Determinate Structure And Antitumor Activity Of Glycosaminoglycans Produced By Thick Shell Mussels

In this paper, water-soluble polysaccharide produced from Mytilus coruscus. The traditional extraction methods of water extract-alcohol precipitation was replaced by enzyme extraction, and optimize the extracting conditions. In addition, Physical and chemical properties, antioxidant and antitumor activity in vitro of the polysaccharides were determinated. The main research results are as follows: 1.The optimization of the extraction process of polysaccharideExtrect conditions of MT produced by Mytilus coruscus were optimized according to one-factor-at-a-time method and the orthogonal test. In the condition of single enzyme, compara the effects of enzyme amount, extraction time, extraction temperature and p H on the extraction rate. orthogonal test was applied to optimize these factors by considered the interactions between the variables. Then, Two-step enzyme extraction was adopted. Finally the optimal extraction solution was determined by comparing the extraction yield of three methods. The optimal papain extraction conditions were as follows:enzyme amount 0.5%,enzymolysis time 4h, enzymolysis temperature 40℃ and p H 7. And the optimal trypsin extraction conditions were : enzyme amount 0.7%,enzymolysis time 4h, enzymolysis temperature 40℃ and p H 9. Under these conditions, the extraction yield was 5.02%, 5.38%, the extraction yield of Two-step enzyme extraction was 6.02%. 2.Polysaccharide separation, purification and the basic character of construction materialsFour components: MT0, MT0.1-1, MT0.1-2 and MT1.2 were obtained after purified with Q Sepharose Fast Flow ion—exchange chromatography and verified with Sephacryl S-300 High Resolution. The total sugar content, protein content, amino sugar, uronic acid content of four components were determined. And the result were as follow: total sugar content are 61.00%, 62.47%, 62.25 and 62.47%; protein content are 0, 0, 0, 1.15%; Amino sugar content are 0.24%, 0.42%, 0.24% and 1.64%; The uronic acid content are 16.15%, 20%, 11.83% and 16.15%. In addition, the structure of polysaccharide were analyzed by the IR. The preliminary result showed: The structure of MT0, MT0.1-1, MT0.1-2 were α-D- Grape pyranose, the structure of MT1.2 was B – furanose. 3.Study on anti-oxidation activity and anti-tumor In vitroStudy on crude polysaccharide antioxidant activity and pure polysaccharide antitumor activity in vitro. The results of antioxidant activity indicated that crude polysaccharide has antioxidant capacity, and dose-dependent manner, but much weaker than ascorbic acid. At the concentration of crude polysaccharide was 3mg/m L, clearance of superoxide anion was 63.20%; the scavenging rates on DPPH was 19.60%; The hydroxyl free radical clearance was 27.00%; and the reducing power absorbance was 0.231. The results of MTT showed that all of MT0, MT0.1-1, MT0.1-2 and MT1.2 had antitumor capacity, MT1.2 was better than other components. The concentration of polysaccharide was 2 mg/m L, and action time was 60 h, the inhibition rate of four components on SPC-A-1 were 20.15%, 28.95%, 25% and 30.87%; the inhibition rate on DU-145 were 26.53%, 24.92%, 30.14% and 34.80%. HE staining results showed that, the morphology of apoptotic cells has changed. And ELISA experiment validated that polysaccharide from Mytilus coruscus inhibits IL-6 and IL-10 expression at protein level.