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Purification and detection method for keratan sulfate in chondroitin sulfate

A technology of chondroitin sulfate and keratan sulfate, which is applied in the field of medicine, can solve the problems of difficult complete degradation, expensive enzymes, and long time consumption, and achieve the effects of short detection time, small sample consumption, and simple operation

Active Publication Date: 2014-04-30
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used method to obtain KS disaccharide is enzymatic hydrolysis, and the enzymes used include keratan sulfate degrading enzyme II or β-galactosidase, but these two enzymes are expensive and cannot completely degrade KS into disaccharides. For samples mixed with KS in glycans, the enzymatic hydrolysis method can only use a variety of enzymes to degrade and analyze different glycosaminoglycans one by one. This degradation and detection method is not only time-consuming, but also difficult to achieve complete degradation
Existing methods cannot meet the needs of current structural analysis, therefore, it is urgent to find a simple, efficient and accurate detection method

Method used

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  • Purification and detection method for keratan sulfate in chondroitin sulfate
  • Purification and detection method for keratan sulfate in chondroitin sulfate
  • Purification and detection method for keratan sulfate in chondroitin sulfate

Examples

Experimental program
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Effect test

example 1

[0045] The chemical degradation of example 1 chondroitin sulfate mixture

[0046] (1) Dissolve 0.5 mg of chondroitin sulfate mixture in 0.25 mL containing 10% N 2 h 4 ·H 2 SO 4 N 2 h 4 ·H 2 O solution, heated to dissolve, then sealed and heated to 98 ° C, reacted for 7 hours, after the reaction was completed, freeze-dried to remove N 2 h 4 Deacetylated products are obtained;

[0047] (2) Completely dissolve the deacetylated product obtained in step (1) in 50uL water, add 50uL sodium nitrite aqueous solution with a pH of 1.5, adjust the pH to 4.0 after reacting at 0-5°C for 10 minutes, and add 50uL sodium nitrite with a pH of 4.0 Nitric acid, continue to react at 0-5°C for 10 minutes, and add 30uL ammonia water to terminate the reaction.

example 2

[0048] The chemical degradation of example 2 chondroitin sulfate mixture

[0049] (1) Dissolve 0.5 mg of chondroitin sulfate mixture in 0.5 mL containing 10% N 2 h 4 ·H 2 SO 4 N 2 h 4 ·H 2 O solution, heated to dissolve, then sealed and heated to 80 ° C, reacted for 4 hours, after the reaction was completed, freeze-dried to remove N 2 h 4 Deacetylated products are obtained;

[0050] (2) Completely dissolve the deacetylated product obtained in step (1) in 50uL water, add 50uL sodium nitrite aqueous solution with a pH of 1.5, adjust the pH to 4.0 after reacting at 0-5°C for 10 minutes, and add 50uL sodium nitrite with a pH of 4.0 Nitric acid, continue to react at 0-5°C for 10 minutes, and add 30uL ammonia water to terminate the reaction.

example 3

[0051] The chemical degradation of example 3 chondroitin sulfate mixture

[0052] (1) Dissolve 0.5 mg of chondroitin sulfate mixture in 0.5 mL containing 10% N 2 h 4 ·H 2 SO 4 N 2 h 4 ·H 2 O solution, heated to dissolve, then sealed and heated to 105 ° C, reacted for 16 hours, after the reaction was completed, freeze-dried to remove N 2 h 4 Deacetylated products are obtained;

[0053] (2) Completely dissolve the deacetylated product obtained in step (1) in 50uL of water, add 50uL of sodium nitrite aqueous solution with a pH of 1.5, adjust the pH to 4.0 after reacting at 5°C for 10 minutes, and add 50uL of nitrous acid with a pH of 4.0 , continue to react at 5°C for 10 min, and add 30 uL of ammonia water to terminate the reaction.

[0054] The chemical degradation products of the chondroitin sulfate mixture in Examples 2-4 were detected, and the results are shown in Table 2.

[0055] Table 2 Analysis of chemical degradation products of chondroitin sulfate mixture

[00...

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Abstract

Belonging to the field of medicines, the invention relates to a purification and detection method for keratan sulfate in chondroitin sulfate. The method includes the steps of: (1) detecting the keratan sulfate in a chondroitin sulfate mixture; (2) separating and purifying the keratan sulphate in chondroitin sulfate; and (3) detecting the content of keratan sulfate. The method provided by the invention can degrade the glycosaminoglycan in the chondroitin sulfate mixture into disaccharide completely, can save all uronic acid structural information, and can accurately and totally obtain the structural information of chondroitin sulfate and keratan sulfate disaccharide. The trace keratan sulfate in the chondroitin sulfate mixture can be qualitative and quantitative. The chemical degradation conditions are mild, the cost is low, the operation is simple, and the requirements for experimental instruments are low. With a wide application range, the method is suitable for detection of keratan sulfate in chondroitin sulfate from any source. The detection needs short time, the sample consumption is small, the sensitivity is high, the analysis result has good repeatability, and the detection limit is ng level.

Description

technical field [0001] The invention belongs to the field of medicine and relates to a method for purifying and detecting keratan sulfate in chondroitin sulfate. Background technique [0002] Keratan sulfate (KS) is a kind of glycosaminoglycan, which is composed of different sulfated modified N-acetylglucosamine (GlcNAc) and D-galactose (Gal) disaccharide repeating units, and the backbone connection method is -[3Ga1β1 → 4GlcNAcβ1] n -, KS exists in the cornea, intervertebral plate, cartilage and arteries of animals in the form of proteoglycans. Studies have found that the content of KS in cartilage polysaccharides in the fetal period is very low, but its content gradually increases with age. KS has biological activities such as anti-inflammation, anti-allergy, immune regulation, cell differentiation induction, and apoptosis induction. [0003] KS exists in a large amount in the cartilage and cornea of ​​sharks and other cartilaginous fishes and mammals such as whales and c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00
Inventor 张丽娟曾洋洋韩章润兰莹于广利邱培菊
Owner OCEAN UNIV OF CHINA
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