Application of marine-derived oligoguluronic acid

A technology of guluronic acid and source, applied in the field of biomedicine, can solve problems such as less reports on oligomer activity

Active Publication Date: 2014-10-08
DALIAN SHENLAN PEPTIDE TECH R & D CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzymatic oligosaccharides have many important biological activities such as anti-tumor, anti-inflammatory, and immune-enhanci

Method used

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  • Application of marine-derived oligoguluronic acid
  • Application of marine-derived oligoguluronic acid
  • Application of marine-derived oligoguluronic acid

Examples

Experimental program
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preparation example Construction

[0020] Preparation of oligomeric guluronic acid from marine sources

[0021] Sodium alginate (sodium alginate) is a water-soluble polysaccharide derived from the cell wall of brown algae. It is a linear acidic polysaccharide formed by connecting D-mannuronic acid and L-guluronic acid through 1→4 glycosidic bonds. The marine-derived oligoguluronic acid (marine-derived oligoguluronic acid, MdOA) of the present invention is obtained from brown algae-derived sodium alginate through H 2 o 2 Cracked, the structure is as follows:

[0022]

Embodiment 1

[0023] Example 1 Detection of NO generation in cells by Griess method

[0024] RAW264.7 cells (2×10 5 per well) adhered to the wall in a 96-well plate, discarded the supernatant after 4-6h, added 1mg / ml of MdOA, used serum-free RPMI1640 medium as the negative control group (Con), and added only 1μg / ml LPS as the negative control group (Con). Positive control group (LPS), with the addition of 1 μg / ml LPS and different concentrations of MdOA (0.1mg / ml, 0.5mg / ml, 1.0mg / ml, 2.0mg / ml) as the experimental group (LPS (1μg / ml) + MdOA), after stimulation for 24h, the production of NO in the medium was detected by the Griess method.

[0025] Such as Figure 1A As shown, the CCK-8 cell viability detection kit showed that compared with the Con group, LPS and different concentrations of MdOA had no toxicity to the cells.

[0026] In the concentration gradient experiment, different concentrations of MdOA (0.1mg / ml, 0.5mg / ml, 1.0mg / ml, 2.0mg / ml) stimulated RAW264.7 cells for 24 hours, and ...

Embodiment 2

[0027] Example 2 Detection of the production of inflammation-related genes in cells by reverse transcription method (RT-PCR)

[0028] RAW264.7 cells (2×10 6 Each well) attached to the wall in a 6-well plate, after 4-6 hours, discard the supernatant, add different concentrations of MdOA, use serum-free RPMI1640 medium as the negative control group (Con), and add only 1 μg / ml LPS as positive For the control group (LPS), after being stimulated for 12 hours, total RNA was extracted according to the total RNA cell rapid extraction kit. Then, the total RNA was reverse-transcribed into cDNA by a reverse transcription kit, and then the genes of iNOS and COX-2 were obtained by PCR.

[0029] iNOS primers are: FE: 5'-CAACCAGTATTATGGCTCCT-3',

[0030] RE: 5'-GTGACAGCCCGGTCTTTCCA-3';

[0031] Primer for β-actin: FE: 5'-GGAGAAGATCTGGCACCACACC-3';

[0032] RE: 5'-CCTGCTTGCTGATCCACATCTGCTGG-3');

[0033] COX-2 primer: FE5'-CCACTTCAAGGGAGTCTGGA-3';

[0034] ...

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Abstract

The invention relates to the field of biological medicine, and discloses an application of marine-derived oligoguluronic acid. MdOA obtained by oxidation and degradation of sodium alginate can inhibit excessive NO production by an RAW264.7 cell induced by LPS, and the inhibition effect is concentration-dependent. According to the application, by adoption of an RT-PCR method, it is found that over-expressed mRNAs of iNOS and COX-2 in the RAW264.7 cell are reduced along with the increase of MdOA stimulation time and the relationship is concentration-dependent. By adoption of a Western Blot method, it is found that overexpression of iNOS protein and COX-2 protein can be inhibited by the MdOA. By adoption of an ELISA method, it is found that the MdOA can inhibit expression of intracellular related inflammatory cytokines comprising TNF-alpha, IL-6, IL-1beta, and the like. Therefore, the marine-derived oligoguluronic acid MdOA can be used for preparation of anti-inflammatory medicines.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of marine source oligomeric guluronic acid in the preparation of anti-inflammatory drugs. Background technique [0002] Inflammation is an extremely common and important pathological process, which has been proven to be closely related to many acute and chronic diseases, such as obesity, diabetes, atherosclerosis, hypertension, stroke, tumor, polycystic ovary syndrome, dementia, etc. Anti-inflammatory therapy is one of the research hotspots in immunotherapy. Oligosaccharides have been proven to have various important biological activities such as anti-tumor, anti-inflammatory, and immune-enhancing effects. In recent years, with the remarkable progress in the determination of oligosaccharide structure and biological activity research, the research on oligosaccharides and carbohydrate complexes has attracted more and more attention in the world. [0003] Sodium alginate...

Claims

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Application Information

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IPC IPC(8): A61K31/734A61K31/702A61P29/00A23L1/29A23L33/00
Inventor 续旭史旭阳周瑞王青青张晗雪万敏
Owner DALIAN SHENLAN PEPTIDE TECH R & D CO LTD
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