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Prepn. process of and use of thrombus dissolving enzyme

A technology for dissolving enzymes and thrombus is applied in the field of preparation of thrombolytic enzymes to achieve the effect of good anticoagulant and thrombolytic effects

Inactive Publication Date: 2006-09-20
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report about the development and application of P. monoringensis, especially the report of using it to prepare thrombolytic enzyme and its application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0009] Take 1,000 g of fresh and alive C. monoringensis, cut open the body wall, take a total of 480 ml of body cavity fluid, filter with silk cloth to remove insoluble matter such as the digestive tract, and centrifuge the obtained filtrate at 10,000 rpm for 30 min at 4°C to obtain 350 ml of centrifuged supernatant. The supernatant is loaded into a membrane ultrafiltration device to remove small molecular substances with a molecular weight below 8KD to obtain 200ml of ultrafiltrate, which is freeze-dried to obtain 5.5g of dry powder. This dry powder is dissolved with the 0.01mol / L phosphate buffered aqueous solution of pH7.4 and is formulated into 25ml liquid, then centrifuges 30min at 15000rpm under 4 ℃ through refrigerated centrifuge, gets supernatant, goes on SephadexG-50 gel separation column (Φ5×100cm ), eluted and separated with 0.01mol / L phosphate buffered aqueous solution of pH 7.4, collected the eluted peak part with enzyme activity, combined, and then freeze-dried to...

Embodiment 2

[0011] Take 500 g of frozen C. monoringensis, thaw it at room temperature, put it into a tissue masher, and use 300 ml of ice-cold 0.01 mol / L phosphate buffer solution of pH 7.4 to mash the tissue, filter with silk cloth to remove insoluble matter, and The obtained filtrate was centrifuged at 10,000 rpm at 4°C for 30min to obtain 350ml of centrifuged supernatant liquid, put the liquid in an ice bath, cooled to 0°C, slowly added acetone solution at -30°C to a concentration of 45% while stirring, and immediately collected the precipitate by suction filtration , add ice-cold pH7.4 0.01mol / L phosphoric acid to slowly dissolve, volume 20ml, freeze and centrifuge at 15000rpm at 4°C for 30min, take the supernatant, put it on a Sephadex G-50 gel separation column (Φ5×100cm), and use 0.01mol / L phosphate buffer for elution and separation, and the elution peaks with enzyme activity were collected, combined, and then freeze-dried to obtain 0.46 g of a purified thrombolytic enzyme product....

Embodiment 3

[0013] Anticoagulant and thrombolytic tests in vitro

[0014] (1) Anticoagulation test

[0015] Weigh 50 mg of C. monocircleum thrombolytic enzyme purified in Example 1 and dissolve it in 10 ml of 0.01 mol / L phosphate buffer (containing 0.8% NaCl) at pH 7.4 to obtain C. monocycle thrombus-dissolving enzyme solution for future use. Take 6 clean glass test tubes respectively, add 1.0ml of thrombolytic enzyme to 3 tubes respectively, and add 1.0ml 0.01mol / L phosphate buffer solution of pH7.4 to the other 3 tubes as a control group, take whole blood of New Zealand rabbits, and immediately pour them into Add 1.0ml to each tube, seal, shake gently, place at 37°C, and observe the anticoagulant status of each tube. The results showed that the thrombus-dissolving enzyme of E. monocircleum had obvious anticoagulant effect, and the results are shown in Table 1.

[0016] group

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control group

single-ringed weed

Thrombo...

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PUM

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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 [0001] The present invention relates to a preparation method of a thrombolytic enzyme and its application, in particular to a method for preparing a thrombolytic enzyme from the marine organism Echinacea monoringensis, which is applied to the preparation of anticoagulant or anticoagulant for the treatment of cardiovascular and cerebrovascular diseases. Dissolves blood clots. Background technique [0002] Commonly known as "Sea Intestines", the marine organisms are benthic organisms that live in coastal sediments. They have strong fecundity and abundant resources. The single-ringed weed belongs to the phylum Echiurioidea, the class Echiurida, the order Xenopneusta, and the family Urchidae. At present, the research on E. monoringensis is limited to related basic biology, such as morphological structure, embryonic development, life history, artificial seedling technology and nutritional composition analysis. There has been no report on the development and appl...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N9/64C12N9/68A61K38/48A61P9/00A61P7/02
Inventor 刘万顺韩宝芹王佃亮杨艳
Owner OCEAN UNIV OF CHINA
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