Study On Polysaccharide From Hemicentrotus Pulcherrimus

Polysaccharide is the richest biomacromolecules in nature,it has many kinds of significant bioactivities, such as antitumor activity, hypoglycemic effect, antilipemic function, anti-oxidative effect, anti-virus, radioprotection and so on.Sea urchins, a kind of marine invertebrates, are sorted as Phylum Echinodermata, Class Echinoidea. Sea urchin roes or eggs are a kind of favorite seafood for their high nutrition and medicinal values in China. Considerable advances have been made at home and abroad in isolation of natural products from sea urchins. Studies on polysaccharides from Hemicentrotus pulcherrimus are not yet reported.Hemicentrotus pulcherrimus, collected for research, is one of the economic sea urchins in China.This paper reported two novel sulfated polysaccharide named as XB-1%和XB-3 was isolated and fractionated from Hemicentrotus pulcherrimus. Its physic-chemical properities were analyzed, and its structure was determined by chemical analysis and spectrum technology, indicating XB-1和XB-3 was the novel polysaccharide purified from sea urchin for the first time and evaluation of the antitumor activities.This dissertation consists of five parts, which are Optimization of extraction process of polysaccharide, Purification of the crude polysaccharide, Structure elucidation of XB-1 & XB-3,Antitumor and immunological activity of XB-1 & XB-3.The results are shown as following.1.Optimization of extraction process of polysaccharideThe active crude polysaccharides were obtained by acetone treatment, hot water extraction, removing protein and nucleic acid and ethanol precipitation.According to the orthogonal experiment and antitumor activities assay, the optimum conditions of polysaccharide of shellspines extract were 90℃,five hours, four times and 15mL H2O per gram shellspines. the optimum conditions of polysaccharide of eggs extract were 90℃,five hours, four times and 10mL H2O per gram eggs.Antitumor activities of crude polysaccharides in vitro were evaluated against human cancer cell A549,MCF-7,Hela by MTT method.The growth inhibitory rate of shellspines was 47.9%,38.1% repectively at the dose of 5.0 ug/mL.It has hardly no inhibition effect to Hela cells.The growth inhibitory rate of eggs was 47.2% at the dose of 5.0ug/mL.It has a certain inhibition effect to Hela and has no inhibition effect to MCF-7 cells.2.Isolation and purification of the active polysaccharide Novel sulfated polysaccharide named as XB-1 & XB-3 was isolated from the crude polysaccharides by hot water extraction and gel-permeation. The homogeneity of XB-1 & XB-3 was verified by HPLC,sepharose CL--4B gel chromatography.The polysaccharide XB-1 & XB-3 has a weight-average molecular weight of about 1.90×106 Da和2.0×106 Da by HPLC in reference to standard T-series Dextran. The specific rotation of XB-1 & XB-3 is +105°,+210°(c0.1,H2O).The total sugar content of XB-1 & XB-3 was determined to be 99.1% and 99.3% by phenol-sulfuric acid mthod.3.Structure elucidation of XB-1 and XB-3The structure of XB-1 and XB-3 has been determined by G.C, Periodate oxidation, Smith degradation, Methylation analysis, IR,1H-NMR, enzyme hydrolysis, SO4 determination and atomic force microscopy etc experiments. The composition was analysed by GC.Results showed that XB-1 was composed of sulfated Fucans. XB-3 was composed of the monosaccharide of galactose and glucose, and the molar ratio is 5:2.The results of periodate oxidation, Smith degradation and fully Methylated showed that XB-1 was composed of(1→2) Fucose and (1→), and 2/3 of 0-4 is S04. XB-3 showed that(1→6) galactose is main chain and(1→4) glucose is side chain,0-4 and 0-2 link of SO42-.The FT-IR spectrum showed that XB-1 and XB-3 had the hydroxyl stretching vibration of the polysaccharide and that the pyranose residues. Its sulfur content is determined by barium sulfide turbidity analysis.Sulfur content of XB-1 and XB-3 was 57.7%.enzyme hydrolysis showed that(1→2)Fucose is the main chain of XB-1.On the basis of the above-mentioned results,it can be concluded that XB-1 and XB-3 is composed of a repeating unit having the possible structure as shown: 4.Antitumor of polysaccharide XB-1 and XB-3Antitumor activities of polysaccharides in vitro were evaluated against human cancer cell A549, MCF-7, Hela by a MTT method.The XB-1 has certain inhibition in A549,MCF-7 cells and Hela cell growth, and showed strongly dependent on the dose.The growth inhibitory rate of XB-1 was 63.2%,18.32 %,42.08% repectively at the dose of 50 ug/mL.The XB-3 has a certain inhibition in Hela cell growth, The growth inhibitory rate was 35.15% at the dose of 50 ug/mL. It has no inhibition effect to MCF-7 and hep-2 cells.5.Immunological activity of polysaccharide XB-1 and XB-3By means of MTT, The immuno-modulating assay in vitro showed that XB-3 has no obvious inhibition effect to spleen lymphocyte. Within the range of 1000～1.95μg/mL, With the decreasing concentration of XB-1,the positive effect is weaker gradually.Effect of XB-3 to the propagation of T, B lymphocyte induced by ConA or LPS.XB-1 didn't co-stimulate spleen lymphocyte proliferation with ConA at 62.5～1.95μg/mL,XB-1 co-stimulated spleen lymphocyte proliferation with LPS(10μg/mL) at the dosage of 125～1000μg/mL remarkably. The effect of XB-3 to the propagation of T lymphocyte induced by ConA is more pronounced with decreasing concentration. Within the concentration of 1.95～1000μg/mL, XB-3 has a pronounced increasing effect to the propagation of B lymphocyte induced by LPS.