Extraction,Separation,Structural Characterization Of Polysaccharide And Analysis On Nucleoside In Stichopus Japonicus

The twenty-first century is the new century for manking to develop and utilize marine resources.In recent years,the study found that sea cucumber contains many substances with important biological activity including anti-tumor,anticoagulant,anti-thrombosis and hypolipidemic.In this paper,Stichopus Japonicus from Xiapu aquaculture base in Fujian province and Weihai in Shandong province were used as research sample to analyze the polysaccharides and nucleoside substances of Stichopus Japonicus.These investigations would provide the material basis for the further study the exploration of medicinal value of Stichopus Japonicus.This thesis contains three chapters:1.Optimization on Ultrasound-microwave Assisted Extraction Technology of Stichopus Japonicus PolysaccharideObjective:To optimize the extraction technology of polysaccharide in Stichopus Japonicus(SJPS)with the extraction rate and purity as evaluation indicator.Method:The extraction technology of SJPS was optimized by orthogonal test with the extraction rate and purity determined by phenol-sulfuric acid method as evaluation indicator.Result:With D-glucuronic acid as reference substance,the optimal microwave-ultrasound assisted extraction conditions of SJPS were as follows:the ratio of solid and liquid was 1:15,microwave power was 80 W,extraction times was four times and extraction time was 30 min.Conclusion:The optimal extraction technology are rational,feasible and higher extraction rate.2.Separation,purification,structural characterization and protective effects against alcoholic hepatic injury in mice of Stichopus Japonicus PolysaccharideObjective:To establish a method of separation,purification,structural characterization of polysaccharides from Stichopus Japonicus and to research the protective effects against alcoholic hepatic injury.Method:At first,the crude polysaccharides from SJPS was prepared by water extraction and alcohol precipitation and purified by DEAE-Sepharose FF.Then,the relative molecular mass of SJPS was determined by HPGPC.Futhermore,the momosaccharide compositions were analyzed by HPLC and LC/MS and the structures were characterized by UV,IR and Congo red test.Finally,the protective effects against alcoholic hepatic injury in mice was researched by the determination of AST and ALT in serum and SOD and MDA values in liver in mice.Conclusion:The refined polysaccharide from Stichopus Japonicus was the light yellow solid,with an average molecular weight of 2.291×10~5 Da;It was mainly composed of glucose and fucose,and the molar ratio was 1:8.01.Pharmacological experiments showed that polysaccharides from Stichopus Japonicus could protect acute alcoholic hepatic injury in mice.Conclusion:This study may provide the theoretical basis for further research on the structural characterization and biological activity of polysaccharides in SJPS.3.Determination of five nucleoside compounds in Stichopus Japonicus by LC-MSObjective:To establish the HPLC-ESI/MS method for simultaneous determination of five nucleoside compounds(uracil,hypoxanthine,inosine,guanosine and adenosine)from Stichopus Japonicus under different extraction technology.Methods:The HPLC-ESI/MS separation was performed on an Agilent ZORBAX XB-C18 column(250 mm×4.6 mm,5?m),gradient eluted with the mixture of acetonitrile and 20mmol/L ammonium acetate solution as mobile phase,the flow rate was 1.0 mL/min.The detection wavelength was 254 nm and the column temperature was 25?.The injection volume was 20?L.The mass spectra were obtained in positive ion mode with electrospray ionization.Results:Stichopus Japonicus which extracted by different techniques both contain uracil,hypoxanthine,inosine,guanosine and adenosine components.Conclusion:The method used for analysis of the five kinds of nucleoside compounds from Stichopus Japonicus is fast and convenient and could be used for analysis of nucleoside compounds.