雲芝胞外多醣?基因毒理及生物活性分析 鄭崇孝、孫芳君
E-mail: [email protected]
摘 要
雲芝(Trametst versicolor)為腐生真菌，屬於一種珍貴藥材，目前在中國及日本已有利用雲芝所萃取出的多醣?做為臨床用藥
，其具有可以抑制腫瘤、增加免疫調節等功效。本研究為利用本土雲芝LH1所萃取出的雲芝胞外多醣?ePSP進行基因毒理 及生物活性分析。基因毒理實驗包含安姆測試、鼷鼠淋巴瘤tk+/-分析法及囓齒類周邊血液之微核測試法。生物活性分析則 進行抗致突變試驗、抑癌測試及抗氧化功能評估。 在基因毒理試驗中，雲芝胞外多醣?ePSP不論是在細菌、動物細胞及動 物的篩檢模式下，使用最高劑量後皆沒有導致基因突變或傷害的可能性；在抗致突變試驗中，ePSP可以使Salmonella typhimurium TA98及TA100在不含S9時，抑制率為71.9%及84.4%；而在含S9時，TA98及TA100的抑制率為50.9%及76.5%
。在腫瘤抑制中，ePSP在 1.25 mg/ml濃度下，對偏正常細胞 (CHO-K1)沒有毒殺性，但對HepG2 (肝癌細胞)及MCF-7 (乳癌 細胞)則分別有66%及48%的細胞生長抑制率。在抗氧化活性中，ePSP的濃度越高，對活性氧自由基(ROS)的抑制率會增加
，同時可以讓細胞內的Glutathione (GSH)具有明顯上升的趨勢，已達清除氧化壓力的目的。
關鍵詞 : 雲芝、胞外多醣?、安姆測試、鼷鼠淋巴瘤tk+/-分析法、微核試驗、抗致突變、抗氧化、抗腫瘤 目錄
封面內頁 簽名頁 中文摘要...iii 英文摘
要...iv 誌謝...v 目 錄...vi 圖目錄...viii 表目 錄...ix 1.前言...1 2.文獻回 顧...2 2.1 雲芝介紹...2 2.2雲芝多醣介 紹...3 2.3雲芝多醣結構...4 2.4雲芝多醣藥 理作用...4 2.5健康食品安全性評估方法-基因毒性測
試...5 2.6 抗突變測試...17 2.7抗氧化系
統...18 3.材料方法...21 3.1 實驗試 藥...21 3.2 樣品製備...23 3.3 安姆試驗 (Ames test)...24 3.4 鼷鼠淋巴瘤tk+/-分析法...27 3.5 動物活體基因 毒性分析...33 3.6 抗致突變試驗...35 3.7細胞存活試驗 (WST-1測試法)...37 3.8抗氧化功能評估...38 3.9 統計分 析...39 4.結果討論...40 4.1 安姆試驗結 果...40 4.2 鼷鼠淋巴瘤tk+/-分析結果...45 4.3微核試驗 分析結果...50 4.4 抗致突變分析結果...54 4.5 細胞生 長抑制率測試...60 4.6 抗氧化功能評估結果...63 5.結 論...66 參考文獻...69 附
錄...77 參考文獻
王子慶，沈盈貝，林麗娟，蔡佳宏，吳明昌，林聖宗。2011。靈芝乙醇萃取物抗氧化與抗致突變性評估。大仁學報，39，17-34。 王伯 徹，陳啟楨。1994。常見食藥用菇介紹33-34。食品工業發展研究所。 李俊鋒。2003。雲芝的生物學特性。藥理作用及應用前景，31(3)
，509-510。 李進。2008。雲芝糖?及其復方的免疫調節和抗腫瘤作用研究。 林芳儀。2009。中草藥萃取液對雲芝胞外多醣??產量、化學 特性及免疫活性之影響。生物產業科技學系博士論文，大葉大學。 林蔚任。2007。由草莓粕製備鞣花酸濃縮物之方法及鞣花酸濃縮物 之安全性評估。農業化學研究所碩士論文，臺灣大學。 張宏，徐惠波，丁濤，溫富春，張殿文，孫曉波。2008。雲芝多醣的藥理研究
。長春中醫藥大學學報，24(1)，24-25。 周海華、馬海樂。2008。雲芝多醣得體外抗氧化活性研究。食飲研究與開發，29(3)，44-48。 周 梅、陳瓊。1997。雲芝多醣對小鼠腹腔巨噬細胞NO釋放及抗氧化?活性的影響。第一軍醫大學學報，17(2)。 陳士瑜。1998。食品菌生產 大全。農業出版社，北京。 陳勁初。2006。樟芝功能性及安全性研究。生命科學系博士論文，國立清華大學。 葉?村。2010。培養基添 加枸杞萃取液發酵雲芝胞內多醣?及胞外多醣?紅外線光譜及免疫活性分析。生物產業科技學系碩士論文，大葉大學。 莫永炎，姜勇，陳
瑗，周玫。2001。雲芝多醣對腦、肝組織的抗氧化作用研究。中國藥理學通報，6(17)，4。 鄒巧根，朱玲，王偉，相秉仁。2003。雲芝 糖?的研究進展，中成藥，7。3 婁寧、周玫、陳媛。1996。雲芝多醣對小鼠心肝腎皮和洪細胞抗氧化能力之影響。中國藥理學通訊
，12(5)，425-426。 劉怡初。2005。利用鼷鼠淋巴癌細胞tk分析法檢測蛋黃油與紅麴米丙酮萃出物之基因毒性。微生物與生化學研究所 碩士論文，臺灣大學。 劉遠嶸。2008。雲芝糖?鎮痛作用研究進展。 衛生職業教育，26-27。 劉燕，林瑞超，李波。2001。雲芝多糖抗 腫瘤作用研究進展。中成藥，23，10-12。 賴佩君。2008。雲芝(Trametes versicolor)在不同發酵槽及不同培養條件對菌絲體及多醣體之影 響。食品科學系碩士論文，東海大學。 韓銳 2003。抗致突變與抑制腫瘤的保健功效。 Ames, B.N., McCann, J., Yamasaki, E. 1975.
Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat Res, 31(6), 347-64. Arteel, G.E., Sies, H. 2001. The biochemistry of selenium and the glutathione system. Environ Toxicol Pharmacol, 10(4), 153-8. Chan, S.L., Yeung, J.H.K. 2006. Effects of polysaccharide peptide (PSP) from Coriolus versicolor on the pharmacokinetics of cyclophosphamide in the rat and cytotoxicity in HepG2 cells. Food and Chemical Toxicology, 44(5), 689-694. Cheng, H.Y., Lin, T.C., Yu, K.H., Yang, C.M., Lin, C.C. 2003.
Antioxidant and free radical scavenging activities of Terminalia chebula. Biol Pharm Bull, 26(9), 1331-5. Chu, K.K., Ho, S.S., Chow, A.H. 2002.
Coriolus versicolor: a medicinal mushroom with promising immunotherapeutic values. J Clin Pharmacol, 42(9), 976-84. Clements, J. 2000. The mouse lymphoma assay. Mutat Res, 455(1-2), 97-110. Clive, D., Flamm, W.G., Machesko, M.R., Bernheim, N.J. 1972. A mutational assay system using the thymidine kinase locus in mouse lymphoma cells. Mutat Res, 16(7), 77-87. Collins, R.A., Ng, T.B. 1997. Polysaccharopeptide from Coriolus versicolor has potential for use against human immunodeficiency virus type 1 infection. Life Sci, 60(25), PL383-7. Cui, J., Chisti, Y. 2003.
Polysaccharopeptides of Coriolus versicolor: physiological activity, uses, and production. Biotechnology Advances, 21(2), 109-122. Dertinger, S.D., Bishop, M.E., McNamee, J.P., Hayashi, M., Suzuki, T., Asano, N., Nakajima, M., Saito, J., Moore, M., Torous, D.K., Macgregor, J.T. 2006. Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: I. Intra- and interlaboratory comparison with microscopic scoring. Toxicol Sci, 94(1), 83-91. Dong, Y., Yang, M.M.P., Kwan, C.Y. 1997. In vitro inhibition of proliferation of HL-60 cells by tetrandrine and Coriolus versicolor peptide derived from Chinese medicinal herbs. Life Sciences, 60(8), PL135-PL140. Fan, Y., Schreiber, E.M., Giorgianni, A., Yalowich, J.C., Day, B.W. 2006. Myeloperoxidase-catalyzed metabolism of etoposide to its quinone and glutathione adduct forms in HL60 cells. Chem Res Toxicol, 19(7), 937-43. Halliwell, B., Chirico, S. 1993. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr, 57(5 Suppl), 715S-724S; discussion 724S-725S. Harhaji, L., Mijatovi?, S., Maksimovi?-Ivani?, D., Stojanovi?, I., Mom?ilovi?, M., Maksimovi?, V., Tufegd?i?, S., Marjanovi?, ?., Mostarica-Stojkovi?, M., Vu?ini?, ?., Sto?i?-Gruji?i?, S. 2008. Anti-tumor effect of Coriolus versicolor methanol extract against mouse B16 melanoma cells: In vitro and in vivo study. Food and Chemical Toxicology, 46(5), 1825-1833. Hayashi, M., MacGregor, J.T., Gatehouse, D.G., Adler, I.D., Blakey, D.H., Dertinger, S.D., Krishna, G., Morita, T., Russo, A., Sutou, S. 2000. In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring.
Environ Mol Mutagen, 35(3), 234-52. Hayashi, M., Morita, T., Kodama, Y., Sofuni, T., Ishidate, M., Jr. 1990. The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides. Mutat Res, 245(4), 245-9. Ho, C.Y., Lau, C.B.S., Kim, C.F., Leung, K.N., Fung, K.P., Tse, T.F., Chan, H.H.L., Chow, M.S.S. 2004. Differential effect of Coriolus versicolor (Yunzhi) extract on cytokine production by murine lymphocytes in vitro. International Immunopharmacology, 4(12), 1549-1557. Hor, S.Y., Ahmad, M., Farsi, E., Lim, C.P., Asmawi, M.Z., Yam, M.F. 2011. Acute and subchronic oral toxicity of Coriolus versicolor standardized water extract in Sprague-Dawley rats. Journal of
Ethnopharmacology, 137(3), 1067-1076. Horn, R.C., Vargas, V. M.2003. Antimutagenic activity of extracts of natural substances in the Salmonella/microsome assay. Mutagenesis,18(2),113-118. Kim, H.S., Kacew, S., Lee, B.M. 1999. In vitro chemopreventive effects of plant polysaccharides (Aloe barbadensis miller, Lentinus edodes, Ganoderma lucidum and Coriolus versicolor). Carcinogenesis, 20(8), 1637-40.
Kobayashi, H., Matsunaga, K., Fujii, M. 1993. PSK as a chemopreventive agent. Cancer Epidemiol Biomarkers Prev, 2(3), 271-6. Kogan, G. 2000.
(1→3,1→6)-β-D-glucans of yeasts and fungi and their biological activity. in: Studies in Natural Products Chemistry, (Ed.) R. Atta ur, Vol. Volume 23, Elsevier, pp. 107-152. Krishna, G., Hayashi, M. 2000. In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutat Res, 455(1-2), 155-66. Lai, C.H., Teng, J.F., Hsu, T.H., Lin, F.Y., Yang, P.W., Lo, H.C. 2011. 28-day oral safety evaluation of extracellular polysaccharopeptides produced in submerged culture from the turkey tail medicinal mushroom Trametes versicolor (L.:Fr.) Pilat LH-1 in mice. Int J Med Mushrooms, 13(3), 227-36. Lau, C.B.S., Ho, C.Y., Kim, C.F., Leung, K.N., Fung, K.P., Tse, T.F., Chan, H.H.L., Chow, M.S.S. 2004.
Cytotoxic activities of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma cells by induction of apoptosis. Life Sciences, 75(7), 797-808. Lee, C.L., Yang, X., Wan, J.M.F. 2006. The culture duration affects the immunomodulatory and anticancer effect of polysaccharopeptide derived from Coriolus versicolor. Enzyme and Microbial Technology, 38(1–2), 14-21. Lin, F.Y., Lai, Y.K., Yu, H.C., Chen, N.Y., Chang, C.Y., Lo, H.C., Hsu, T.H. 2008. Effects of Lycium barbarum extract on production and immunomodulatory activity of the extracellular
polysaccharopeptides from submerged fermentation culture of Coriolus versicolor. Food Chemistry, 110(2), 446-453. Lloyd, M., Kidd, D. 2012.
The mouse lymphoma assay. Methods Mol Biol, 817, 35-54. MacGregor, J.T., Bishop, M.E., McNamee, J.P., Hayashi, M., Asano, N., Wakata, A., Nakajima, M., Saito, J., Aidoo, A., Moore, M.M., Dertinger, S.D. 2006. Flow cytometric analysis of micronuclei in peripheral blood reticulocytes:
II. An efficient method of monitoring chromosomal damage in the rat. Toxicol Sci, 94(1), 92-107. Maehara, Y., Tsujitani, S., Saeki, H., Oki, E., Yoshinaga, K., Emi, Y., Morita, M., Kohnoe, S., Kakeji, Y., Yano, T., Baba, H. 2012. Biological mechanism and clinical effect of protein-bound polysaccharide K (KRESTIN((R))): review of development and future perspectives. Surg Today, 42(1), 8-28. Moore, M.M., Honma, M., Clements, J., Awogi, T., Douglas, G.R., van Goethem, F., Gollapudi, B., Kimura, A., Muster, W., O'Donovan, M., Schoeny, R., Wakuri, S. 2011. Suitable top concentration for tests with mammalian cells: mouse lymphoma assay workgroup. Mutat Res, 723(2), 84-6. Moore, M.M., Honma, M.,
Clements, J., Bolcsfoldi, G., Burlinson, B., Cifone, M., Clarke, J., Clay, P., Doppalapudi, R., Fellows, M., Gollapudi, B., Hou, S., Jenkinson, P., Muster, W., Pant, K., Kidd, D.A., Lorge, E., Lloyd, M., Myhr, B., O'Donovan, M., Riach, C., Stankowski, L.F., Jr., Thakur, A.K., Van Goethem, F. 2007. Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment. Mutat Res, 627(1), 36-40. Mortelmans, K., Zeiger, E. 2000. The Ames Salmonella/microsome mutagenicity assay. Mutat Res, 455(1-2), 29-60. Ng, T.B., Chan, W.Y. 1997. Polysaccharopeptide from the mushroom Coriolus versicolor possesses analgesic activity but does not produce adverse effects on female reproductive or embryonic development in mice. Gen Pharmacol, 29(2), 269-73.
Ng. T.B. 1998. A review of research on the protein-bound polysaccharide (polysaccharopeptide, PSP) from the mushroom Coriolus versicolor (basidiomycetes: Polyporaceae). General Pharmacology: The Vascular System, 30(1), 1-4. OECD. 1997. Bacterial Reverse Mutation Test. OECD GUIDELINE FOR THE TESTING OF CHEMICALS, 471, 10. OECD. 1997. In Vitro Mammalian Chromosome Aberration Test. OECD GUIDELINE FOR THE TESTING OF CHEMICALS, 473, 10. OECD. 1997. Mammalian Erythrocyte Micronucleus Test. OECD GUIDELINE FOR THE TESTING OF CHEMICALS, 474, 10. Pathak, N., Khandelwal, S. 2006. Oxidative stress and apoptotic changes in murine splenocytes exposed to cadmium. Toxicology, 220(1), 26-36. Schramke, H., Meisgen, T.J., Tewes, F.J., Gomm, W., Roemer, E. 2006. The mouse lymphoma thymidine kinase assay for the assessment and comparison of the mutagenic activity of cigarette mainstream smoke particulate phase. Toxicology, 227(3), 193-210. Soriano, C., Creus, A., Marcos, R. 2007. Gene-mutation induction by arsenic compounds in the mouse lymphoma assay. Mutat Res, 634(1-2), 40-50. Torous, D.K., Dertinger, S.D., Hall, N.E., Tometsko, C.R. 2000. Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study. Mutat Res, 465(1-2), 91-9. Torous, D.K., Hall, N.E., Murante, F.G., Gleason, S.E., Tometsko, C.R., Dertinger, S.D. 2003. Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy. Toxicol Sci, 74(2), 309-14. Tsang, K.W., Lam, C.L., Yan, C., Mak, J.C., Ooi, G.C., Ho, J.C., Lam, B., Man, R., Sham, J.S., Lam, W.K. 2003. Coriolus versicolor polysaccharide peptide slows progression of advanced non-small cell lung cancer. Respiratory Medicine, 97(6), 618-624. Tsukagoshi, S., Hashimoto, Y., Fujii, G., Kobayashi, H., Nomoto, K., Orita, K. 1984. Krestin (PSK). Cancer Treatment Reviews, 11(2), 131-155. Wang, J., Sawyer, J.R., Chen, L., Chen, T., Honma, M., Mei, N., Moore, M.M. 2009. The mouse lymphoma assay detects
recombination, deletion, and aneuploidy. Toxicol Sci, 109(1), 96-105. Yang, J.P., Hsu, T., Lin, F., Hsu, W., Chen, Y. 2012. Potential antidiabetic activity of extracellular polysaccharides in submerged fermentation culture of Coriolus versicolor LH1. Carbohydrate Polymers(0). Yeung, J.H.K., Or, P.M.Y. 2007. Effects of polysaccharide peptides from COV-1 strain of Coriolus versicolor on glutathione and glutathione-related enzymes in the mouse. Food and Chemical Toxicology, 45(6), 953-961.
