A kind of chitosan oligosaccharide with specific structure and its preparation method and application

A chitosan oligosaccharide and a specific technology, applied in the field of chitosan oligosaccharide and its preparation, can solve problems such as differences in biological activity of chitosan oligosaccharides, differences in substrate recognition, etc., and achieve the effects of good pharmacological activity and inhibiting the growth of liver cancer cells

Active Publication Date: 2019-02-01
ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Even though the hydrolyzed substrate is the same low deacetylation degree chitosan, but because these different enzymes have differences in the recognition of the substrate, different chitosan hydrolases can be used to obtain chitosan oligosaccharides of different structural types, such as The degree of polymerization and the distribution of acetyl sites are different, and these structurally different chitosan oligosaccharides may have large differences in specific biological activities

Method used

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  • A kind of chitosan oligosaccharide with specific structure and its preparation method and application
  • A kind of chitosan oligosaccharide with specific structure and its preparation method and application
  • A kind of chitosan oligosaccharide with specific structure and its preparation method and application

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Effect test

Embodiment 1

[0032] Embodiment 1: Preparation of chitosan oligosaccharides with low deacetylation degree of different structural types

[0033] Refer to the method of Liu Dasheng et al. Acetyl chitosan, the specific method is as follows: Weigh 30g of ultra-finely pulverized chitin powder, add it to 10 times of 45% sodium hydroxide solution, stir evenly, then heat up to 60°C for 2h deacetylation reaction, and react After centrifugation, the precipitate is washed with 60% ethanol-water mixed solution until it is free of alkalinity, and the product is obtained after drying. use 1 H-NMR measures its degree of deacetylation (see figure 1 ),according to figure 1 of 1 The H-NMR spectrum confirmed that the degree of deacetylation was 62%. Two portions of prepared chitosan were weighed, 10 g each, respectively added to a constant-temperature reaction kettle filled with 200 mL of 1.5% acetic acid aqueous solution, fully stirred to make it completely dissolved. Adjust the reaction temperature t...

Embodiment 2

[0034] Embodiment 2: Composition and structural identification of different types of chitosan oligosaccharides

[0035] The chitosan oligosaccharide components in the above three chitosan oligosaccharides were identified by MALDI-TOF mass spectrometry. The specific method is: weigh three prepared or purchased chitosan oligosaccharide samples COS-62-NP, COS-62-PA and COS-MP-162, and prepare an aqueous solution with a concentration of 2 mg / mL with ultrapure water, and draw each 1 μL was applied to the sample plate, and after it was naturally dried, 1 μL of matrix 2,5-dihydroxybenzoic acid (DHB) solution was added, and after drying, the autoflexⅢ smartbeam MALDI-TOF mass spectrometer (Bruker Company) was used for detection ( positive ion reflectance mode). Mass spectrometry results such as figure 2 Shown: Mass spectra corresponding to COS-62-NP, COS-62-PA and COS-MP-162 respectively; for easy distinction, in figure 2 A stands for N-acetylglucosamine, D stands for glucosamine...

Embodiment 3

[0037] Embodiment 3: Comparison of anti-liver cancer activity of different structural types of chitosan oligosaccharides

[0038] Digest the human liver cancer cell line HepG2 cultured to the logarithmic phase and add MEM medium (10% FBS, S / P, 1% NAEE) to dilute to 1×10 4 Cells / ml, 100μl / well inoculated three 96-well cell culture plates, 37℃5%CO 2 Culture overnight in an incubator until the cells are completely attached. Three kinds of chitosan oligosaccharides (COS-62-NP, COS-62-PA and COS-MP-162) aqueous solutions (10 mg / ml) were prepared and sterilized by filtration with a 0.22 μm filter membrane in an intercellular ultra-clean bench. Dilute the three kinds of chitosan oligosaccharide aqueous solutions prepared above to 200 μg / ml with MEM medium (take 60 μl of 10 mg / ml oligosaccharide solution and add MEM medium 2940 μl to 3ml), 100 μl / well inoculates three 96-well cell culture plates ( Chitooligosaccharide final concentration is 100μg / ml), 37 ℃ 5% CO 2 Continue to grow ...

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Abstract

Provided are a chitooligosaccharide of a specific structure, a preparation method therefor and use thereof. The reducing and non-reducing ends of all components of the chitooligosaccharide are glucosamine, and the degree of deacetylation is 50% - 80%. The chitooligosaccharide may be obtained by means of enzymatic hydrolysis of a chitosan substrate, and the enzyme used is capable of specifically recognizing and hydrolyzing glycosidic bonds formed from glucosamine and glucosamine, such that the reducing and non-reducing ends of all components of the chitooligosaccharide, which are obtained by hydrolysis, are both glucosamine. Compared with chitooligosaccharides of common structures, the chitooligosaccharide of the specific structure has a higher inhibitory activity on liver cancer cells, and can be applied to the fields of prevention and adjuvant therapy of liver cancer and the like.

Description

technical field [0001] The invention belongs to the technical field of chitosan oligosaccharide application, and in particular relates to a chitosan oligosaccharide with a specific structure and its preparation method and application. Background technique [0002] Chitooligosaccharides (COS) are oligomers with a degree of polymerization less than 20 formed by connecting glucosamine and N-acetylglucosamine through β-1,4 glycosidic bonds, and have biological activities such as anti-inflammatory, anti-tumor and immune regulation. Studies have shown that the structure of chitosan oligosaccharides, including the degree of polymerization, degree of deacetylation, and the distribution of acetyl sites in oligosaccharides, plays a decisive role in its biological activity. The traditional process of preparing chitosan involves high temperature and strong alkali deacetylation of chitin, and the product chitosan has good acid solubility only when the degree of deacetylation is >80%. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/08A61K31/722A61P35/00A61P1/16C12P19/26C12P19/14
CPCA61K31/722C08B37/003C12P19/14C12P19/26
Inventor 杜昱光程功贾培媛孙明焦思明任立世冯翠
Owner ZHONGKE RUNXIN SUZHOU BIOLOGICAL TECH CO LTD
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