Extracting Preparation And Quality Control Of Fucoidan

In this thesis, a new fucoidan was isolated from sea cucumber Thelenata anana, which was collected from the South China Sea. Its low mocular weight derivatives were first prepared by free radical depolymerization with hydrogen peroxide in the presence of copper(II) ion and acid depolymerization. The fractions were determined, their chemical structures of were elucidated and their anticoagulant activities were evaluated. The thesis can be used for the quantitative determination and quality control of fucoidan, and provide a theoretical base for developing new anticoagulant medicine.Free polysaccharides were extracted by using the method of combining of alkaline hydrolysis and enzymatic hydrolysis from sea cucumber T. anana, and then a new fucoidan was purified by precipitation, salting and other purification procedures. The combining approach of alkaline hydrolysis and enzymatic hydrolysis was superior to alkaline hydrolysis, enzymatic hydrolysis, and combining approach of enzymatic hydrolysis and alkaline hydrolysis. Orthogonal design was adopted to search for the appropriate reaction conditions of alkaline hydrolysis and enzymatic hydrolysis. A large number of proteins were effectively removed by the isoelectric point method. Finally, the best conditions to select the optimum extraction and depuration procedure for fucoidan from sea cucumber Thelenata anana was confirmed: 8 times amout of 0.5 M NaOH to crude medical material(v/w) was added to exteract for 3 h at 40±2 °C, then 0.5% of papainwas added with its pH value to 6.5 and extracted at 50±2 °C for 6h, The extracted solution was standed for 4 h at pH value of 2.5, then the supernatant was collected by centrifugation and adjusted its pH value to neutral. Finally, the solution was purified by ethanol deposit-salting method followed by purifiedand used freezing-dry technique to obtain FUC(fucoidan).Ten low mocular weight derivatives were first prepared by free radical depolymerization with hydrogen peroxide in the presence of copper(II) ion and acid depolymerization. The method has the advantages of simple operation, mild conditions and high yield.Anticoagulant activities of fucoidan and the derivatives were evaluated. It shows that all of them have certain anticoagulant activity, but fucoidan has the better ability to extend APTT than the derivatives, which suggests that the low mocular weight derivative, dFUC-1, may be a a good anticoagulant drug.The molecular weight of fucoidan and the derivatives were analyzed by HPSEC and sulfate content was determined by barium chloride-gelatin method respectively. The monosaccharides composition of fucoidan and the derivatives, analyzed by pre-column derivatization-HPLC, were mainly fucose, and also a small amount of glucose, galactose, and galactosamine. Then HILIC-ELSD method for the determination of fucose in fucoidan and the derivatives was developed. The method, simple, quick, and accurate, can be used for the quantitative determination and quality control of fucoidan.