Journal of Applied Phycology

, Volume 24, Issue 6, pp 1537–1546 | Cite as

Acidic polysaccharides of Arthrospira (Spirulina) platensis induce the synthesis of TNF-α in RAW macrophages

  • María L. Parages
  • Rosa M. Rico
  • Roberto T. Abdala-Díaz
  • Mariana Chabrillón
  • Theodore G. Sotiroudis
  • Carlos Jiménez
Article

Abstract

The study of the enhancement of the immune system by administration of algal cell components is a current research field of great interest for future development of algal biotechnology. Arthrospira (Spirulina) platensis is one of the key organisms, showing interesting results in the treatment of certain tumors, viral infection, and immunodeficiency. Polysaccharides from Arthrospira, together with phycocyanin, seem to be responsible for most of these positive effects. In this work, we isolated the acidic polysaccharide fraction from A. platensis and tested its capacity to induce the production of the proinflammatory cytokine tumor necrosis factor alpha in macrophages. For this purpose, we modified a previous isolation method developed by one of us, which includes several depigmentation steps, as well as differential partitioning with N-cetylpyridinium bromide (Cetavlon). Infrared spectroscopy of the acidic polysaccharide fraction indicates the presence of hydroxyl radicals, aliphatic residues, carbonyl groups, sulfate groups, and sulfate esters, as well as amine residues. Liquid chromatography confirmed the polysaccharidic nature of the fraction, revealing its high purity, essentially free of lipopolysaccharide (LPS) contamination (0.0017% w/w), and complying with international pharmacological standards. The results indicate that a very high production of tumor necrosis factor- α (TNF−α) occurred in macrophages in the presence of the polysaccharides in the range 5–100 μg mL−1, reaching values of 8 ng TNF-α mL−1 after 24 h and 30 ng TNF-α mL−1 after 48 h. These data demonstrate that acidic polysaccharides from Spirulina elicit TNF-α production levels comparable to LPS at ~100× higher concentration than LPS, but without significantly increasing the risk of septic shock or deleterious pyrogenesis.

Keywords

Spirulina Acidic polysaccharides Immune system Macrophages Tumor necrosis factor-α 

Notes

Acknowledgments

This work has been supported in part by a binational project (Acción Integrada HG2004-0025 to C.J. and to T.G.S.). We thank Dr. Ma Ángeles Vargas and Dr. Luis Alemany (Chemical Engineering Department, UMA) for their technical assistance in the FT-IR analysis, Prof. Francisco R. Sarabia and Miss Francisca Martín-Gálvez (Organic Chemistry Department, UMA) for HPLC analysis, Mr. Casimiro Cárdenas (Cell Culture Division, SCAI, UMA) for technical help during macrophage cultivation, and Mrs. Azucena Muñoz García (Pharmacology Section, CIMES, UMA) for LPS contamination assays.

References

  1. Abdala Díaz RT, Chabrillón M, Cabello-Pasini A, Gómez-Pinchetti JL, Figueroa FL (2010) Characterization of polysaccharides from Hypnea spinella (Gigartinales) and Halopithys incurva (Ceramiales) and their effect on RAW 264.7 macrophage activity. J Appl Phycol 23:523–528CrossRefGoogle Scholar
  2. Adams DO, Hamilton TA (1986) Regulation of macrophage activation at the molecular level. Ann Inst Past Immunol 137:229–234CrossRefGoogle Scholar
  3. Beer S, Eshel A (1985) Determining phycoerythrin and phycocyanin concentrations in aqueous crude extracts of red algae. Aust J Mar Freshwat Res 36:785–792CrossRefGoogle Scholar
  4. Belay A, Kato T, Ota Y (1996) Spirulina (Arthrospira): potential application as an animal feed supplement. J Appl Phycol 8:303–311CrossRefGoogle Scholar
  5. Belay A (2002) The potential application of Spirulina (Arthrospira) as a nutritional and therapeutic supplement in health management. J Am Nutr Ass 5:27–48Google Scholar
  6. Bird KT, Dawes CJ, Romeo JT (1981) Light quality effects on carbon metabolism and allocation in Gracillaria verrucosa. Mar Biol 64:219–223CrossRefGoogle Scholar
  7. Bird KT, Habig C, De Busk T (1982) Nitrogen allocation and storage patterns in Gracillaria tikvahiae (Rhodophyta). J Phycol 18:344–348CrossRefGoogle Scholar
  8. Bitler CM, Viale TM, Damaj B, Crea R (2005) Hydrolyzed olive vegetation water in mice has anti-inflammatory activity. J Nutr 135:1475–1479PubMedGoogle Scholar
  9. Cheng A, Wan F, Wang J, Jin Z, Xu X (2008) Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis fish. Internat Immunophar 8:43–50CrossRefGoogle Scholar
  10. Clayton JR, Dortch Q, Thorensen S, Ahmed SI (1988) Evaluation of methods for the separation and analysis of proteins and free amino acids in phytoplankton samples. J Plank Res 10:341–358CrossRefGoogle Scholar
  11. Cood GA, Bell SG, Brooks WP (1995) Cyanobacterial toxins in water. Wat Sci Tech 21:1–13Google Scholar
  12. Desai V, Ramkrishnan R, Chintalwar G, Sainis KB (2007) G1-4A, an immunomodulatory polysaccharide from Tinospora cordifolia, modulates macrophage responses and protects mice against lipopolysaccharide induced endotoxic shock Internat. Immunopharmacol 7:1375–1386CrossRefGoogle Scholar
  13. Folch J, Lees M, Sloane-Stanley GH (1957) A simple method for de isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509PubMedGoogle Scholar
  14. Fox R.D. 1996. Spirulina. Production and potential. Edisud, Aix-en-Provence. 232 pp.Google Scholar
  15. Gershwin ME, Belay A (eds) (2008) Spirulina in human nutrition and health. CRC Press, Boca Raton, 312 ppGoogle Scholar
  16. Hasko G, Szabó C, Németh ZH, Kvetan V, McCarthy PS, Vizi ES (1996) Adenosine receptor agonists differentially regulate IL-10, TNF-α, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J Inmunol 157:4634–4640Google Scholar
  17. Hayakawa Y, Hayashi T, Hayashi K, Hayashi T, Ozawa T, Niiya T, Sakuragawa N (1996) Heparin cofactor II-dependent antithrombin activity of calcium spirulan. Blood Coagul Fibrinol 7:554–560CrossRefGoogle Scholar
  18. Hayakawa Y, Hayashi T, Hayashi K, Ozawa T, Niiya K, Sakuragawa N (1997) Calcium spirulan as an inducer of tissue-type plasminogen activator in human fetal lung fibroblasts. Biochim Biophys Acta 1355:241–247PubMedCrossRefGoogle Scholar
  19. Hayakawa Y, Hayashi T, Lee JB, Ozawa T, Sakuragawa N (2000) Activation of heparin cofactor II by calcium spirulan. J Biol Chem 275:11379–11382PubMedCrossRefGoogle Scholar
  20. Hayashi O, Katoh T, Okuwaki Y (1994) Enhancement of antibody production in mice by dietary Spirulina platensis. J Nutr Sci Vitaminol 40:431–441PubMedCrossRefGoogle Scholar
  21. Hayashi T, Hayashi K, Maeda M, Kojima I (1996a) Calcium spirulan, an inhibitor of enveloped virus replication from a blue-green alga Spirulina platensis. J Nat Prod 59:83–87PubMedCrossRefGoogle Scholar
  22. Hayashi K, Hayashi T, Kojima I (1996b) A natural sulphated polysaccharide, calcium spirulan, isolated from Spirulina platensis: in vitro and ex vivo evaluation of anti-herpes simplex virus and anti-human immunodeficiency virus activities. AIDS Res Hum Retrov 12:1463–1471CrossRefGoogle Scholar
  23. Hayashi O, Hirahashi T, Katoh T, Miyajima H, Hirano T, Okuwakim Y (1998) Class specific influence of dietary Spirulina platensis on antibody production in mice. J Nutr Sci Vitaminol 44:841–851PubMedCrossRefGoogle Scholar
  24. Hirahashi T, Matsumoto M, Hazeki K, Saeki Y, Ui M, Seya T (2002) Activation of the human innate immune system by Spirulina: augmentation of interferon production and NK cytotoxicity by oral administration of hot water extract of Spirulina platensis. Int Immunophar 2:423–434CrossRefGoogle Scholar
  25. Kochert G (1993) Carbohydrate determination by the phenol sulphuric acid method. In: Hellebust JA, Craigie JS (eds) Handbook of phycological methods. Physiological and biochemical methods. Cambridge University Press, Cambridge, pp 95–97Google Scholar
  26. Lee JB, Hayashi T, Hayashi K, Sankawa U (2000) Structural analysis of calcium spirulan (Ca-SP)-derived oligosaccharides using electrospray ionization mass spectrometry. J Nat Prod 63:136–138PubMedCrossRefGoogle Scholar
  27. Martínez C, Delgado M, Pozo D, Leceta J, Calvo JR, Ganea D, Gomariz RP (1998) Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide modulate endotoxin-induced IL-6 production by murine peritoneal macrophages. J Leukoc Biol 63:591–601PubMedGoogle Scholar
  28. Mao TK, Van de Water J, Gershwin ME (2005) Effects of a Spirulina-based dietary supplement on cytokine production from allergic rhinitis patients. J Med Food 8:27–30PubMedCrossRefGoogle Scholar
  29. Meager A (1998) The molecular biology of cytokines. Wiley, ChichesterGoogle Scholar
  30. Meager A (2004) Cytokines: interleukins. In: Meyers RA (ed) Encyclopedia of molecular cell biology and molecular medicine, vol 3. Wiley-VCH, Weinheim, pp 115–151Google Scholar
  31. Meager A (2005) Viral inhibitors and immune response mediators: the interferons. In: Meyers RA (ed) Encyclopedia of molecular cell biology and molecular medicine, vol 15. Wiley-VCH, Weinheim, pp 387–421Google Scholar
  32. Mishima T, Murata J, Toyoshima M, Fujii H, Nakajima M, Hayashi T, Kato T, Saiki I (1998) Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue-green alga. Spirulina platensis Clin Exp Metastasis 16:541–550CrossRefGoogle Scholar
  33. Morris Quevedo HJ, Martínez Manrique CE, Abdala Díaz RT, Pupo C (2000) Evidencias preliminares de la actividad inmunomoduladora de la fracción polisacárida de origen marno PC-1. Rev Cubana Oncol 16:171–176Google Scholar
  34. Oh S-H, Ahn J, Kang D-H, Lee H-Y (2011) The effect of ultrasonificated extracts of Spirulina maxima on the anticancer activity. Mar Biotechnol 13:205–214PubMedCrossRefGoogle Scholar
  35. Pérez-Loyola M., Popowski Casaña G, Pérez-Castillo G., Alonso Romero H. 2003. Caracterización biológica y química del biogel del balneario San Diego de los Baños. Rev Cubana Plant Med 8 (3). Available at: http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=S1028-47962003000300011&lng=es
  36. Pouria S, de Andrade A, Barbosa J, Cavalcanti RL, Barreto VTS, Ward CJ, Preiser W, Poon GK (1998) Fatal microcystin intoxicaction in haemodialysis unit in Caruaru, Brazil. Lancet 352:21–26PubMedCrossRefGoogle Scholar
  37. Qureshi MA, Ali RA (1996) Spirulina platensis exposure enhances macrophage phagocytic function in cats. Immunoph Immunotox 18:457–463CrossRefGoogle Scholar
  38. Qureshi MA, Kidd MT, Ali RA (1995) Spirulina platensis extract enhances chicken macrophage functions after in vitro exposure. J Nutr Inmunol 3:35–43Google Scholar
  39. Qureshi MA, Garlich JD, Kidd MT (1996) Dietary Spirulina platensis enhances humoral and cell-mediated immune functions in chickens. Immunoph Immunotox 18:465–476CrossRefGoogle Scholar
  40. Schwartz J, Shklar G (1987) Regression of experimental oral cancer by beta carotene and algae extracts. J Oral Maxillofac Surg 45:510–515PubMedCrossRefGoogle Scholar
  41. Shklar G, Schwartz J (1988) Tumor necrosis factor in experimental cancer regression with alphatocopherol, beta-carotene, canthaxanthin and algae extracts. Eur J Cancer Clin Oncol 24:839–850PubMedCrossRefGoogle Scholar
  42. Thorpe R, Wadhwa M, Page C, Mire-Sluis A (1999) Bioassays for the characterisation and control of therapeutic cytokines; determination of potency. Dev Biol Stand 97:61–71PubMedGoogle Scholar
  43. Trabelsi L, M’sakni NH, Ben OH, Bacha H, Roudesli S (2009) Partial characterization of extracellular polysaccharides produced by cyanobacterium Arthrospira platensis. Biotech Biopr Eng 14:27–31CrossRefGoogle Scholar
  44. Trinchieri G (1993) Interleukin-12 and its role in the generation of TH1 cells. Immunol Tod 14:335–339CrossRefGoogle Scholar
  45. Ushio S, Namba M, Okura T, Hattori K, Nukada Y, Akita K, Tanabe F, Konishi K, Micallef M, Fujii M, Torigoe K, Tanomoto T, Fukuda S, Ikeda M, Okamura H, Kurimoto M (1996) Cloning of the cDNA for human IFN-g-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol 156:4274–4282PubMedGoogle Scholar
  46. Zarrouk, C. 1966. Contribution á l’étude d’une cyanophycée: influence de divers facteurs physiques et chimiques sur la croissance et la photosynthèse de Spirulina maxima (Setch et Gardner) Geitler. Thèse de doctorat, Faculté des Sciences de l’Université de Paris.Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • María L. Parages
    • 1
  • Rosa M. Rico
    • 1
  • Roberto T. Abdala-Díaz
    • 1
  • Mariana Chabrillón
    • 1
  • Theodore G. Sotiroudis
    • 2
  • Carlos Jiménez
    • 1
  1. 1.Department of Ecology, Faculty of SciencesUniversity of MálagaMálagaSpain
  2. 2.Institute of Biological Research and BiotechnologyNational Hellenic Research FoundationAthensGreece

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