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Purification process of bupleurun chinense polysaccharide fragment and application thereof in preparation of anti-inflammatory drug

A kind of Bupleurum polysaccharide and fragment technology, which is applied in the field of natural medicinal chemistry and achieves the effect of good application prospect

Active Publication Date: 2016-06-29
WENZHOU UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, research at home and abroad has mainly focused on the pharmacodynamic function of saikosaponin, while there are few research reports on polysaccharides from Bupleurum chinensis, especially the purification of polysaccharide fragments that have the function of antagonizing P-selectin in Bupleurum chinensis and their Drug use not reported

Method used

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  • Purification process of bupleurun chinense polysaccharide fragment and application thereof in preparation of anti-inflammatory drug
  • Purification process of bupleurun chinense polysaccharide fragment and application thereof in preparation of anti-inflammatory drug
  • Purification process of bupleurun chinense polysaccharide fragment and application thereof in preparation of anti-inflammatory drug

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Preparation of Bupleurum chinense polysaccharide fragments that antagonize the function of P-selectin:

[0017] (1) Separation and extraction of total polysaccharides from Bupleurum chinense. The dried Bupleurum humilis was crushed and sieved. After degreasing with ethanol reflux, the residue was leached in hot water at 60~90℃ for 3 to 5 times, each extraction was carried out for 2 to 4 hours, and the filtrate was combined by suction and filtered. Concentrate under reduced pressure at ~70℃ and vacuum degree -0.03~-0.09MPa, add 2~5 times volume of absolute ethanol for precipitation, let stand overnight at low temperature, centrifuge, precipitate with distilled water to dissolve, repeat the steps 2~5 of ethanol precipitation again Second, the last obtained precipitate was rinsed with ethanol, acetone, ether and dried under reduced pressure, and then dissolved in distilled water. After repeated freezing and thawing, the supernatant was digested with streptomycin for 4 to 24 h...

Embodiment 2

[0021] Bupleurum polysaccharide fragments antagonize the adhesion of P-selectin to HL-60 cells: flow cytometry was used to evaluate the blocking of P-selectin and its physiological ligand PSGL-1 (P-selectinglycoproteinligand-1) The binding ability. PSGL-1 is highly expressed on the surface of HL-60 cell membrane, so HL-60 cells are used as the donor of P-selectin physiological ligand in this experiment. Firstly, rhP-Fc was incubated with 10, 50, 200μg / ml Bupleurum polysaccharide fragments, and then HL-60 cell suspension was added to the reaction system to continue incubating, and then the rhP was labeled with FITC-conjugated anti-human IgG fluorescent antibody -Fc, using flow cytometry to detect the average fluorescence intensity and percentage of positive cells in each experimental group. The results show that the polysaccharide fragment of Bupleurum chinense can effectively block the binding of rhP-Fc to HL-60 cells. The inhibitory effects of the low, medium and high dose gr...

Embodiment 3

[0023] Bupleurum polysaccharide fragment inhibits the interaction of P-selectin and PSGL-1: In vitro protein interaction analysis was used to evaluate the ability of polysaccharide fragment of Bupleurum fragrans to antagonize the interaction between P-selectin and PSGL-1. After the rhP-Fc is bound to the SepharoseProteinA solid phase matrix, it is fully incubated with Bupleurum chinense polysaccharide fragment, and the solid phase matrix is ​​incubated with the HL-60 cell membrane lysate. After repeated washing with PBS, the P- The PSGL-1 co-precipitated with selectin was analyzed to evaluate the ability of the polysaccharide fragment of Bupleurum chinense to block the binding of P-selectin to PSGL-1. Experimental results show that P-selectin can firmly bind to PSGL-1 expressed by HL-60 cells, and the intervention of Bupleurum chinense polysaccharide fragment can effectively block the interaction between P-selectin and PSGL-1. When it is 50 and 200μg / ml, the blocking rate is 66...

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Abstract

The invention belongs to the field of natural medicinal chemistry and particularly relates to a separation and purification process of natural polysaccharide type anti-inflammatory drug molecules and application thereof. The separation and purification process utilizes water extraction and alcohol sedimentation, protein removal, acid hydrolysis, freeze drying, desalination, affinity chromatography and other methods to purify a polysaccharide fragment having a P-selectin antagonism function from bupleurun chinense. Flow cytometry and protein interaction analysis experiment results show that the bupleurun chinense polysaccharide fragment can effectively prevent the combination between P-selectin and HL-60 cells and the antagonism to the mutual effect of the P-selectin and PSGL-1. The experimental results of a parallel-plate flow chamber and an acute abdominal inflammation mouse model show that the bupleurun chinense polysaccharide fragment has the remarkable inhibitory effect on the HL-60 cells mediated by the P-selectin in a shear stress state and can effectively inhibit leukocytic exudation from the peritoneal fluid of a mouse. The bupleurun chinense polysaccharide fragment prepared by means of the method has good anti-inflammatory activity and has a broad application prospect by serving as a novel anti-inflammatory drug.

Description

Technical field [0001] The invention belongs to the field of natural medicinal chemistry, and particularly relates to the separation and purification of a natural anti-inflammatory drug molecule with specific targeting and its application. Background technique [0002] The inflammatory response plays a very important role in the body’s defense against pathogen invasion and repair of its own damaged tissues. It is highly finely regulated by the immune system. A moderate inflammatory response can mobilize the body’s defense forces to overcome the damage to the body. Abnormal ability will cause a series of uncontrollable inflammatory reactions. Recent studies have found that the large and continuous infiltration of white blood cells into tissues is a variety of acute and chronic inflammatory diseases, such as psoriasis, systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, chronic obstructive pneumonia, and atherosclerosis. The prevention and treatment of the above-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00A61K31/715A61P29/00
CPCA61K31/715C08B37/0003
Inventor 佟海滨孙新
Owner WENZHOU UNIVERSITY
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