Studies On Preparation And Antitumor Activity Of Low Molecular Weight Fucoidin From Laminaria Japonica

Fucoidan, a kind of polysaccharides containing substantial percentages of L–fucoseand sulfate ester groups and different proportion of xylose, glucose, galactose, arabinoseand uronic acid etc, has varied biological activities, including antivirus, anticoagulant,antitumor, regulating immune function, and other biological activities. And the activitiesare associated with the fucoidan structure, such as the content of sulfate, monosaccharidecomposition, molecular weight and so on. The main research contents and results in thispaper are as follows:1. Purification and composition analysis of fucoidan from Laminaria japonica. Thecrude Laminaria japonica fucoidan purchased from Baoju Seaweed Company, Yantai,China was purified. The yield rate of purified fucoidan reached10.77%by stepwiseaddition of ethanol. Composition analysis showed that the content of sugar in the purifiedfucoidan was68.80±0.45%. The content of fucose in the purified fucoidan was30.20±1.44%. The content of protein in the purified fucoidan was1.30±0.32%. The contentof glucuronic acid in the purified fucoidan was11.49±0.64%. The content of sulfate in thepurified fucoidan was9.20±1.02%. The result of thin layer chromatography analysis (TLC)showed the purified fucoidan was composed of D–galactose, L–fucose, possiblyD–mannose and/or D–arabinose. GC analysis showed monosaccharides constituents in thepurified fucoidan were L–fucose, L–arabinose, D–xylose, D–mannose, D–glucose,D–galactose. And their contents were31.33%,15.16%,12.83%,10.72%,20.99%,8.97%,respectively.2. Preparation and property identification of the highest antitumor component: Thelower weight molecular fucoidan FS was prepared by the microbial enzymolysis; and sixcomponents (F1, F2, F3, F4, F5, F6) were obtained by DEAE-Sepharose Fast Flow ionexchange column. The antitumor activities of the products were detected by MTT assaysand Hep-2cells were selected as detected cells. The results showed that the low weightmolecular fucoidan fraction5has the higest antitumor activity, with a inhibition rate48.05±0.73%. GC analysis results showed that F5was composed mainly of L–fucose andsuspect spots of L–arabinose, D–xylose, D–mannose, D–glucose, D–galactose. And theircontents were42.42%,8.71%,9.28%,11.50%,10.61%,17.48%, respectively. Themolecular weight of the F5was ranged from12107to33329Da by high performance liquid chromatography method. The content of the sulfate was16.21%±0.58%. Thus wehad got fucoidan composed mainly of L–fucose with low molecular weight and highcontent of sulfate.3. Pilot study of the antitumor activity of F5. The inhibitory capability of F5wasdose-related and time-related by MTT assays, and the IC50is0.149mg/mL. Hep-2cellstreated by F5appeared chromatin condensation, cell shrinkage, looking like cell apoptosis.The comets were found under the fluorescent microscope by single cell gelelectrophoresis assay, which suggested that the DNA of Hep-2cells were damaged by F5.And the longer F5were treated the more severe the Hep-2cells were damaged.