Reevaluation of bactericidal, cytotoxic, and macrophage-stimulating activities of commercially available Fucus vesiculosus fucoidan

  • cc icon
  • ABSTRACT

    Polysaccharides prepared from marine algae sometimes contain contaminants such as polyphenols and endotoxins that may mislead their bona fide biological activities. In this study, we examined bioactive contaminants in commercially available fucoindan from Fucus vesiculosus, along with ascophyllan and fucoidan from Ascophyllum nodosum. F. vesiculosus fucoidan inhibited the growth of Vibrio alginolyticus in a concentration-dependent manner (0-1,000 μg mL-1). However, the antibacterial activity of the fucoidan significantly reduced after methanol-extraction, and the methanol-extract showed a potent antibacterial activity. The extract also showed cytotoxicity to RAW264.7 and U937 cells, and induced apoptotic nuclear morphological changes in U937 cells. These results suggest that the antibacterial activity of the fucoidan is partly due to the methanol-extractable contaminants that can also contribute to the cytotoxicity on RAW264.7 and U937 cells. On the other hand, the activities to induce secretion of nitric oxide and tumor necrosis factor-α from RAW264.7 cells were observed in the fucoidan even after methanol extraction, and the extract had no such activities. Our observations suggest that commercially available fucoidan should be purified prior to biochemical use.


  • KEYWORD

    antibacterial activity , contaminants , cytotoxic activity , Fucus vesiculosus , fucoidan

  • 1. Ahmed S. A., Gogal R. M. Jr., Walsh J. E. 1994 A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay [J. Immunol. Methods] Vol.170 P.211-224 google doi
  • 2. Baba M., Schols D., Pauwels R., Nakashima H., De Clercq E. 1990 Sulfated polysaccharides as potent inhibitors of HIV-induced syncytium formation: a new strategy towards AIDS chemotherapy [J. Acquir. Immune Defic. Syndr.] Vol.3 P.493-499 google
  • 3. Chotigeat W., Tongsupa S., Supamataya K., Phongdara A. 2004 Effect of fucoidan on disease resistance of black tiger shrimp [Aquaculture] Vol.233 P.23-30 google doi
  • 4. Clement M. J., Tissot B., Chevolot L., Adjadj E., Du Y., Curmi P. A., Daniel R 2010 NMR characterization and molecular modeling of fucoidan showing the importance of oligosaccharide branching in its anticomplementary activity [Glycobiology] Vol.20 P.883-894 google doi
  • 5. Collins L., Franzblau S. G. 1997 Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium [Antimicrob. Agents Chemother.] Vol.41 P.1004-1009 google
  • 6. Costa L. S., Fidelis G. P., Cordeiro S. L., Oliveira R. M., Sabry D. A., Camara R. B., Nobre L. T., Costa M. S., Almeida- Lima J., Farias E. H., Leite E. L., Rocha H. A. 2010 Biological activities of sulfated polysaccharides from tropical seaweeds [Biomed. Pharmacother.] Vol.64 P.21-28 google doi
  • 7. Croci D. O., Cumashi A., Ushakova N. A., Preobrazhenskaya M. E., Piccoli A., Totani L., Ustyuzhanina N. E., Bilan M. I., Usov A. I., Grachev A. A., Morozevich G. E., Berman A. E., Sanderson C. J., Kelly M., Di Gregorio P., Rossi C., Tinari N., Iacobelli S., Rabinovich G. A., Nifantiev N. E. 2011 Fucans but not fucomannoglucuronans determine the biological activities of sulfated polysaccharides from Laminaria saccharina brown seaweed [PLoS One] Vol.6 P.e17283 google doi
  • 8. Cumashi A., Ushakova N. A., Preobrazhenskaya M. E., Incecco A., Piccoli A., Totani L., Tinari N., Morozevich G. E., Berman A. E., Bilan M. I., Usov A. I., Ustyuzhanina N. E., Grachev A. A., Sanderson C. J., Kelly M., Rabinovich G. A., Iacobelli S., Nifantiev N. E. 2007 A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds [Glycobiology] Vol.17 P.541-552 google
  • 9. Damonte E. B., Matulewicz M. C., Cerezo A. S. 2004 Sulfated seaweed polysaccharides as antiviral agents [Curr. Med. Chem.] Vol.11 P.2399-2419 google doi
  • 10. De Souza M. C. R., Marques C. T., Dore C. M. G., Da Silva F. R. F., Rocha H. A. O., Leite E. L. 2007 Antioxidant activities of sulfated polysaccharides from brown and red seaweeds [J. Appl. Phycol.] Vol.19 P.153-160 google doi
  • 11. Ellouali M., Boisson-Vidal C., Durand P., Jozefonvicz J. 1993 Antitumor activity of low molecular weight fucans extracted from brown seaweed Ascophyllum nodosum [HeliAnticancer Res.] Vol.13 P.2011-2019 google
  • 12. Heinzelmann M., Polk H. C., Miller F. N. 1998 Modulation of lipopolysaccharide-induced monocyte activation by heparin-binding protein and fucoidan [Infect. Immun.] Vol.66 P.5842-5847 google
  • 13. Jiang Z., Okimura T., Yamaguchi K., Oda T. 2011 The potent activity of sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum to induce nitric oxide and cytokine production from mouse macrophage RAW2647 cells: comparison between ascophyllan and fucoidan [Nitric Oxide] Vol.25 P.407-415 google doi
  • 14. Jiang Z., Okimura T., Yokose T., Yamasaki Y., Yamaguchi K., Oda T. 2010 Effects of sulfated fucan ascophyllan from the brown alga Ascophyllum nodosum on various cell lines: a comparative study on ascophyllan and fucoidan [J. Biosci. Bioeng.] Vol.110 P.113-117 google doi
  • 15. Jin J. -O., Song M. -G., Kim Y. -N., Park J. -I., Kwak J. -Y. 2010 The mechanism of fucoidan-induced apoptosis in leukemic cells: involvement of ERK1/2 JNK glutathione and nitric oxide [Mol. Carcinog.] Vol.49 P.771-782 google
  • 16. Kang S. -M., Kim K. -N., Lee S. -H., Ahn G., Cha S. -H., Kim A. -D., Yang X. -D., Kang M. -C., Jeon Y. -J. 2011 Anti-inflammatory activity of polysaccharide purified from AMG-assistant extract of Ecklonia cava in LPS-stimulated RAW 2647 macrophages [Carbohydr. Polym.] Vol.85 P.80-85 google doi
  • 17. Karmakar P., Pujol C. A., Damonte E. B., Ghosh T., Ray B. 2010 Polysaccharides from Padina tetrastromatica: structural features chemical modification and antiviral activity [Carbohydr. Polym.] Vol.80 P.513-520 google doi
  • 18. Kim E. J., Park S. Y., Lee J. -Y., Park J. H. 2010 Fucoidan present in brown algae induces apoptosis of human colon cancer cells [BMC Gastroenterol] Vol.10 P.96 google doi
  • 19. Kloareg B., Demarty M., Mabeau S. 1986 Polyanionic characteristics of purified sulphated homofucans from brown algae [Int. J. Biol. Macromol.] Vol.8 P.380-386 google doi
  • 20. Koyanagi S., Tanigawa N., Nakagawa H., Soeda S., Shimeno H. 2003 Oversulfation of fucoidan enhances its anti-angiogenic and antitumor activities [Biochem. Pharmacol.] Vol.65 P.173-179 google doi
  • 21. Kusaykin M., Bakunina I., Sova V., Ermakova S., Kuznetsova T., Besednova N., Zaporozhets T., Zvyagintseva T. 2008 Structure biological activity and enzymatic transformation of fucoidans from the brown seaweeds [Biotechnol. J.] Vol.3 P.904-915 google doi
  • 22. Leiro J. M., Castro R., Arranz J. A., Lamas J. 2007 Immunomodulating activities of acidic sulphated polysaccharides obtained from the seaweed Ulva rigida C [Agardh. Int. Immunopharmacol.] Vol.7 P.879-888 google doi
  • 23. Li L. -Y., Li L. -Q., Guo C. -H. 2010 Evaluation of in vitro antioxidant and antibacterial activities of Laminaria japonica polysaccharides [J. Med. Plants Res.] Vol.4 P.2194-2198 google
  • 24. Lins K. O., Bezerra D. P., Alves A. P., Alencar N. M., Lima M. W., Torres V. M., Farias W. R., Pessoa C., De Moraes M. O., Costa- Lotufo L. V. 2009 Antitumor properties of a sulfated polysaccharide from the red seaweed Champia feldmannii (Diaz-Pifferer) [J. Appl. Toxicol.] Vol.29 P.20-26 google doi
  • 25. Lustigman B., Brown C. 1991 Antibiotic production by marine algae isolated from the New York/New Jersey coast [Bull. Environ. Contam. Toxicol.] Vol.46 P.329-335 google doi
  • 26. Medcalf D. G., Larsen B. 1977 Fucose-containing polysaccharides in the brown algae Ascophyllum nodosum and Fucus vesiculosus [Carbohydr. Res.] Vol.59 P.531-537 google doi
  • 27. Nagayama K., Iwamura Y., Shibata T., Hirayama I., Nakamura T. 2002 Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome [J. Antimicrob. Chemother.] Vol.50 P.889-893 google doi
  • 28. Nakayasu S., Soegima R., Yamaguchi K., Oda T. 2009 Biological activities of fucose-containing polysaccharide ascophyllan isolated from the brown alga Ascophyllum nodosum [Biosci. Biotechnol. Biochem.] Vol.73 P.961-964 google doi
  • 29. Nishino T., Nishioka C., Ura H., Nagumo T. 1994 Isolation and partial characterization of a novel amino sugar-containing fucan sulfate from commercial Fucus vesiculosus fucoidan [Carbohydr. Res.] Vol.255 P.213-224 google doi
  • 30. Niwano Y., Sato E., Kohno M., Matsuyama Y., Kim D., Oda T. 2007 Antioxidant properties of aqueous extracts from red tide plankton cultures [Biosci. Biotechnol. Biochem.] Vol.71 P.1145-1153 google doi
  • 31. Noda H., Amano H., Arashima K., Nisizawa K. 1990 Antitumor activity of marine algae [Hydrobiologia] Vol.204/205 P.577-584 google doi
  • 32. Pereira M. S., Mulloy B., Mourao P. A. 1999 Structure and anticoagulant activity of sulfated fucans: comparison between the regular repetitive and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae [J. Biol. Chem.] Vol.274 P.7656-7667 google doi
  • 33. Pierre G., Sopena V., Juin C., Mastouri A., Graber M., Maugard T. 2011 Antibacterial activity of a sulfated galactan extracted from the marine alga Chaetomorpha aerea against Staphylococcus aureus [Biotechnol. Bioprocess. Eng.] Vol.16 P.937-945 google doi
  • 34. Raghavendran H. R., Srinivasan P., Rekha S. 2011 Immunomodulatory activity of fucoidan against aspirin-induced gastric mucosal damage in rats [Int. Immunopharmacol.] Vol.11 P.157-163 google doi
  • 35. Shibata H., Kimura-Takagi I., Nagaoka M., Hashimoto S., Sawada H., Ueyama S., Yokokura T. 1999 Inhibitory effect of Cladosiphon fucoidan on the adhesion of Helicobacter pylori to human gastric cells [J. Nutr. Sci. Vitaminol. (Tokyo)] Vol.45 P.325-336 google doi
  • 36. Sinha S., Astani A., Ghosh T., Schnitzler P., Ray B. 2010 Polysaccharides from Sargassum tenerrimum: structural features chemical modification and anti-viral activity [Phytochemistry] Vol.71 P.235-242 google doi
  • 37. Tissot B., Daniel R. 2003 Biological properties of sulfated fucans: the potent inhibiting activity of algal fucoidan against the human compliment system [Glycobiology] Vol.13 P.29G-30G google doi
  • 38. Wang H., Chiu L. C. M., Ooi V. E. C., Ang P. O. 2010 A potent antitumor polysaccharide from the edible brown seaweed Hydroclathrus clathratus [Bot. Mar.] Vol.53 P.265-274 google
  • 39. Witvrouw M., De Clercq E. 1997 Sulfated polysaccharides extracted from sea algae as potential antiviral drugs [Gen. Pharmacol. Vasc. Syst.] Vol.29 P.497-511 google doi
  • 40. Yoon J. -S., Yadunandam A. K., Kim S. -J., Woo H. -C., Kim H. -R., Kim G. -D. 2013 Dieckol isolated from Ecklonia stolonifera induces apoptosis in human hepatocellular carcinoma Hep3B cells [J. Nat. Med.] Vol.67 P.519-527 google doi
  • [Fig. 1.] Effects of polysaccharides on Vibrio alginolyticus. (A) Effects of F-fucoidan (○), A-fucoidan (△), and ascophyllan (□) on V. alginolyticus as measured by Alamar blue assay. (B) Effects of methanol-extraction on the antibacterial activity of F-fucoidan (○), the methanol-extract (△), and the methanol-insoluble residual fraction (▲) toward V. alginolyticus by Alamar blue assay. Data represent the average of triplicate measurements and bars indicate the standard deviation. Asterisks indicate significant differences between with and without polysaccharide samples (p < 0.01).
    Effects of polysaccharides on Vibrio alginolyticus. (A) Effects of F-fucoidan (○), A-fucoidan (△), and ascophyllan (□) on V. alginolyticus as measured by Alamar blue assay. (B) Effects of methanol-extraction on the antibacterial activity of F-fucoidan (○), the methanol-extract (△), and the methanol-insoluble residual fraction (▲) toward V. alginolyticus by Alamar blue assay. Data represent the average of triplicate measurements and bars indicate the standard deviation. Asterisks indicate significant differences between with and without polysaccharide samples (p < 0.01).
  • [Table 1.] Antibacterial activities of F-fucoidan, the methanol-insoluble fraction, and the methanol-extract on Vibrio alginolyticus, Escherichia coli, and Staphylococcus aureus as measured by colony formation assay
    Antibacterial activities of F-fucoidan, the methanol-insoluble fraction, and the methanol-extract on Vibrio alginolyticus, Escherichia coli, and Staphylococcus aureus as measured by colony formation assay
  • [Fig. 2.] Effects of heat-treatment and dialysis on the antibacterial activity of F-fucoidan on Vibrio alginolyticus as measured by colony formation assay. The data represent the average of triplicate measurements and the bars indicate the standard deviation. Asterisks indicate significant differences between with and without test samples (p < 0.01).
    Effects of heat-treatment and dialysis on the antibacterial activity of F-fucoidan on Vibrio alginolyticus as measured by colony formation assay. The data represent the average of triplicate measurements and the bars indicate the standard deviation. Asterisks indicate significant differences between with and without test samples (p < 0.01).
  • [Table 2.] Chemical composition analysis of the methanol-extract of F-fucoidan
    Chemical composition analysis of the methanol-extract of F-fucoidan
  • [Fig. 3.] Cytotoxic effects of F-fucoidan, the methanol-extract, and the methanol-insoluble fraction on RAW264.7 and U937 cells. (A) MTT assay of varying concentrations of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲) on RAW264.7 cells. (B) Alamar blue assay of U937 cells treated with varying concentrations of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲). Data represent the average of triplicate measurements and the bars indicate standard deviations. Asterisks indicate significant differences between with and without test samples (p < 0.01).
    Cytotoxic effects of F-fucoidan, the methanol-extract, and the methanol-insoluble fraction on RAW264.7 and U937 cells. (A) MTT assay of varying concentrations of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲) on RAW264.7 cells. (B) Alamar blue assay of U937 cells treated with varying concentrations of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲). Data represent the average of triplicate measurements and the bars indicate standard deviations. Asterisks indicate significant differences between with and without test samples (p < 0.01).
  • [Fig. 4.] Nuclear morphological changes in U937 cells treated with F-fucoidan, the methanol-extract, or the methanol-insoluble fraction. (AD) Nuclear morphological changes in U937 cells treated with medium alone (A) or with the methanol-insoluble fraction (B), F-fucoidan (C), or the methanol-extract (D) stained cells observed by a fluorescent microscope. (E) U937 cells were incubated with indicated concentrations of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲) for 24 h at 37℃. The populations of the cells with apoptotic nuclear morphological changes were scored as described in the text. (F) U937 cells were incubated with 1,000 μg mL-1 of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲) for the indicated periods of time at 37℃ and the populations of the cells with apoptotic nuclear morphological changes were scored as described in the text. Data represent the average of triplicate measurements and the bars indicate standard deviations. Asterisks indicate significant differences between with and without test samples (p < 0.01). Scale bar represents: A-D, 20 μm.
    Nuclear morphological changes in U937 cells treated with F-fucoidan, the methanol-extract, or the methanol-insoluble fraction. (AD) Nuclear morphological changes in U937 cells treated with medium alone (A) or with the methanol-insoluble fraction (B), F-fucoidan (C), or the methanol-extract (D) stained cells observed by a fluorescent microscope. (E) U937 cells were incubated with indicated concentrations of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲) for 24 h at 37℃. The populations of the cells with apoptotic nuclear morphological changes were scored as described in the text. (F) U937 cells were incubated with 1,000 μg mL-1 of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲) for the indicated periods of time at 37℃ and the populations of the cells with apoptotic nuclear morphological changes were scored as described in the text. Data represent the average of triplicate measurements and the bars indicate standard deviations. Asterisks indicate significant differences between with and without test samples (p < 0.01). Scale bar represents: A-D, 20 μm.
  • [Fig. 5.] Nitric oxide (NO)- and tumor necrosis factor-α (TNF-α)-inducing activities of F-fucoidan, the methanol-extract, and the methanol-insoluble fraction in RAW264.7 cells. Adherent RAW264.7 cells were incubated with varying concentrations of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲). (A) The NO levels in the culture medium from the treated cells were estimated by Griess assay. (B) The levels of TNF-α in the culture supernatants of the treated cells were measured by enzyme-linked immunosorbent assay as described in the text. Data represent the average of triplicate measurements and the bars indicate standard deviations. Asterisks indicate significant differences between with and without test samples (p < 0.01).
    Nitric oxide (NO)- and tumor necrosis factor-α (TNF-α)-inducing activities of F-fucoidan, the methanol-extract, and the methanol-insoluble fraction in RAW264.7 cells. Adherent RAW264.7 cells were incubated with varying concentrations of F-fucoidan (○), the methanol-extract (△), or the methanol-insoluble fraction (▲). (A) The NO levels in the culture medium from the treated cells were estimated by Griess assay. (B) The levels of TNF-α in the culture supernatants of the treated cells were measured by enzyme-linked immunosorbent assay as described in the text. Data represent the average of triplicate measurements and the bars indicate standard deviations. Asterisks indicate significant differences between with and without test samples (p < 0.01).