| Apostichopus japonicus Selenka is widely distributed in the sea of north China,and it is famous for high nutritional value and economic value. Polysaccharide, with avariety of biological functions and pharmacological activity, is a kind of importantactive ingredient in sea cucumber. A. japonicus glycosaminoglycan (HGAG) showsbroad prospects for drug development, due to the unique molecular structure andprominent immunomodulatory activity. There are some progress on the research ofsea cucumber polysaccharides, but mostly are the initial activity screening ofcucumber extract or coarse polysaccharides. The reports about purified HGAGanalyze and immune activity are still rare. The main purpose of this thesis is to getpurified HGAG, study the immune regulation activity both on non-specific immunityand specific immunity, and lay the foundation for the development and application ofHGAG.In the experiments, A. Japonicus is firstly hydrolyzed by enzymes, deposited byethanol to get coarse HGAG. Then get rid of the pigments by hydrogen peroxide,remove protein by trichloroacetic acid to get refined HGAG. Q-FF anion exchangechromatography is used to get the purified HGAG. The uniformity of purified HGAGis good quality through GPC testing, and weight average molecular weight is94,091.Established the gas chromatographic methods to detect the residual acetate andethanol respectively. The method is rapid, accurate, reliability, sensitive andreproducible. The residual acetate and ethanol in HGAG was determined by applyingthe methods, and the results were0.18%and0.013%respectively. The residual levelof the two organic solvents in HGAG samples were all up to the standard stipulated inChina Pharmacopeia. In order to investigate the non-specific immune regulation of HGAG, studied theeffects of HGAG on the function of mice peritoneal macrophages (Mφ). Differentconcentrations of isolated HGAG were added to Mφ in vitro. Morphological changeswere observed by inverted microscope, Metabolic activity of Mφ was measured byMTT assay, the phagocytosis of neutral red was detected by colorimetric assay, lactatedehydrogenase (LDH) and acid phosphatase (ACP) activity were analyzed, nitricoxide (NO) was detected by Griess reagent, concentrations of TNF-α and IL-1β weredetermined by ELISA. The results showed that, HGAG have a certain role inpromoting the activity of Mφ within the range of0.1~25μg·mL-1. HGAG couldenhance the metabolism and phagocytosis (P <0.01), increase LDH, ACP activity andNO production (P <0.05), improve TNF-α and IL-1β production (P <0.01) within arange of1~10μg·mL-1compared with the control group. The phagocytosis, TNF-αand IL-1β production were increased significantly within a range of1~10μg·mL-1compared with the positive control group (P <0.05). The Mφ function parameters ofhigh concentration (100μg·mL-1) group were lower than the control group. It issuggested that within the range of1~10μg·mL-1, HGAG can promote the activity ofMφ, and better than the positive control group to a certain extent, strengthen thebody’s non-specific immune function.In order to investigate the specific immune regulation of HGAG, determined thesplenic lymphocyte transformation by MTT assay, measured the swelling of feet toreflect the delayed type hypersensitivity reaction, detected hemolysin generation andquantity of antibody-forming cells to evaluate the production of the antibody and theactivation of B lymphocytes. The results showed that, HGAG can be significantlyenhance splenic lymphocyte transformation compared with the control group withinthe range of0.05~25μg·mL-1in vitro (P<0.05), the enhancement is more significantlythan the positive control group within the range of0.5~10μg·mL-1(P <0.05). Low,medium and high doses of HGAG could promote the delayed type hypersensitivity,increase the serum hemolysin generation and quantity of antibody-forming cells invivo (P <0.05). The effects of high-dose group are more significantly compared withthe low-dose group, with a certain dose-effect relationship. It is suggested that HGAG can activate T, B lymphocyte, increase the production of antibody, promote the body’sspecific immune function.In this paper, the purified HGAG can promote the body’s immune system inmany aspects including non-specific and specific immunity. Indicated that HGAG hasgood immunomodulatory activity. |
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