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A kind of preparation method and application of high molecular weight deacetylated hyaluronic acid

A molecular weight, high-efficiency liquid phase technology, applied in the field of polysaccharide modification, can solve the problems of glycosidic bond breakage, hydrazine hydrate deacetylation effect is not very ideal, high molecular weight products cannot be obtained, etc., to achieve the effect of easy operation and short time

Inactive Publication Date: 2017-08-29
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing deacetylation methods of hyaluronic acid include sodium hydroxide deacetylation and hydrazine hydrate deacetylation. Experiments have proved that the sodium hydroxide method is too alkaline, and the high concentration of sodium hydroxide agrees to cause The sharp breakage of glycosidic bonds cannot obtain high-molecular-weight products, and low-concentration sodium hydroxide cannot achieve good deacetylation effects. Similarly, the deacetylation effect of hydrazine hydrate is not ideal, and high deacetylation effects cannot be obtained. Zwitterionized hyaluronic acid with high molecular weight

Method used

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  • A kind of preparation method and application of high molecular weight deacetylated hyaluronic acid
  • A kind of preparation method and application of high molecular weight deacetylated hyaluronic acid
  • A kind of preparation method and application of high molecular weight deacetylated hyaluronic acid

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Embodiment 1

[0030] Embodiment 1 Preparation of high molecular weight deacetylated hyaluronic acid

[0031] like figure 1 Shown, a kind of preparation method of high molecular weight deacetylated polysaccharide specifically comprises the following steps:

[0032] 1) Preparation of anhydrous hydrazine: Add 12 grams of potassium hydroxide to 30 mL of hydrazine monohydrate, overnight at 4°C, take the supernatant, add 12 grams of sodium hydroxide, protect with nitrogen, heat and reflux for 2 hours, change to a distillation device, and collect the distillate , to obtain 90-94% anhydrous hydrazine.

[0033] 2) Dissolving hyaluronic acid with anhydrous hydrazine containing hydrazine sulfate at a weight concentration of 1%, reacting at 60° C. for 72 hours after deoxygenated nitrogen protection;

[0034] 3) The reaction product is precipitated with cold ethanol, and the precipitate is dissolved in glacial acetic acid with a concentration of 5%. Add iodic acid with a concentration of 5g / L, and pla...

Embodiment 2

[0037] Example 2 Proliferation of polysaccharide spleen lymphocytes

[0038] 1) Acquisition of spleen lymphocytes: Take 5 B6 mice, kill them with nitrogen gas, soak them in 70% ethanol for 10 minutes, open the abdominal cavity of the mice under aseptic conditions to take the spleen, grind them through a 70-micron sieve with the inner core of a syringe, and pour them into PBS Flush with liquid, pass through a 40-micron sieve, collect the separated splenocyte suspension and divide it into five tubes, centrifuge at 1000r / min for 5min, and discard the supernatant. Add 12mL of ammonium chloride erythrocyte lysate to each centrifuge tube, mix the spleen cells, let stand for 5-6min, wait for the erythrocytes to be completely broken, centrifuge at 1000r / min for 5min, discard the supernatant to remove the erythrocytes, wash 1-2 times with PBS, and use RPMI -1640 (containing 10% fetal bovine serum) resuspended cells, counted to adjust the cell concentration to 8~10×10 6 a / mL;

[0039]...

Embodiment 3

[0042] Example 3 Spleen Cell Proliferation Experiment Using TLR2- / - and TLR4- / - Deficient Mice

[0043] 1) The acquisition of splenic lymphocytes was the same as in Example 2, and the spleen cells of TLR2- / - and TLR4- / - and their control mice B6 and B10 were obtained respectively;

[0044] 2) Drug-stimulated splenocyte proliferation experiment: the same as in Example 2, plating, adding drugs, culturing, and detecting;

[0045] 3) Calculate the proliferation rate according to the absorbance graph, and compare whether the proliferation effect of the drug on splenocytes depends on the TLR2 or TLR4 pathway, such as Figure 5 As shown, the results showed that the proliferation of splenic lymphocytes by HA-72 was dependent on the TLR4 pathway, whereas the proliferation of splenic lymphocytes by dHA-72 was dependent on the TLR2 pathway.

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Abstract

The invention discloses a preparation method and application of high-molecular-weight deacetylated hyaluronic acid. The preparation method comprises the following steps: dissolving hyaluronic acid in anhydrous hydrazine containing 1% of hydrazine sulfate, reacting in a deoxidized nitrogen protective atmosphere at 60 DEG C for 72 hours, precipitating the reaction product with cold ethanol, dissolving the precipitate in 5% glacial acetic acid, adding 0.5M of iodic acid, standing at 4 DEG C for 2 hours, and adding excessive iodic acid into 57% hydriodic acid; and carrying out ether extraction to remove generated iodine, neutralizing, and carrying out freeze-drying to obtain the high-molecular-weight deacetylated hyaluronic acid. The zwitterion hyaluronic acid polysaccharide disclosed in the invention is used for immunological diseases, and has the advantages of obvious curative effect and no side effect.

Description

technical field [0001] The invention belongs to the technical field of polysaccharide modification, and in particular relates to a preparation method and application of high molecular weight deacetylated hyaluronic acid. Background technique [0002] Polysaccharides are polymers linked by monosaccharides. The initial research on polysaccharides can be traced back to Shear's discovery of the antitumor activity of polysaccharides in 1936. By the 1950s, it was found that some fungal polysaccharides and higher plant polysaccharides had obvious antitumor activity. Since the 1970s, scientists have discovered that polysaccharides and sugar complexes participate in and mediate the regulation of various life phenomena of cells, especially the regulation of immune function. [0003] Hyaluronic acid (Hyaluronic acid, HA) is an acidic polysaccharide formed by the alternating connection of acetylglucosamine and glucuronic acid through β-1, 4 and β-1, 3, and is the main component of extr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/08A61P37/00
Inventor 段金友张武霞李鹏王冬冬
Owner NORTHWEST A & F UNIV
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