Structural Characterization And Biological Activities Of Polysaccharide MP-Ⅰ From Mytilus Coruscus

Polysaccharide is an important biological macromolecule which widely exist in animals, plants and microbials. With the progress in molecular biology research, people have gradually found that different sources of polysaccharides have extensive and complex biological activities. Polysaccharide has attached great importance in medicine and become one of the hottest research in life sience. In 2001, the United States launched the "Functional Glycomics plan", while Japan has launched the " Sugar project forward plan ". The overall goal is to clarify the biological function of the polysaccharide chain, and to design carbohydrate drugs which can cure cardiovascular diseases, cancer, infection and other diseases. Though the starting in polysaccharide research of our country was late, the progress is rapid, especially in the biological functions of polysaccharide.Ocean is an important source of biopolysaccharides. With the exploration of land plants polysaccharides and fungi polysaccharides, marine polysaccharides caught more and more attention. At present, the research of the marine polysaccharides is concentrated on marine plants, and the structures of some seaweed polysaccharides have been clarified,which found to have activities such as enhancing immune system, anti-tumor, anti-radiation and anti-aging. But the polysaccharides from marine animals have attached less attention.Mytilus coruscus belonged to the marine mollusks door, widely cultured in China's Bohai Sea, Yellow Sea and other waters. It is called Haihong in northern China, while it is called Dancai in Zhejiang areas. Researches have shown that Mytilus coruscus extract has functions such as anti-tumor, anti-aging, anti-viral and anti-bacterial, enhancing immunity and lowering blood lipid content. Polysaccharide is one of the most important active ingredients in it.Our lab has many year's research experience in marine animal polysaccharide. This paper mainly concentrate on the separation, purification, structure elucidation and biological activity of polysaccharide MP - I (Mytilus coruscus polysaccharide MP-â… ) from Mytilus coruscus.1. Separation and purification of MP-â… . We got crude polysaccharide through homogenate, hot-water extraction and Sevag method. Then the crude Polysaccharide was separated through DEAE-Sepharose anion exchange resin, and purified through Sepharose CL-6B. The purity of MP-â… was inspected by HPLC detection and optical measurement. The molecular weight was determined using HPLC. We also optimizated the separation and purification conditions.2. Structure characteration of MP-â… . The accurate structure of MP-â… was determined by UV scanning (200-400nm) , infrared scanning, monosaccharide components determination (gas chromatography and silica TLC) , methylation analysis, periodate oxidation, NMR analysis, uronic acid and sulfate contents determination.3. Biological activity of polysaccharide MP-â… . (1) Anti-acute liver injury activity of MP-â… in mice. The animals were grouped into six groups:â‘ Normal group (saline+olive oil) ;â‘¡negative control group (saline+0.2% CCl₄ olive oil solution) ;â‘¢positive control group (Kurarinone, 30 mg·kg^-1·d^-1+0.2% CCl₄ olive oil solution) ;â‘£MP-â… high concentration group (MP-â… 100 mg·kg^-1·d^-1+0.2% CCl₄ olive oil solution) ;⑤MP-â… middle concentration group (MP-â… 50 mg·kg^-1·d^-1+0.2% CCl₄ olive oil solution) ;â‘¥MP-â… low concentration group (MP-â… 25 mg·kg^-1·d^-1+0.2% CCl₄ olive oil solution) . The animals were pre-treated for 7d by intraperitoneal injection respectively (0.2ml per animal) . Then the normal group was treated with olive oil and other groups were treated with 0.2% CCl₄ olive oil solution (0.2ml per 20g body weight) by intraperitoneal injection after 2 h of the last dose to cause acute liver injury. Liver index, serum alanine aminotransferase (ALT) , serum aspartate aminotransferase (AST) , liver superoxide dismutase (SOD) and liver malondialdehyde (MDA) levels were measured after 24h of the administration. The histopathological changes of liver was also observed through histopathological slice. (2) Growth inhibition on tumor cells of polysaccharide MP-â… . The inhibition rate on HO-8910, MCF-7, K562, SMMC-7721, HL-60 and Hela tumor cells by MP-â… (500μg/ml,100μg/ml,20μg/ml) was measured using MTT method.MP-â… appears to be white powder, easily soluble in water, has a molecule weight of 1350KDa. After optimizating the separation conditions, the time of separation and purification was shortened,and the yield of polysaccharide was reached to 2.14% of the fresh mussel tissue.Structure characteration showed that MP-â… was a glucan with a backbone ofα-D-(1→4) Glc,branched withα-D-(1→6) Glc. Its chemical structure was as below:The results of anti-acute liver injury in mice showed that MP-â… high concentration (100 mg·kg^-1·d^-1) could significantly reduce serum ALT and AST levels (P <0.01) , reduce liver MDA level (P<0.01) , increase liver SOD level (P<0.01) in CCl₄-induced liver injury mice. MP-â… middle concentration (50 mg·kg^-1·d^-1) could significantly reduce serum ALT (P<0.01) and AST (P<0.05) levels, reduce liver MDA level (P<0.05) , increase liver SOD level (P<0.01) in CCl₄-induced liver injury mice. MP-â… low concentration (25 mg·kg^-1·d^-1) could significantly reduce serum ALT levels (P<0.01) in CCl₄-induced liver injury mice. But the effect on serum AST, liver MDA and SOD was not so significant (P>0.05) . Kurarinone(30 mg·kg^-1·d^-1) could significantly reduce serum AST level (P<0.01) , reduce liver MDA level (P<0.05) , increase liver SOD level (P<0.05) in CCl₄-induced liver injury mice. But the effect on serum ALT is not so significant (P>0.05) . MP-â… could prevent cell necrosis and invasion of inflammatory cell, significantly improve liver histopathological damage, which is dose related. But the difference of liver index in mouse was not so obvious.The results of growth inhibition on tumor cells in vitro showed that MP-â… had significant effect on inhibiting the growth of HO-8910, MCF-7, K562, SMMC–7721 in vitro(P<0.01) . Inhibition rates of MP-â… high concentration to HO-8910, MCF-7 tumor cells were 30.55% and 36.38%.Therefore, we concluded that MP-â… had an average molecule weight of 1350KDa. MP-â… was a glucan, which had a backbone ofα-D-(1→4) Glc, branched withα-D-(1→6) Glc. MP-â… had a protective effect on CCl₄-induced liver injury in mice, which was dose-related. MP-â… could inhibit the growth of tumor cells in vitro. The growth inhibition on HO-8910 and MCF-7 were most notable.