Structural Characterization And Antioxidation Activity In Vitro Of Polysaccharides From Pinctada Martensi Dunker

Aims:Physicochemical properties, primary structure and the antioxidant activity in vitro of polysaccharides, which was isolated and purified from Pinctada martensi Dunker by enzymatic hydrolysis and column chromatography, was researched to explore structure-function relationship among purified polysaccharides.Methods:Use ion exchange chromatography and sephadex gel filtration chromatography to get pure polysaccharide fractions. Use chemical method to determine basic composition. Use high performance liquid chromatography to test purity and get relative molecular mass.Use pre-column performance liquid chromatography(PMP-HPLC) to determine monosaccharide composition. Use infrared spectrometry(IR), nuclear magnetic resonance(NMR), periodate oxidation and smith degradation to analyze primary structure.Use beta elimination method and congo red test to analyze linkage-bond between sugar chain and peptide chain and three-strand spiral structure. Use chemical method such as phenanthroline and pyrogallol autoxidation spectrophotometric method to determine antioxidant activity in vitro.Results:1. Crude polysaccharides were extracted from Pinctada martensi Dunker by enzymatic hydrolysis and alcohol precipitation,and purified polysaccharides were got by isoelectric precipitation. PMPS-1, PMPS-2 and PMPS-3 were from refined polysaccharides from Pinctada martensi Dunker by DEAE-52 cellulose column chromatography and Sephadex G-100 column chromatography. Three fractions were different homogeneous polysaccharides, and those relative molecular mass were respectively 1.31×104 Da,3.02×106 Da,2.26×106 Da.As to CPS, RPS, PMPS-1, PMPS-2 and PMPS-3, total sugur content was 34.76 %,46.30 %, 82.84 %, 60.72 % and 43.89 % respectively. Glycosaminoglycan content was12.93 %, 4.31 %, 2.16 %, 6.64 % and 41.25 % respectively. Protein content was 32.85 %,9.20 %, 0.77 %, 8.10 % and 16.01 % respectively. Sulfate radical content was 3.65 %,4.85 %, 1.57 %, 1.64 % and 5.77 % respectively. Uronic acid content was 2.53 %, 3.55 %,13.64 %, 8.85 % and 2.49 % respectively. Total hexosamine content was 15.44 %, 15.92 %,4.56 %, 18.4 % and 21.28 % respectively.2. PMPS-1, composed of glucosamine, glucose, galactosamine, fucose, galactose,xylose, glucuronic acid and arabinose with a molar ratio of 1.60:94.98:0.21:0.58:1.19:0.73:0.20:0.5, was neutral polysaccharose with the highest content of glucose. The main chain of PMPS-2,which was acidic polysaccharides, was hexosamine and Ara, and their molar ratio was Glc N:Glc:Gal N:Fuc:Gal:Xyl:Glc UA:Ara:Gal UA=28.71:7.82:12.09:2.53:16.81:12.62:4.61:13.07:1.74. PMPS-3 was also acidic polysaccharose with hexosamine as main chain, and their molar ratio was Glc N:Glc:Gal N:Fuc:Gal:Xyl:Glc UA:Ara=57.95:4.82:19.16:5.84: 6.98:2.51:1.63:1.1.In UV and IR spectrum indicated that characteristic absorptions of polysaccharides,carboxyl radical, pyranoid ring were detected in CPS、RPS、PMPS-1、PMPS-2 和 PMPS-3.E specially, PMPS-3 had stronger absorption peaks of protein and sulfuric acid radical than other polysaccharide fractions.PMPS-1 was linked with backbone chain of →4)-α-D-Glcp-(1→ or→4,6)-α-D-Glcp-(1→ and branched chain of α-Glc-(1→, →3)-α-D-Glcp-(1→ or→3,6)-α-D-Galp-(1→. PMPS-2 was linked with backbone chain of →4)-β-D-Glc N(1→,→2)-α-L-Arap-(1→ or →4)-α-D-Xylp-(1→ and branched chain of →3)-β-D-Glcp-(1→,→2)-β-D-Glcp-(1→ or →2)-β-D-Galp-(1→. PMPS-3, without suger chain by 1→4、1→4,6 、1→3、1→3,4 and 1→3,6 glycosidic bond, was linked with backbone chain of→6)-β-D-Gal NAc-(1→ and branched chain of →2,6)-β-D-Glcp-(1→ or →2)-α-Fuc(1→.The results of beta elimination method showed that PMPS-2 had three-strand spiral structure, While PMPS-1 and PMPS-3 did not. Linkage-bond between sugar chain and peptide chain is N-glycopeetide linking bond in PMPS-2, while O-glycopeetide linking bond in PMPS-3.3. Pinctada martensi polysaccharides had strong scavenging ability of DPPH free radicals, superoxide anion free radicals and hydrogen peroxide, certain hydroxyl radical scavenging ability, however weak reduction potential. Antioxidant activity was gradually increased with the increase of polysaccharide concentrations from 0 mg/m L to 4 mg/m L.Antioxidant activity of polysaccharides in vitro from strong to low was PMPS-3﹥PMPS-2﹥PMPS-3.Conclusion:PMPS-1, PMPS-2 and PMPS-3 were different polysaccharide fractions purified from Pinctada martensii, and their relative molecular mass were 1.31×104 Da、3.02×106 Da、2.26×106 Da respectively.PMPS-1 was neutral polysaccharides, with backbone chain of α-(1→4) glucopyranose and branched chain of α-(1→3) glucopyranose. PMPS-2 was acidic proteoglycan with triple helical structure linked by N-glycopeetide, with backbone chain of β-(1→4)glucosamine and α-(1→2) arabinose and branched chain of β-(1→3) glucopyranose andβ-(1→2) galactose. PMPS-3 was acidic proteoglycan linked by O-glycopeetide, with backbone chain of β-(1→6) galactosamine and branched chain of β-(1→2) glucopyranose and α-(1→2) fucose.PMPS-1, PMPS-2, and PMPS-3 had the ability of scavenging free radicals in vitro.The antioxidant activity of PMPS-3 was stronger than others, because their contain of sulfuric acid and amino hexose was more high.