菇類已經在全世界被廣泛的用來治療疾病有數千年的歷史。其中樟芝為台灣民間之傳統用藥，相信其有某種程度的治療效果，特別是保肝方面。因此本研究藉由MTT assay來評估樟芝萃取物對肝癌細胞（包括：Hep 3B, Hep G2及Alexander cells）生長的抑制效果；另外，以硫代乙醯胺（TAA）誘導大鼠肝纖維化或肝硬化模式作為評估樟芝是否有保肝效果，其功效以Masson trichrome的染色方法評估肝纖維化或肝硬化的程度。此外，也利用體外試驗的方式，以anti-HBsAg assay評估樟芝對anti-HBsAg 的效果。
結果顯示，樟芝水萃物或酒萃物對肝癌細胞Hep 3B, Hep G2及Alexander 細胞增生的抑制率分別為76、77 and 68 %。在動物實驗進行十二週後，TAA成功的誘導極嚴重的肝硬化產生，且在給予樟芝水萃物後能有效的降低肝損傷。此外，在anti-HBsAg assay中，樟芝萃取物也能有效的抑制Alexander cell
所分泌的HBsAg。
此研究結果證明，樟芝水萃物有保護肝臟之能力，此可提供作為在另類醫療中的參考。

Mushrooms have been widely used to prevent and cure diseases for thousand years. The fruit bodies of “niu-chang chih” (Antrodia camphorata) are believed to have certain medical effects in Taiwanese folk medicine, especially for
hepatoprotection.
In this study, the inhibitory effect of Antrodia camphorata on hepatoma cell lines growth (such as Hep 3B, Hep G2 and Alexander cells) was conducted by MTT assay. To evaluate whether there is the hepatoprotective ability of Antrodia camphorata, the rodent model of liver fibrosis, induced by thioacetamide (TAA), has also been set up. The collected livers were stained with Masson trichrome in order to evaluate the degree of liver fibrosis. Furthermore, anti-HBsAg assay was used to investigate the hepatoprotective effect of Antrodia camphorata in reducing the secretion of HBsAg on Alexander cells.
The results demonstrated that there were significantly inhibitory effects of water extract or alcohol extract of mycelium from Antrodia camphorata on Hep 3B, Hep G2 and Alexander cells growth, and the inhibitory rate were 76, 77 and 68% respectively. After twelve week’s TAA-induced, serious liver fibrosis or liver cirrhosis rats have been successfully set up, and the water extract of mycelium from Antrodia camphorata can reduce the liver damage by treated with TAA. Moreover, the secretion of HBsAg on Alexander cells was also significantly inhibited by the water
extract of Antrodia camphorata.
These results suggest that water extract of mycelium from Antrodia camphorata has hepatoprotective ability. The results derived from this study offer some information in alternative medicine.

目 次
中文摘要………………………………………………………………………………. ii
英文摘要…………………………………………………………………………….....iii
目次……………………………………………………………………………………v
表目錄………………………………………………………………………………....vii
圖目錄………………………………………………………………………………...viii
第一章 文獻回顧……………………………………………………………………….1
1-1 肝臟的簡介…………………………………………………………………...1
1-2 肝硬化的簡介………………………………………………………………...2
1-3 硫代乙烯胺（Thioacetamide）的介紹……………………………………..3
1-4 樟芝的介紹…………………………………………………………………...4
1-4-1 分類學………………………………………………………………….....4
1-4-2 外觀與型態特性……………………………………………………….....5
1-4-3 化學成分研究………………………………………………………….....5
1-4-5 安全性評估…………………………………………………………….....6
1-4-6 藥理作用探討………………………………………………………….....7
1-5 水飛薊（Silymarin）的簡介…………………………………………………9
第二章 研究動機與目的……………………………………………………………...11
第三章 材料與研究方法……………………………………………………………..14
3-1 實驗材料…………………………………………………………………….14
3-1-1 樟芝………………………………………………………………………14
3-1-2 水飛薊……………………………………………………………………14
3-1-3 細胞株……………………………………………………………………14
3-1-4 實驗動物…………………………………………………………………14
3-2 實驗藥品………………………………………………………………….....15
3-3實驗套組試劑……………………………………………………………….16
3-4 實驗儀器…………………………………………………………………....16
3-5 實驗方法………………………………………………………………….....17
3-5-1 MTT assay……………………………………………………………......17
3-5-2 Anti-HBsAg assay……………………………………………………......18
3-5-3 Thioacetamide誘導肝硬化model之建立…………………………….20
3-5-4 動物實驗設計…………………………………………………………20
3-5-5 血清測定…………………………………………………………………21
3-5-6 蘇木紫與伊紅（Hematoxylin-Eosin stain）……………………………21
3-5-7 Masson trichrome stain…………………………………………………22
3-5-8 組織病理病變程度的定量……………………………………………23
3-5-9 二維電泳…………………………………………………………………23
3-5-10 統計分析………………………………………………………………..25
第四章 結果…………………………………………………………………………...25
4-1 樟芝水萃物及酒萃物對腫瘤細胞影響…………………………………26
4-2 Silymarin水萃物對腫瘤細胞影響………………………………………28
4-3 樟芝水萃物及酒萃物對HBsAg 的影響……………………………….29
4-4 Thioacetamide誘導肝硬化model之建立………………………………30
4-5 樟芝萃取物對TAA誘導肝損傷之保肝效果…………………………...30
4-6 二維電泳…………………………………………………………………31
第五章 討論…………………………………………………………………………...32
第六章 參考文獻……………………………………………………………………...35
附錄…………………………………………………………………………………….59

王伯徹. 具有開發潛力食藥用菇的介紹. 食品工業. 2000;30:1-36.
行政院衛生署統計室. http://www.doh.gov.tw/statistic/index.htm. 2004.
邱建中. 張隆仁. 保健植物－水飛薊. 台中區農業專訊. 2003;43:20-21.
林文川、郭淑卿、吳岳文. 樟芝菌株(CCRC 93032)發酵液對大鼠28天餵食的毒性作用. J. Chin. Med. 2001;12:293-303.
吳家駒、盧珍妏、洪鳴遠、黃瑞玉、陳如茵、蔡美珠. 樟芝（BCRC 930032）濃縮發酵液之安全性評估. 台灣農業化學與食品科學. 2003;41:268-276.
徐敬衡. 文榮輝. 賴秋梅. 翁偉恆. 利用深層發酵生產樟芝菌絲體及其抗菌研究. 2000;23-32.
陳啟楨. 李曉雯. 謝慈丰. 林威伸. 蘇慶華. 藍明煌. 樟芝固體栽培及其生物活性之研究. 第十七屆全國技職教育研討會. 2002;33-40.
許益超. 丹參對於肝硬化大鼠之轉型生長因子及原膠原蛋白基因表現的效應. 國立陽明大學傳統醫藥研究所碩士論文, 2002.
郭淑卿. 樟芝發酵液對大鼠肝臟纖維化及胃腸功能之改善作用. 中國醫藥學院中國藥學研究所碩士論文. 2001.
雷永耀. 周嘉揚. 吳秋文. 彭芳谷. 肝臟腫瘤學.(1~11)合記圖書出版社. 2004.
潘一紅. 秋惜禾. 牛奶薊中的黃金物質. 化工資訊. 2000;54-57.
魏宏吉.龍膽瀉肝湯等藥物對肝硬化大鼠的血流動力效應研究. 國立陽明大學傳統醫藥研究所碩士論文, 2001.
戴宇昀. 樟芝菌絲體與子實體對四氯化碳及酒精誘導之慢性及急性肝損傷之保肝功能評估. 國立中興大學食品科學研究所碩士論文.2000.
Berger, Jose., Kowdley, Kris V. Is Silymarin Hepatoprotective in Alcoholic Liver Disease? J Clin Gastroenterol. 2003;37: 278-9.
Chang, T. T., and Chou, W. N. Antrodia Cinnamomea sp. nov. on Cinnamomum kamehirai in Taiwan. Mycol. Res. 1995;99: 756-758.
Chen, J. C., Lin, W. H., Chen, C. N., Sheu, S. J., Huang, S. J., and Chen, Y. L. Development of Antrodia camphorata mycelium with submerge culture. Fung. Sci. 2001;16: 7-22.
Cherng, I. H. and Chiang, H. C. Three new triterpenoids from Antrodia cinnamomea. J. Natural Products. 1995;58: 365-371.
Cherng, I. H., Wu, D. P., and Chiang, H. C. Triterpenoids from Antrodia cinnamomea. Phytochemistry. 1996;41: 263-267.
Chiang, H. C., Wu, D. P., Cherng, I. H., and Ueng, C. H. A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. Phytochemistry. 1995;39: 613-616.
Chieli, E., and Malvaldi, G. Role of the microsomal FAD-containing monooxygenase in the liver toxicity of thioacetamide s-oxide. Toxicology. 1984;31: 41-52.
Dai, Y. Y., Chuang, C. H., Tsai, C. C., Sio, H. M., Huang, S. C., Chen, J. C., and Hu, M. L. The protection of Antrodia camphorate against acute hepatotoxicity of alcohol in rats. Journal of Food and Drug Analysis. 2003;11: 177-185.
Dashti, H., Jeppsson, B., Hagerstrand, I., Hultberg, B., Srinivas, U., and Abdulla, M. et al. Thioacetamide- and carbon tetrachloride-induced liver cirrhosis. Eur Surg Res 1989;21: 83–91.
Ding, T. M., Tian, S. J., Zhang, Z. X., Gu, D. Z., Chen, Y. F., Shi, Y. H., and Sun, Z. P. Determination of active component in silymarin by RP-LC and LC/MS. Journal of Pharmaceutical and Biomedical Analysis. 2001;26: 155-161.
Fitzhugh, O. G., and Nelson, A. A. Liver tumors in rats fed thiourea or thioacetamide. Science. 1948;108: 626-628.
Gabriele, B. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology. 1997; 26: 643-649.
Girish S. Achliya, Sudhir G. Wadodkar, Avinash K. Dorle evaluation of hepatoprotective effect of Amalkadi Ghrita against carbon tetrachloride-induced hepatic damage in rats. Journal of Ethnopharmacology. 2004;90: 229–232.
Green, J., and Better, O. S. Systemic hypotension and renal failure in obstructive jaundice-mechanistic and therapeutic aspects. J Am Soc Nephrol. 1995; 5: 1853-1871.
Hananske-Abel HM. Fibrosis: representive molecular elements, a basic concept, and emerging targets for suppressive treatment. In: Hepatology: A textbook of liver disease. 1996.
Hsiao, G.., Shen, M. Y., Lin, K. H., Lan, M. H., Wu, L. Y., Chou, D. S., Lin, C. H., Su, C. H., and Sheu, J. R. Antioxidative and hepatoprotective effects of Antrodia camphorata extract. Journal of Agricultural and Food Chemistry. 2003;51: 3302-3308.
Hu, C. M., and Cheng, H. W. Method development for evaluation of hepatoprotection functionality of health food. Journal of Food and Drug Analysis. 2000;8: 174-179.
Jeong, D. H., Jang, J. J., Lee, S. J., Lee, J. H., Lim, I. K., Lee, M. J., and Lee. Y. S. Expression patterns of cell cycle-related proteins in a rat cirrhotic model induced by CCl4 or thioacetamide. J Gastroenterol. 2001;36: 24–33.
Jezequel, A. M., Mancini, R., Rinaldesi, M. L., Macrri, G., Ventruini, C., and Orlandi, F. A morphological study of the early stages of hepatic fibrosis induced by low doses of dimethylnitrosamine in the rats. J. Hepatol. 1987;5: 174-181.
Lee, I. H., Huang, R. L., Chen, C. T., Chen, H. C., Hsu, W. C., and Lu, M. K. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS
Microbiology Letters. 2002;209: 63-67.
Lee, T. Y., Mai, L. M., Wang, G. J., Chiu, J. H., Lin, Y. L., and Lin, H. C. Protective mechanism of salvia miltiorrhiza on carbon tetrachloride-induced acute hepatotoxicity in rats. Journal of Pharmacological Sciences. 2003;91: 202-210.
Letteron, P., Labbe, G., Degott, C., Berson, A., Fromenty, B., Delaforge, M., Larrey, D., and Pessayre, D. Mechanism for the protective effects of silymarin againstcarbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice. Evidence that silymarin acts both as an inhibitory of metabolic activiationand as a chain-breaking antioxidant. Biochem. Pharmaco. 1990;39: 2027-2034.
Li. X., Benjamin. I. S., Alexander. B. Reproducible production of thioacetamide
-induced macronodular cirrhosis in the rat with no mortality. Journal of Hepatology.
2002;36: 488–493.
Magee, P. N. Toxic liver necrosis. Lab. Invest. 1966;15: 111-131.
Noda, S., Masumi, S., Moriyama, M., Kannan, Y., Ohta, M., Sugano, T., et al. Population of hepatic macrophages and response of perfused liver to platelet-Activatingfactor during production of thioacetamide-induced cirrhosis in rats.
Hepatology. 1996;24: 412-418.
Porter, W. R., and Neal, R. A. Metabolism of thioacetamide and thioacetamide s-oxide by rat liver microsomes. Drug Metab Dispos. 1978;6: 179-388.
Porter, W. R. Thioacetamide induced hepatic necrosis pharmacokinetics of thioacetamide and thioacetamide-oxide in the rat. J Pharmcol Exp Ther. 1979;208(3):
386-391.
Poyer, J. L., and Mccay, P. B. Confirmation of assignment of the trichloromethyl radical spin adduct detected by spin-trapping during 13C-carbon tetrachloride metabolism in vitro and in vivo. Biochem Biophys Res Commun. 1980;94: 1154-1160.
Quanren He, Jiyoung Kim, and Raghubir P. Sharma, Silymarin Protects Against Liver Damage in BALB/c Mice Exposed to Fumonisin B1 Despite Increasing Accumulation of Free Sphingoid Bases. Toxicological sciences. 2004;80: 335-342.
Rosen, H.R., and Keeffe, E.B. Evaluation of abnormal liver enzymes, use of liver tests, and the serology of viral hepatitis. In: Bacon BR, Di Bisceglie AM, eds. LiverDisease: Diagnosis and Management. Philadelphia: Churchill Livingstone; 2000;
24-35.
Ruwart, M. J., Wilkinson, K. F., Rush, B. D. et al. The integrated value of serum procollagen Ⅲ peptide over time predicts hepatic hydroxyproline content and stainable collagen in a model of dietary cirrhosis in the rat. Hepatology. 1989;10: 801-806.
Shen, C. C., Kuo, Y. C., Huang, R. L., and Lin, L. C. New ergostane and lanostane from Antrodia camphorata. J. Chin. Med. 2003;14: 247-258.
Song, T. Y., and Yen, G. C. Protective Effects of fermented filtrate from Antrodia camphorata in submerged culture against CCl4-induced hepatic toxicity in Rats. Journal of Agricultural and Food Chemistry. 2003;51: 1571-1577.
Vorobioff, J., Bredfeldt, J. E., and Groszmann, R. J. Increased blood flow through the portal system in cirrhotic rats. Gastroenterology. 1984;87: 1120-1126.
Wu, S. H., Leif, R., and Chang, T. T. Antrodia camphorata (“ niu-chang-chih”), new combination of a medicinal fungus in Taiwan. Bot. Bull. Acad. Sin.1997;38: 273-275.
Yang, S. W., Shen, Y. C., and Chen, C. H. Steroids and triterpenoids of Antrodia cinnmomea-A fungus parasitic on Cinnamomum micranthum. Phytochemistry. 1996;41(5): 1389-1392.
Zang, M., and Su, Q. Ganoderma camphoratum a new taxon in genus Ganoderma from Taiwan, China. Acta Bot. Yunnanica. 1990;12:395-396.
Zimmermann, T., Muller, A., Machnik, G., Franke, H., Schubert, H., and Dargel, R. Biochemical and morphological studies on production and regression of experimental liver cirrhosis induced by thioacetamide in Uje:WIST rats. Z Versuchstierkd 1987;30: 165-180.