A clam polysaccharide with immune-enhancing activity, its preparation method and application

A technology for enhancing immunity and green clams, applied in the application field of functional food or medicine, can solve problems such as limited sources

Active Publication Date: 2021-03-09
DALIAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sources of polysaccharides with immunological activity are extremely limited at present. Therefore, it is urgent to develop new polysaccharides with high immune activity to solve the urgent need

Method used

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  • A clam polysaccharide with immune-enhancing activity, its preparation method and application
  • A clam polysaccharide with immune-enhancing activity, its preparation method and application
  • A clam polysaccharide with immune-enhancing activity, its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] A preparation method of clam polysaccharide with enhanced immune activity, comprising the steps of:

[0066] S1, preparation of defatted powder: take fresh green clam meat, freeze-dry (cold trap temperature-60 ℃, vacuum degree 1~10Pa), described green clam meat freeze-drying time ≥ 4h, break into green clam powder (300g); Add 1000mL of acetone to 300g of green clam powder, stir with a magnetic stirrer at 100rpm for 30min, remove the upper layer of acetone after standing for 24h, and dry the resulting precipitate in a fume hood until the mass is constant to obtain a defatted powder (280g);

[0067] S2, enzymatic hydrolysis: take 280g of degreasing powder described in step S1, add 700mL, 0.05mol / L cysteine-EDTA disodium solution and 5740mL 0.05mol / L, pH=8 K 2 HPO 4 Buffer solution, then add 2.8g 250U / mg trypsin, 37°C water bath, 100rpm shaking enzymolysis for 4h, to get mixed solution 1; add 2.8g 2000U / mg papain to the mixed solution 1, 65°C water bath, 100rpm shaking en...

Embodiment 2

[0081] This example is used to evaluate the toxicity analysis of clam polysaccharide CSP prepared in Example 1 to cells. The specific experimental method is as follows:

[0082] Take 100 μL logarithmic growth phase density of 50 × 10 4 Individual / mL RAW 264.7 cells were inoculated in 96-well culture plates, and the complete medium added by the 7 dose treatment groups (the complete medium was: RPMI-1640 medium (VWR, 45000-604) contained 1% (v / v) The concentrations of CSP in the penicillin-streptomycin mixed solution (Corning, MT30002CI), 10% (v / v) premium fetal bovine serum (VWR, 340B17) are 0.8mg / mL, 0.5mg / mL, 0.2mg respectively / mL, 0.1mg / mL, 0.05mg / mL, 0.02mg / mL and 0.01mg / mL, the positive control contains 5μg / mL LPS complete medium, the volume of the medium containing or not containing CSP in each well is 200μL, Set up 6 replicate wells for each group; at 37°C, 5% CO 2 After incubating in the incubator for 24 hours, add 200 μL of MTT (0.5 mg / mL) phosphate buffer solutio...

Embodiment 3

[0085] Activated macrophages secrete NO is an important way to kill tumor cells and pathogenic microorganisms. Polysaccharides can stimulate macrophages, induce macrophages to release NO, and then affect their immune regulation. In this experiment, the Griess method was used to measure the effect of CSP on the NO production of RAW 264.7 cells. The specific operation method is as follows:

[0086] Count the RAW 264.7 cells in the logarithmic growth phase, and adjust the cell suspension density to 1x10 6 / mL, add 100 μL / well into a 96-well plate, 37°C, 5% CO 2 Cultured for 24 hours to allow the cells to fully adhere to the wall; divided into 6 dose treatment groups with CSP concentrations of 0.2 μg / mL, 0.4 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL and 10 μg / mL, blank control group and positive control group , each group is provided with 6 duplicate wells; the concentration of CSP in the complete medium added by the 6 dosage treatment groups is 0.2 μg / mL, 0.4 μg / mL, 1 μg / mL, 2 μg / mL, 5 ...

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Abstract

The invention discloses a clam polysaccharide with enhanced immune activity, the main chain of the clam polysaccharide is composed of →4)-α-D-Glcp(1→, in the structure of the polysaccharide →4)Glc( 1→and→4,6)Glc(1→The ratio is 6.8:1, the molecular weight is 380KDa, and the monosaccharide composition is mannose:glucose=1:35; using green clam as raw material, after degreasing, enzymatic hydrolysis, complex combined precipitation, alcohol precipitation to remove impurities, and DEAE-cellulose column purification to obtain clam polysaccharide; the obtained clam polysaccharide can promote the production of NO by RAW 264.7 cells, and the clam polysaccharide is within the range of 1 to 10 μg / mL It can significantly improve the NO secretion ability of RAW 264.7 cells and is positively correlated with the dose; the clam polysaccharide can be used as an important source for the development of immunomodulatory drugs and functional foods, and provides theoretical basis and practical guidance for the high-value transformation of marine shellfish.

Description

technical field [0001] The invention relates to the technical field of active polysaccharides, in particular to a clam polysaccharide with immune-enhancing activity, an extraction method and its application in immune-enhancing functional foods or medicines. Background technique [0002] Green clam (Cyclina sinensis), also known as black clam, iron clam, round clam, clam, ox-eye clam, is widely distributed in Liaoning, Hebei, Shandong, Jiangsu, Zhejiang, Fujian, Guangdong, Guangxi and Hainan Island. The common and main edible bivalve molluscs in the north and south coasts occupy an important economic position in the tidal flat shellfish farming in my country. Green clam not only has edible value, but also has medicinal value, and is a very important marine drug; "Shen Nong's Herbal Classic" and "Compendium of Materia Medica" all include green clam as a commonly used drug. Green clam has high nutritional and medicinal value, so the development and utilization of green clam res...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/00A61K31/715A61P37/04A23L33/125
CPCA23V2002/00A61K31/715A23L33/125A61P37/04C08B37/0003C08B37/006A23V2200/324A23V2250/51
Inventor 宋爽曹春阳宫悦闫春红艾春青温成荣付颖寰董秀萍祁立波
Owner DALIAN POLYTECHNIC UNIVERSITY
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