Preparation method of unsaturated hyaluronic acid odd oligosaccharides

A technology of hyaluronic acid and hyaluronidase, applied in the field of biomedicine, can solve the problems of low reaction efficiency, complex process, non-single reaction product structure, etc., and achieve the effects of single structure, simplified preparation process and improved preparation efficiency.

Active Publication Date: 2019-05-03
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the above two methods can successfully prepare HA odd-numbered oligosaccharides, there are the following disadvantages: 1. Both of these two methods use CH3COONH4 as the enzymatic hydrolysis working solution. CH3COONH4 will affect the subsequent degradation process, and it will take time to 2. The GlcNAc at the reducing end of HA oligosaccharides falls off under acidic or alkaline conditions, thereby obtaining HA odd-numbered oligosaccharides, but the reaction efficiency is low; 3. Degradation by-products-isomers will also appear during the reaction. And it cannot be separated by existing separation technology, and the structure of the reaction product is not single
Therefore, the whole process of preparing HA odd-numbered oligosaccharides takes a long time, the process is relatively complicated, and the yield is low, so it has not been widely used and paid attention to.

Method used

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  • Preparation method of unsaturated hyaluronic acid odd oligosaccharides
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  • Preparation method of unsaturated hyaluronic acid odd oligosaccharides

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Embodiment 1

[0037] Step 1) Prepare the hyaluronic acid (HA) solution with a concentration of 10 mg / mL from the lyophilized powdered hyaluronic acid (HA) with pure water, and prepare the lyophilized powdered microbial hyaluronidase with an enzymatic hydrolysis working solution 200IU / mL hyaluronidase solution;

[0038] Further, the enzymolysis working solution is pure water;

[0039] Further, the microbial hyaluronidase is derived from Streptomyces hyalurolyticus;

[0040] Step 2) Take 5 parts of the hyaluronic acid (HA) solution, add 1 part of the hyaluronidase solution under ice bath conditions, shake and mix, and incubate in a 37°C water bath for 24 hours to prepare a sample; The sample was moved to a water bath at 100°C, heated for 5 minutes, centrifuged, and precipitated;

[0041] Step 3) using a low-pressure chromatography column (2.6 × 100cm) with a filler of Bio-gel P10 to separate and purify the sample after deprecipitation, wherein the mobile phase concentration is 0.2M sodium chl...

Embodiment 2

[0044] Step 1) Prepare lyophilized powdered hyaluronic acid (HA) with pure water to form a hyaluronic acid (HA) solution with a concentration of 8 mg / mL, and prepare lyophilized powdered microbial hyaluronidase with enzymatic hydrolysis working solution 150IU / mL hyaluronidase solution;

[0045] Further, the enzymolysis working solution is pure water;

[0046] Further, the microbial hyaluronidase is derived from Streptomyces hyalurolyticus;

[0047] Step 2) Take 8 parts of the hyaluronic acid (HA) solution, add 2 parts of the hyaluronidase solution under ice bath conditions, shake and mix, and incubate in a 37°C water bath for 30 hours to prepare a sample; The sample was moved to a water bath at 100°C, heated for 7 minutes, centrifuged, and precipitated;

[0048] Step 3) using a low-pressure chromatography column (2.6 × 100cm) filled with Bio-gel P6 to separate and purify the sample after deprecipitation, wherein the mobile phase concentration is 0.1M potassium chloride, and ...

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Abstract

The invention discloses unsaturated odd-numbered hyaluronan oligosaccharides and a preparation method thereof. A series of unsaturated odd-numbered hyaluronan oligosaccharides are prepared from hyaluronic acid by enzymatic hydrolyzing with microbial hyaluronidase, separating and purifying with a low-pressure chromatographic column and desalting with size exclusion chromatography finally, the molecular formula of the unsaturated odd-numbered hyaluronan oligosaccharides is C6+14nH8+21nO6+11nNn, the molecular weight is 176.0320 plus (379 .1146)n Da, and the polymerization degree is 2n plus 1, wherein n is larger than or equal to 1 and smaller than or equal to 5. The preparation method is simple and convenient to operate, consumes shorter time and is high in sugar yield, the purity reaches 100%, and the defect that the series of unsaturated odd-numbered hyaluronan oligosaccharides cannot be prepared rapidly with the conventional synthesis and degradation technologies is overcome. The prepared unsaturated odd-numbered hyaluronan oligosaccharides provide important value in further study of structure-activity relationship of the hyaluronic acid with low polymerization degree and guidance of the application of the hyaluronan oligosaccharides in the fields of clinics, food, cosmetics and the like in future.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a preparation method of unsaturated hyaluronic acid odd-numbered oligosaccharides (that is, unsaturated hyaluronic acid odd-numbered oligosaccharides whose reducing and non-reducing ends are glucuronic acid). Background technique [0002] Hyaluronic acid (HA) is a linear macromolecule composed of D-glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) disaccharide units linked alternately by β-1,3 and β-1,4 glycosidic bonds Acid mucopolysaccharides. HA is widely distributed in extracellular matrix, soft connective tissue and body fluids. HA is involved in many pathological and physiological processes by regulating cellular activities. Hyaluronic acid has good hydrophilicity, viscoelasticity, and lubricity, and is widely used in clinical, cosmetic, food and other fields. [0003] It has been reported that HA with different molecular weights have different physicochemical...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H7/033C07H1/06C12P19/26C08B37/08
CPCC07H1/06C07H7/033C08B37/0072C12P19/26
Inventor 张真庆陶蕾李笃信
Owner SUZHOU UNIV
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