Study On Effect Of Different Molecular Weight Sargssum Fucoidan On Thrombosis And Vascular Endothelial Cells

This paper prepared different levels of Sargassum fucoidan by ultrasonic hot water extraction,DEAE Cellulose-52 anion exchange chromatography,free radical oxidation,Superdex 75 gel column chromatography and analysized the chemical composition of fucoidan;analyze the structure of fucoidan by GC-MS,gel filtration chromatography and infrared spectroscopy;preliminarily evaluated Sargassum fucoidan antithrombotic activity by blood clot lysis assay in vitro and mice pulmonary embolism experiment,and investigated the hemorrhagic risk of Sargassum fucoidan by mice tail bleeding experiment;cultured human umbilical vein endothelial cells?HUVEC?,studied on the effects of different levels of fucoidan on proliferation and secretion function of HUVEC,to investigate the antithrombotic pathway and mechanism,in order to provide data support for Sargassum fucoidan as antithrombotic functional food,and provide the basis for improving utility value of Sargassum and development of characteristic Seaweed resources.The main research contents are as follows:?1?Prepared crude Sargassum fucoidan?F?by ultrasonic hot water extraction,the two fraction F?F1 were purified by DEAE-C-52 anion exchange chromatography.DF1?DF2were prepared by using different conditions of free radical oxidative degradation to degradate F1.Then DF1?DF2 were purified by Superdex 75 prep grade gel column,and the DF11,DF12,DF21,DF22 four components were obtained by collection according to peaks.Detected the polysaccharide contents of F,F1,F2,DF1,DF2 were 32.86%,58.62%,50.74%,49.27%,54.38%;sulfate contents were 12.76%,20.78%,21.08%,17.52%,17.73%;the fucose contents were 15.62%,24.87%,19.43%,24.92%,28.34%;the contents of uronic acid were 11.93% 13.86%,9.52%,10.85%,9.56% respectively.Protein contents of F before and after removal of protein and F1 were 9.41%,6.02%,3.61%.Detected the sulfate contents of DF12,DF21,DF11,DF22 were 15.07%,9.66%,15.15%,10.48%;the fucose contents were 18.97%,13.37%,24.81%,10.48%.The UV spectra showed that the protein content of F1 was significantly lower than that of F,and the degradated fucoidan had almost no protein.?2?Determinated monosaccharide components F and F1 by GC-MS,found the two are complex material composed of fucose,glucose,galactose,mannose and other various monosaccharide,but the type and the proportion of each monosaccharides are different,thetype of monosaccharides of F1 is more abundant than F.Measured the average relative molecular weight of F?F1 were 567.7 K and 439.3 K.DF11,DF12,DF21,DF22 are more homogeneous component,average relative molecular weight were 2027,6166,19953,7493 respectively.Infrared spectrum showed that the several fucoidan fraction contained characteristics absorption peak of polysaccharide and sulfate,and the glycosidic bond type were beta glycosidic bond.¹H NMR and ¹³C NMR showed that Sargassum fucoidan are composed of fucose and other various monosaccharide,and contained sulfate group,the anomeric carbon existence of alpha and beta two configurations.?3?Compared and evaluated antithrombotic activity of crude fucoidan F and F1 preliminarily by blood clot lysis assay in vitro,and pulmonary embolism test in mice.And evaluated evaluation risk of the two fucoidan by the bleeding experiment.The results showed that,the middle?high dose of F,F1 had significant effect of blood clot dissolution?P < 0.05?,the high dose of F1 can significantly improve the survival rate of pulmonary embolism model mice?P < 0.05?.Bleeding time of two kinds of fucoidan were significantly different with that of the saline control group?P < 0.05?,and were significantly less than that of heparin positive control group?P < 0.05?.Experiments indicate fucoidan have good antithrombotic activity,less hemorrhagic risk,and in a dosedependent manner.?4?Comparative studied the protective function of F1,DF1,DF12,DF21,DF22 the five Sargassum fucoidans to HUVEC injury model which induced by adrenalin?Adr?,and studied the effect on endothelial cell secretion of vWF,t-PA.PAI-1,TF,TFPI by enzymelinked immunosorbent assay?ELISA?,in order to explore the possible ways of antithrombosis.The results showed that 5 kinds of fucoidan had protective effects on endothelial cells,can antagonize the damage of endothelial cell caused by adrenalin,can enhance proliferative activity of damaged endothelial cells;can reduce the secretion of vWF by endothelial,block platelet bind with the exposed collagen of damaged endothelial cells,thereby inhibiting platelet activation,inhibiting the platelet adhesion and aggregation;can reduce the the secretion and release of PAI-1 by damaged endothelial cells,raise the ratio of t-PA/PAI-1,increase the fibrinolytic activity of endothelial cells;can reduce the increation of TF which caused by Adr,inhibit the extrinsic coagulation system and play anticoagulant effection;but it does not significantly increase the release of TFPI,has less effect on exogenous blood coagulation system.Sargassum fucoidan can play important role of antithrombosis through the above multiple pathways.