Extraction And Chemical Component Analysis Of Polysaccharides From Different Sea Cucumbers

Ten species of sea cucumbers with different price and quality were selected from the market for chemical, spectrum and hydrolyzation analysis. Polysaccharides from different sea cucumbers were separated by papain, precipitated by ethanol, analyzed with six chemical indexes using traditional method and HPLC, and eluted on Q-Sepharose FF, recording the elution curve. Sulfation position was determined by IR and NMR; hydrolyzation with H2SO4 was analyzed by TLC and HPLC.The result shows that chemical component of polysaccharides from ten species of sea cucumbers are obviously diversified, and the elution curves of these polysaccharides on Q-Sepharose FF are distinct. For the yield of ten polysaccharides, polysaccharide from Apostichopus japonicus arrested in China achieves the highest, 7.16%, and the lowest goes to polysaccharide from Apostichopus japonicus arrested in Japan, 1.31%. The molecular weight of ten polysaccharides falls into three groups: >700,000, 160,000-250,000, 5,000-12,000. The molecular weight of the main fraction of polysaccharides from Holothuria vagabunda,Holothuria mexicana, Cucumaria frondosa, Stichopus tremulus and Apostichopus japonicus arrested in Japan are in the second group, and the rest five polysaccharides are in the first group. Content of total sugars, protein, sulfate, uronic acid and aminosugar of polysaccharides from ten species of sea cucumbers are fluctuated within 40.33%-62.11%,0.44%-19.48%, 19.54%-29.95%, 9.85%-13.38% and 4.92%-10.18%, respectively. Neutral monosaccharide analysis shows that fucose and galactose exist in all the ten polysaccharides while mannose and glucose existed partly, and the content of fucose is very high. Elution curve of ten polysaccharides on Q-Sepharose FF differs obviously. All the polysaccharides are eluted at the salinity of 3.2 mol/L, except for those from Apostichopus japonicus. A fast and sensitive method is developed for simultaneous determination of neutral sugars, amino sugars and uronic acids in polysaccharides extracted from sea cucumbers, which is best hydrolyzed under the condition of 2mol/L TFA at 110℃for 8h, using HPLC with 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization and Variable Wavelength Detection (VWD). Compared with the result determined in traditional method, the chromatography result seems exact for aminosugars and neutral sugars, and lower for uronic acid, but the latter method gives more precise result and can be applied for monosaccharide component analysis in polysaccharides from S. fusiforme, squid ink, shark bone, shark fin and seaweeds with satisfying result.The IR analysis of ten polysaccharides shows that each polysaccharide contains groups of -OH,-COOH,-NHAc,-SO4,-CH3, and is sulfated at different positions. Sulfate groups of polysaccharides from Pearsonothria graeffei,Apostichopus japonicus arrested in China,Holothuria scabra,Holothuria vagabunda,Holothuria mexicana and Isostichopus fuscus are substituted at C-4 position of fucose residues or C-4 position of galactosamine residues; Sulfate groups of polysaccharides from Cucumaria frondosa and Isostichopus badionotus are substituted at C-4 position of fucose residues or C-2/4 position of galactosamine residues; and those for Stichopus tremulus and Apostichopus japonicus arrested in Japan are at C-2/3/4 position of fucose residues or C-4/6 position of galactosamine residues. The NMR analysis shows that sulfate groups are disubstitued at C-2,4 positions in all the polysaccharides except for that of Holothuria scabra, and at C-3,4 positions in Pearsonothria graeffei,Holothuria scabra,Holothuria vagabund,Holothuria mexicana,Cucumaria frondosa and Apostichopus japonicus arrested in Japan. Fucose without sulfation existed in all the polysaccharides except for those from Stichopus tremulu,Isostichopus badionotus and Isostichopus fuscus. The best hydrolytic condition for sea cucumber polysaccharides is with 0.05 mol·/LH2SO4 at 80℃for 18h. Four to seven component are found in ten hydrolyzates after TLC separation. Component from Holothuria mexicana,Stichopus tremulus,Isostichopus badionotus,Isostichopus fuscus and Apostichopus japonicus arrested in Japan are suitable for further analysis. HPLC analysis shows that the molecular weight of ten hydrolyzates is between 920 to 2000, and the possible polymeric degree is 5 to 11.