牛樟芝(Antrodia cinnamomea)為台灣特有種藥用真菌菇類，已多篇研究報告證實牛樟芝因含有三萜類及多醣體等成份故具有解毒、消炎、治療肝臟疾病及抗癌等功用，據說白牛樟芝為白色變種牛樟芝是更稀少的菌種、功效更佳及價格更昂貴，為了確定紅、白牛樟芝的成份及藥效，故本研究進行椴木白牛樟芝BWAC(Basswood White AC)、自製白牛樟芝HMWAC(Homemade medium white AC)、自製白牛樟芝HMWAC+P1 (Homemade medium white AC+P1)、市售白牛樟芝CMWAC(Commercial medium white AC)、自製紅牛樟芝(15cm dish)HMRAC(Homemade medium Red AC +15cm dish)、自製野生紅牛樟芝HMNTAC+P1(Homemade medium Native Type AC)、野生紅牛樟芝NTAC(Native Type AC)等上述自製改良培養基及與市售培養基培育紅牛樟、白牛樟，並以野生型紅牛樟芝及椴木白牛樟芝做樟芝酸指標成份之比較及進一步萃取各式培育牛樟芝之成份分析測量包括總三萜類、多醣體、總多酚類及抗氧化力及抗人類肝癌細胞HepG2細胞存活檢測(MTT assay)效果比較，以尋找出最佳之生產方式以滿足產業需求。本研究結果發現皿培牛樟芝確可得到相當量的三萜類，而多醣體含量自製培養基之紅白牛樟均高於市售培養基，而總多酚類為皿培牛樟芝高於椴木白牛樟，在抗氧化力比較亦如此。另外研究發現皿培培養基添加重要元素P1的確可增加牛樟芝之指標成份如Antcin A、B、H、DeSA和DeEA等含量。在抗肝癌細胞試驗發現皿培牛樟芝在62.5ug/ml即有約50%抑制效果，而野生紅牛樟濃度高於125ug/ml以上，有超過80%抑制效果，本研究結果顯示野生紅牛樟有不錯抑癌效果，然而取得不易及培育時間長等缺點，故研發適合的配方以生產皿培牛樟芝產品對B肝病人的健康保護是相當具有潛力生技健康產品。

Antrodia cinnamomea (AC) is a specially and locally medicinal fungus in Taiwan. Many studies have been confirmed that AC is rich in triterpenoids, polysaccharides and other nutrients that may carry functional properties like detoxification, anti-inflammatory, improve liver function and anti-cancer. It is said that white AC is relative rare and more expensive and more functional contents than regular form of AC. Thus, studies to develop the best culture medium, comparing the bioactive compounds include total triterpenes, polysaccharides, total polyphenols and medicinal functions like to free radicals(DPPH) and anti-B liver cancer (MTT assay) on the regular form(red AC), white variant of AC, wild type(red AC) and various AC species in petri-dish culture medium (HMWAC、HMWAC+P1、CMWAC、HMRWAC、HMNTAC+P1 and NTAC) and basswood AC(BWAC) should be conducted. This study results showed that the homemade medium can enhance crude triterpenoid of mycelia and basidiomatal formation of AC in petri-dish solid-state fermentation. The polysaccharides content of homemade medium for regular and white AC were higher than the commercial medium. The total polyphenols of AC in the petri-dish culture was more than basswood white AC and DPPH was also the case. In addition, it was found that the addition of P1 element to the culture medium could increase the contents of Antcin A 、B、H 、DeSA and DeEA. In the anti- liver cancer cell HepG2 assay showed that about 50% inhibition was achieved at 62.5 μg / ml. The wild type AC (NTAC) fruiting bodies extracted concentration was higher than 125μg/ml, and the anti-liver cancer cell effect was over 80%. The results showed that NTAC also had good anti-cancer effects. But it’s not easy to obtain and need long-time about 2-3 years to culture and other shortcomings like infection issues to overcome. This study demonstrates that the development of culture medium formula to produce solid–state fermentation of AC products on the B liver health protection is considerable potential for health products and is the best making way to fit the needs of the industry.

謝誌 I
中文摘要 II
ABSTRACT III
目錄 V
表目錄 VII
圖目錄 VIII
第一章 前言 1
1.1 研究背景 1
1.2 研究動機 2
第二章 文獻回顧 4
第三章 材料與方法 6
3.1 實驗架構 6
3.2 藥品試劑與設備 6
3.2.1 藥品試劑 7
3.2.2 設備儀器 8
3.3 牛樟芝菌種製備 8
3.4 牛樟芝菌種之培養基製備 8
3.5 培養基固態發酵牛樟芝子實體之育成 9
3.6 利用HPLC 定量分析牛樟芝樟芝酸相對含量分析 9
3.7 育成牛樟芝總三萜類含量測定: 9
3.8 培養基育成牛樟芝總多醣體含量定量 10
3.9 牛樟芝總多酚含量定量: 10
3.10 各種培養基育成牛樟芝抗氧化能力測定 10
3.11 肝癌細胞培養 11
3.12 MTT 反應分析 11
3.13 統計分析(Statistical analysis) 12
第四章 結果 13
4.1 紅牛樟芝與白牛樟芝之培育 13
4.2 白牛樟芝之椴木培養 14
4.3 建立HPLC樟芝酸八大指標成分分析圖譜 15
4.4 白牛樟芝指標成分分析 16
4.5 樟芝酸成分統計分析 18
4.6 牛樟芝總三萜類含量之測定 19
4.7 各式牛樟芝試驗樣品之總多醣體含量之測定 21
4.8 各式牛樟芝試驗樣品總酚類之測定 22
4.9 各式牛樟芝試驗樣品抗氧化力測定 23
4.10 牛樟芝萃取物對HepG2細胞株之細胞毒殺試驗 25
第五章 討論 32
第六章 參考文獻 35

高曉薇 1992。 臺灣靈芝屬新種樟芝之三萜類成份研究。臺北醫學院天
然物醫學研究所碩士論文。
劉嘉玲、張秀芳、黃志傑、周玉民 臺灣B型肝炎防治。2016。 疫情報
導 32(14):290-300。
魏育慧 利用動物細胞進行抗癌藥物篩選。2000。食品工業。32(11):27-35。
Allan, A. L., Vantyghem, S. A., Tuck, A. B., Chambers, A.
F., Chin-Yee, I. H. and Keeney, M. 2005. Detection and quantification of circulating tumor cells in mouse
models of human breast cancer using immunomagnetic
enrichment and multiparameter flow cytometry.
Cytometry Part A. 65:4-14.
Chang, T. T., and Chou W. N. 1995. Anrodia cinnamomea sp.
Nov. on Ginnamomum Kanehirai in Taiwan. Mycological
Research 99(6) :756-758.
Chang, F.R., Lu, M.C., Hwang, S.L., Chen, Y.H., Chang,
T.T., Hung. C.S., Wang, C.L., Chu, Y.H., and Pan, S.H.
2009. Immunostimulatory effect of Antrodia camphorata
extract on functional maturation of dendritic cells.
Food Chemistry. 4:1049-1057
Chang, T. T., and Wang, W. R. 2005. Basidiomatal
formation of Antrodia cinnamomea on artificial agar
media. Bot. Bull. Acad. Sin. 46:151-154.
Chen, C. H., and Yang, S. W. 1995 New steroid acids from
Antrodia cinnamomea, a fungal parasite of Cinnamomum
micranthum. J Nat Prod 58:1655–1661.
Chen, W. L., Ho, Y. P. and Chou, J. C. 2016. Phenologic
variation of major triterpenoids in regular and white
Antrodia cinnamomea. Bot. Stud. 57:33-41.
Chen, C.C., Lin, W.H., Chen, C.N., Hsu, S.J., Huang,
S.C., and Chen, Y.L., 2001. Development of Antrodia
camphorata mycelium with submerge culture. Fungi
Science 16:7– 22.
Chen, T. I., Chen, C. C., Lin, T.W., Tsai, Y. T., and Nam
,M. K. 2011. A 90-day subchronic toxicological
assessment of Antrodia cinnamomea in Sprague–Dawley
rats. Food Chem Toxicol 49:429–433
Cherng, I. H., and Chiang, H. C.. 1995. Three new
triterpenoids from Antrodia cinnamomea. J. Nat. Prod.
58: 365-371.
Cherng, I. H., Wu, D.P., and Chiang, H. C. 1995.
Triterpenoids from Antrodia cinnamomea. Phytochemistry
41: 263-267.
Denizot, F., and Lang, R. 1986. Rapid colorimetric assay
for cell growth and survival modifications to the
tetrazolium dye procedure giving improved sensitivity
and reliability. Journal of Immunological Methods.
89:271-7.
Doong, S. L., Tsai, C. H., Schinazi, R. F., Liotta, D.
C., and Cheng, Y. C. 1991. Inhibition of the
replication of hepatitis B virus in vitro by 2’,3’-
dideoxy-3’-thiacytidine and related analogues Proc.
Natl. Acad. Sci. USA. Vol(88):8495-8499.
Ferrari, M., Fornasiero, M. C., and Isetta, A. M. 1990.
MTT colorimetric assay for testing macrophage
cytotoxicity activity in vitro. Journal Immunological
Methods. 131:165-72.
Gerschenson, L. E., and Rotello, R. J. 1992. Apoptosis: a
different type of cell death. FASEB Journal.
6(7):2450-2455.
Hseu, Y. C., Chang, W.C., Hseu, Y. T., Lee, C. Y., Yech,
Y. J., Chen, P. C., Chen, J. Y., and Yang, H. L. 2002.
Protection of oxidative damage by aqueous extract from
Antrodia camphorata mycelia in normal human
erythrocytes. Life Sci 71:469–482.
Hseu, Y. C., Wu, F.Y., Wu, J. J., Chen, J. Y., Chang, W.
H., Lu F. J., Lai, Y. C., and Yang, H. L. 2005.
Antiinflammatory potential of Antrodia Camphorata
through inhibition of iNOS, COX-2 and cytokines via
the NF-kappaB pathway. Int Immunopharmacol 5:1914–
1925.
Hseu, Y. C., Chen, S. C., Chen, H. C., Liao, J. W., and
Yang, H. L. 2008. Antrodia camphorata inhibits
proliferation of human breast cancer cells in vitro
and in vivo. Food Chem Toxicol 46: 2680-2688.
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. 2003.
Antioxidative and hepatoprotective effects of Antrodia
camphorata extract. J Agric Food Chem. 51:3302–3308.
Hsieh, C., Hsu, T.H., and Yang, F.C. 2005. Production of
polysaccharides of Ganoderma lucidum (CCRC36021) by
reusing thin stillage, Process Biochem. 40:909–916.
Hsieh, Y. C., Rao Y. K., Wu C. C., Huang C. Y.,
Geethangili M., Hsu S. L., and Tzeng, Y.M., 2010.
Methyl antcinate A from Antrodia camphorata induces
apoptosis in human liver cancer cells through oxidant-
mediated cofilin- and Bax-triggered mitochondrial
pathway. Chem Res Toxicol 23(7):1256-67.
Hsu, Y. L., Kuo, Y. C., Kuo, P.L., Ng, L. T, Kuo, Y. H.,
and Lin, C. C. 2005. Apoptotic effects of extract from
Antrodia camphorata fruiting bodies in human
hepatocellular carcinoma cell lines. Cancer Lett 221:
77-89.
Huh, D., Gu, W., Kamotani, Y., Grotberg, J. B., and
Takayama, S. 2005. Micro-fluidics for flow cytometric
analysis of cells and particles . Physiological
Measurement. (26):173-198.
Jennings, C. D., and Foon, K. A. 1997. Recent advances in
flow cytometry: application to the diagnosis of
hematologic malignancy. Blood. 90:2863-2892.
Kerr, J. F. R., Winterford, C. M., and Harmon, B. V.
1994. Apoptosis: Its significance in cancer and cancer
therapy. Cancers. 73(8):2013-2026.
Ko, H. C., Tsai, T. H., Chou, C. J., Hsu, S. Y., L, S.
Y., and Chen, C. F. 1994. High-performance liquid
chromatographic determination of rutaecarpine in rat
plasma: application to a pharmacokinetic study.
Journal of Chromatography. 655:27-31.
Lai, C. I., Chu Y. L., Ho C. T., Su Y. C., Kuo Y. H., and
Sheen, L.Y., 2015. Antcin K, an active triterpenoid
from the fruiting bodies of basswood cultivated
Antrodia cinnamomea, induces mitochondria and
endoplasmic reticulum stress-mediated apoptosis in
human hepatoma cells. J Tradit Complement Med.
4;6(1):48-56.
Lee, I. H., Huang, R. L., Chen, C. T., Chen, H. C., Hsu,
W. C., and Lu, M. K. 2002. Antrodia camphorate
polysaccharides exibit anti-hepatitis B virus effects.
FEMS Microbiology Letters 209:63-67.
Lee, Y. P., Tsai, W. C., Ko, C. J., Rao, Y. K., Yang, C.
R., Chen, D. R., Yang, M. H., Yang, C. C., and Tzeng,
Y. M. 2012. Anticancer effects of eleven triterpenoids
derived from Antrodia camphorat. Anticancer Research
32:2727-2734.
Lin, J. Y., Wu, T. Z., and Chou, J. C. 2006. In vitro
induction of fruiting body in Antrodia cinnamomea- a
medicinally important fungus. Botanical Studies
47:267-272.
Lu, Y. C., Wu, P. F., Chiang, T. A., Chang, S. N., Wang,
S. C., Wu, H. C., Huang, Y. F., Lin, H. T., Chao, T.
P., and Shih, S. L. 2014. The signal transduction
pathway of apoptosis by Antrodia camphorate extracts
in human breast cancer MCF-7 cells. Medical Journal of
South Taiwan. 10:11-29.
Meerloo J., Kaspers J. L., and Cloos, J. 2011. Cell
Sensitivity Assays: The MTT Assay. Cancer Cell
Culture: Methods and Protocols, Methods in Molecular
Biology, vol. 731.
Singleton, V. L., and Rossi, J. A., 1965. Colorimetry of
total phenolics with phosphomolybdic-phosphotungstic
acid reagents. Am. J. Enol. Viticult. 16: 144-158.
Song, T. Y., and Yen, G. C. 2002. Antioxidant properties
of Antrodia camphorata in submerged culture. J. Agric.
Food Chem. 50: 3322-3327.
Song, T. Y., and Yen, G. C. 2003. Protective effects of
fermented filtrate from Antrodia camphorata in
submerged culture against CCl4-induced hepatic
toxicity in rats. J. Agric. Food Chem. 51: 1571-1577.
Song, T. Y., Hsu, S.L., Yeh, C. T., and Yen, G. C.2005.:
Mycelia from Antrodia camphorata in submerged culture
induce apoptosis of human hepatoma HepG2 cells
possibly through regulation of Faspathway. J Agri Food
Chem 53: 5559-5564.
Yang, H. L., Chen, C. S., Chang, W. H., Lu, F. J, Lai,
Y. C., Chen, C. C, Hseu, T. H., Kuo, C. T., and Hseu,
Y. C. 2006. Growth inhibition and induction of
apoptosis in MCF-7 breast cancer cells by Antrodia
camphorata. Cancer Lett 231: 215-227,
Peng, C. C., Chen, K. C., Peng, R. Y, Su, C. H., and
Hsieh, Li. H. M.2006: Human urinary bladder cancer T24
cells are susceptible to the Antrodia camphorata
extracts, Cancer Lett 243: 109-119.
Tsujikura, Y, Higuchi, T., Miyamoto, Y., Sato, S. and Jpn
Kokai Tokkyo Koho. 1992. Manufacture of ganoderic acid
by fermentation of Ganoderma lucidum; JP04304890.
Wang, G. J., Tseng, H. W., Chou, C. J, Tsai, T. H, Chen,
C. T, Lu, M. K .2003. The vasorelaxation of Antrodia
camphorata mycelia: involvement of endothelial Ca(2+)-
NO-cGMP pathway. Life Sci 73:2769–2783.
Wang, H. F., Yih, K. H. and Huang, K. F. 2010.
Comparative study of the antioxidant activity of
forty-five commonly used essential oils and their
potential active components. Journal of Food and Drug
Analysis, 18 (1):24-33.
Wu, D. P. and Chiang, H. C. 1995. Constituents of
Antrodia cinnamomea. J. Chin. Chem. Soc. 42: 797-800.
Wu, M.F, Peng, F.C, Chen, Y.L., Lee, C.S., Yang, Y.Y.,
Yeh, M.Y., Liu, C.M., Chang, J.B., Wu, R.S., Yu, C.C.,
Lu, H.F., Chung, J. G. 2011. Evaluation of
genotoxicity of Antrodia cinnamomea in the Ames test
and the in vitro chromosomal aberration test.In Vivo.
25:419-423.
Wu, T. Y., Du, Y. C., Hsu, Y. M., Lu, C. Y., Singab, A.
N. B., Shazly, M. E., Hwang, T. L., Lin, W. Y., Lai,
K. H., Lu, M. C., Tsai, Y. H., Hou, M. F., Duh, T. H.,
and Chang, F. R. 2013.New approach to the
characterization and quantification of Antrodia
cinnamomea benzenoid components utilizing HPLC-PDA,
qNMR and HPLC-tandem Ms: Comparting the wild fruiting
bodies and its artificial cultivated commercial
products. Food Research International 51:23-31.
Yang, F. C., Ma, T. W., and Lee, Y. H. 2013. Reuse of
citrus peel to enhance the formation of bioactive
metabolite-triterpenoid in solid-state fermentation of
A. cinnamomea. Biochemical Engineering Journal. 78:59-
66.
Yeh, C. T., Rao, Y. K, Yao, C. J., Yeh, C. F., and Li, C.
H, l. 2009. Cytotoxic triterpenes from Antrodia
camphorata and their mode of action in HT-29 human
colon cancer cells. Cancer Lett 285: 73-9.
Yen, I. C., Yao, C. W., Kuo, M. T., Chao, C. L., Pai, C.
Y., and Chang ,W. L. 2015. Anti-cancer agents derived
from solid-state fermentated Antrodia camphorate
mycelium. Fitoterapia 102:115-119.