The Study On The Structure,Molecular Modification And Macrophage-stimulating Activities Of Enzymolysis Fragments From Ascophyllan

In this study,firstly,the low molecular weight fragments of ascophyllan were prepared by degrading ascophyllan with enzymes.Secondly,the molecular weights,monosaccharide compositions and chemical compositions of these low molecular weight fragments were analyzed with HPSEC,GC-MS and some chemical methods,respectively.Then,the fragments possessed strong macrophage-stimulating activities were selected by the analysis of macrophage-stimulating activities.Further,to study the relation between bioactivities and structures,the glycosidic bond types of these fragments were measured.Finally,to study the effects of different modified group on cell signal pathway that related activating macrophage-stimulating activities,these fragments with strong macrophage-stimulating activity were chemical modified and the macrophagestimulating activity of their derivatives were also analyzed.The results as following:Six fragments were obtained by enzymatically degraded ascophyllan and by separated with ultrafiltration,i.e As-A,As-AH,As-C,As-CH,As-X and As-XH.And their molecular weight were 3.5 KDa、105.9 KDa,2.5 KDa,72.2 KDa,2.7 KDa and 103.4 KDa,respectively.The six fragment both were consisted of Man,Glc,Gal,Xyl and Fuc,however,the contents of monosaccharides,sulfate and uronic acid were different among these fragments.What’smore,As-A and As-AH were analyzed as the fragments with strong macrophage-stimulating activity.According to the results of methylation,the As-A mainly contained six different glycosidic linkages types,i.e T-D-GlcpA,1,3-D-Fucp,1,4-D-Xylp,1,4-D-Manp,1,2-D-Galp and 1,2-D-Glcp;the As-AH mainly contained four different glycosidic linkages types,i.e T-D-GlcpA,1,3-D-Fucp,1,4-D-Xylp,and 1,4-D-Manp.In addition,the results of periodate oxidation and Smith degradation demonstrated that As-A consists mainly of(1→2)-glycosidic linkages and may contain a small amount(1→3),(1→4)-glycosidic bond;As-AH consists mainly of 1→3,1→4-glycosidic linkages and may contain a small amount of 1→glycosidic bond.As-A and As-AH were chemical modified with acylation,carboxymethylation,phosphorylation and sulfation,respectively.The derivatives were As-AA(DS=0.090),As-AHA(DS=0.030),As-AC(DS=0.860),As-AHC(DS=0.010),As-AP(DS=0.013),As-AHP(DS=0.012),As-AS(DS=0.620)and As-AHS(DS=1.310),respectively.The results of macrophagestimulating activity indicated that acetyl group can significantly improve the macrophagestimulating activity of polysaccharide fragment;phosphate group and sulfate group have no significant effect on the macrophage-stimulating activity of polysaccharide fragments;however,carboxymethyl group can significantly reduce the macrophage-stimulating activity of polysaccharide fragments.Furthermore,the results of modified groups effect cell signal pathway suggested that acetyl group and carboxymethyl group can effectively activate the JNK-MAPK signal pathway;the phosphate group can effectively activate NF-κB signal pathway;while the activation of the signal pathway by the sulfate group is complex and may be related to other factors.