| Agaricus blazei murill polysaccharide(ABMP) has many kinds of biological activities,such as immunity improvement, blood sugar reduction, tumor growth resistance,anti-oxidation, anti-radiation, anti-mutation, etc. Studies have found that activities of polysaccharides have a close relationship with their own molecular structures. Chemical modification can increase the solubility of polysaccharide and enhance the biological activity of the polysaccharide itself. This study explores the process of ABMP phosphorylation and the pharmacological effects of the phosphorylated ABMP, and further lays the theoretical foundation for development of new products.(1) ABMP was purified by macroporous resin adsorption, followed by single factor test to observe the influences from material ratio, phosphorylation reagent ratio, temperature, time and pH value on the phosphate-group grafting amount of ABMP. And then the optimum conditions for ABMP phosphorylation were obtained by orthogonal design method. Results showed that material ratio of polysaccharide / phosphorylation reagent was 1:3, and the STPP/STMP ratio was 6:1, temperature was 95 ℃, time was 5.5h, and pH value was 9, and the phosphate-group grafting amount at this moment was 9.35%, which was the optimal reaction condition for ABMP phosphorylation.(2) Intraperitoneal injection was used to conduct the acute toxicity test for mice to be dosed with phosphorylated ABMP. Among the determinations within safe dose range, the mice survival rate was 100% when the dose range was 0.25g/kg-5g/kg. Among the maximum dose tests, the dosage were 5g/kg, 10g/kg and 15g/kg; histological examination and blood biochemical test found no significant difference between mice of the test group and that of the control group, demonstrating that phosphorylated ABMP was non-toxic to the mice.(3)Comparative tests were conducted to test the antibacterial effects from the phosphorylated ABMP and the ABMP on four types of bacteria- E. coli, salmonella,diplococcus, and streptococcus. Results showed that both of the two showed antibacterial effects on E. coli and salmonella, and that the phosphorylated ABMP was better.(4) Three groups of mice were respectively fed with phosphorylated ABMP, ABMP, and saline. Results showed that the mice fed with phosphorylated ABMP showed rapider weight gains which were significantly higher than that of the group fed with ABMP and that of the control group; spleen vs body weight ratio showed that phosphorylated ABMP played apositive role in regulating the immune function of the mice; all organs of the three groups of mice were normal and good, and there was no significant difference. Three groups of mice were fed twice with E. coli, and eyeball blood samplings were conducted thirteen days after each feed for determinations, expression levels of IL-2, IL-4, and INF-γin mouse spleens were determined by RT-PCR..Results showed that, after the first immunity, the antibody(IgG) amounts of the phosphorylated-ABMP group were significantly higher than that of the saline control group; after the second immunity, the antibody(IgG) amounts of the phosphorylated-ABMP group were significantly higher than that of the other two groups.After the first immunity, there was no significant differences between the mice’s gene expression levels of the polysaccharide group and the control group; however, after the second immunity,there were significant differences between the mice’s gene expression levels of both polysaccharide groups, indicating that the immune effect of the second immunity was better than that of the first immunity.By comparison,both ABMP before and after phosphorylation could significantly increase the serum antibody levels in mice after the second immunity, and significantly increase the expression of IL-2, IL-4, and INF-γ in spleens of the mice after the second immunity, and that the phosphorylated ABMP always showed a better effect. |
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