Study On Physicochemical Properties And Bioactivities Of Glycoprotein From Jellyfish (Rhopilema Esculentum Kishinouye) Oral-arms

Rhopilema esculentum jellyfish, a species of large and edible jellyfish, having the high economic, nutritional and medicinal value, is a big and important fishery resource in China. It was found that jellyfish(R.e)had an effective cure for hypertension, chronic tracheitis, asthma, gastric ulcer and struma. Considering its nutrient and curative value, R. esculentum jellyfish should be further investigate and exploited for its chemical compositions and pharmacological characters. Although some bioactivity components such as protein, toxic, polypeptides and carbohydrates were researched, no report on the biological activity of glycoprotein from jellyfish (R.e) oral-arms had yet been made.Having fresh jellyfish (R. esculentum, captured in Yellow Sea near Qingdao city) as material in this dissertation and utilizing a series of modern technologies such as isolation and purification technology, instrument analysis technology, medical analysis technology and molecular biological technology, the author had systematically studied the purification, physicochemical prosperities, radical scavenging activity and immunity of glycoprotein (JGP-Ⅲ) from jellyfish(R. e) oral-arms. Main results achieved in this research as follows:1 In order to optimize extraction technology for the glycoprotein from jellyfish(R.e) oral-arms, on the basis of single factor experiments, the effects of operating conditions such as solvent pH,material ratio, ultrasonic time, ultrasonic power and extraction time on the yield of glycoprotein were analyzed by response surface methodology. The optimized extraction conditions as follows: solvent pH was 7.26, material ratio was 1:4, ultrasonic time was 15min, ultrasonic power was 300W, and extraction time was 60min. under the above mentioned conditions, the actual yield of target glycoprotein was 9.14%.2 Three grades of jellyfish glycoprotein JGP-Ⅱ, JGP-Ⅲ, JGP-Ⅳwere isolated and purified from jellyfish oral-arms through ethanol fractionated precipitation, Sp Sephadex C-25 column. The fraction (JGP-Ⅲ) with high carbohydrate content, protein content and strong radical scavenging activity was further studied. By means of Sephacryl S300HR, Sepharose CL-6B and HPLC, JGP-Ⅲwas sole peak. A blue band and a pink band appeared on the correspondence site of SDS-PAGE gel of JGP-Ⅲstained by Coomssie brilliant blue R-250 and PAS respectively. All indicated JGP-Ⅲwas not a mixture of carbohydrate and protein but a homogeneous glycoprotein. The yield of JGP-Ⅲwas 0.5%(in dry state).3 JGP-Ⅲwas white floccules, freely soluble in water, and not soluble in organic solvents such as ethanol, acetone. JGP-Ⅲcontain 12.61% total suger,74.34% protein, 8.47% amide suger,0.84% uronic acid, 1.06% sulfate group and 0.92% sialic acid. The molecular weight of JGP-Ⅲwas estimated to be 109.7kDa by SDS-PAGE and 85.3 kDa by HPLC. The denaturation temperature (Td) and shrinkage temperature (Ts) were 31.06℃and 61.74℃respectively. Monosaccharides composition of JGP-Ⅲwas Rha, Fuc, Ara, Man, Glc, GalNAc, and GlcNAc determined by GC. Amino acid composition of JGP-Ⅲwas rich in glycine, valine, glutanmic acid, alanine, proline, methionine and asparagic acid, and lacking in histidine. The existence of O-glycosidic and N-glycosidic linkage in the glycoprotein was demonstrated withβ–elimination reaction and peptide N-glycosidase F reaction. IR and NMR spectrum of the glycoprotein indicated the strcture characterization of JGP-Ⅲ. These results provided the theoretical basis for further research.4 Scavenging activities on superoxide and hydroxyl radicals of JGP-Ⅲwere estimated by chemiluminescence method, and the structure-function relationship were initial studied by chemical and enzymatic hydrolysis methods. Radical scavenging activities of JGP-Ⅲhad significant dose-dependent relationship. The carbohydrate moiety and protein moiety of JGP-Ⅲwere both involved in Scavenging radicals. The N-linked oligosaccharides played an important role in the Scavenging radicals of JGP-Ⅲ. Different structure of JGP-Ⅲhad different radicals scavenging activities. These results indicated that JGP-Ⅲhad strong free radical scavenging activities, and which had close relations with its three dimensional structure.5 The regulating effect of JGP-Ⅲon immunological function in normal and immunosuppression mice were also investigated. The results showed that JGP-Ⅲof different dosages could obviously enhance spleen and thymus indexes, increase hemolysin conten(tP<0.01)and quantity of antibody forming cells in vivo(P<0.05), heighten delayed hypersensitivity level ( P<0.05, P<0.01, and promote the phagocytosis ability of celiac macrophage(P<0.05, P<0.01). It is suggested that JGP-Ⅲcan enhance immune function by activating specific and nonspecific immunity in organism.6 The immunomodulatory of JGP-Ⅲwas investigated by the methods of molecular biology and cellular biology. JGP-Ⅲwas found to significantly increase the proliferation of total spleen lymphocytes cell populations and more strongly increases that of T cells. However, JGP-Ⅲhad less influence on the proliferation of B cells. JGP-Ⅲexerted its immunomodulating activity at an optimal dose of 50μg/mL. At this concentration, JGP-Ⅲpromoted farthest proliferation of spleen lymphocyte. Time-dependence analysis showed some differences action of JGP-Ⅲon T cells among four kinds of cytokines. IL-2 and IFN-γresponded rapidly to JGP-Ⅲ, whereas IL-4 and IL-6 were affected after a few hours treatment with JGP-Ⅲ, Accordingly, this suggested that Th1 cells, which secret IL-2 and IFN-γcytokines, were primary cellular targets directly affected by JGP-Ⅲon T lymphocyte. Whereupon, secondary response of Th2 cells related with IL-4 and IL-6 mRNA expression were followed.