Study of Macrophage Activation and Structural Characteristics of Purified Polysaccharides from the Fruiting Body of Hericium erinaceus

  • Lee, Jong-Seok (Department of Bioengineering and Technology, Kangwon National University) ;
  • Min, Kyoung-Min (Department of Bioengineering and Technology, Kangwon National University) ;
  • Cho, Jae-Youl (School of Bioscience and Biotechnology, and Institute of Bioscience and Biotechnology, Kangwon National University) ;
  • Hong, Eock-Kee (Department of Bioengineering and Technology, Kangwon National University)
  • Published : 2009.09.30


Most, if not all, Basidiomycetes mushrooms have biologically active polysaccharides showing potent antitumor activity with immunomodulating properties. These polysaccharides have various chemical compositions and belong primarily to the $\beta$-glucan group. In this study, the crude water-soluble polysaccharide HEF-P, which was obtained from the fruiting body of Hericium erinaceus by hot water extraction and ethanol precipitation, was fractionated by DEAE-cellulose and Sepharose CL-6B column chromatographies. This process resulted in four polysaccharide fractions, named HEF-NP Fr I, HEF-NP Fr II, HEF-AP Fr I, and HEF-AP Fr II. Of these fractions, HEF-AP Fr II was able to upregulate the functional events mediated by activated macrophages, such as production of nitric oxide and expression ofcytokines (IL-1${\beta}$ and TNF-${\alpha}$). The molecular mass of HEF-AP Fr II was estimated by gel filtration to be 13 kDa. Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Results indicate that HEF-AP Fr II is a low-molecular-mass polysaccharide with a laminarin-like triple helix conformation of a ${\beta}$-1,3-branched-${\beta}$-1,6-glucan.



  1. Benjamini, E. and S. Leskowitz. 1991. Immunology: A Short Course, pp. 51-58. Wiley-Liss Inc
  2. Blumenkrantz, N. and G. Asboe-Hansen. 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54: 484-489
  3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254
  4. Chaplin, M. F. and J. F. Kennedy (eds.). 1986. Carbohydrate Analysis. A Practical Approach, pp. 3. Oxford IRL Press
  5. Hamuro, J. and G. Chihara. 1985. Lentinan, a T-cell oriented immunopotentiator: Its experimental and clinical applications and possible mechanism of immune modulation, pp. 409-436. In R. L. Fenichel and M. A. Chirigos (eds.). Immunomodulation Agents and Their Mechanisms. Dekker, New York
  6. Ciucanu, I. and F. Kerek. 1984. A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. 131: 209- 217
  7. Cui, S., J. S. Reichner, R. B. Mateo, and J. E. Albina. 1994. Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or -independent mechanisms. Cancer Res. 54: 2462-2467
  8. Dennert, G. and D. Tucker. 1973. Antitumor polysaccharide lentinan. A T cell adjuvant. J. Natl. Cancer Inst. 51: 1727- 1729
  9. Dische, Z. 1962. Color reactions of hexosamines, pp. 507-512. In: Methods in Carbohydrate Chemistry I. Academic Press
  10. Drapier, J. C. and J. B. Hibbs Jr. 1988. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial ironsulfur enzymes in the macrophage effector cells. J. Immunol. 140: 2829-2838
  11. Franz, G. 1989. Polysaccharides in pharmacy: Current applications and future concepts. Planta Med. 55: 493-497
  12. Fujimiya, Y., Y. Suzuki, R. Katakura, and T. Ebina. 1999. Tumorspecific cytocidal and immunopotentiating effects of relatively low molecular weight products derived from the basidiomycete, Agaricus blazei Murrill. Anticancer Res. 19: 113-118
  13. Fujimiya, Y., Y. Suzuki, K. I. Oshiman, H. Kobori, K. Moriguchi, H. Nakashima, et al. 1998. Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murrill, mediated via natural killer cell activation and apoptosis. Cancer Immunol. Immunother. 46: 147-159
  14. Gao, Q. P., R. Z. Jiang, H. Q. Chen, E. Jensen, and R. Seljelid. 1996. Characterization and cytokine stimulating activities of heteroglycans from Tremella fuciformis. Planta Med. 62: 297- 302
  15. Goossens, V., J. Grooten, K. De Vos, and W. Fiers. 1995. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc. Natl. Acad. Sci. U.S.A. 92: 8115-8119
  16. Kabat, E. A. and A. E. Bezer. 1958. The effect of variation in molecular weight on the antigenicity of dextran in man. Arch. Biochem. Biophys. 78: 306-318
  17. Kawagishi, H., M. Ando, and T. Mizuno. 1990. Hericenone A and B as cytotoxic principles from the mushroom Hericium erinaceum. Tetrahedron Lett. 31: 373-376
  18. Kawagishi, H., A. Shimada, R. Shirai, K. Okamoto, F. Ojima, H. Sakamoro, Y. Ishiguro, and S. Furukawa. 1994. Erinacines A, B and C, strong stimulators of nerve growth factor (NGF) synthesis from the mycelia of Hericium erinaceum. Tetrahedron Lett. 35: 1569-1572
  19. Keller, R., M. Geiges, and R. Keist. 1990. L-Arginine-dependent reactive nitrogen intermediates as mediators of tumor cell killing by activated macrophages. Cancer Res. 50: 1421-1425
  20. Kim, D. M., C. W. Pyun, H. G. Ko, and W. M. Park. 2000. Isolation of antimicrobial substances from Hericium erinaceum. Mycobiology. 28: 33-38
  21. Kroncke, K. D., K. Fehsel, T. Schmidt, F. T. Zenke, I. Dasting, J. R. Wesener, H. Bettermann, K. D. Breunig, and V. Kolb- Bachofen. 1994. Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9. Biochem. Biophys. Res. Commun. 200: 1105-1110
  22. Kuwahara, S., E. Morihiro, A. Nemoto, and A. Hiromatsu. 1992. Synthesis and absolute configuration of a cytotoxic fatty acid isolated from the mushroom, Hericium erinaceum. Biosci. Biotechnol. Biochem. 56: 1417-1419
  23. Kwon, S. H., C. N. Kim, C. Y. Kim, S. T. Kwon, K. M. Park, and S. Hwangbo. 2003. Antitumor activities of protein-bound polysaccharide extracted from mycelia of mushroom. Kor. J. Food Nutr. 16: 15-21
  24. Lee, D. G., J. W. Hyun, K. A. Kang, J. O. Lee, S. H. Lee, B. J. Ha, J. M. Ha, E. Y. Lee, and J. H. Lee. 2004. Ulva lactuca: A potential seaweed for tumor treatment and immune stimulation. Biotechnol. Bioprocess Eng. 3: 236-238
  25. Lee, E. W., K. Shizuki, S. Hosokawa, M. Suzuki, H. Suganuma, T. Inakuma, et al. 2000. Two novel diterpenoids, erinacines H and I from the mycelia of Hericium erinaceum. Biosci. Biotechnol. Biochem. 64: 2402-2405
  26. Lee, J. Y., J. Y. Kim, Y. G. Lee, M. H. Rhee, E. K. Hong, and J. Y. Cho. 2008. Molecular mechanism of macrophage activation by exopolysaccharides from liquid culture of Lentinus edodes. J. Microbiol. Biotechnol. 18: 355-364
  27. Liu, C. P., J. N. Fang, X. Y. Li, and X. Q. Xiao. 2002. Structural characterization and biological activities of SC4, an acidic polysaccharide from Salvia chinensis. Acta Pharmacol. Sin. 23: 162-166
  28. Mackay, R. J. and S. W. Pussell. 1986. Protein changes associated with stages of activation of mouse macrophages for tumor cell killing. J. Immunol. 137: 1392-1398
  29. Mau, J. L., H. C. Lin, and S. F. Song. 2002. Antioxidant properties of several specialty mushrooms. Food Res. Int. 35: 519-526
  30. Mizuno, T., H. Saito, T. Nishitoba, and H. Kawagishi. 1995. Antitumor-active substances from mushrooms. Food Rev. Int. 11: 23-61
  31. Mizuno, T., T. Wasa, H. Ito, C. Suzuki, and N. Ukai. 1992. Antitumor active polysaccharides isolated from the fruiting body of Hericium erinaceus, an edible and medicinal mushroom called yamabushitake or houtou. Biosci. Biotechnol. Biochem. 56: 347-348
  32. Ogawa, K. and M. Hatano. 1978. Circular dichroism of the complex of a (1$\rightarrow$3)-$\beta$-D-glucan with Congo red. Carbohydr. Res. 67: 527-535
  33. Ogawa, K., J. Tsurugi, and T. Watanabe. 1973. The dependence of the conformation of a (1$\rightarrow$3)-$\beta$-D-glucan on chain-length in alkaline solution. Carbohydr. Res. 29: 397-403
  34. Ogura, T., M. Tatemichi, and H. Esumi. 1997. TNF-alpha mediates inducible nitric oxide synthase expression in human neuroblastoma cell line by cisplatin. Biochem. Biophys. Res. Commun. 233: 788-791
  35. Ohno, N. (2005) Structural Diversity and Physiological Functions of beta-Glucans. International Journal of Medicinal Mushrooms. 7: 167-174
  36. Ohno, N., I. Suzuki, S. Oikawa, K. Sato, T. Miyazaki, and T. Yadomae. 1984. Antitumor activity and structural characterization of glucans extracted from cultured fruit bodies of Grifola frondosa. Chem. Pharm. Bull. 32: 1142-1151
  37. Okamoto, K., A. Shimada, R. Shirai, H. Sakamoto, S. Toshida, F. Ojima, Y. Ishiguro, T. Sakai, and H. Kawagishi. 1993. Antimicrobial chlorinated orcinol derivatives from mycelia of Hericium erinaceum. Phytochemistry. 34: 1445-1446
  38. Park, S. S., K. H. Yu, and T. J. Min. 1998. Antioxidant activities of extracts from fruiting bodies of mushrooms. Kor. J. Mycol. 26: 69-77
  39. Porcheray, F., S. Viaud, A. C. Rimaniol, C. Leone, B. Samah, N. Dereuddre-Bosquet, D. Dormont, and G. Gras. 2005. Macrophage activation switching: An asset for the resolution of inflammation. Clin. Exp. Immunol. 142: 481-489
  40. Richter, C., V. Gogvadze, R. Schlapbach, M. Schweizer, and J. Schlegel. 1994. Nitric oxide kills hepatocytes by mobilizing mitochondrial calcium. Biochem. Biophys. Res. Commun. 205: 1143-1150
  41. Rolf, D. and G. R. Gray. 1982. Reductive cleavage of glycosides. J. Am. Chem. Soc. 104: 3539-3541
  42. Suffys, P., R. Beyaert, F. Van Roy, and W. Fiers. 1988. Involvement of a serine protease in tumor-necrosis-factor-mediated cytotoxicity. Eur. J. Biochem. 178: 257-265
  43. Yadomae, T. and N. Ohno. 1996. Structure-activity relationship of immunomodulating (1-3)-$\beta$-D-glucans. Recent Res. Dev. Chem. Pharm. Sci. 1: 23-33
  44. Yanaki, T., W. Ito, and K. Tabata. 1986. Correlation between antitumor activity of schizophyllan and its triple helix. Agric. Biol. Chem. 509: 2415-2426
  45. Yang, B. K., J. B. Park, and C. H. Song. 2002. Hypolipidemic effect of exo-polymer produced in submerged mycelial culture of five different mushrooms. J. Microbiol. Biotechnol. 12: 957-961