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Method for identifying uronic acid-containing polysaccharide in biological tissue

A technology of biological tissue and alkydose polysaccharides, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of difficult wide application, expensive degrading enzymes and oligosaccharide standard products, and difficult to accurately identify types, so as to avoid separation and purification The effect of the process

Inactive Publication Date: 2015-04-29
DALIAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some chromatographic methods, such as electrophoresis, gel filtration chromatography, etc., can separate polysaccharides according to molecular weight and charge, but it is difficult to accurately identify their species
It is a shortcut to analyze the oligosaccharide fragments produced by degradation to determine the type of polysaccharide, but only a few polysaccharides with corresponding degrading enzymes have applied this method, such as heparin and chondroitin sulfate, but the prices of degrading enzymes and oligosaccharide standard products are all low. Very expensive and difficult to apply widely

Method used

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  • Method for identifying uronic acid-containing polysaccharide in biological tissue
  • Method for identifying uronic acid-containing polysaccharide in biological tissue
  • Method for identifying uronic acid-containing polysaccharide in biological tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] (1) Prepare a series of TFA solutions containing 2mg / mL chondroitin sulfate and 2mg / mL abalone polysaccharide AGSP, the TFA concentrations are 0.10, 0.30, 0.90, 1.30, 1.70mol / L, hydrolyze at 110℃ for 2h ;

[0041] (2) Prepare a series of 1mol / L TFA solutions containing 2mg / mL chondroitin sulfate and 2mg / mL abalone polysaccharide AGSP, and hydrolyze them at 90°C, 100°C, 110°C, 120°C, and 130°C for 2 hours;

[0042] (3) Prepare a series of 1mol / L TFA solutions containing 2mg / mL chondroitin sulfate and 2mg / mL abalone polysaccharide AGSP, and hydrolyze them at 110°C for 1h, 2h, 3h, 4h, 5h respectively;

[0043] (4) Use L 4 (2 3 ) Orthogonal table Orthogonal experiments were performed on TFA concentration (0.9mol / L1.3mol / L), acid hydrolysis temperature (105°C, 115°C), acid hydrolysis time (2h, 3h).

[0044] (5) Add 1 mL of water to the acid hydrolyzate obtained in steps (1)-(4), remove TFA by rotary evaporation, and repeat three times to achieve the purpose of acid remova...

Embodiment 2

[0055] (1) Take fresh equilateral shallow clams, square clams, Chinese clams, razor clams, and mussels with thick shells, remove the shells and viscera, and homogenize. Weigh 15g of the homogenized raw materials and put them in 75mL of KH with pH=8. 2 PO 4 In the buffer solution, add 0.5% trypsin to digest for 4 hours, then add 0.5% papain to digest for 3 hours, then inactivate the enzyme with boiling water at 100°C for 5 minutes; centrifuge the hydrolyzate at 10,000 r / min for 20 minutes to take the supernatant solution; add 1.5 times the volume of ethanol to the supernatant for alcohol precipitation overnight; after alcohol precipitation, centrifuge at 4000r / min for 15min to collect the precipitate, and freeze-dry the precipitate to obtain crude polysaccharide dry powder.

[0056] (2) Accurately weigh 50 mg of crude polysaccharide dry powder into a 5 mL hydrolysis tube, and add 1 mL of 1.3 mol / L trifluoroacetic acid to the hydrolysis tube for hydrolysis at 105 °C for 3 hours;...

Embodiment 3

[0062] (1) Take 1 g of sea cucumber boiled liquid freeze-dried powder in 25 mL of KH with pH=8 2 PO 4 In the buffer solution, add 0.5% trypsin to digest for 4 hours, then add 0.5% papain to digest for 3 hours, then inactivate the enzyme with boiling water at 100°C for 5 minutes; centrifuge the hydrolyzate at 10,000 r / min for 20 minutes to take the supernatant solution; add 1.5 times the volume of ethanol to the supernatant for alcohol precipitation overnight; after alcohol precipitation, centrifuge at 4000r / min for 15min to collect the precipitate, and freeze-dry the precipitate to obtain crude polysaccharide dry powder.

[0063] (2) Accurately weigh 20 mg of crude polysaccharide dry powder into a 5 mL hydrolysis tube, and add 1 mL of 1.3 mol / L trifluoroacetic acid to the hydrolysis tube for hydrolysis at 105 °C for 3 hours; add 1 mL of water to the acid hydrolyzate, and use a rotary The evaporator removes the solvent under reduced pressure, repeats three times, and removes t...

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Abstract

The invention discloses a method for identifying uronic acid-containing polysaccharide in a biological tissue. The method comprises the following steps: 1, extracting crude polysaccharide in the biological tissue; 2, hydrolyzing the crude polysaccharide with trifluoroacetic acid, and removing acid from the obtained hydrolysate; 3, carrying out derivatization on the residues obtained from the step 2 with 1-phenyl-3-methyl-5-pyrazolone to prepare derivatives of sugar; 4, preparing a reference solution from the identified uronic acid-containing polysaccharide by the methods in the step 2 and the step 3; and 5, analyzing and extracting the derivatives of sugar from the step 3 and the reference substance from the step 4 by a liquid chromatogram-mass-spectrometric technique, and identifying the uronic acid-containing polysaccharide in the biological tissue by comparison. According to the method, the polysaccharide in the biological tissue can be directly detected; fussy separation and purification processes are avoided; the used sample amount is relatively low; and the result can be obtained within a relatively short period of time.

Description

technical field [0001] The invention relates to a method for detecting biological macromolecules, more specifically, a method for identifying polysaccharides containing uronic acid in biological tissues. Background technique [0002] Polysaccharides and their conjugates, such as glycoproteins, glycolipids, glycosaminoglycans, etc., participate in various life phenomena and physiological processes as information molecules, and they widely exist in living organisms. Studies have confirmed that many polysaccharides have species and organ specificity, and the polysaccharides in the organism are not only related to genetic factors, but also related to the physiological state of the organism. Therefore, the identification of polysaccharides can provide information for the source of biological samples, physiological state, etc. Moreover, polysaccharides are also an important indicator for evaluating food nutrition. It can not only provide energy for the human body, but may also ha...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/06
Inventor 宋爽王洪旭宋亮林心萍曹九零张立鹏朱容仟朱蓓薇
Owner DALIAN POLYTECHNIC UNIVERSITY
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