CN1834238A - Prepn. process of and use of thrombus dissolving enzyme - Google Patents
Prepn. process of and use of thrombus dissolving enzyme Download PDFInfo
- Publication number
- CN1834238A CN1834238A CN 200610043371 CN200610043371A CN1834238A CN 1834238 A CN1834238 A CN 1834238A CN 200610043371 CN200610043371 CN 200610043371 CN 200610043371 A CN200610043371 A CN 200610043371A CN 1834238 A CN1834238 A CN 1834238A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- thrombus
- thrombus dissolving
- dissolving enzyme
- urechis uniconctus
- 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.)
- Granted
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
This invention discloses a method for manufacturing a fibrinolytic enzyme from whole-body or coelomic fluid of Urechis unicinctus, which comprises the steps of: (1) grinding and/or separating the tissues, (2) centrifuging to obtain the clear supernatant, (3) ultrafiltrating, (4) freeze-drying to obtain freeze-dried powders, or precipitating with acetone at a low temperature and vacuum filtrating to obtain the precipitate, (5) dissolving, purifying through a gel permeation column, and freeze-drying to obtain the fibrinolytic enzyme. The enzyme has good anticoagulation effects, and can be used to manufacture drugs for treating cardiovascular and cerebrovascular diseases.
Description
Technical field
The present invention relates to a kind of preparation method and application thereof of thrombus dissolving enzyme, particularly relate to and a kind ofly prepare the method for thrombus dissolving enzyme, and be applied to prepare the anticoagulation or the thrombus for the treatment of cardiovascular and cerebrovascular diseases with the marine organisms Urechis uniconctus.
Background technology
The marine organisms Urechis uniconctus is commonly called as " extra large intestines ", is the bottom-dwelling that lives in coastal silt substrate, and reproductivity is strong, aboundresources.Urechis uniconctus is Echiuroidea (Echiurioidea), and Yi guiding principle (Echiurida) does not have pipe Yi order (Xenopneusta), thorn Yi section (Urchidae).The fundamental biological knowledge research that Urechis uniconctus is only limited to be correlated with at present is as morphological structure, fetal development, the life history, artificial breeding technique and Analysis of Nutritional etc.Application and Development about Urechis uniconctus is not appeared in the newspapers as yet, does not particularly see the report with its preparation thrombus dissolving enzyme and application thereof.
Summary of the invention
The purpose of this invention is to provide and a kind ofly prepare the method for thrombus dissolving enzyme with the marine organisms Urechis uniconctus, and with it prepare a kind of novel anti blood coagulation, dissolved thrombus medicine is developed, to remedy
The above-mentioned deficiency of prior art.
A kind of preparation method of thrombus dissolving enzyme is characterized in that with the whole of Urechis uniconctus or its coelomic fluid be material, smashs to pieces and/or filtration, centrifugal by tissue, gets centrifuged supernatant, through ultrafiltration; Again through lyophilize, make dry powder or through the low temperature acetone precipitation, suction filtration makes throw out; Through dissolving, gel column separation and purification, again through lyophilize.
Above-mentioned thrombus dissolving enzyme is in the anticoagulation of preparation treatment cardiovascular and cerebrovascular diseases or the application in the dissolved thrombus medicine.
Thrombus dissolving enzyme of the present invention has good anti-freezing thrombus dissolving effect, has the research and development prospect.
Embodiment
Embodiment 1
Get fresh and alive Urechis uniconctus 1000g, cut body wall open, get coelomic fluid and amount to 480ml, remove by filter insolubless such as digestive tube with silk, gained filtrate in 4 ℃ of centrifugal 30min of following 10000rpm, is got centrifuged supernatant 350ml, with this supernatant liquor membrane ultrafiltration device of packing into, remove the following small-molecule substance of molecular weight 8KD, get ultrafiltrated 200ml, obtained by freeze drying dry powder 5.5g.This dry powder is mixed with 25ml liquid with the dissolving of the 0.01mol/L phosphoric acid buffer aqueous solution of pH7.4, again through 4 ℃ of centrifugal 30min of following 15000rpm of refrigerated centrifuge, get supernatant liquor, (Φ 5 * 100cm) for last SephadexG-50 gel separation post, 0.01mol/L phosphoric acid buffer aqueous solution wash-out with pH7.4 separates, and collects the elution peak part with enzymic activity, merges, through lyophilize, get the thrombolysis enzyme preparation 0.8g of purifying again.
Embodiment 2
Get refrigerated Urechis uniconctus 500g, thaw under the room temperature, put into tissue mashing machine, the 0.01mol/L phosphoric acid buffer of the pH7.4 that 300ml is ice-cold is organized and is smashed to pieces, removes by filter insolubles with silk, with gained filtrate in 4 ℃ of centrifugal 30min of following 10000rpm, get centrifuged supernatant body 350ml, liquid is carried out ice bath, be cooled to 0 ℃, stir acetone solution to 45% concentration that slowly adds-30 ℃ down, suction filtration collecting precipitation thing immediately adds the slow dissolving of 0.01mol/L phosphoric acid of ice-cold pH7.4, volume 20ml, 4 ℃ of following 15000rpm frozen centrifugation 30min, get supernatant liquor, (Φ 5 * 100cm), separate with 0.01mol/L phosphoric acid buffer wash-out for last Sephadex G-50 gel separation post, collection has the elution peak part of enzymic activity, merge,, get the thrombolysis enzyme preparation 0.46g of purifying again through lyophilize.
Embodiment 3
External anticoagulation, thrombus dissolving test
(1) anticoagulation test
The Urechis uniconctus thrombus dissolving enzyme that takes by weighing purifying among the 50mg embodiment 1 is dissolved in the 0.01mol/L phosphoric acid buffer (containing 0.8%NaCl) of 10mlpH7.4, gets Urechis uniconctus thrombus dissolving enzyme liquid, and is standby.Get 6 clean glass test tubees respectively, wherein 3 pipes add thrombus dissolving enzyme 1.0ml respectively, other 3 0.01mol/L phosphoric acid buffers that add 1.0ml pH7.4 are respectively organized in contrast, get the new zealand rabbit whole blood, in every pipe, add 1.0ml respectively immediately, seal, shake up gently, place 37 ℃, the anticoagulation situation of observing each pipe.The result shows that the Urechis uniconctus thrombus dissolving enzyme has tangible blood coagulation resisting function, the results are shown in Table 1.
Table 1 Urechis uniconctus thrombus dissolving enzyme blood coagulation resisting function (37 ℃)
| Group | 5min | 10min | 30min | 60min |
| Control group Urechis uniconctus thrombus dissolving enzyme group | Be and solidify clot and respectively manage little clot is arranged in the blood, blood has flowability | Be and solidify clot and respectively manage little clot is arranged in the blood, blood has flowability | The upper strata has serum to separate out, blood is respectively managed in the clot deepening flowability, and clot diminishes | The upper strata has serum to separate out, blood is respectively managed in the clot deepening flowability, and clot disappears |
(2) thrombus test
Get 6 clean glass test tubees respectively, add new zealand rabbit fresh blood 1.0ml respectively, solidify 30min under the room temperature and form clot, in 3 test tubes wherein, add the Urechis uniconctus thrombus dissolving enzyme 1.0ml that is prepared in the anticoagulation test (1) respectively, all the other 3 test tubes add phosphoric acid buffer (the containing 0.8%NaCl) 1.0ml of 0.01mol/L pH7.4, seal, the variation of clot is observed in vibration (100rpm) on 37 ℃ of following shaking tables.The result shows that Urechis uniconctus thrombus dissolving enzyme 3 hours energy under this experiment condition all dissolve clot, and red corpuscle is not had obvious influence, the results are shown in Table 2.
Table 2 Urechis uniconctus thrombus dissolving enzyme dissolving clot effect (37 ℃)
| Group | 60min | 120min | 180min |
| Control group Urechis uniconctus thrombus dissolving enzyme group | Clot does not have considerable change clot dissolution 25% | Clot does not have considerable change clot dissolution 60% | Clot does not have considerable change clot dissolution 100% |
2, thrombus test in the rat body
Get body weight and be 20 of the Wistar rats of 250 ± 20g,, carry out intraperitoneal injection of anesthesia by the dosage of 0.1ml/100g body weight with the Sodital aqueous solution of 1% (weight percentage).Anesthesia back row neck median incision is isolated right carotid and is about 10mm, successively presss from both sides with bulldog clamp earlier and closes artery distal end and proximal part, makes two ends at a distance of 5mm, and has blood full in artery.Two ends lead to direct current 5mA 20min, and the electricity Shu Songkai bulldog clamp that condenses is made the rat carotid artery thrombus model.Getting the Urechis uniconctus thrombus dissolving enzyme of purifying among the embodiment 2, is the Urechis uniconctus thrombus dissolving enzyme 10ml of 10mg/ml with injection physiological saline compound concentration, sterile filtration.Get 10 of carotid artery thrombus model rats, every animal intravenous injection Urechis uniconctus lytic enzyme liquid 2ml is as experimental group.Get 10 of carotid artery thrombus model rats again, the physiological saline of the same volume of intravenous injection, group in contrast.Experimental result shows that behind the 72hr, it is normal that the action of experimental group animal limb recovers, and the control animals limb activity does not have obvious improvement.Illustrate that the Urechis uniconctus thrombus dissolving enzyme has good thrombolytic effect.
Above experimental result shows, according to the enzyme product of embodiment 1 and 2 preparations, through external anticoagulation, thrombus dissolving test and animal thrombus dissolving model trial, proves good anti-freezing thrombus dissolving effect, has the research and development prospect.
Claims (2)
1, a kind of preparation method of thrombus dissolving enzyme is characterized in that with the whole of Urechis uniconctus or its coelomic fluid be material, smashs to pieces and/or filtration, centrifugal by tissue, gets centrifuged supernatant, through ultrafiltration; Again through lyophilize, make dry powder or through the low temperature acetone precipitation, suction filtration makes throw out; Through dissolving, gel column separation and purification, again through lyophilize.
2, thrombus dissolving enzyme as claimed in claim 1 is in the anticoagulation of preparation treatment cardiovascular and cerebrovascular diseases or the application in the dissolved thrombus medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100433713A CN100457897C (en) | 2006-03-28 | 2006-03-28 | Prepn. process of and use of thrombus dissolving enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100433713A CN100457897C (en) | 2006-03-28 | 2006-03-28 | Prepn. process of and use of thrombus dissolving enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1834238A true CN1834238A (en) | 2006-09-20 |
| CN100457897C CN100457897C (en) | 2009-02-04 |
Family
ID=37002135
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100433713A Expired - Fee Related CN100457897C (en) | 2006-03-28 | 2006-03-28 | Prepn. process of and use of thrombus dissolving enzyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100457897C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101015606B (en) * | 2006-12-14 | 2011-08-10 | 刁勇 | Traditional Chinese medicine composition for preventing and treating cardiovascular and cerebrovascular diseases and preparing process thereof |
| CN104404019A (en) * | 2014-11-11 | 2015-03-11 | 陈良海 | Preparation technology of interventional thrombolytic biological product namely urechis unicinctus plasmin |
| CN106399282A (en) * | 2016-06-28 | 2017-02-15 | 华侨大学 | Method for extracting thrombus plasmin from nephridium of urechis unicinctus |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199615B (en) * | 2011-03-08 | 2013-01-23 | 中国海洋大学 | cDNA sequence coding fibrinolysin, amino acid sequence and applications of sequences |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0656887A (en) * | 1991-08-23 | 1994-03-01 | Suntory Ltd | Neuropeptide |
| CN1306086A (en) * | 2000-09-29 | 2001-08-01 | 武汉迪普生物技术有限公司 | Process for preparing recombinant cumic kinase and its bioactivity |
| CN100424171C (en) * | 2004-08-16 | 2008-10-08 | 北京赛生药业有限公司 | High purity venom fibrinolysin prepartion method and its pharmaceutical formulation |
-
2006
- 2006-03-28 CN CNB2006100433713A patent/CN100457897C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101015606B (en) * | 2006-12-14 | 2011-08-10 | 刁勇 | Traditional Chinese medicine composition for preventing and treating cardiovascular and cerebrovascular diseases and preparing process thereof |
| CN104404019A (en) * | 2014-11-11 | 2015-03-11 | 陈良海 | Preparation technology of interventional thrombolytic biological product namely urechis unicinctus plasmin |
| CN106399282A (en) * | 2016-06-28 | 2017-02-15 | 华侨大学 | Method for extracting thrombus plasmin from nephridium of urechis unicinctus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100457897C (en) | 2009-02-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5048673B2 (en) | A kind of extract that prevents or treats thrombotic diseases | |
| US6268119B1 (en) | Method for separating cells | |
| CN100457897C (en) | Prepn. process of and use of thrombus dissolving enzyme | |
| CN104152405B (en) | The method of separation and Extraction hematopoietic stem cell from Placenta Hominis | |
| AU8165698A (en) | Method and apparatus for the production of purified plasma proteins | |
| US20040152190A1 (en) | Method of separating and concentrating cells for kidney regfneration | |
| CN101433553A (en) | Method for preparing deproteinized extract of calf blood and freeze-dried powder thereof | |
| CN103285031B (en) | The application of depolymerization glycosaminoglycan extracted from sea cucumber in preparation control thromboembolic disorders medicine | |
| CN101879274B (en) | Medicine composition containing red ginseng saponin extract and radix ophiopogonis saponin extract as well as preparation method and application thereof | |
| CN104342422A (en) | Method for extracting high-activity lumbrukinase from earthworm | |
| CN106267418B (en) | Female tire blood group incompatibility antibody adsorbing therapy instrument | |
| CN111035766A (en) | Application of erythrocyte and activated platelet cell membrane as carrier in preparing thrombus treating medicine | |
| CN101942040A (en) | Method for removing oversulfated chondroitin sulfate in heparin sodium | |
| CN113244458B (en) | Composite material for repairing articular cartilage damage and preparation method thereof | |
| CN108578791A (en) | A kind of hirudin is modified the preparation method of anticoagulant material | |
| CN108660126A (en) | A kind of preparation process of freeze dried human zymoplasm | |
| KR102298179B1 (en) | Method for producing unfractionated heparin | |
| Kalowski et al. | Multinucleated giant cells in antiglomerular basement membrane antibody-induced glomerulonephritis | |
| CN114306713B (en) | Absorbable hemostatic wound-protecting dressing and preparation method thereof | |
| CN111840527B (en) | Preparation method and application of shear-responsive nano drug delivery system | |
| CN1247271C (en) | Apparatus for producing fibrin adhesive & the like blood products and its medical use | |
| CN114788874B (en) | Nanometer micelle coated with baicalein and application thereof | |
| WO1986000622A1 (en) | Hexapeptide | |
| CN1404825A (en) | Novel decanoy acetaldelzy sodium intravenous preparation and its preparing method thereof | |
| CN110585210B (en) | Application of sparganium stoloniferum A in preparation of anti-ischemic cerebral apoplexy medicines |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090204 Termination date: 20210328 |