Journal of Aquaculture (한국양식학회지)

The Korean Society of Fisheries and Aquatic Science (한국양식학회)
권호 표지

Isolation and Purification of Anticoagulant Polysaccharide Compound from Fermented Edible Brown Seaweed, Laminaria ochotensis

  • Published : 2006.02.25
Download PDF
⟨ Previous Next ⟩

Abstract

Anticoagulant activities of a fermented edible brown alga, Laminaria ochotensis was investigated. L. ochotensis was fermented with 15% sugar (w/v) at $25^{\circ}C$ for 10 weeks. Anticoagulant activity was measured from the supernatant of algal mixture at biweekly intervals up to $10^{th}$ week by activated partial thromboplastin (APTT), prothrombin time (PT) and thrombin time (TT) assay using citrated human plasma. Sample having high APTT activity $(6^{th}\;week)$ was filtered, ethanol precipitated and freeze-dried. The polysaccharide compound having anticoagulant activity was purified by DEAE ion exchange chromatography followed by Sepharose-4B gel filtration chromatography. Anticoagulant activity, polysaccharide concentration, and heparin like activity were determined for the collected fractions by APTT, $phenol-H_2SO_4$, and glycosaminoglycan assay, respectively. The anticoagulant activity assay showed that the activity was increased up to $6^{th}$ week, and decreased thereafter. The concentration of our purified compound was $31.0{\mu}g/ml$ and showed higher APTT activity than commercial heparin. At the same concentration of $31.0{\mu}g/ml$, the heparin showed 186.5 sec activity while our purified compound showed an activity of 386 sec. Single spot on agarose gel electrophoresis showed that the compound was purified and polyacrylamide gel electrophoresis (PAGE) results revealed that the molecular mass of the purified polysaccharide compound was between 60 and 500 kDa. Therapeutic interest of the algal polysaccharide as an anticoagulant has recently been in highlighted. This purified anticoagulant compound from fermented L. ochotensis can be used as a model for anticoagulant agent or could be developed as an anticoagulant agent. This study can be extended to identify the structure and chemical composition of the purified polysaccharide, and to establish a relationship between structure and the function of the identified anticoagulant compounds.

Keywords

References

  1. Abdel-Fattah, A. F., M. M. Hussein and H. M. Salem, 1974. Studies of the purification and some properties of sargassan, a sulfated heteropolysaccharide from Sargassum linifolium. Carbohydr. Res., 33, 9-17 https://doi.org/10.1016/S0008-6215(00)82935-3
  2. Albuquerque, I. R. L., K. C. S. Queiroz, L. G. Alves, E. A. Santos, E. L. Leite and H. A. O. Rocha, 2004. Heterofucans from Dictyota menstrualis have anticoagulant activity. Braz. J. Med. Biol. Res., 37, 167-171 https://doi.org/10.1590/S0100-879X2004000200002
  3. Atlas, R. M., 1995. Cellular metabolism: The generation of ATP. (in) R. M. Atlas (ed.), Principles of Microbiology. Mosby-Year book, St. Louis, pp. 147-154
  4. Chaplin, M. F. 1994. Monosachcharides. (in) M. F. Chaplin and J. F. Kennedy (ed.), Carbohydrate analysis. Information Press Ltd., Oxford, pp. 1-40
  5. Chargaff, E., F. W. Bancroft and M. Stanley-Brown, 1936. Studies on the chemistry of blood coagulation. II. On the inhibition of blood clotting by substances of high molecular weight. J. Biol. Chem., 115, 155-161
  6. Chevolot, L., A. Foucault, F. Chaubet, N. Kervarec, C. Sinquin, A. Fisher and C. Boisson-Vidal, 1999. Further data the structure of brown seaweed fucans: relationships with anticoagulant activity. Carbohydr. Res., 319, 154-165 https://doi.org/10.1016/S0008-6215(99)00127-5
  7. Church, F. C., J. B. Meade, R. E. Treanor and H. C. Whinna, 1989. Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin. J. Biol. Chem., 264, 3618-3623
  8. Colliec, S., A. M. Fisher, J. Tapon-Bretaudiere, C. Boisson, P. Durant and J. Jozefonvicz, 1991. Anticoagulant properties of a fucoidan fraction. Thromb. Res., 64, 143-154 https://doi.org/10.1016/0049-3848(91)90114-C
  9. Dietrich, C. P., G. G. M. Farias, L. R. D. Abreu, E. L. Leite, L. F. Silva and H. B. Nader, 1995. A new approach for characterization of polysaccharides from algae: Presence of four main acidic polysaccharides in three species of the class Phaeophyceae. Plant Sci., 108, 143-153 https://doi.org/10.1016/0168-9452(95)04142-H
  10. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith, 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-356 https://doi.org/10.1021/ac60111a017
  11. Farndale, R. W., D. J. Buttle and A. J. Barret 1986, Improved quantitation and discrimination of sulfated glycosaminoglycans by use of dimethylmethylene blue. Biochem. Biophys. Acta., 883, 173-177 https://doi.org/10.1016/0304-4165(86)90306-5
  12. Grauffel, V., B. Kloareg, S. Mabeau, P. Durand and J. Josefonficz, 1989. New natural polysaccharides with potent antithrombotic activity: fucans from brown algae. Biomaterials, 10, 363-369 https://doi.org/10.1016/0142-9612(89)90127-0
  13. Kurachi, K., K. Fujikawa, G. Schmer and E. W. Davie, 1976. Inhibition of bovine factor IXa and factor Xa by antithrombin III. Biochemistry, 15, 373-377 https://doi.org/10.1021/bi00647a021
  14. Leite, E. L., M. G. L Medeiros, H. A. O Rocha, G. G. M. Farias, L F.da Silva, S. F. Chavante, L. D. de Abreu, C. P. Dietrich and H. B. Nader, 1998. Structure and pharmacological activities of a sulfated xylofucoglucuronan from the alga Spatoglossum schroederi. Plant Sci., 132, 215-228 https://doi.org/10.1016/S0168-9452(98)00012-0
  15. Matsubara, K., Y. Matsuura, A. Bacic, M. Liao, K. Hori and K. Miyazawa, 2001. Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum. Int. J. Biol. Macromol., 28, 395-399 https://doi.org/10.1016/S0141-8130(01)00137-4
  16. Matsubara, K., Y. Matsuura, K. Hori and K. Miyazawa, 2000. An anticoagulant proteoglycan from the marine green alga, Codium pugniformis. J. Appl. Phycol., 12, 9-14 https://doi.org/10.1023/A:1008174115350
  17. Percival, E., 1979. The polysaccharides of green, red, and brown seaweeds: their basic structure, biosynthesis, and function. Br. Phycol. J., 14, 103-117 https://doi.org/10.1080/00071617900650121
  18. Percival, E. G. V. and R. H. McDowell, 1967. Chemistry and enzymology of marine algal polysaccharides. Academic Press, London
  19. Pereira, M. S., B. Mulloy and P. A. S. Mourao, 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., 274, 7656-7667 https://doi.org/10.1074/jbc.274.12.7656
  20. Shanmugam, M. and K. H. Mody, 2000. Heparinoid-active sulfated polysaccharides from marine algae as potential blood anticoagulant agents. Curr. Sci., 9, 1672-1683
  21. Siddhanta, A. K., M. Shanmugam, K. H. Mody, A. M. Goswami and B. K. Ramavat, 1999. Sulfated polysaccharides of Codium dwarkens Boergs: from the west coast of India: chemical composition and blood anticoagulant activity. Int. J. Biol. Macromol., 26, 151-154 https://doi.org/10.1016/S0141-8130(99)00079-3
  22. Srivastava, R. and D. K. Kulshreshtha, 1989. Bioactive polysaccharides from plants. Phytochemistry, 28, 2877-2883 https://doi.org/10.1016/0031-9422(89)80245-6
  23. Uchida, M. and M. Murata, 2004. Isolation of a lactic acid bacterium and yeast consortium from a fermented material of Ulva spp. (Chloropyta). J. Appl. Microbiol., 97, 1297-1310 https://doi.org/10.1111/j.1365-2672.2004.02425.x
  24. Vehar, G. A. and E. W. Davie, 1977. Formation of a serine enzyme in the presence of bovine factor VIII (antihemophilic factor) and thrombin. Science, 197, 374-376 https://doi.org/10.1126/science.877560
  25. Wen, Z., E. C. O. Vincent, K. S. C. Paul and O. A. J. Put, 2003. Isolation and characterization of a sulfated polysaccharide from the brown alga Sargassum patens and determination of its anti-herpis activity. Biochem. Cell Biol., 81, 25-33 https://doi.org/10.1139/o02-169
  26. Yang, J. Y., Y. Du, R. Huang, Y. Wan and T. Li, 2002. Chemical modification, characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides. Bio. Macromol., 31, 55-62 https://doi.org/10.1016/S0141-8130(02)00066-1
  27. Yoon, S. J., M. S. Periera, M. S. G. Pavao, J. K. Hwang, Y. R. Pyun and P. A. S. Mourao, 2002. The medical plant Porana volubilis contains polysaccharides with anticoagulant activity mediated by heparin cofactor II. Thromb. Res., 106, 51-58 https://doi.org/10.1016/S0049-3848(02)00071-3