US-12618813-B2 - Method for detecting glycosidic bonds of plant polysaccharides

US12618813B2US 12618813 B2US12618813 B2US 12618813B2US-12618813-B2

Abstract

The present invention relates to a method for detecting glycosidic bonds of plant polysaccharides. The method includes taking 100 μL of NaOH-DMSO suspension (1 mg/mL) of plant polysaccharides containing 20 or more kinds of substances such as galactan, araban, polygalacturonic acid, rhamnogalacturonan-I, rhamnogalacturonan-II, xyluronic acid, arabogalacturonic acid I and arabogalacturonic acid II, shaking at room temperature for 30 minutes, adding 40 μL of iodomethane and shaking for 1 hour. Such process is repeated three times to obtain methylated plant polysaccharides. The method further includes using an automatic detection equipment for methylation combined with PMP derivatization to analyze glycosidic bonds in polysaccharides. Not only the glycosidic bond information of neutral sugars can be analyzed in the subsequent LC-MS mass spectrometry analysis, but also the glycosidic bond information of acidic sugars can be resulted, and the analysis results are faster, more detailed and more reliable.

Inventors

Shiguo Chen
Kai Zhu
Xingqian YE
Zhiqiang Hou
Jinghua Wu

Assignees

ZHEJIANG UNIVERSITY

Dates

Publication Date: 20260505
Application Date: 20230927
Priority Date: 20230306

Claims (2)

1 . A method for detecting glycosidic bonds of plant polysaccharides, comprising steps of: (a) performing methylation of the plant polysaccharides; (b) derivatizing glycosidic residues of the methylated plant polysaccharides with PMP reagent (1-phenyl-3-methyl-5-pyrazolone); and (c) performing mass spectrometry analysis by LC-MS; and wherein the steps (a)-(c) comprise steps of: (1) dispersing the plant polysaccharides in 100 μL of NaOH-DMSO suspension to a concentration of 1 mg/mL and shaking at room temperature for 30 minutes, and adding 40 μL of iodomethane and shaking for 1 hour; repeating three times to obtain the methylated and substituted polysaccharides; (2) placing the methylated and substituted polysaccharides in an ice bath, adding 500 μL of water of 0° C. to terminate reaction, and standing for 10 minutes; (3) adding 800 μL of dichloromethane and shaking for 30 seconds, and removing supernatant; (4) repeating steps (2) and (3) three times; then drying a lower organic layer with nitrogen to obtain partially methylated glycoside residues; (5) adding 100 μL of 2 M trifluoroacetic acid to a product resulted from step (4), hydrolyzing at 100° C. for 8 hours, and blow drying with nitrogen after hydrolysis; (6) adding 100 μL of 0.2 M PMP methanol solution to a product resulted from step (5), and heating in a 70° C. water bath for 30 minutes to obtain partially methylated PMP-derived glycoside residues; and (7) taking a product resulted from step (6) out, cooling at room temperature, drying in vacuum, dissolving in 1 mL of 70% methanol aqueous solution, and analyzing qualitatively and quantitatively by LC-MS in MRM mode.
2 . A method for detecting glycosidic bonds of plant polysaccharides, comprising steps of: (a) performing methylation of the plant polysaccharides; (b) derivatizing glycosidic residues of the methylated plant polysaccharides with PMP reagent (1-phenyl-3-methyl-5-pyrazolone); and (c) performing mass spectrometry analysis by LC-MS; and wherein the steps (a)-(c) comprise steps of: (1) dispersing the plant polysaccharides in 100 μL of NaOH-DMSO suspension to a concentration of 1 mg/mL and shaking at room temperature for 30 minutes, and adding 40 μL of iodomethane and shaking for 1 hour; repeating three times to obtain the methylated and substituted polysaccharides; (2) placing the methylated and substituted polysaccharides in an ice bath, adding 500 μL of water of 0° C. to terminate reaction, and standing for 10 minutes; (3) adding 800 μL of dichloromethane and shaking for 30 seconds, and removing supernatant; (4) repeating steps (2) and (3) three times; then drying a lower organic layer with nitrogen to obtain partially methylated glycoside residues; (5) adding 100 μL of 2 M trifluoroacetic acid to a product resulted from step (4), hydrolyzing at 100° C. for 8 hours, and blow drying with nitrogen after hydrolysis; (6) adding 100 μL of 0.2 M PMP methanol solution to a product resulted from step (5), and heating in a 70° C. water bath for 30 minutes to obtain partially methylated PMP-derived glycoside residues; and (7) taking a product resulted from step (6) out, cooling at room temperature, drying in vacuum, dissolving in 1 mL of 70% methanol aqueous solution, and analyzing qualitatively and quantitatively by LC-MS in MRM mode, and wherein the plant polysaccharides are citrus pectin or araban.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority of Chinese Patent Application No. 202310235420.7 filed on Mar. 6, 2023, the entire contents of which are hereby incorporated by reference. TECHNICAL FIELD The invention relates to a method for detecting glycosidic bonds of plant polysaccharides. DESCRIPTION OF THE PRIOR ART Plant polysaccharide, an important component of plant cell wall, is a kind of natural high molecular polymer formed by aldose or ketose linked by glycosidic bonds. The rich biological activity of polysaccharide has a close relation to its complex structure. Polysaccharide structure includes molecular weight and distribution, monosaccharide composition and molar ratio, glycosidic bond connection mode, repeating structural unit and branching degree, etc. There are many methods to characterize the structure of polysaccharide, including chemical methods, instrumental analysis methods, etc. However, the structure of polysaccharide is complex and requires a combination of multiple methods for analysis. Glycosidic bond is a specific type of chemical bond that connects an aglycon to a sugar group or a sugar group to a sugar group in a glycoside molecule. Analyzing the glycosidic bonds of plant polysaccharides is an important step for analyzing the fine structure of polysaccharides. Common chemical analysis methods include methylation derivatization, acid hydrolysis, etc., using gas chromatography-mass spectrometry (GC-MS) for analysis. However, such methods have problems such as time-consuming, cumbersome operation, and loss of fine structure. LC-MS detection (liquid chromatography-mass spectrometry detection) is a cutting-edge technology in the field of sugar analysis. It has the advantages of high separating capacity, high sensitivity, high efficiency, and small sample volume, and realizes the separation and fine structure identification of complex oligosaccharides, showing great potential in the study of the fine structure of polysaccharides. By combining methylation with LC-MS technology, it is possible to quickly and efficiently determine the glycosidic bonds of plant polysaccharides in a micro-volume. However, the existing data show that it can only analyze the glycosidic bond information of neutral sugars, but cannot give the glycosidic bond information of acidic sugars. SUMMARY OF THE DISCLOSURE The purpose of the present invention is to address the deficiencies in the prior art, to provide a method for detecting glycosidic bonds of plant polysaccharides to comprehensively analyze more than 90 glycosidic residues, cover all glycosidic bond types in plant polysaccharides, especially to give the glycosidic bond information of acidic sugars, and the analysis results are more authentic and reliable. The present invention adopts the following technical scheme: a method for detecting glycosidic bonds of plant polysaccharides, comprising the following steps: (1) performing rapid methylation of the plant polysaccharides in a micro-volume;(2) derivatizing glycosidic residues of the methylated plant polysaccharides with PMP reagent (derivatization reagent); and(3) performing mass spectrometry analysis by LC-MS in MRM mode. Further, the method includes the following steps: (1) dispersing the plant polysaccharides in 100 μL of NaOH-DMSO suspension to a concentration of 1 mg/mL and shaking at room temperature for 30 minutes, and adding 40 μL of iodomethane and shaking for 1 hour; repeating three times to obtain the methylated and substituted polysaccharides;(2) placing the methylated and substituted polysaccharides in an ice bath, adding 500 μL of water of 0° C. to terminate reaction, and standing for 10 minutes;(3) adding 800 μL of dichloromethane and shaking for 30 seconds, and removing supernatant;(4) repeating steps (2) and (3) three times; then drying a lower organic layer with nitrogen to obtain partially methylated glycoside residues;(5) adding 100 μL of 2 M trifluoroacetic acid to a product resulted from step (4), hydrolyzing at 100° C. for 8 hours, and blow drying with nitrogen after hydrolysis;(6) adding 100 μL of 0.2 M PMP methanol solution to a product resulted from step (5), and heating in a 70° C. water bath for 30 minutes to obtain partially methylated PMP-derived glycoside residues; and(7) taking a product resulted from step (6) out, cooling at room temperature, drying in vacuum, dissolving in 1 mL of 70% methanol aqueous solution, and analyzing qualitatively and quantitatively by LC-MS in MRM mode. Further, the plant polysaccharide is citrus pectin, as citrus pectin has a complex structure and diverse composition, and is representative. By subjecting the citrus pectin to methylation derivation with the present inventive method, it can be concluded that the present inventive method has a general applicability for plant polysaccharides. The plant polysaccharide can also use apple pectin, mulberry polysaccharide, pomelo peel pectin, wolfberry polysaccharide,