Method for extracting collagen type II by taking agkistrodon as raw material

A technology of collagen and viper, which is applied in the field of extracting type II collagen, can solve the problems of complex operation, difficult chemical methods, and differences in results, and achieve the effect of simple operation and high purity

Inactive Publication Date: 2013-05-08
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Application Information

AI Technical Summary

Problems solved by technology

Although CⅡ is abundant in the human body, the amount available in clinical materials is limited; CⅡ forms a tight combination with proteoglycans, making it difficult to extract by simple chemical methods
The methods reported so far are complex in operation and under different conditions, resulting in different results.

Method used

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  • Method for extracting collagen type II by taking agkistrodon as raw material
  • Method for extracting collagen type II by taking agkistrodon as raw material
  • Method for extracting collagen type II by taking agkistrodon as raw material

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Embodiment 1

[0029] Methods as below:

[0030] 1. Crush Viper (purchased from Huangshan Snake Science Research Institute) into a powder with a particle size of 2 mm;

[0031] 2. Suspend 150 grams of Viper powder in 2000 mL of 4 M guanidine hydrochloride-50 mM Tris-HCl mixed solution (pH7.5), control the temperature at 4 ° C, stir for 24 h; 4 ° C, 14000 × Centrifuge at g for 1 h, remove the supernatant, wash the precipitate with 50 mM Tris-HCl solution to remove guanidine hydrochloride, centrifuge at 14,000×g for 1 h at 4°C, and remove the supernatant;

[0032] 3. The precipitate obtained in the previous step was resuspended with 20 times the volume of the precipitate in 0.5 M acetic acid, and the pH was adjusted to 2.8 with 70% formic acid, and 5 g of pepsin (purchased from Wuxi Zhongtian Food Additives Co., Ltd., unit Enzyme activity 15000U / g), stirred gently at 4°C for 48 h;

[0033] 4. Centrifuge to remove the precipitate: 4°C, 14000×g, centrifuge for 1 h, discard the precipitate (if...

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Abstract

The invention provides a method for extracting collagen type II by taking agkistrodon as a raw material. The method comprises the following steps of: removing protein polysaccharide from agkistrodon powder by a mixed guanidine hydrochloride-Tris-HCl solution; collecting precipitates after the polysaccharide is removed; dissolving the precipitates with an acetic acid solution; releasing the collagen type II from the solution by pepsase enzymolysis; centrifuging enzymatic hydrolysate to obtain supernatant, and salting out the supernatant to fully precipitate the collagen type II; centrifuging and collecting the precipitated collagen type II, and dissolving the precipitated collagen type II with the acetic acid solution again, and dialyzing the dissolved solution to remove the pepsase and other impure proteins; dissolving the collagen type II in a dialysis bag by the acetic acid solution again, and dialyzing the collagen type II to remove salt ions; and freezing the dialyzed liquid in the bag at a low temperature, and drying the liquid in vacuum to obtain the collagen type II of the agkistrodon. The method provided by the invention mainly has the beneficial effects that the operation is simple, and the purity of the obtained collage type II is high.

Description

(1) Technical field [0001] The invention relates to a method for extracting type II collagen from vipers. (2) Background technology [0002] Type II collagen (collagen type II, CII) is the main organic component of cartilage matrix and one of the main extracellular matrices in articular cartilage. Recent studies have shown that the pathogenesis of rheumatoid arthritis is related to CⅡ autoimmune response [Deyl Z, Miksik I, Eckhardt A. Preparative procedures and purity assessment of collagen proteins [J]. Chromatography B, 2003,79(12): 245-275. Smolen JS, Hayer S, Schett G, et al. Immunosenescence autoimmunity and Rheumatoid Arthritis [J]. Autoimmunity and rheumatoid arthritis, 2004, 31(1): 23-25.], some animals can be immunized with CⅡ Induced experimental arthritis [Sweeney SE, Firestein GS. Rheumatoid arthritis: regulation of synovial inflammation [J]. Intern J Biochemical Cell Bio, 2004, 36(3): 372-378.], oral CⅡ induced immune tolerance to Rheumatoid Arthritis (Rheumat...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K1/34
Inventor 丁兴红谷恒存丁志山蒋福升范永升
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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