang Provinces agkistrodon halyx pallas venom lyophilized powder 20 grams, be dissolved in the 0.05M Tris-HCl of 20ml, the pH7.8 damping fluid, centrifugal 10 minutes in 3000 rev/mins of refrigerated centrifuges, remove insolubles, supernatant liquor is added DEAE Mierocrystalline cellulose DE-52 chromatography column, 3 * 80cm, chromatography column needs elder generation through Tris-HCl damping fluid balance, and elutriant is to carry out gradient elution with the concentration 0 to 0.5M that contains NaCl, collects to contain the active component of arginine esterase (detecting at the 280nm wavelength with ultraviolet spectrophotometer), merge and collect liquid, through with molecular weight cut-off being the ultra-filtration membrane ultrafiltration and concentration more than 10,000, dialysis is desalted, and again through the CM-Sepharose column chromatography for separation, the size of post is 2 * 40cm; Sodium-acetate buffer balance with 0.05M pH5.0, after adding sample, carry out gradient elution with the sodium-acetate buffer that contains 0 to 0.5M NaCl, collection contains the component of arginine esterase, through dialysis, freeze-drying is again through the Sephadex-75 column chromatography for separation, the size of post is 2 * 150cm, and elutriant is 0.05M NaHCO
3, contain the solution of 0.1MNaCl, collect active ingredient, desalt, after the freeze-drying product.Through degerming, the thermal source accreditation, the dress pin be 100 units/.
Claims (2)
1, a kind of preparation method of arginine esterase is characterized in that this method comprises the steps:
Step a: get Jiangsu and Zhejiang Provinces agkistrodon halyx pallas venom lyophilized powder and be dissolved in 0.05Mtris-HCl, in the pH7.8 damping fluid, 4 ℃ of following centrifugal insolubless of removing are got supernatant liquor;
Step b: supernatant liquor is earlier through DEAE Mierocrystalline cellulose DE-52 column chromatography, and chromatography column with after the Tris-HCl damping fluid balance, carries out gradient elution with the damping fluid that contains NaCl from 0 to 0.5M earlier, collection contains the active component of arginine esterase, concentrate, after desalting, carry out next step separation;
Step c: use the CM-Sepharose column chromatography for separation, balance liquid is a 0.05M sodium-acetate pH5.0 damping fluid, and elutriant contains the sodium-acetate buffer gradient elution of 0 to 0.5MNaCl pH5.0, collects to contain the arginine ester enzyme active component, concentrate, do next step separation after desalting again;
Steps d: doubt the glue filtering separation with the Sephadex-75 column chromatography, elutriant is 0.05M NaHCO
3Contain 0.1M NaCl solution, collect the arginine esterase component, concentrate, desalt, get product after the freeze-drying.
2, method according to claim 1 is characterized in that said desalting is to adopt dialysis method, concentrates the ultrafiltration and concentration method that adopts.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011443170A CN1143893C (en) | 2001-12-14 | 2001-12-14 | Prepn of active haparin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011443170A CN1143893C (en) | 2001-12-14 | 2001-12-14 | Prepn of active haparin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1357623A CN1357623A (en) | 2002-07-10 |
| CN1143893C true CN1143893C (en) | 2004-03-31 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011443170A Expired - Fee Related CN1143893C (en) | 2001-12-14 | 2001-12-14 | Prepn of active haparin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1143893C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100390276C (en) * | 2006-04-11 | 2008-05-28 | 修志明 | A method for extracting esterase from animal liver |
| CN102021160B (en) * | 2010-11-11 | 2012-07-18 | 中国农业大学 | Snake venom serine protease and coding gene and application thereof |
-
2001
- 2001-12-14 CN CNB011443170A patent/CN1143893C/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| CN1357623A (en) | 2002-07-10 |
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