Preparation method of heparin oligosaccharides

A technology of heparin oligosaccharides and heparin, which is applied in the field of preparation of heparin oligosaccharides, can solve the problems of complex process, high yield, and low cost, and achieve the effects of simple process, high yield, and convenient operation

Inactive Publication Date: 2011-10-05
GUANGYUAN HAIPENG BIOLOGICAL TECH
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Problems solved by technology

The existing technologies include chemical cleavage and enzymatic hydrolysis. The chemical cleavage method of heparin reacts violently, the process is complicated, and the activity and function of heparin are destroyed to varying degrees.
The enzymatic hydrolysis method has mild reaction conditions, high yield, no toxicity to the environment, simple operation and easy control, and has become a hot spot in current biological research. Some related researches have been done at home and abroad, but there is no enzymatic hydrolysis production method with relatively low cost. craft

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  • Preparation method of heparin oligosaccharides
  • Preparation method of heparin oligosaccharides

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Embodiment Construction

[0010] One embodiment of the present invention, a method for preparing heparin oligosaccharides, comprises the following steps:

[0011] 1) Enzymatic degradation of heparin: Take 20 mg of heparin, dissolve it in 1 ml of reaction solution Digestion Buffer (25 mM ammonium acetate, 25 μM CaCl2, 0.25 μg / ml BSA, pH 7.4), add 500 uL of heparinase I, II, and III each, 37°C constant temperature shaking reaction for 72 hours, in which heparanase was supplemented every 12 hours, after the reaction was completed, heated in a water bath at 100°C for 15 minutes, and centrifuged at 12000 rpm for 10 minutes at high speed, and the supernatant was taken for later use.

[0012] 2) Fractional purification of heparin oligosaccharides: the oligosaccharide fragments obtained in step 1 are subjected to fractional purification through a Bio-gel P2 column, collected and freeze-dried to obtain a finished product.

[0013] The degraded heparin oligosaccharide fragment has an unsaturated double bond at t...

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Abstract

A preparation method of heparin oligosaccharides comprises the following steps: 1, carrying out enzymic degradation on heparin: dissolving 20 mg of heparin in 1 ml of a reaction solution Digestion Buffer, adding 500 [mu]l of heparinase I, 500 [mu]l of heparinase II and 500 [mu]l of heparinase III, carrying out an oscillatory reaction for 72 h at a constant temperature of 37 DEG C with heparinase supplement every 12 h, heating in a water bath with the temperature of 100 DEG C for 15 min after ending the reaction, carrying out high speed centrifugation for 10 min with a speed of 12000 rpm, taking out a supernatant and setting aside; and 2, carrying out stage purification on heparin oligosaccharides: carrying out stage purification through passing oligosaccharide fragments obtained in step 1 through a Bio-gel P2 column, collecting and lyophilizing. So the finished products are obtained.

Description

technical field [0001] The invention relates to a preparation method of heparin oligosaccharide. Background technique [0002] Compared with unfractionated heparin, heparin oligosaccharide has less anticoagulant effect than heparin at the same dose, but significantly stronger antithrombotic effect than heparin. Stable dose-effect relationship, less binding with platelets, less likely to cause thrombocytopenia. Therefore, heparin oligosaccharides can not only effectively prevent thrombosis, but also reduce bleeding and other adverse reactions. It is a safe and effective antithrombotic drug and can be used as a substitute for heparin. Heparin oligosaccharides with different degrees of polymerization can interact with different protein factors, thus presenting different biological effects. Therefore, the production of heparin oligosaccharides is of great significance. The prior art includes chemical cleavage method and enzymatic hydrolysis method. The chemical cleavage metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/14C12P19/00
Inventor 王香李海生
Owner GUANGYUAN HAIPENG BIOLOGICAL TECH
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