Preparation method and application of high-molecular-weight deacetylated hyaluronic acid

A technology of hyaluronic acid and high molecular weight, which is applied in the field of polysaccharide modification, can solve the problems of glycosidic bond breakage, hydrazine hydrate deacetylation effect is not very ideal, and can not achieve good deacetylation effect, etc. It takes a short time to achieve and is easy to operate Effect

Inactive Publication Date: 2015-09-16
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 bon

Method used

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  • Preparation method and application of high-molecular-weight deacetylated hyaluronic acid
  • Preparation method and application of high-molecular-weight deacetylated hyaluronic acid
  • Preparation method and application of high-molecular-weight deacetylated hyaluronic acid

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Embodiment 1

[0030] The preparation of embodiment 1 high molecular weight deacetylated hyaluronic acid

[0031] like figure 1 As 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 g of potassium hydroxide to 30 mL of hydrazine monohydrate, overnight at 4°C, take the supernatant, add 12 g of sodium hydroxide, protect with nitrogen, heat under reflux for 2 hours, change to a distillation apparatus, and collect the distillate , to obtain 90-94% of anhydrous hydrazine.

[0033] 2) Dissolving hyaluronic acid with anhydrous hydrazine containing 1% hydrazine sulfate by weight, and reacting at 60°C for 72h after deoxygenation nitrogen protection;

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

Embodiment 2

[0037] Example 2 Proliferation of polysaccharide spleen lymphocytes

[0038] 1) Acquisition of splenic lymphocytes: Take 5 B6 mice, kill them with nitrogen, soak them in 70% alcohol for 10 minutes, open the abdominal cavity of the mice under aseptic conditions to take the spleens, grind them through a 70-micron mesh with the inner core of a syringe, PBS The spleen cell suspension was collected and divided into five tubes, centrifuged at 1000 r / min for 5 min, and the supernatant was discarded. Add 12 mL of ammonium chloride erythrocyte lysate to each centrifuge tube, mix spleen cells evenly, let stand for 5-6 min, until the erythrocytes are completely broken, centrifuge at 1000 r / min for 5 min, discard the supernatant to remove the erythrocytes, wash 1-2 times with PBS, and rinse with RPMI -1640 (containing 10% fetal bovine serum) to resuspend the cells, count and adjust the cell concentration to 8-10×10 6 pcs / mL;

[0039] 2) Drug-stimulated splenocyte proliferation experimen...

Embodiment 3

[0042] Example 3 Using TLR2- / - and TLR4- / - deficient mice to do splenocyte proliferation experiment

[0043] 1) The acquisition of spleen lymphocytes was the same as that 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) According to the absorbance plot, calculate the proliferation rate, 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 show that the proliferation of splenic lymphocytes by HA-72 is dependent on the TLR4 pathway, whereas the proliferation of splenic lymphocytes by dHA-72 is 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 composed of monosaccharides linked together. The initial research on polysaccharides can be traced back to Shear's discovery of the antitumor activity of polysaccharides in 1936. In the 1950s, some fungal polysaccharides and higher plant polysaccharides were found to have obvious antitumor activity. Since the 1970s, scientists have found that polysaccharides and sugar complexes participate in and mediate the regulation of various life phenomena in cells, especially the regulation of immune function. [0003] Hyaluronic acid (HA) is an acidic polysaccharide composed of acetylglucosamine and glucuronic acid alternately linked by β-1,4 and β-1,3, and is the main component of extracellular matrix and in...

Claims

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Application Information

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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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