Study On Purification, Structural Characterization And Immunoregulation Of Glycosaminoglycans From Pinctada Martensii

The glycosaminoglycans extracted from Pinctada martensii have good biological activity, and the annual output of Pinctada martensii meat(the visceral mass, software department) reached more than 2000 t in recent years. To make better use of these resources, we carried on separation, purification, structural elucidation and biological activities of glycosaminoglycan in-depth step. This research aims to carry out systematic research of glycosaminoglycans about extraction, separation, purification,structural analysis and immunoregulation activity, in order to achieve the function value of Pinctada martensii meat.To make sure the optimization of extraction process for glycosaminoglycan from Pinctada martensii by Response Surface Methodology. Glycosaminoglycan was achieved from the whole viscera of Pinctada martensii via enzymatic hydrolysis by pancreatin and neutral protease of Bacillus subtilis. Enzymatic hydrolysate was deproteinized twice by sevage and subsequent ethanol precipitation. The pure product(GAG1, GAG2) of glycosaminoglycans were achieved through DEAE-52 column.This research analyzed chemical properties and the primary structure of glycosaminoglycans(GAG1, GAG2) by phenol-sulfuric acid method, 1,9-dimethyl methylene blue method, Bradford method, gelatin-chloride barium method, Orcinol,Carbzole, Wagner method, Agarose gel electrophoresis, HPGPC, infrared analysis method, ultra high performance liquid phase.Finally, we explored the in vivo biological effects of glycosaminoglycan from Pinctada martensii via Cyclophosphamide-treated immunosuppression mice model.The following conclusions are obtained.(1) The product Y/(×10-4) of the yield of glycosaminoglycan and content of glycosaminoglycan was response value. Liquid-solid ratio, enzyme dosage andenzymolysis time were impact factors. The optimum extraction conditions for glycosaminoglycan were got by response surface, and the optimal enzyme dosage,liquid-solid ratio and enzymolysis time were of 1.14%(the ratio of neutral protease and pancreatin at 7:8), 3:1 and 4.1 h, respectively. Under these conditions, the yield and content of glycosaminoglycan were of 0.518%, 40.9%. By further purification the content of GAG1 and GAG2 was 82.2%, 80.3% through DEAE-52 column.(2) We got these conclusions by chemical analysis. The total sugar contents were12.87%, 10.78%(GAG1, GAG2). Protein content were 6.01%, 5.86%(GAG1, GAG2).The hexuronic acid contents were 16.89%, 21.57%(GAG1, GAG2). Sulfate radical content were 15.35%, 18.72%(GAG1, GAG2). Hexosamine content were 37.93%,11.14%(GAG1, GAG2).The ratio of Ah/Al, GAG1 and GAG2 were 10.13 and 9.85, respectively. The ratio of C/O of GAG1 and GAG2, were 0.531 and 0.96, respectively. Through the above data we can draw the following conclusions. Hexuronic acid of GAG1 was iduronic acid. Hexuronic acid of GAG2 was glucuronic acid. Amino hexose of GAG1 and GAG2 were glucosamine. Sulfate radical may be O-sulfated groups, but does not exclude the possibility of a small amount of N-sulfate.From the Agarose gel electrophoresis map can be seen that the two substances in crude glycosaminoglycan were separated into homogeneous components effectively by DEAE-52 ion column.Via the standard curve drawn by different molecular weight dextran standards,the third order linear regression equation was calculated. After that, Mn and Mp of GAG1 was 8358076 and 16356464, respectively. While Mn and Mp of GAG2 was25992 and 44597, respectively. It showed that GAG1 had higher degree of polymerization than GAG2, which indicated that may GAG1 had high biological activity.The result of infrared spectroscopy characterized the main functional groups of glycosaminoglycans from Pinctada martensii, which showed that they were accord with the structural feature of acidic mucopolysaccharides.The samples were decomposed into monosaccharide by hydrolysis, and thecomponents in the samples were determined by ultra performance liquid chromatography. The retention time of standard monosaccharide which can be purchased can be determined in order to determine the composition of monosaccharide in the sample. By the measurement results, we could find D-amino glucose, mannose,glucose, galactose, xylose and fucose in GAG1, as well as D-amino glucose, mannose,D- glucuronic acid, rhamnose, glucose, xylose and fucose in GAG2. Due to the lack of standard products, several peaks still failed to determine what kind of monosaccharide.This is also the lack of this study.(3) The study demonstrated that chronic treatment with glycosaminoglycans from Pinctada martensii resulted in accelerating recovery of immunosuppression in Cyclophosphamide-treated mice, including accelerating recovery dose-dependently of spleen index, peripheral white blood cell and bone marrow cell counts, enhancing T cell and B cell proliferation responses, as well as splenic nature killer cell activity and peritoneal macrophage phagocytosis, and restoring the level of interleukin-2 in the serum. It indicated that glycosaminoglycans from Pinctada martensii were competent in both precautions and remedy on the immune organs, immunocytes and immune cytokines for the immunoregulation effect.