Immune Enhancing Effects of Intracellular and Extracellular Polysaccharides Extracted from Mycelial Cultivate of Agaricus blazei Murill

신령버섯(Agaricus blazei Murill) 균사체내외 다당체의 면역활성효과

  • Published : 2007.12.30

Abstract

This study was performed to compare in vitro immune enhancing effects of polysaccharides extracted from cultivated mycelia of Agaricus blazei Murill. Carbohydrate contents of semi-purified polysaccharides were 85.6% and 95.3%, while ${\beta}$-glucan conents were 67.9% and 88.1% for intracellular and extracellular polysaccharide, respectively. Samples were adjusted to the same in their carbohydrate contents before efficacy tests. Both intracellular and extracellular polysaccharide increased nitric oxide (NO) synthesis of macrophage RAW 264.7 in dose dependent manner, and the maximum increase rate was 53.9 and 53.1% in intracellular and extraceltular polysaccharide, respectively. The polysaccharides also increased synthesis of cytokines such as interleukin (IL)-$1{\beta}$, IL-6 and tumor necrosis factor (TNF)-${\alpha}$ in RAW 264.7. For all the 3 cytokines, the increase rate of synthesis was much higher in extracellular polysaccharide compared to intracellular polysaccharide, especially at low concentration. Both polysaccarides increased the proliferation of splenocytes in vitro, intracellular polysaccharide showed increase in dose dependent manner while extraceltular polysaccharide showed increase untill medium concentration ($250\;{\mu}g/ml$). They did not show direct cytotoxicity against cancer cells such as B16F0 melanoma. As results, it was regarded that the both intracellular and extracellular polysaccharide from A. blazei showed immune enhancing effects in vitro, but the activity is higher in extracellular polysaccharide compared to intracellular polysaccharide.

신령버섯(Agaricus blazei Murill)의 액체 배양으로 다당체의 균사체외 분비를 유도하였으며, 버섯 균사체의 새포내 다당체와 세포외 분비 다당체의 면역증진활성을 in vitro 시험으로 비교하였다. 부분 정제된 세포내 다당체와 세포외 다당체의 총당 함량은 각각 85.6%와 95.3%였으며, ${\beta}$-glucan 함량은 각각 67.9%와 88.1%로 측정되었다. 면역활성 실험에는 시료의 닥 함량을 동일하게 맞추어 사용하였다. In vitro에서 균사체내외 다당체는 대식세포 주인 RAW 264.7을 활성화시켜 nitric oxide (NO) 생성을 농도 의존적으로 증가시켰으며, 각각 최대 53.9%, 53.1%의 비슷한 증가활성을 나타내었다. 또 균사채내외 다당체는 모두 RAW 264.7을 활성화시켜 염중성 cytokine류인 interleukin (IL)-$1{\beta}$, IL-6, tumor necrosis factor (TNF)-${\alpha}$의 생성을 증가시켰으며, 이때 3종의 cytokine 모두에서, 세포내 다당체에 비해 세포외 다당체를 처리했을때 저농도에서 높은 증가률을 나타내었다. 두 다당체는 in vitro 상에서 비장세포를 증식시키는 효과를 나타내었으며, 세포내 다당체가 농도 의존적으로 증식효과를 보인데 반해 세포외 다당체는 저농도에서 증식이 높았고, $250\;{\mu}g/ml$ 농도 이상에서는 더 높아지지 않았다. 두 다당체 모두 암세포인 B16F0 melanoma에 대한 직접적인 세포독성 효과는 나타내지 않았다. 신령버섯 균사체 배양으로 생성된 세포내외 다당체는 in vitro에서 모두 면역활성을 증가시키는 것으로 나타났으며 전반적으로 그 활성은 세포내 다당체보다 세포외 다당체가 우수한 것으로 판단되었다.

Keywords

References

  1. Bohn, J.A. and J.N. BeMiller. 1995. (13)-$\beta$-D-Glucans as biological response modifiers: a review of structure-functional activity relationships. Carbohydr. Polymers 28, 3-14 https://doi.org/10.1016/0144-8617(95)00076-3
  2. Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350-356 https://doi.org/10.1021/ac60111a017
  3. Fitzpatrick, F.W. and J.F. DiCarlo. 1964. Zymosan, pp. 233-262. In Annals of the New York Academy of Sciences 118
  4. Gregory, F.J., E.M. Healy, H.P.K. Agerborg, Jr., and G.H. Warren. 1966. Studies on antitumor substances produced by Basidiomycetes. Mycologia 58, 80-90 https://doi.org/10.2307/3756990
  5. Kroncke, K.D., V. Kolb-Bachofen, B. Berschick, V. Burkart, and H. Kolb. 1991. Activated macrophages kill pancreatic syngeneic isolet cells via arginine-dependent nitric oxide generation. Biochem. Biophys. Res. Commun. 175, 752-758 https://doi.org/10.1016/0006-291X(91)91630-U
  6. Lorsbach, R.B., W.J. Murphy, C.J. Lowenstein, S.H. Snyder, and S.W. Russel. 1993. Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. J. Biol. Chem. 268, 1908-1913
  7. Lovett, D., B. Kozan, M. Hadam, M. Resch, and D. Gemsa. 1986. Macrophage cytotoxicity: Interleukin 1 as a mediator of tumor cytostasis. J. Immunol. 136, 340-347
  8. Lowenstein, C.J. and S.H. Snyder. 1992. Nitric oxide, novel biological messenger, Cell 70, 705-707 https://doi.org/10.1016/0092-8674(92)90301-R
  9. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1954. Protein measurement with the Folin-phenol reagents. J. Biol. Chem. 193, 265-275
  10. Mano-Hirano, Y., N. Sato, Y. Sawasaki, K. Haranaka, N. Satomi, H. Nariuchi, and T. Goto. 1987. Inhibition of tumor-induced migration of bovine capillary endothelial cells by mouse and rabbit tumor necrosis factor. J. Natl. Cancer. Inst. 78, 115-120 https://doi.org/10.1093/jnci/78.1.115
  11. Marcinkiewicz, J. and B.M. Chain. 1993. Different regulation of cytokine production by nitric oxide. Immunol. 80, 146-150
  12. Megazyme International Ireland Ltd. 2002(10). Mushroom and yeast $\beta$-glucan: Assay procedure
  13. Ohno, N., Y. Emori, T. Yadomae, K. Saito, A. Masuda, and S. Oikawa. 1990. Reactivity of Limulus amoebocyte lysate towards (1$\rightarrow$3)-beta-D-glucans. Carbohydr. Res. 25, 311-318
  14. Peng, Y., L. Zhang, F. Zeng, and Y. Xu. 2003. Structure and antitumor activity of extracellular polysaccharides from mycelium. Carbohydr. Polymers 54, 297-303 https://doi.org/10.1016/S0144-8617(03)00190-5
  15. Roland, J.F., Z.F. Chmielewicz, B.A. Weiner, A.M. Gross, O.P. Boening, J.V. Luck, T.J. Bardos, H. Christine Reilly, K. Sugiura, C. Chester Stock, E.H. Lucas, R.U. Byerrum, and J.A. Stevens. 1960. Calvacin, a new antitumor agent. Science 132, 1897 https://doi.org/10.1126/science.132.3443.1897
  16. Shalaby, M.R., M.A. Palladino, Jr., S.E. Hirabayashi, T.E. Eessalu, G.D. Lewis, H.M. Shepard, and B.B. Aggarwal. 1987. Receptor binding and activation of polymorphonuclear neutrophils by tumor necrosis factor-alpha. J. Leukoc. Biol. 41, 196-204 https://doi.org/10.1002/jlb.41.3.196
  17. Won, S.Y. and E.H. Park. 2005. Anti-inflammatory and related pharmacological activities of cultured mycelia and fruiting bodies of Cordyceps militaris. J. Ethnopharm. 555-561