Extraction, Separation, Structural Characterization And Sulfated Modification Of Polysaccharides From Crassostrea Gigas

Concha Ostreae is a kind of common economic seafood, whose meat not onlydelicious and rich in nutrients but also has unique medicinal value, known as "themilk of seabed". Concha Ostreae meat contains abundant polysaccharides, theresearch found that the Concha Ostreae polysaccharides have many bioactivities,suchas immune function, antioxidation, antiviral activity and hypoglycemic action. In thispaper, the crude polysaccharides were extract by different methods from Crassostreagigas which were from seas of Qingdao and five components with high purity wereobtained by two chromatography separation methods. The structure of the puritiedcomponents CT-11was determined by modern analytical techniques. CT-11was alsosulfated modification, the anticoagulation and antitumor activity in vitro of sulfatedpolysaccharide were studied.Crassostrea gigas polysaccarides were obtained from the acetone powder ofCrassostrea gigas by water extraction at room temperature,60℃and by dilute alkaliextraction sequentially. And the protein of polysaccarides was removed by twoenzymes method, which including pepsin and trypsin. Three kinds polysaccharideswere got, which named CT, RT and JT. The yield of CT, RT and JT were16.1%、1.9%and13.3%. The total sugar content of the polysaccharides was determined accordingto the Phenol-sulfuric acid method using glucose as a stangard, and protein contentwas determined with method of Folin-phenol. The suger contens of CT, RT and JTwere87.4%,67.9%and42.9%.The protein contens of CT, RT and JT were10.4%,21.8%and47.7%. The monosaccharide compositions were analyzed by highperformance liquid chromatography and their monosaccharide composition wassimple, only including Glc. The molecular weights of the three samples were2.1×106Da,2.0×106Da�'�9.8×105Da, which determined with high performance gelpermeation chromatogram and eighteen angles laser light scattering method. CT, RT and JT were further purified by DEAE Sepharose FF anion exchangechromatography and Sepharcryl S-400, five kinds of pure polysaccharides CT-11,CT-21, RT-11, RT-21and JT-11were acquired. CT-11was researched more and itscontent of total suger and protein were95.1%and4.7%. Also, CT-11showed asingle peak on high performance gel permeation chromatography, and its relativemolecular mass was1299kDa. The structure of CT-11was characterized by FT-IR,methylation analysis, GC/MS, ESI-MS and NMR, the date showed that the structurewas characterized to be�'4)-α-D-Glc-(1�', with little�'3,4)-β-D-Glc-(1�'and�'2,4)-β-D-Glc-(1�'branched units.CT-11was sulfated with method of SO3-pyridine and chlorosulfonic–formamide,two kinds of derivatives were got and named as CT-S1and CT-S2. The sulfate degreeof CT-S1was36.2%and CT-S2was43.1%. The properties of CT-S2wereinvestigated, its molecular weight was11.2kDa. IR and NMR further confirmed itsstructure, the IR and NMR spectra indicated that sulfation mainly occurred at the6-position of (1�'4)-Glc, parially at C2and C3.Anticoagulant activity assay of CT-S2showed it had no obvious effect on PT, butprolong APTT and TT, which showed its anticoagulant effect was mainly achieved byendogenous clotting system. The antitumour in vivtro activities of CT-S2wasevaluated MTT assay or SRB assay against five cancer cell lines.CT-S2had evidentlyinhibitory effects on HCT-116cells, and also had some inhibitory effects on HL-60and A-549cells, the inhibitory effects expressed dose-effect relationship. But therewere no inhibitory effects on hela and K562cells.There are seldom reports about the systematic research of the physicochemicalproperties, purification, structure and chemical modification of Concha Ostreaepolysaccharide, though the preliminary research of physicochemical properties andbiological activity about Concha Ostreae polysaccharide. These results will providebasis for further research on relationships of structure and activity of Crassostreagigas polysaccharides, and also will be helpful for the research of Concha Ostreaeand other mollusc.