Isolation,Purification And Biological Activity Of Polysaccharide From Enteromorpha Prolifera

This research is based on the study of Enteromorpha prolifera.First,the nutrient composition of crude fiber,amino acids and elemental content were analyzed,then the crude polysaccharides WEP and AEP were extracted from E.prolifera by hot water extraction and alkaline extraction,respectively.The crude polysaccharides were sequentially purified by chromatography of DEAE-52 Cellulose and Sephadex G-100.10 purified polysaccharides's primary structures were analyzed by infrared spectroscopy(FT-IR),high-performance gel permeation chromatography(HPGPC),high performance liquid chromatography(HPLC),nuclear magnetic resonance(NMR),The surface morphology of purified polysaccharides were analyzed by scanning electron microscopy(SEM).Congo red test was used to conducted a preliminarily study on their advanced structures.Their antioxidant activity was tested by DPPH assay,superoxide anion radical assay(·O₂^-)and ABTS radical assay in vitro.To evaluate the The inhibity activity of polysaccharides against four tumor cell lines(MGC-803,Bel-7402,Hep G2 and T24)was investigated by MTT method.Water-extracted crude polysaccharide WEP was purified by DEAE-Cellulose 52 and Sephadex G-100 to yield six purified polysaccharides,coded PWEP-1,PWEP-2,PWEP-3,PWEP-4,PWEP-5and PWEP-6.The structure identification and pharmacological activity of the four water-extracted polysaccharides isolated in large quantities were studied.HPGPC results showed that the molecular weights of the four water-extracted sugar polymers(PWEP-1,PWEP-3,PWEP-5,and PWEP-6)were4259 Da,186056 Da,225267 Da,and 136817 Da,respectively.Alkali-extracted crude polysaccharide AEP was purified to yielded six fractions,named PAEP-1,PAEP-2,PAEP-3,PAEP-4,PAEP-5 and PAEP-6,with molecular weights of 34377 Da,22905 Da,64151 Da,120062 Da,48161 Da and105283 Da respectively.HPLC results showed that the main monosaccharide component of PWEP-1,PAEP-4 and PAEP-5 were rhamnose and the main monosaccharide component of PWEP-6,PAEP-1,PAEP-2 and PAEP-3 was galacturonic acid.PWEP-5 mainly contained glucuronic acid,PWEP-3mainly contained glucose and PAEP-6 mainly contained xylose.The monosaccharide composition analysis showed that PAEP-5 did not contain aldehydic acid monosaccharides indicating that was a neutral polysaccharide,while the rest were acidic polysaccharide.Preliminary studies of the advanced structure of the polysaccharides by Congo red test and SEM,Congo red test showed that no triple helical structure was detected in the 10 polysaccharides.The results of SEM showed that the microstructure of these 10 purified polysaccharides were different,which may be related to the extraction method,the concentration of the eluate and the molecular weight,monosaccharide components and other factors are relevant.NMR results showed that PWEP-5 contained?-D-Glcp A,?-D-Glcp,?-D-Galp,?-D-Manp and?-D-Rhap residues;PAEP-2 had a?4)-?-Galp A-6-OMe-(1?main chain backbone,the branch contains?-D-Manp-6-OMe-(1?,?3,5)-?-D-Araf-(1?and?-D-Glc A-(1?residues,and there are acetylated and methylated residues,indicating that PAEP-2 was a kind of pectic polysaccharide isolated from the cell wall;PAEP-4 had?-L-Rhap-(1?skeleton.In vitro antioxidant activity of purified polysaccharides were evaluated by DPPH,·O₂^-and ABTS radical assay.All of the 10 polysaccharides exhibited good antioxidant activity and showed a dose-dependent relationship to scavenge free radicals.Among the water-extracted purified polysaccharides,PWEP-5 had the strongest scavenging ability against the three radicals and the scavenging rates of DPPH,·O₂^-and ABTS were 57.27%,52.92%and 66.53%,respectively.Among the alkali-extracted purified polysaccharides,PAEP-1 and PAEP-2 had the highest scavenging rates as high as 56.40%,54.27%and 42.07%and the scavenging rates of PAEP-2 were 48.67%,52.92%and 48.20%.In vitro antioxidant results indicated that polysaccharides with lower molecular weight and larger uronic acid content have stronger antioxidant activity.The MTT results of antitumor activity showed that the inhibitory rate of purified polysaccharides against MGC-803,Bel-7402,T24 and Hep G2 were less than 50%,indicating that purified polysaccharides did not have significant inhibitory effect on tumor cells,which is related to the absence of triple helical structure.