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A method for degrading, obtaining and detecting chondroitin sulfate and hyaluronic acid disaccharide

A chondroitin sulfate and hyaluronic acid technology, applied in the field of medicine, can solve the problems of loss of relative content information, poor repeatability of analysis results, cumbersome detection steps, etc., and achieve the effects of short detection time, low requirements for experimental instruments and high sensitivity

Active Publication Date: 2015-08-05
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when chondroitin sulfate is degraded by enzymes, glucuronic acid (GlcA) and iduronic acid (IdoA) are dehydrated to form anhydrouronic acid with the same structure, thereby losing glucuronic acid (GlcA) and glycosides in chondroitin sulfate. Relative content information of duronic acid (IdoA), and the lyase cannot fully degrade CS or HA into disaccharides
Enzymes, strong anion exchange columns and disaccharide standards are very expensive, and the enzymes are prone to inactivation, unstable properties, cumbersome detection steps, large sample consumption, long detection time, and poor repeatability of analysis results

Method used

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  • A method for degrading, obtaining and detecting chondroitin sulfate and hyaluronic acid disaccharide
  • A method for degrading, obtaining and detecting chondroitin sulfate and hyaluronic acid disaccharide
  • A method for degrading, obtaining and detecting chondroitin sulfate and hyaluronic acid disaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The chemical degradation method of embodiment 1 chondroitin sulfate class sample disaccharide

[0036] (1) Dissolve 1mg chondroitin sulfate in 500uL 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 9 hours, after the reaction was completed, freeze-dried to remove N 2 h 4 Deacetylated chondroitin sulfate;

[0037] (2) Dissolve the deacetylated chondroitin sulfate 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 solution with a pH of 4.0 Nitrous acid, react at 0-5°C for 10 minutes, add 30uL ammonia water to terminate the reaction.

Embodiment 2

[0038] The chemical degradation method of embodiment 2 chondroitin sulfate class sample disaccharide

[0039] (1) Dissolve 1 mg of chondroitin sulfate in 1 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 90 ° C, reacted for 5 hours, after the reaction was completed, freeze-dried to remove N 2 h 4 Deacetylated chondroitin sulfate;

[0040](2) Dissolve the deacetylated chondroitin sulfate obtained in step (1) in 50uL of water, add 50uL of sodium nitrite aqueous solution with a pH of 1.5, react at 0-5°C for 10 minutes, adjust the pH to 4.0, add 50uL with a pH of 4.0 The nitrous acid was reacted at 0-5°C for 10 minutes, and 30uL of ammonia water was added to terminate the reaction.

Embodiment 3

[0041] The chemical degradation method of embodiment 3 chondroitin sulfate class sample disaccharide

[0042] (1) Dissolve 1 mg of chondroitin sulfate in 1 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 110°C, reacted for 16 hours, after the reaction was completed, freeze-dried to remove N 2 h 4 Deacetylated chondroitin sulfate;

[0043] (2) Dissolve the deacetylated chondroitin sulfate 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 solution with a pH of 4.0 Nitrous acid, react at 0-5°C for 10 minutes, add 30uL ammonia water to terminate the reaction.

[0044] Table 1 Analysis of chondroitin sulfate chemical degradation products

[0045]

[0046] The results show that CS can be completely chemically degraded in Examples 1 and 3, and the time spent in Example 1 is 43.8% l...

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Abstract

The invention belongs to the field of medicine, and relates to a method for degrading chondroitin sulfate and hyaluronic acid to obtain chondroitin sulfate disaccharide and hyaluronic acid disaccharide and detecting the chondroitin sulfate disaccharide and the hyaluronic acid disaccharide. The method comprises the following steps: (1) deacetylating chondroitin sulfate or the hyaluronic acid to obtain a deacetylation product; (2) performing degradation on the deacetylation product, namely nitrite, to obtain chondroitin sulfate disaccharide or hyaluronic acid disaccharide; (3) completely concentrating disaccharide, dissolving disaccharide in water, regulating the pH value as alkaline, adding pyrazolone derivatization reagents, and after reaction is completed, performing three times of extraction through chloroform to remove redundant derivatization reagents; (4) detecting the derived disaccharide through LC (liquid chromatography) or LC-MS (liquid chromatography-mass spectrometry). According to a chemical degradation method, chondroitin sulfate or hyaluronic acid can be completely degraded as the disaccharide, all pieces of uronic acid information can be stored, the degradation condition is mild, the cost is low, the operation is easy, the requirement on experiment instruments can be met, consumed detection time is short, the sample consumption is low, the sensitivity is high and the analysis result repeatability is good.

Description

technical field [0001] The invention belongs to the field of medicine and relates to a method for degrading, obtaining and detecting chondroitin sulfate and hyaluronic acid disaccharide. Background technique [0002] Chondroitin sulfate (CS) is a class of sulfate-modified disaccharide repeating units consisting of uronic acid (glucuronic acid, GlcA or iduronic acid, IdoA) and N-acetylgalactosamine (GalNAc). Linear polymer polysaccharides, GlcA and GalNAc are connected by β-1,3 glycosidic bonds, IdoA and GalNAc are connected by α-1,3 glycosidic bonds, and disaccharide units are connected by β-1,4 glycosidic bonds, including chondroitin sulfate A (CSA), chondroitin sulfate B (CSB), chondroitin sulfate C (CSC), chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), etc. Related drugs or health care products have been listed, such as chondroitin sulfate tablets, chondroitin sulfate injection, chondroitin sulfate eye drops, chondroitin sulfate capsules, etc. Different typ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
Inventor 张丽娟韩章润曾洋洋兰莹邱培菊
Owner OCEAN UNIV OF CHINA
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