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Antithrombotic heparin extracted from short necked clam and preparation method and application of antithrombotic heparin

An anti-thrombotic and anti-thrombotic technology, applied in the field of anti-thrombotic heparin and its preparation, can solve the problems of bleeding effect thrombocytopenia, bleeding side effects, etc.

Active Publication Date: 2020-04-24
GUANGDONG OCEAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the heparin used clinically comes from porcine small intestine and bovine lung, but the heparin derived from porcine small intestine and bovine lung has too strong anticoagulant effect, which inevitably causes side effects such as bleeding effect and thrombocytopenia.
In view of the problem that heparin used in the treatment of thrombotic diseases can cause bleeding side effects, it is urgent to develop a heparin or heparin-like that does not rely on anticoagulation and exerts antithrombotic function

Method used

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  • Antithrombotic heparin extracted from short necked clam and preparation method and application of antithrombotic heparin
  • Antithrombotic heparin extracted from short necked clam and preparation method and application of antithrombotic heparin
  • Antithrombotic heparin extracted from short necked clam and preparation method and application of antithrombotic heparin

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0034] The preparation method of the antithrombotic heparin extracted from Guifei clam comprises the following steps:

[0035] 1. Raw material processing: Wash the raw materials of Guifei mussels, remove the shells, homogenize the shellfish meat, and freeze-dry them into powder;

[0036] 2. Enzyme hydrolysis: take 100g of dried mussel powder and add 8L of distilled water to dissolve it, add NaOH to adjust the pH to 8.0 to obtain a raw material solution, add 0.5% of the dry powder of mussel with 2709 alkaline protease and 0.5% of papain, at 50°C Enzymolysis for 20 hours to obtain an enzymolysis solution;

[0037] 3. Enzyme inactivation centrifugation: put the enzymatic solution in boiling water to inactivate the enzyme for 10 minutes, then centrifuge at 8000 rpm for 20 minutes to obtain the supernatant;

[0038] 4. Sample loading: transfer the supernatant to the equilibrated chromatographic column equipped with AMBERLITE FPA98 CI macroporous ion exchange resin, the solvent in ...

specific Embodiment 2

[0046] Structural Characterization of Guifei Mussel Heparin

[0047] 1. Ultraviolet Spectrum Scanning of Guifei Mussel Heparin

[0048] The Guifei mussel heparin prepared in the specific example 1 was prepared into a solution of 1-3 mg / ml with distilled water, and the distilled water was used as a zero adjustment tube, and the ultraviolet spectrum scanning of the sample was performed on an ultraviolet-visible spectrophotometer. Such asfigure 1 As shown, the ultraviolet spectrum scanning diagram shows that Guifei mussel heparin is of high purity and does not contain protein and nucleic acid.

[0049] 2. Electrophoresis analysis of Guifei mussel heparin acetate

[0050] Take 3 mg of the concubine mussel heparin prepared in the specific example 1, and make it 3 mg / mL with distilled water. Soak the cellulose acetate membrane (8×2 cm) in 0.1mol / L pyridine-0.47 mol / L formic acid (pH3.0) electrophoresis buffer solution, take it out after 30min, and absorb the excess buffer solution...

specific Embodiment 3

[0065] Anticoagulant Activity of Guifei Mussel Heparin

[0066] Specific Example 1 Effects of the Prepared Guifei Mussel Heparin on the Three Indexes of Plasma Coagulation (APTT, PT, TT)

[0067] Dissolve mussel heparin and heparin standard in physiological saline, and prepare 10ug / ml, 20ug / ml, 40ug / ml, 80ug / ml, 500ug / mL respectively, with normal saline as negative control and heparin standard solution as positive control , APTT, PT and TT were determined according to the kit instructions.

[0068] 1. Determination of activated partial thromboplastin time (APTT value): Add 20uL of sample, 0.1mL of sheep plasma and 0.1mL of APTT reagent to the test tube, mix well, place in a water bath at 37°C for 3min; shake gently ; Add 0.1 mL of 0.025 mol / L calcium chloride solution, shake it up immediately and start timing, and place it in a pot of water for continuous shaking; when it is about 30 s, slowly tilt the test tube from time to time to observe the flow state of the test tube, wh...

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Abstract

The invention discloses antithrombotic heparin extracted from short necked clam and a preparation method and application of the antithrombotic heparin. The invention is characterized in that the structural formula of the heparin tetrasaccharide is as shown in (I), and the preparation method comprises the following steps: adding distilled water into short necked clam meat powder to dissolve a sample, regulating the pH value to 7.8-8.2, adding alkaline protease and papain to carryi out enzymolysis to obtain an enzymatic hydrolysate, carrying out enzyme deactivation, centrifuging, and taking a supernatant; transferring the supernatant into a balanced chromatographic column with macroporous ion exchange resin, taking distilled water as a solvent in the column, and combining and collecting elution components of a 1.5 M NaCl solution; concentrating the collected eluted component, adding ethanol, standing, centrifuging, taking a precipitate, alternately washing the precipitate with acetone and ethanol, dissolving the precipitate with water, centrifuging to remove insoluble substances, and repeating the operation for 3-4 times; and finally, dialyzing, and freeze-drying to obtain the antithrombotic heparin product extracted from short necked clam. The invention has the advantages of low bleeding side effect, mild anticoagulation effect and strong fibrinolysis effect.

Description

technical field [0001] The present invention relates to an antithrombotic heparin, in particular to an antithrombotic heparin extracted from Guifei mussel and its preparation method and application. Background technique [0002] Thrombotic disease is a serious threat to human life and health, and its incidence rate ranks first among various diseases. Although the incidence of thrombotic disease has been reduced after the introduction of antithrombotic drugs such as heparin, compared with malignant tumors, thrombotic disease is still an important cause of death, and it has gradually increased in recent years. One of the key points and hot spots. [0003] Heparin is a glycosaminoglycan that exists in animals. It has various biological activities and is widely used clinically, especially as an anticoagulant. At present, there is no product that can completely replace it. Therefore, heparin used in the treatment of thrombotic diseases mainly relies on its powerful anticoagulan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H11/00C07H1/08
CPCC07H11/00C07H1/08
Inventor 钟赛意杜振兴陈菁周斯仪贾学静陈建平刘晓菲曹笑皇洪鹏志
Owner GUANGDONG OCEAN UNIVERSITY
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