Study On Extraction,Purification,Structure And Bioactivity Of Glycosaminoglycan From Ruditapes Philippinarum

As one of the four major cultured shellfish in China,Ruditapes philippinarum is abundant and has high nutritional value,but it has not been fully exploited.As an important bioactive substance in Ruditapes philippinarum,glycosaminoglycan(GAG)has many bioactive properties,including anti-tumor,anti-oxidation,strengthening immune system,etc.With the improvement of human health,nutritional requirements and the transformation of marine biological industry growth,high value utilizations of glycosaminoglycan from Ruditapes philippinarum and the further study on its mechanism in vivo are significant to the development of resource utilization to a virtuous cycle.The current stage of research on Ruditapes philippinarum glycosaminoglycan is studied in various aspects:extraction,purification,characterization and biological activities,which provides a reference for the development and utilization of Ruditapes philippinarum.The main contents and results are shown as follows:1)To hydrolyze Ruditapes philippinarum,four proteases were used as follows:trypsin,neutral protease,papain and pepsin,and then neutral protease that had the highest yield of glycosaminoglycan was choosen for the further experiment.Based on the single-factor experiments,response surface method of neutral protease was adopted to gain the optimal parameter of enzymolysis process:solid-liquid ratio,1:1(w:v);hydrolysis temperature,50?;hydrolysis time,5 hours;enzyme concentration,9800 U/g.Under these conditions,the yield and content of glycosaminoglycan from extract was 1.04%and 7.13%respectively.2)Two components were obtained after DEAE-650M column chromatography:GAG-1 and GAG-2.The glycosaminoglycan content of GAG-1 was 89.56%,which was higher than that of extract by 82.43%.The GAG extract and GAG-1 could significantly promote the proliferation of mouse spleen lymphocytes in vitro in a certain concentration range(20?100 mg/mL and 2.5?200 pg/mL).And the proliferation effect of GAG-1 was significantly better than that of extract.The analysis of ultraviolet spectrum,infrared spectrum and nuclear magnetic resonance showed that the structure of GAG-1 contained trace nucleic acid and protein,and it also showed that carboxyl,acetyl amino and sulfate groups existed in the molecule whose monomer was pyranose,with ?-D-Glc(1?4)-as the main connection.3)Based on the criterion of testing and evaluating health foods(2003),the immune activity of GAG extract from Ruditapes philippinarum in immunosuppressed mice was studied.We found that this extract could enhance the proliferation of T spleen lymphocytes induced by ConA,the activity of NK cells,the proliferation of spleen lymphocyte subsets(CD4+,CD8+),IL-2 and IFN-y levels,the phagocytic activity of mice peritoneal macrophages against neutral red.It also could increase the rate of carbon clearance and the serum hemolysin level.The results showed that GAG extract from Ruditapes philippinarum could improve immune ability of immunosuppressed mice induced by CTX in many ways and aspects.4)Based on the criterion of testing and evaluating health foods(2003),the hypoglycemic effect of GAG extract from Ruditapes philippinarum in diabetic mice was studied as well.Compared with the diabetic model group,the decrease of fasting blood glucose and the ability of glucose tolerance in middle and high group were significant(P<0.01),which were similar to those in positive control group.These two indicators were all positive,and the high group of GAG extract had no adverse reaction to normal mice.The results showed that GAG extract from Ruditapes philippinarum had hypoglycemic activity by effectively reducing the blood glucose of diabetic mice.