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Analysis method of disaccharides containing uronic acid based on reduction amine derivatization and preparation method thereof

A technology of uronic acid disaccharide and analysis method, which is applied in the analysis method and preparation field of uronic acid disaccharide-containing uronic acid disaccharide based on derivatization of reductive amine, can solve the problems of subtle difference in molecular structure, large polarity of oligosaccharide, etc. Removal, simple operation, chemically stable effect

Inactive Publication Date: 2017-06-13
DALIAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, due to the high polarity of oligosaccharides and subtle differences in molecular structure, the separation and purification technology of oligosaccharides is still challenging.

Method used

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  • Analysis method of disaccharides containing uronic acid based on reduction amine derivatization and preparation method thereof
  • Analysis method of disaccharides containing uronic acid based on reduction amine derivatization and preparation method thereof
  • Analysis method of disaccharides containing uronic acid based on reduction amine derivatization and preparation method thereof

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[0054] The invention provides an analysis method and preparation method based on derivatization of uronic acid-containing disaccharides based on reducing amines. The specific steps are as follows:

[0055] An analytical method based on derivatization of uronic acid disaccharides based on reducing amines:

[0056] S1. The polysaccharide in the sample is extracted by alcohol precipitation after enzymatic hydrolysis by double enzyme method;

[0057] S2. Dissolve the polysaccharide sample in water, treat with anion exchange resin, elute with 1.3M and 3M NaCl solution respectively, and then ultrafilter the collected eluate with ultrafiltration membrane (molecular weight cut-off: 3000-10000Da) Desalting, the intercepted part is acidic polysaccharide, and finally freeze-dried to obtain uronic acid-containing polysaccharide.

[0058] S3, dissolving the uronic acid-containing polysaccharide obtained in S2 in 1.3M trifluoroacetic acid solution, hydrolyzing at 105°C for 3 hours; adding ...

Embodiment 1

[0071] (1) Wash, shell and crush fresh green clams (Cyclina sinensis), take 10 g and place them in 100 mL of 0.05 mol / L phosphate buffer solution with pH=8, add 25 mL of 0.05 mol / L cysteine- EDTA-2Na solution, then add 0.5% (m / m) trypsin, 37 ° C water bath shaking enzymolysis for 4 hours, then add 0.5% (m / m) papain 65 ° C water bath shaking enzymolysis 3 h. Then inactivate the enzyme at 100°C for 5 min. Centrifuge at 12300×g for 20min. Take the supernatant, add 1.5 times of absolute ethanol to it, ethanol precipitate overnight, centrifuge at 7800×g for 15min, and take the precipitate. Ethanol was evaporated to dryness, and deionized water was added to redissolve, and then freeze-dried at low temperature to obtain crude polysaccharide.

[0072] (2) Accurately weigh 2.0g of crude polysaccharide dry powder and dissolve in 50mL water, handle with strong anion exchange resin, elute with the NaCl solution of 1.3M and 3M respectively, then use ultrafiltration membrane (molecular we...

Embodiment 2

[0082] (1) Wash, shell and crush fresh green clams (Cyclina sinensis), take 10 g and place them in 100 mL of 0.05 mol / L phosphate buffer solution with pH=8, add 25 mL of 0.05 mol / L cysteine- EDTA-2Na solution, then add 0.5% (m / m) trypsin, 37 ° C water bath shaking enzymolysis for 4 hours, then add 0.5% (m / m) papain 65 ° C water bath shaking enzymolysis 3 h. Then inactivate the enzyme at 100°C for 5 min. Centrifuge at 12300×g for 20min. Take the supernatant, add 1.5 times of absolute ethanol to it, ethanol precipitate overnight, centrifuge at 7800×g for 15min, and take the precipitate. Ethanol was evaporated to dryness, and deionized water was added to redissolve, and then freeze-dried at low temperature to obtain crude polysaccharide.

[0083] (2) Accurately weigh 2.0g of crude polysaccharide dry powder and dissolve in 50mL water, handle with strong anion exchange resin, elute with the NaCl solution of 1.3M and 3M respectively, then use ultrafiltration membrane (molecular we...

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Abstract

The invention discloses an analysis method of disaccharides containing uronic acid based on reductive amine derivatization. The analysis method comprises the following specific steps: extracting polysaccharides in animal tissue and collecting polysaccharides containing uronic acid; carrying out acid hydrolysis to obtain disaccharide products; adding a reduction amine reagent solution, preparing reduction amine derivatives of disaccharides of polysaccharides containing uronic acid, detecting by using a high performance liquid chromatography-multistage mass spectrometry, and indirectly achieving qualitative analysis on the polysaccharides containing uronic acid. The invention also provides a preparation method of the disaccharides containing uronic acid based on reductive amine derivatization. The method is suitable for analyzing and preparing disaccharides of two types of hexuronic acid-hexosamine and hexuronic acid-hexose; acid degradation is adopted; compare with enzymatic degradation, acid hydrolyzation is low in cost and simple to operate, and has degradation effects on various polysaccharides; ammonium hydroxide is used for replacing common NaOH; ammonium hydroxide is volatilizable, is easy to remove, and can facilitate the mass spectrometry analysis in the later period.

Description

technical field [0001] The invention relates to an analysis and preparation method of disaccharides, and more specifically, to an analysis method and preparation method of disaccharides containing uronic acid based on derivatization of reducing amines. Background technique [0002] There are some uronic acid-containing polysaccharides in animal tissues, which are composed of uronic acid and hexosamine or hexose alternately linked. Glycosaminoglycans such as heparin, chondroitin sulfate, and hyaluronic acid are composed of repeating disaccharide fragments linked by uronic acid and hexosamine. In addition, in some shellfish and other organisms, polysaccharides composed of repeated disaccharide fragments connected by uronic acid and hexose, such as abalone gonad sulfate polysaccharide AGSP, etc., have also been found. Studies have confirmed that these uronic acid-containing polysaccharides have multiple functional activities. Due to their significant functional activity, some...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H7/033C07H1/00C08B37/00G01N30/02
CPCC07H7/033C07H1/00C08B37/0003G01N30/02
Inventor 宋爽刘斌艾春青温成荣孙玉姣鲁姣姣张豹曹春阳
Owner DALIAN POLYTECHNIC UNIVERSITY
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