Method for separating heparan sulfate from animal lungs

A heparan sulfate and lung technology, applied in the field of separating heparan sulfate, to achieve the effect of easy acquisition, simple process conditions and easy realization

Inactive Publication Date: 2015-03-11
SHANDONG CHENZHONG BIOPHARM +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Method for separating heparan sulfate from animal lungs

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Embodiment 1

[0029] A method for isolating heparan sulfate from animal lungs, comprising the following steps:

[0030] 1) Take 3Kg of pig lung and mash it, then add 120L of 2% NaCl solution, stir at 90°C for 5 hours, centrifuge, transfer the supernatant and collect the precipitate, add 300L of 2% NaCl purified aqueous solution to the precipitate, repeat the above operation twice ;

[0031] 2) Combine the supernatants in step 1), adjust the pH to 9 with NaOH solution, add 60 g of trypsin at 40°C, stir for 24 hours, then raise the temperature to 90°C for 3 hours, centrifuge, discard To remove the precipitate, add ethanol to the supernatant, and precipitate for 18 hours to obtain 626 g of precipitate;

[0032] 3) Add 2% NaCl aqueous solution 60 times its weight to the precipitate obtained in step 2), stir and dissolve to obtain a solution; control the temperature of the solution to 40° C., adjust the pH of the solution to 9, and add a volume of the solution of 0.1 % KMnO 4 and 0.2% activat...

Embodiment 2

[0041] A method for isolating heparan sulfate from animal lungs, comprising the following steps:

[0042] 1) Take 2.5Kg bovine lung and mash it, then add 100L 2% NaCl solution, stir at 95°C for 6 hours, centrifuge, transfer the supernatant and collect the precipitate. Add 200L of 2% NaCl solution to the precipitate, and repeat 2 times;

[0043] 2) Combine the supernatants in step 1), adjust the pH to 11 with NaOH solution, add 20 g of trypsinase and 13 g of ribonuclease II at 50° C. and stir for 22 hours, then raise the temperature to 95° C. for 3 hours, Centrifuge, discard the precipitate, add ethanol to the supernatant, and precipitate for 17 hours to obtain 532 g of precipitate;

[0044] 3) Add 2% NaCl aqueous solution 50 times its weight to the precipitate obtained in step 2), stir and dissolve to obtain a solution; control the temperature of the solution to 50° C., adjust the pH of the solution to 11, and add 0.1% of the volume of the solution KMnO 4 and 0.2% activated...

Embodiment 3

[0053] A method for isolating heparan sulfate from animal lungs, comprising the following steps:

[0054] 1) Take 2Kg and mash it, then add 80L 2% NaCl solution, stir at 92°C for 5 hours, centrifuge, transfer the supernatant and collect the precipitate. Add 200 L of 2% NaCl purified aqueous solution to the precipitate, and repeat 2 times;

[0055] 2) Combine the supernatants in step 1), adjust the pH to 10 with NaOH solution, add 36 g of trypsinase and 24 g of ribonuclease II at 45°C and stir for 24 hours, then raise the temperature to 92°C for 3 hours, Centrifuge, discard the precipitate, add ethanol to the supernatant, and precipitate for 15 hours to obtain 626 g of precipitate;

[0056] 3) Add 2% NaCl aqueous solution 50 times its weight to the precipitate obtained in step 2), stir and dissolve to obtain a solution; control the temperature of the solution to 45°C, adjust the pH of the solution to 10, and add 0.1% of the volume of the solution KMnO 4 and 0.2% activated ca...

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Abstract

The invention discloses a method for separating heparan sulfate from animal lungs, belonging to the technical field of biological pharmacy. The method comprises the following steps: by using animal lungs as the raw material, carrying out composite enzyme degradation on an animal lung brine extract by using hydrolases, adding an oxidizer and activated carbon into the enzymolysis solution to perform oxidative adsorption decolorization, carrying out fractional precipitation on the oxidized solution by using acetone, dehydrating to obtain a heparan sulfate crude product, sequentially carrying out membrane separation and anion exchange chromatography on the crude product heparan sulfate water solution to separate the heparan sulfate from dermatan sulfate, chondroitin sulfate, heparin and other impurities, carrying out ultrafiltration concentration on the eluate through a 5000-7000Da ultrafiltration membrane, desalting by gel filtration chromatography, and freeze-drying to obtain the heparan sulfate refined product. The method has advantages of simple technological conditions, accessible raw materials and low cost, and is easy to implement; and the obtained heparan sulfate has high purity.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, in particular to a method for separating heparan sulfate from animal lungs. Background technique [0002] Heparan sulfate (HS) is a mucopolysaccharide compound, named for its similarity to heparin in the composition of glycosyl groups and glycosidic linkages. However, its sugar chain is composed of disaccharides linked by β-D-glucuronic acid and 2-N-acetylamino 2-deoxyα-D-glucose (including C6-O-sulfate substitution) through (1→4) Unit-based. HS is a class of glycosaminoglycans closely related to but independent from heparin (Casu, 2005). Compared with heparin, HS has a lower degree of sulfation and a higher degree of acetylation. [0003] Tumor is a disease with the highest incidence rate in the world and a great threat to human health and life. According to statistics, in recent years in China, the number of people who died of cancer accounted for more than 25% of the total numbe...

Claims

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Application Information

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IPC IPC(8): C08B37/10
Inventor 周霞郭维雷晓刚林勇乔德强郭恩中
Owner SHANDONG CHENZHONG BIOPHARM
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