High-sulfating chondroitin sulfate (CS) and preparing method and application thereof

A technology of chondroitin sulfate and high sulfuric acid, applied in the biological field, to achieve mild reaction conditions, strong anti-tumor metastasis activity, and high market value

Active Publication Date: 2016-09-07
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no relevant report on the research and utilization of the cartilage of the American red squid

Method used

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  • High-sulfating chondroitin sulfate (CS) and preparing method and application thereof
  • High-sulfating chondroitin sulfate (CS) and preparing method and application thereof
  • High-sulfating chondroitin sulfate (CS) and preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A preparation method of highly sulfated chondroitin sulfate, comprising the steps of:

[0058] (1) After crushing 1 kg of American red squid (Dosidicus Gigas) cartilage, add 3 times the volume of absolute ethanol to dehydrate and degrease for 24 hours, filter, repeat the steps of dehydration and degreasing 3 times, then dry and weigh at 50°C to obtain defatted cartilage ;

[0059] (2) Submerge the degreased cartilage prepared in step (1) with water, treat it in a water bath at 100°C for 30 minutes, cool to room temperature and filter, add 20 times the volume of calcium chloride borate buffer solution (0.1M boric acid, 10mM calcium chloride , pH8.0) and mix well, add Aspergillus niger protease (purchased from TCI company) with a dry weight of 2‰ of raw materials, react at 60°C for 72h, add enzyme once every 24h, and the amount of enzyme added is gradually reduced to the last enzyme added 0.5 times the amount, add twice in total, inactivate the enzyme at 100°C for 10 min...

Embodiment 2

[0062] The purity analysis of embodiment 2, DG-CS

[0063] 2.1 Analysis of protein and glycosaminoglycan content

[0064] Using carbazole reaction, BCA protein assay kit, full-wavelength scanning and other technical means to test the purity of the prepared DG-CS, it was found that the protein content in the sample was less than 2%, and the protein absorption peak near 280nm was extremely small, such as figure 1 Shown; The glycosaminoglycan content measured by the carbazole reaction is more than 97%, and the recovery rate is 20%.

[0065] 2.2 Content analysis of other glycosaminoglycans

[0066] It is well known that the types of polysaccharide samples can be determined by using properties such as substrate specificity of biological enzymes. Chondroteinase ABC mainly degrades chondroitin sulfate, and Chondroteinase B can specifically degrade dermatan sulfate (DS), a glycosaminoglycan lyase disclosed by Chinese patent document CN103923899A (Patent No. 201410160095.3) rHCLase ...

Embodiment 3D

[0068] The molecular weight analysis of embodiment 3DG-CS

[0069] Average molecular weight is respectively the dextran standard of 11.6KD, 48.6KD, 147KD, 273KD, 409.8KD and the highly sulfated chondroitin sulfate DG-CS (mass concentration 1%) that embodiment 1 makes, get 100 μ l respectively and carry out HPLC analysis .

[0070] HPLC analysis conditions for gel column: Ultrahydrogel TM 100 7.8×300mm (Water); mobile phase: 0.02MNa 2 HPO4, 0.02M NaH 2 PO 4 , 0.02% NaN 3 , pH 7.0; flow rate: 0.6mL / min; detection condition: Shimadzu differential detector (RID-10A).

[0071] The result is as image 3 shown, according to image 3 It can be calculated that the average molecular weight of the prepared DG-CS is about 623.2KD.

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Abstract

The invention relates to a high-sulfating chondroitin sulfate (CS) and a preparing method and application thereof. According to the high-sulfating CS, the molecular weight is 100 KDa to 800 KDa, the sulfating degree is 0.7 to 1.43, and disaccharide compositions are O-units, C-units, A-units and E-units. The high-sulfating CS is a CS which is extracted from cartilage of dosidicus gigas and is rich in E-unit, and is a DG-CS with the specific disaccharide compositions, specific carbohydrate chain arranging and the specific molecular weight. An experiment proves that the high-sulfating CS has quite good anti-tumor metastasis activity.

Description

technical field [0001] The invention relates to a highly sulfated chondroitin sulfate and its preparation method and application, in particular to the preparation of a new type of natural high sulfated chondroitin sulfate derived from giant squid cartilage and its application in anti-tumor, belonging to the technical field of biotechnology . Background technique [0002] Chondroitin sulfate (CS for short) is an acidic mucopolysaccharide widely present in bovine bone, pork bone, chicken bone, shark bone, and squid bone. molecular. Its basic unit is a disaccharide formed by linking D-glucuronic acid and N-acetyl-D-galactosamine through β-1,3 glycosidic bonds (→4GlcUaβ1→3GalNAcβ1→) [1] . Most CS in nature exists in the form of sulfated 4- or 6-position of N-acetyl-D-galactosamine, which we call CS-A and CS-C respectively. In addition to CS-A and CS-C, according to the different sulfation modes of D-glucuronic acid and N-acetyl-D-galactosamine, there are also CS-O, CS-E, CS-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/08A61K31/737A61P35/00
CPCA61K31/737C08B37/0003C08B37/0069
Inventor 李福川王庆彬彭春娥陈翔雪韩乃寒
Owner SHANDONG UNIV
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