A Novel Procedure Of The Extraction Of Fucoidan With A High Yield From Laminaria Japonica And Its Bio Activity

In this study,a novel method was developed to extract fucoidan from brown seaweed Laminaria japonica.The extraction procedure was optimized.The purification of crude fucoidans was carried out.The composition of purified fucoidans was analyzed,and the bioactivity of fucoidans was further investigated.The findings and conclusions are as follows:0.5%ethylenediaminetetraacetic acid(EDTA)solution as the solvent was used to extract fucoidans from dried L.japonica.The optimal conditions for fucoidan extraction are as the following:the ratio of algae to solvent at 1:30,constant stirring at 70? for 3 h,pH 7 before precipitation with ethanol.The collected precipitate dissolved in water was dialyzed against water using a membrane with MWCO 3600 Da to remove to the remains of EDTA,which further removed by the activated carbon adsorption method.The yield of crude fucodian(CF)is around 16%,in which the contents of total sugar and sulfate are 73.00%and 27.95%.The crude fucoidan was purified by DEAE sephadex A25 anion exchange chromatography.Three fractions,F1,F2 and F3 with the yields of 13.00%,52.95%and 7.08%were obtained by step-wise elution with 0?2 M NaCl solution.The contents of total sugar and sulfate in the F1,F2 and F3 are 64.82%,68.40%,45.42%and 17.77%,30.38%,13.20%,respectively.The molar ratios of the sulfate content to fucose in F1,F2 and F3 are 2.09,2.02 and 1.82,respectively.The major monosaccharide composition in the fractions of F2 and F3 is fucose,whereas a high amount of glucose and glucuronic acid is present in the fractions of CF and F3.The molecular weights of CF,F1,F2 and F3 are 64.04 KDa,28.95 KDa,46.17 KDa and 30.01 KDa,respectively,using a high performance size exclusion chromatography.The result of IR spectrum shows that absorption peaks of O-H stretching vibration(3416-3440 cm-1),C-H stretching vibration(2920-2988 cm-1)and S=O stretching vibration(1254-1296 cm-1)existed in each fraction,indicating that all fractions are fucoidans with the sulfate ester.The band at around 820 cm-1 of CF,F1 and F2 is probably due to the sulfate group mainly located at the position of C-2 or C-3,less at the location of C-4 of the side chain,but the F3 was not found.F1 and F2 have nearly the same antioxidant activity,much higher than CF and F3.The high antioxidant activities of the crude fucoidans and its fractions are due to a combination of several factors associated with high sulfate content,high molar ratio of sulfate content to fucose and monosaccharide composition in extracted fucoidans.F3 exhibits less cytotoxicity than F1 and F2 with around 30%inhibition rate at 2 mg·ML-1,indicating that the low sulfate content and the monosaccharide composition play an important role in the anticancer activity.