Horse Manure, Sea Urchin Gonads (hemicentrotus Pulcherrimus) Polysaccharide Extraction, Purification And Anti-tumor Activity Study

Hemicentrous pulcherrimus, which belongs to Echinodermata, is the special species in Chinese and Japanese sea. They mainly exist in the coast of Huanghai and Bohai, the coast of Zhejiang, Fujian and Japan. This paper used the the gonad of Hemicentrous pulcherrimus as the experimental material. First, the total nutrition components were determined. Second, the extraction process of polysaccharide from the gonad of Hemicentrous pulcherrimus (HpPS) was researched and then the extraction of polysaccharide, separation and purification were also researched. This paper have provided the necessary data and theoretical basis for the exploration and application of Hemicentrous pulcherrimus.The analyses of water, ash, protein, total sugar, rude fat, fatty acid, amino acid and inorganic element of the gonad of Hemicentrous pulcherrimus have been done. The concentration of water, ash, protein, total sugar, rude fat are 64.20%,12.70%,12.25%,5.59% and 2.34%, respectively. And among the fatty acid, the concentration of arachidonic acid and EPA were high. The concentration of Ca, Mg, Fe were high among the inorganic elements. This indicates that it has abundant nutrition and varies of inorganic elements and unsaturated fatty acids, so it has well eatable, healthy and applicable prospectOrthogonal experiment has been used to investigate the extraction process of HpcPS. The extraction temperature, extraction time, ratio of the material to water and times of extraction were selected as variable parameters. Their different effects on the yield of polysaccharide that extracted from the gonad of Hemicentrotus pulcherrimus were studied by the L9 (34) experimental design method. In addition, a multiple linear regression(MLR) model, in which the yield of polysaccharide was as the dependent and the experiment conditions of the orthogonal experiment was as the independent, was firstly constructed to predict the yield of polysaccharide of two experiment conditions in the orthogonal experiment. The statistic parameters were R=0.922, F=2.854. And the MLR model was used to predict yields of polysaccharide of two experiments. The predicted results were in good agreement with the experimental values, indicating that the model can be used to predict the yield of polysaccharide.The traditional method of water extraction and alkali extraction were used to extract the polysaccharide. The fraction was after the process of the protein exclusion. And then rude HpcPS was obtained. The rude HpcPS which were extracted by alkali and precipitated by 70% ethanol were through super-filtered, DEAE-cellulose anion exchange columns. Finally, the polysaccharide from the gonad of Hemicentrous pulcherrimus were gained. The purity and molecular mass were determined by high-performance gel permeation chromatography (HPGPC) and the results indicated the molecular mass of the polysaccharide was 32.494kD and the polysaccharide was pure.To study the anti-tumor effect of alkali-extracted HpPS in vitro and in vivo. Cytotoxic effect of human liver cancer cell SMMC-7721 and human lung cancer cell A549 were assayed using MTT method in vitro. The results show that alkali-extracted HpPS has strong anti-tumor activities to human lung cancer cell A549 and has no obvious cytotoxicity to human liver cancer cell SMMC-7721. It indicates that the Cytotoxic effect of alkali-extracted HpPS have some selection. The effect in vivo was studied by injecting alkali-extracted HpPS to the bearing H22 and Lewis mice for 10d. The results indicate that alkali-extracted HpPS do not have remarkable inhibition to the tumor of H22 and Lewis.