Degradation,Isolation,Purification And Biological Evaluation Of Polysaccharide From Pophyra Yezoensis Sargassumfusiforme

Porphyra yezoensis is a kind of economic brown algae,mainly distributed along the coast of Jiangsu Province.It contains rich nutrients such as proteins,polysaccharides,lipids,vitamins and minerals.Polysaccharides derived from Porphyra yezoensis were reported to be capable of providing relief to inflammation,anti-tumor,antioxidation,and some other biological effects.The objective of this thesis is to provide theoretical foundation for the development of Porphyra yezoensis functional foods possessing antioxidant,activities.Generally speaking,the biological activities of polysaccharides are considered to be dependent on the molecular weight,degree of branching and space structures.In the present study,in order to improve the biological activity of polysaccharide extracted from porphyra yezoensis(PSPY),it was degraded by ascorbic acid in combination with hydrogen peroxide.The degraded polysaccharide DPSPY was further isolated and purified.Chemical composition,structure and activity of the obtained homogeneous polysaccharide were preliminary investigated.The main results that we have obtained are as follows:1.Crude polysaccharide(PSPY)was extracted from porphyra yezoensis with hot water and was obtained by alcohol precipitation.PSPY was degraded by ascorbic acid in combination with hydrogen peroxide.The degradation conditions were optimized using a Box-Behnken response surface design.The optimum conditions were established as:4.1 mM of ascorbic acid and hydrogen peroxide,degradation temperature 52.4℃ and degradation time 2.1 h.2.The contents of chemical component of PSPY and DPSPY are different.It was found that the content of carbohydrate and sulfate increased significantly,while the content of protein decreased,after degradation.The molecular weight of PSPY and DPSPY were determined to be 4.52×105 and 1.70×104 by high performance gel permeation chromatography(HPGPC).FT-IR spectra of DPSPY had no significant different with those of PSPY.The in vitro antioxidant activities of PSPY and DPSPY were evaluated by determining their ferric iron reducing power and radical(DPPH,hydroxyl and superoxide anion radicals)-scavenging abilities.It was found that the antioxidant activity of DPSPY was greatly increased when compared to those of PSPY.Basing on the simulation of human digestive environment in vitro and using the cholestyramine as a positive control and cellucose as a negative control,the binding capacity of DPSPY to five bile acid derivatives was found to be stronger than that of PSPY.3.In this study,DPSPY was isolated and purified with the cellulose DEAE-52 and Sephadex G-100.Four fractions were isolated,one neutral polysaccharide being named DPSPY-0 and three acidic polysaccharide fractions being named DPSPY-0.1M,DPSPY-0.3M and DPSPY-0.5M,respectively.The molecular weight of DPSPY-0,DPSPY-0.1M,DPSPY-0.3M and DPSPY-0.5M was determined to be 1.08×104,1.07×104,1.87×104and 3.55×104 respectively.As to DPSPY-0,DPSPY-0.1M,DPSPY-0.3M and DPSPY-0.5M,polysaccharides content was 75.86%,78.40%,78.54%and 79.20%respectively,protein content was 0.72%,not detected,0.24%and not detected,respectively,sulfate content was 4.9%,16.93%,17.10%and 17.76%respectively.GC-MS was employed to analyze the monosaccharide composition,which indicated that DPSPY-0,DPSPY-0.1M,DPSPY-0.3M and DPSPY-0.5M were mainly made up of galactose as well as small portions of mannose,rhamnose,glucose and xylose.UV detection inferred that DPSPY-0.1M and DPSPY-0.5M had no absorption peaks at 260～280 nm and 400～700 nm indicating that the two samples do not contained nucleic acid,protein and pigments,while the absorption of DPSPY-0 and DPSPY-0.3M at the 280 nm is very weak,indicating that these polysaccharide fractions may have a small amount of bound glycoprotein.The IR spectra showed that the four purified components had the characteristic absorption peaks of polysaccharides,all of which were pyranoses.4,Immunomodulating activities in vitro of PSPY,DPSPY,DPSPY-0,DPSPY-0.1M,DPSPY-0.3M and DPSPY-0.5M were evaluated by using the cell model experiments.Results showed that porphyra yezoensis polysaccharides could promote proliferation of RAW264.7 macrophages,and increase ability of devouring neutral red of the peritoneal macrophages in vitro.DPSPY and DPSPY-0.5M have the ability to promote the secretion of cytokine nitric oxide by RAW264.7 phagocytes.5.Ferric iron reducing power and radical(DPPH,hydroxyl and superoxide anion radicals)-scavenging ability assays indicated that DPSPY-0.5M possessed the strongest antioxidant activity,and DPSPY-0 the weakest among the four purified polysaccharide fractions.The order of the antioxidant activity of the four fractions basically agrees with that of their sulfate content.