Isolation, Fractionation, And Preliminary Characterization Of A Polysaccharide From Apostichopus Japonicus

In this study, we selected Apostichopus japonicas as the material for theisolation, fractionation and preliminary characterization. We have established amethod of precolumn derivatization HPCE, which could be used to analyze theneutral saccharide, uronic acid and amino saccharide. This HPCE method has thecharacters of briefness, speediness and high efficiency, that is meaningful for theanalysis of monosaccharide composition of holothurians polysaccharides.Polysaccharides from Apostichopus japonicas was hydrolyzed by papain,precipitated by ethanol and potassium acetate, analyzed with the traditional chemicalmethods and precolumn derivatization HPCE, determined it’s molecular weight byHPGPC, and eluted on Q-FF following the elution curve. Its’ sulfated position wasdetected by IR,1H NMR and MS, meanwhile, the structures of main chain and thebranch were essayed by HPCE and1H NMR after mild hydrolyzation.The results showed the following conclusions: the ratio of polysaccharidesobtained from sea cucumber is1.18‰(calculated with the fresh sea cucumber); themolecular weight got through HPGPC is43092; the content of total sugar, protein,sulfate, uronic acid and aminosugar of polysaccharides are51.54%,5.50%,25.07%,17.20%and13.53%, respectively. The GC results showed that fucose exists mostly inthe polysaccharides with a high content of15.4%.A quick and sensitive method is established for simultaneous analysis of neutralsugars, uronic acids and amino sugar in the polysaccharides from the sea cucumber,which is hydrolyzed under the situation of4mol/L TFA at110℃for6h. Thehydrolyzed product is applied to HPCE at the wavelength of245nm after derivatizedwith1-phenyl-3-methyl-5-pyrazolone (PMP). The consequence of Apostichopusjaponicas showed that the monosaccharides composition of galn, glcn, galnac, glc,man, fuc, gal, glcua is3.23:0.83:0.38:3.97:0.68:1.00, which had a much higherconten of fucose compared with the result of GC. The consequence of IR indicated that the polysaccharide had the carboxyl group,acetamido group, sulfate and methyl group of fucose. Polysaccharide fromApostichopus japonicas was eluted by utilizing Q-FF anion exchange resin with threedifferent concentration of NaCl, and three class of polysaccharides SC-1, SC-2andSC-3were abtained followed by dialysis and purification. The results of theirmonosaccharide composition analyzed by HPCE showed that SC-1was a sulfatedfucan, SC-2and SC-3were two diseparate glycosaminoglycan. According to comparebetween IR and1H NMR, we inferred the sulfation substitute of fucose as follows:only few C-4sulfation existed in SC-1; SC-2consist3,4-disulfation fucose and nativefucose; SC-3had some2,4-disulfation fucose and4-sulfation fucose of lowconcentration.