Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Study of Macrophage Activation and Structural Characteristics of Purified Polysaccharide from the Fruiting Body of Cordyceps militaris

  • Lee, Jong-Seok (Department of Bioengineering and Technology, Kangwon National University) ;
  • Kwon, Jeong-Seok (Department of Bioengineering and Technology, Kangwon National University) ;
  • Won, Dong-Pil (Department of Bioengineering and Technology, Kangwon National University) ;
  • Lee, Jung-Hyun (Department of Bioengineering and Technology, Kangwon National University) ;
  • Lee, Keun-Eok (Department of Bioengineering and Technology, Kangwon National University) ;
  • Lee, Shin-Young (Department of Bioengineering and Technology, Kangwon National University) ;
  • Hong, Eock-Kee (Department of Bioengineering and Technology, Kangwon National University)
  • Received : 2009.10.16
  • Accepted : 2010.04.21
  • Published : 2010.07.28
Download PDF
⟨ Previous Next ⟩

Abstract

Cordyceps militaris, an entomopathogenic fungus belonging to the class Ascomycetes, has been reported to have beneficial biological activities such as hypoglycemic, anti-inflammatory, antitumor, antimetastatic, hypolipidemic, immunomodulatory, and antioxidant effects. In this study, the crude water-soluble polysaccharide CMP, which was obtained from the fruiting body of C. militaris by hot water extraction and ethanol precipitation, was fractionated by DEAE-cellulose and Sepharose CL-6B column chromatographies. This process resulted in three polysaccharide fractions, termed CMP Fr I, CMP Fr II, and CMP Fr III. Of these fractions, CMP Fr II, with an average molecular mass of 127 kDa, was able to upregulate effectively the phenotypic functions of macrophages such as NO production and cytokine expression. The chemical property of the stimulatory polysaccharide, CMP Fr II, was determined based on its monosaccharide composition, which consisted of glucose (56.4%), galactose (26.4%), and mannose (17.2%). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FTIR), and gas chromatography-mass spectrometry (GCMS). Results indicated that CMP Fr II consisted of the (1${\rightarrow}$4) or (1${\rightarrow}$2) linked glucopyranosyl or galactopyranosyl residue with a (1${\rightarrow}$2) or (1${\rightarrow}$6) linked mannopyranosyl, glucopyranosyl, or galactopyranosyl residue as a side chain. The configuration of the ${\beta}$-linkage and random coil conformation of CMP Fr II were confirmed using a Fungi-Fluor kit and Congo red reagent, respectively.

Keywords

References

  1. Allen, L. A. and A. Aderem. 1996. Mechanisms of phagocytosis. Curr. Opin. Immunol. 8: 36-40. https://doi.org/10.1016/S0952-7915(96)80102-6
  2. Benjamini, E. and S. Leskowitz. 1991. Immunology: A Short Course, pp. 51-58. Wiley-Liss Inc.
  3. Beutler, B. 2004. Innate immunity: An overview. Mol. Immunol. 40: 845-859. https://doi.org/10.1016/j.molimm.2003.10.005
  4. Blumenkrantz, N. and G. Asboe-Hansen. 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54: 484-489. https://doi.org/10.1016/0003-2697(73)90377-1
  5. 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. https://doi.org/10.1016/0003-2697(76)90527-3
  6. Carmichael, J., W. G. DeGraff, A. F. Gazdar, J. D. Minna, and J. B. Mitchell. 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res. 47: 936-942.
  7. Chaplin, M. F. and J. F. Kennedy (eds.). 1986. Carbohydrate Analysis. A Practical Approach, pp. 3. Oxford IRL Press.
  8. Chihara, G., Y. Maeda, J. Hamuro, T. Sasaki, and F. Fukuoka. 1969. Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) sing. Nature 222: 687-688. https://doi.org/10.1038/222687a0
  9. Cho, S. M., J. S. Park, K. P. Kim, D. Y. Cha, H. M. Kim, and I. D. Yoo. 1999. Chemical features and purification of immunostimulating polysaccharides from the fruit bodies of Agaricus blazei. Kor. J. Mycol. 27: 170-174.
  10. Ciucanu, I. and F. Kerek. 1984. A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res. 131: 209-217. https://doi.org/10.1016/0008-6215(84)85242-8
  11. Collins, L., T. Zhu, J. Guo, Z. J. Xiao, and C. Y. Chen. 2006. Phellinus linteus sensitizes apoptosis induced by doxorubicin in prostate cancer. Br. J. Cancer 95: 282-288. https://doi.org/10.1038/sj.bjc.6603277
  12. 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.
  13. Dalmo, R. A. and J. Boqwald. 2008. Beta-glucans as conductors of immune symphonies. Fish Shellfish Immunol. 25: 384-396. https://doi.org/10.1016/j.fsi.2008.04.008
  14. Dennert, G. and D. Tucker. 1973. Antitumor polysaccharide lentinan. A T cell adjuvant. J. Natl. Cancer Inst. 51: 1727-1729.
  15. Dische, Z. 1962. Color reactions of hexosamines, pp. 507-512. In: Methods in Carbohydrate Chemistry I. Academic Press.
  16. 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.
  17. Ghobrial, I. M., T. E. Witziq, and A. A. Adjei. 2005. Targeting apoptosis pathways in cancer therapy. CA Cancer J. Clin. 55: 178-194. https://doi.org/10.3322/canjclin.55.3.178
  18. 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. https://doi.org/10.1073/pnas.92.18.8115
  19. Gordon, S. B. and R. C. Read. 2002. Macrophage defences against respiratory tract infections. Br. Med. Bull. 61: 45-61. https://doi.org/10.1093/bmb/61.1.45
  20. 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.
  21. 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. https://doi.org/10.1016/0003-9861(58)90354-0
  22. 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.
  23. Kiho, T., A. Yamane, J. Hui, S. Usui, and S. Ukai. 1996. Polysaccharides in fungi. XXXVI. Hypoglycemic activity of polysaccharide (CF-F30) from the cultural mycelium of Cordyceps sinensis and its effect on glucose metabolism in mouse liver. Biol. Pharm. Bull. 19: 294-296. https://doi.org/10.1248/bpb.19.294
  24. Klimp, A. H., E. G. E. de Vries, G. L. Scherphof, and T. Daemen. 2002. A potential role of macrophage activation in the treatment of cancer. Crit. Rev. Oncol. Hematol. 44: 143-161. https://doi.org/10.1016/S1040-8428(01)00203-7
  25. 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. https://doi.org/10.1006/bbrc.1994.1564
  26. Lee, H. H., J. S. Lee, J. Y. Cho, Y. E. Kim, and E. K. Hong. 2009. Study on immunostimulating activity of macrophage treated with purified polysaccharides from liquid culture and fruiting body of Lentinus edodes. J. Microbiol. Biotechnol. 19: 566-572. https://doi.org/10.4014/jmb.0809.541
  27. Lee, J. S., J. Y. Cho, and E. K. Hong. 2009. Study on macrophage activation and structural characteristics of purified polysaccharides from the liquid culture broth of Hericium erinaceus. Carbohydr. Polym. 78: 162-168. https://doi.org/10.1016/j.carbpol.2009.04.036
  28. Lee, J. S., K. M. Min, J. Y. Cho, and E. K. Hong. 2009. Study of macrophage activation and structural characteristics of purified polysaccharides from the fruiting body of Hericium erinaceus. J. Microbiol. Biotechnol. 19: 951-959. https://doi.org/10.4014/jmb.0901.013
  29. Medzhitov, R. and C. Janeway. 2000. Innate immune recognition: Mechanisms and pathways. Immunol. Rev. 173: 89-97. https://doi.org/10.1034/j.1600-065X.2000.917309.x
  30. Nakamura, K., Y. Yamaguchi, S. Kagota, Y. M. Kwon, K. Shinozuka, and M. Kunitomo. 1999. Inhibitory effects of Cordyceps sinensis on spontaneous liver metastasis of Lewis lung carcinoma and B16 melanoma cells in syngeneic mice. Jpn. J. Pharmacol. 79: 335-341. https://doi.org/10.1254/jjp.79.335
  31. Nardin, A. and J. P. Abastado. 2008. Macrophages and cancer. Front. Biosci. 13: 3494-3505.
  32. Nathan, C. F., S. C. Silrerstein, L. D. Bruker, and Z. A. Lohn. 1979. Extracellular cytolysis by activated macrophages and granulocytes. J. Exp. Med. 149: 100-113. https://doi.org/10.1084/jem.149.1.100
  33. Ng, T. B. and H. X. Wang. 2005. Pharmacological actions of Cordyceps, a prized folk medicine. J. Pharm. Pharmacol. 57: 1509-1520. https://doi.org/10.1211/jpp.57.12.0001
  34. Novak, M. and V. Vetvicka. 2008. Beta-glucans, history, and the present: Immunomodulatory aspects and mechanisms of action. J. Immunotoxicol. 5: 47-57. https://doi.org/10.1080/15476910802019045
  35. 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. https://doi.org/10.1016/S0008-6215(00)84144-0
  36. 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. https://doi.org/10.1016/S0008-6215(00)83025-6
  37. 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. https://doi.org/10.1006/bbrc.1997.6558
  38. Park, C., S. H. Hong, J. Y. Lee, G. Y. Kim, B. T. Choi, Y. T. Lee, et al. 2005. Growth inhibition of U937 leukemia cells by aqueous extract of Cordyceps militaris through induction of apoptosis. Oncol. Rep. 13: 1211-1216.
  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. https://doi.org/10.1006/bbrc.1994.2785
  41. Rolf, D. and G. R. Gray. 1982. Reductive cleavage of glycosides. J. Am. Chem. Soc. 104: 3539-3541. https://doi.org/10.1021/ja00376a065
  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. https://doi.org/10.1111/j.1432-1033.1988.tb14451.x
  43. Vetvicka, V. and J. Vetvickova. 2007. Physiological effects of different types of beta-glucan. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc. Czech. Repub. 151: 225-231. https://doi.org/10.5507/bp.2007.038
  44. Wasser, S. P. 2002. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 60: 258-274. https://doi.org/10.1007/s00253-002-1076-7
  45. Yadomae, T. and N. Ohno. 1996. Structure-activity relationship of immuno-modulating (1-3)-$\beta$-D-glucans. Recent Res. Dev. Chem. Pharm. Sci. 1: 23-33.
  46. Yanaki, T., W. Ito, and K. Tabata. 1986. Correlation between antitumor activity of schizophyllan and its triple helix. Agric. Biol. Chem. 509: 2415-2426.
  47. Yang, B. K., J. Y. Ha, S. C. Jeong, S. Das, J. W. Yun, Y. S. Lee, J. W. Choi, and C. H. Song. 2000. Production of exo-polymers by submerged mycelial culture of Cordyceps militaris and its hypolipidemic effect. J. Microbiol. Biotechnol. 10: 784-788.
  48. Yang, L. Y., A. Chen, Y. C. Kuo, and C. Y. Lin. 1999. Efficacy of a pure compound H1-A extracted from Cordyceps sinensis on autoimmune disease of MRL lpr/lpr mice. J. Lab. Clin. Med. 134: 492-500. https://doi.org/10.1016/S0022-2143(99)90171-3
  49. Yu, R., L. Song, Y. Zhao, W. Bin, L. Wang, H. Zhang, Y. Wu, Y. Ye, and X. Yao. 2004. Isolation and biological properties of polysaccharide CPS-1 from cultured Cordyceps militaris. Fitoterapia 75: 465-472. https://doi.org/10.1016/j.fitote.2004.04.003
  50. Yu, R., W. Yang, L. Song, C. Yan, Z. Zhang, and Y. Zhao. 2007. Structural characterization and antioxidant activity of a polysaccharide from the fruiting bodies of cultured Cordyceps militaris. Carbohydr. Polym. 70: 430-436. https://doi.org/10.1016/j.carbpol.2007.05.005

Cited by

  1. Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris vol.11, pp.9, 2010, https://doi.org/10.1016/j.intimp.2011.04.001
  2. Cordycepin 고함유 동충하초(Cordyceps militaris JLM 0636)의 orotic acid 유발 흰쥐의 지방간 개선효과 vol.21, pp.9, 2010, https://doi.org/10.5352/jls.2011.21.9.1274
  3. Extracts of Cordyceps militaris Lower Blood Glucose via the Stimulation of Cholinergic Activation and Insulin Secretion in Normal Rats vol.26, pp.8, 2010, https://doi.org/10.1002/ptr.3709
  4. Cordyceps militaris and mycelial fermentation induced apoptosis and autophagy of human glioblastoma cells vol.3, pp.None, 2010, https://doi.org/10.1038/cddis.2012.172
  5. Cordycepin이 사염화탄소 유발 간손상 흰쥐의 조직 과산화 지질 농도 및 항산화 활성에 미치는 영향 vol.23, pp.7, 2013, https://doi.org/10.5352/jls.2013.23.7.904
  6. Polysaccharide from Inonotus obliquus inhibits migration and invasion in B16-F10 cells by suppressing MMP-2 and MMP-9 via downregulation of NF-κB signaling pathway vol.31, pp.5, 2010, https://doi.org/10.3892/or.2014.3103
  7. Inonotus obliquus-derived polysaccharide inhibits the migration and invasion of human non-small cell lung carcinoma cells via suppression of MMP-2 and MMP-9 vol.45, pp.6, 2010, https://doi.org/10.3892/ijo.2014.2685
  8. Purification of polysaccharides from Cordyceps militaris and their anti-hypoxic effect vol.11, pp.2, 2010, https://doi.org/10.3892/mmr.2014.2786
  9. 발효 동충하초의 유용성분 및 생리 활성 작용 vol.25, pp.2, 2010, https://doi.org/10.5352/jls.2015.25.2.197
  10. Characterization and In vitro Antioxidant Activity of a Polysaccharide from Cordyceps sobolifera vol.40, pp.3, 2010, https://doi.org/10.1111/jfpp.12622
  11. Polysaccharides isolated from liquid culture broth of Inonotus obliquus inhibit the invasion of human non-small cell lung carcinoma cells vol.22, pp.1, 2010, https://doi.org/10.1007/s12257-016-0458-0
  12. A novel protein from edible fungi Cordyceps militaris that induces apoptosis vol.26, pp.1, 2018, https://doi.org/10.1016/j.jfda.2016.10.013
  13. Structural characterisation and cholesterol efflux improving capacity of the novel polysaccharides from Cordyceps militaris vol.131, pp.None, 2019, https://doi.org/10.1016/j.ijbiomac.2019.03.078
  14. Anti-atopic dermatitis properties of Cordyceps militaris on TNFα/IFNγ-stimulated HaCaT cells and experimentally induced atopic dermatitis in mice vol.24, pp.4, 2010, https://doi.org/10.20463/pan.2020.0022
  15. New Insights Into the Biosynthesis of Typical Bioactive Components in the Traditional Chinese Medicinal Fungus Cordyceps militaris vol.9, pp.None, 2021, https://doi.org/10.3389/fbioe.2021.801721
  16. Antioxidant and Immunostimulatory Activities of a Submerged Culture of Cordyceps sinensis Using Spent Coffee vol.10, pp.8, 2010, https://doi.org/10.3390/foods10081697