Study On The Extraction, Isolation, Structure And Biological Activity Of Polysaccharides From Mytilus Edulis

Mussel is an important aquaculture species worldwide, the ratio of Mytilus edulis (ME) is the largest. But people could not make more profits if only mussel was sold directly. Mussel not only is a traditional tonic food, but also has a high nutritional value and variety of pharmacological effects. According to related materials, both mussel and its extracts have variety of functional effects, such as anti-oxidant, immune regulation, anti-tumor, antihypertensive effect and so on. Polysaccharides are one of the major chemical ingredients of ME. And now more researches were focused on the crude polysaccharides from ME, however, the studies about the extractions, purifications and structures and functional researched systemly have not yet reported. Therefore, ME were obtained from an island named wolfberry in Zhoushan and then employed to extract polysaccharides. Extraction, isolation, structure identification and bioactivity of polysaccharides from ME were further studied. The major results obtained in this study were as follows:Response surface method (RSM) was applied to optimize extractions of polysaccharides from ME by hot-water extraction. The effects of extraction temperature, extraction time and ratio of liquid to solid on the yields of polysaccharides were investigated. The optimum extraction conditions were obtained as follows:extraction temperature 93℃, extraction time 4.2h, ratio of liquid to solid 44.3:1. The yield of polysaccharides can be up to 19.73% under the optimal extraction conditions.Three different deprotein-methods (seveg method, enzymatic method and enzymatic-seveg method) were used to deproteinize crude polysaccharides from MES. The results showed that enzymatic-seveg method was the best method to remove protein. The optimum conditons were as follow:Alkaline protease was as enzyme:the amount of [E]/[S]1.79%, pH9.19, temperature of 61.79℃; Sevag method: chloroform:butanol= 4:1 (V/V), liquid-Solvent ratio= 3:1 (V/V), the times of removing-protein processing was three.In this study, MES-I were isolated and purified by several approaches. After using 0.45μm microporous membrane and ultra-filtration, the obtained polysaccharides were further isolated with Marco-Prep DEAE ion-exchange chromatography and Sephrose 4B gel column chromatography. In the end, three components (MES-I-11, MES-I-12 and MES-I-2) were obtained. They were pure determined by Sephrose 6B gel column chromatography, HPLC and cellulose acetate membrane electrophoresis (CAME).The Physical, chemical properties and the structures of MES-I-11, MES-I-12 and MES-I-2 was analyzed with chemical methods and instrumental analysis methods. According to Phenol-sulfuric acid method, the total sugar contents of MES-I-11, MES-I-12 and MES-I-2 were 95.2%, 91.2% and 94.8% respectively. Color reactions and UV detection inferred that the three homogeneous samples contained no protein and nucleic acids. From HPLC analysis, molecular weight of MES-I-11, MES-I-12 and MES-I-2 were 572.8KD,18.20KD and 69.50KD respectively. From FT-IR, MES-I-11, MES-I-12 and MES-I-2 were polysaccharides, and all their polysaccharides structures contained a-D-glucopyranoside bond. From GC-MS analysis, MES-I-11, MES-I-12 and MES-I-2 were composition of mannose, galactose and glucose with different molar ratios. MES-I-11 was composition of Man, Gal and Glc with the molar ratio of 2.74:1:18.06; MES-I-12 with molar ratio of 2.2:1:19.44; MES-I-2 with molar ratio of 2.87:1:3.63. Nuclear magnetic resonance analysis results was that the structures of MES-I-11, MES-I-12 and MES-I-2 were mainly composed with the presence of a-D-Glc(1→4)-linkage glycoside, and in the C6 where had been replaced.Cellular immunity was tested by MTT colormetry. The results showed that MES-I-1, MES-I-2, MES-I-11 and MES-I-12 could affect the proliferation of Lymphocyte induced by ConA differently. And in high concentrations, all samples could inhibit the proliferation, with the respective significances were P<0.01, P<0.001, P<0.05,P<0.01. In Lymphocyte proliferation induced by LPS, MES-I-1 and MES-I-12 did not show significant proliferation effects. And MES-I-2, at high concentrations, showed promotive effects, also did MES-I-11 at middle concentrations. But it showed depressant effects in low concentrations(P <0.05). The results of antitumor test of K562 tumor cells showed that MES-I-1, MES-I-2, MES-I-11 and MES-I-12 could inhibit proliferation of cancer cell differently. And the effects were enhanced with the increasing of sample doses. MES-I-2, at low concentrations and high concentrations, displayed growth inhibitory effects with 24.48±1.72% and 62.82±8.57% respectively, the results showed that the inhibition effects of MES-I-2 on K562 tumor cells were better than the effects of other samples.