Preparation,Structural Characterization And Biological Activity Of Fucoidan From Laminaria Japonica

The Laminaria japonica from Fujian Province was used as raw material to extract fucoidan by water extraction method,and the extraction process was optimized.Ethanol reprecipitation method was used to purified the crude fucoidan,and the fucoidan was further purified by Q sepharose fast flow?Q FF?anion exchange chromatography.The molecular weight,physical and chemical properties,monosaccharide composition and ratio of different fucoidan components were analysed by HPLC chromatography and the biological activities of different fucoidan were investigated.The main research contents are as follows:?1?After comparing the yield of fucoidan preparated by water extraction and compound enzymes method,and water extraction was selected as the best extraction condition.In order to improve the yield of fucoidan,hot water extraction process of fucoidan from Laminaria japonica was optimized by response surface methodology.The optimum process parameters for hot water extraction process was obtained as:extraction temperature 92?,extraction time 4 h,liquid-material ratio 41?1,and the fucose content of fucoidan was 20.94%.Then,Ethanol reprecipitation method was used to purified the crude fucoidan,and the fucose content of primary fucoidan was 24.13%.We used Q FF to further isolate fucoidan.After that,four fucoidan named SF1,SF2,SF3,SF4 were got.The molecular weights of each component fucoidan were 225.28 kDa,231.72 kDa,234.68 kD and 249.68 kDa,respectively.The monosaccharide composition of different fucoidan fractions was determined by HPLC,the molar ratio of Mannose,Rhamnose,Galactose,Fucose in SF1 was0.6249?0.5056?0.8408?1,the molar ratio of Mannose,Rhamnose,Galactose,Xylose,Fucose in SF2 was 0.4962?0.8136?0.4747?0.0460?1,the molar ratio of Mannose,Rhamnose,Galactose,Xylose,FucoseinSF3was0..2782?0.6289?0.5761?0.0553?1,the molar ratio of Mannose,Rhamnose,Galactose,Fucose in SF4 was 0.7369?0.6787?1.7292?1.?2?Infrared spectroscopy analysis showed that the strong absorption peaks of SF1-SF4 around 1250 cm^-1 were attributed to S=O stretching vibration,indicating that all four fractions were sulfated polysaccharides,1733 cm^-1 has a weak peak indicating that the polysaccharide contains acetyl.It can be seen from the ultraviolet spectrum that four fractions do not contain proteins and nucleic acids.According to results of ¹H and ¹³C NMR spectroscopy,SF4 is partially acetylated and sulfated with its main chain consisting of blocks built up of fucose and galactose residues.?3?The effects of SF1,SF2,SF3 and SF4 on cytotoxicity,NO and cytokine release and MAPK protein expression of RAW 264.7 macrophages were studied.The cell viability results showed that four fractions were not toxic to cells.NO release levels of RAW 264.7 macrophages treated with LPS increased significantly.The results showed that increasing concentrations of SF1 and SF2 increased the NO release in LPS-stimulated RAW 264.7 macrophages,whereas NO release in SF4 showed dose-dependent reduced.RAW 264.7 macrophages were treated with LPS alone significantly increased cytokine secretion compared to the control group.SF1 and SF2 that exhibit the same effect as LPS,the results showed that SF1 and SF2 dose-dependently increased the TNF-?,IL-1?and IL-6 in LPS-stimulated RAW264.7 macrophages.However,pretreatment of RAW 264.7 macrophages with SF3and SF4 significantly inhibited the cytokine sections.According to measure the NO release and cytokine sections on LPS-stimulated RAW 264.7 macrophages with SF1,SF2,SF3 and SF4,it was determined that SF1 has immunostimulatory activity,and SF4 has anti-inflammatory potential among the four fractions.It can be seen from the results that SF1 significantly promotes the expression of ERK 1/2,JNK,and p38phosphorylated protein levels,and the phosphorylation of ERK 1/2,JNK was significantly dose-dependently reduced by SF4,and SF4 inhibit phosphorylation of p38 protein expression.The date show that SF1 had inmmunomodulatory potencial through activating RAW 264.7 macrophages and enhancing host immune system function,which enabled it to be a novel immunomodulator for application in immunological disease of functionl food,and SF4 had the anti-inflammatory properties.