樟芝(Antrodia camphorata)為台灣特有生物，且只生長於台灣特有種牛樟樹(Cinnamomum kanehirai)樹洞中，價格昂貴，民間視為解毒、保肝、抗癌草藥，但栽培困難迄今仍以採集為主，因此對牛樟樹之保育傷害甚大。本研究乃以液態深層培養方式進行樟芝菌絲體之生產，培養條件為1 %之接種量，轉數50 rpm，通氣量為0.5 vvm，溫度為25℃，以50噸槽體進行40噸培養，8天後可得到紅色菌絲，發酵全液於55℃減壓濃縮後進行冷凍乾燥，收率可達1.8 %，多醣含量則在10 %以上。
另樟芝可在僅含牛樟木屑或香桂木屑的瓊脂培養基上生長，且隨木屑添加量之增加生長愈好。添加牛樟木屑於PDA培養基中可促進樟芝之生長速度，此促長成分可溶於熱水、酒精或乙酸乙酯，推測可能為牛樟樹精油，其成分如4-terpineol、α- terpineol 、safrole及camphor對靈芝、雲芝、茯苓、舞茸及猴頭菇等菇菌類有抑菌效果，在此些菇類受抑制之濃度下，樟芝則展現較高之耐性，故推測樟芝之宿主專一性，乃因牛樟樹精油抑制其他菇類之故。
在安全性試驗方面，以五株Salmonella typhimurium進行致突變性試驗，發現在5 mg/plate 以下無論水溶性及脂溶性區分，有無S9存在下，均無致突變性；以體外淋巴瘤細胞株之tk基因突變分析，水溶性成分在4 mg/ml以下，脂溶性成分在0.5 mg/ml以下，不論S9有無，測試結果皆為陰性反應；對ICR雄性鼷鼠之週邊血液進行遺傳基因微核試驗，在5000 mg/kg體重以下餵食，亦為陰性反應；又以SD懷孕雌鼠做致畸胎試驗，證明無任何臨床致畸胎毒性症狀顯現；以SD大白鼠進行急性毒性試驗，連續餵食高劑量30天(4500 mg/kg)及90天(4500 mg/kg)後犧牲並進行解剖，結果均無任何毒性症狀。
在功能性方面以相當於人每日建議劑量（3.78 g/day/人）之 0.5、1或2倍樟芝發酵全液凍乾品對於小鼠生長均無不良的影響。三測試劑量亦均不影響脾臟中淋巴細胞MHC I、MHC II、T及B細胞次群之比率。在非特異性免疫反應方面1及2倍二劑量對於ova特異淋巴細胞之增生均有提升的作用，並可明顯提昇脾臟ova特異性淋巴細胞分泌IL-2及IFN-γ的分泌；三劑量皆可明顯地抑制脾臟ova特異性淋巴細胞分泌IL-4。另從子實體中鑑定出一個COX II specific inhibitor 化合物，且可以在菌絲體培養時，透過 vanillin 之添加而重製此成分。

Antrodia camphorata (Chan-Ku) is a new and exclusive fungus parasitic on the inner wall of the heart wood of endemic evergreen Cinnamomun kanehirai Hay in Taiwan. It has been utilized as traditional herb for the treatment of food and drug intoxications, diarrhea, abnormal pains, hypertension and liver cancer.
A. camphorata could grow on the simple saw-dust agars no matter from Cinnamomum kanehirae or Cinnamomum subavenium, and that is dosage dependent. Saw-dust from C. kanehirae also could promote the growth rate of A. camphorata on PDA. Some promoting ingredients are water soluble, but some are solube in alcohol or ethyl acetate. Some major components of essential oil from C. kanehirae such as 4-terpineol, α-terpineol, safrole and camphor would inhibit the growth of several mushroom at lower concentration, but would make A. camphorata grow better at the same concentration. It is therefore assumed that the host specificity of A. camphorata is due to the selective inhibition of C. kanehirae essential oil.
Solid culture of its fruit body is still difficult, so submerged culture was developed at 50 Ton scale in a 50 rpm agitation, 0.5 vvm aeration, 25℃ condition. Whole broth turned into redness at 8th day after 1 % inoculating was concentrated at 55℃ and lyophilized. The product is proven as safe after evaluation in Ames test, in vitro and in vivo chromosomal damage in mammalian cell, 1-day, 28-day, 90-day feeding toxicity and teratogenicity studies. It also could increase NK cell cytotoxicity of BALB/C mice at double suggested dose after 9 week feeding. IL-2 & INF-γ secretion of spleen cell also could be enhanced even at half dose of suggestion but that of IL-5 be inhibited. After stimulated with ova, IL-2 & INF-γ were enhanced at suggested dose and even at half of the dose, lyophilized A.camphorata broth still could inhibit IL-4 secretion.
The volatile components from the fruiting bodies, the liquid cultured broth of Antrodia camphorata, and Cinnamomum kanehirae wood were separately isolated by steam distillation-solvent extraction and identified by gas chromatography-mass spectrometry. In the fruiting bodies, a COX-2 inhibitor 2, 4, 5-trimethoxybenzaldehyde (TMBA) was found to be the most abundant constituent, but was totally absent in its cultured broth and its natural host, C. kanehirae wood. On feeding with the acid-digested sawdust of C. kanehirae wood or vanillin to the broth for culture, TMBA was produced in both cultured broths.

表次………………………………………………………………………Ⅲ
圖次………………………………………………………………………Ⅴ
中文摘要…………………………………………………………………Ⅵ
Abstract…………………………………………………………………Ⅶ
Chapter 1 緒論…………………………………………………………1
Chapter 2樟芝之培養……………………………………………………4
Chapter 3樟芝安全性研究…………………………………………… 31
Chapter 4樟芝之功能性研究………………………………………… 71
Chapter 5 結論…………………………………………………………84
Chapter 6 參考文獻……………………………………………………86
Chapter 7 附錄…………………………………………………………90

1.王心怡(2004)樟芝深層發酵液之成分分析.實踐大學食品營養研究所.碩士論文.

2.王伯徹、邱世浩和黃仁彰(1998)食用菇保健食品專輯.食品工業30(5),1-36.

3.王伯徹和黃仁彰(2002)靈芝與樟芝之研發與市場面面觀.食品工業34(5),3-17.

4.林讚標(1997)談牛樟與冇樟.在:牛樟生物學及育林技術研討會論文集.林業叢刊第72號.(1997) p.27-34台灣省林業試驗所印行.台北,台灣.

5.行政院衛生署(1999)體外鼷鼠淋巴瘤tk分析法.健康食品安全評估方法.台北,台灣.

6.吳聲華(1999)林中貴客-牛樟芝.博覽台灣28,26-27.

7.高曉薇(1991)台灣靈芝屬新種樟芝之三萜類成分研究.台北醫學院天然藥物醫學研究所.碩士論文.

8.黃惟敏(1999)樟芝微量成分的研究.靜宜大學應用化學系.碩士論文.

9.許勝傑、陳清農和陳勁初(2000)樟芝宿主專一性之探討,台灣農業化學與食品科學,38(6),533-539.

10.陳勁初、林文鑫、陳清農、許勝傑、黃仕政及陳炎鍊(2001)台灣特有真菌-樟芝菌絲體之開發,真菌科學,16(1,2)7-22.

11.曹巧吟(2002)樟芝中免疫調節蛋白的純化與其生理活性之探討.國立台灣大學園藝研究所碩士論文

12.賴鈺菁(2003)樟芝發酵液之抗發炎及其誘導癌細胞凋亡機制之探討.中國醫藥大學營養研究所碩士論文.

13.劉俊仁(2002)中草藥抗癌機制研究.國立臺灣大學生物化學暨分子生物學研究所博士論文.

14.廖英明 (1998) 菇類中的許不了－樟芝.農業世界雜誌176,76-79.

15.劉昌孝和孫瑞元(1999)藥物評價實驗設計與統計學基礎.p.19-20.軍事醫學科學出版社,北京,中國.

16.臧穆和蘇慶華 (1990) 我國台灣產靈芝屬新種一樟芝(形態、定名).雲南植物研究 12(4),395-396.

17.簡秋源、姜宏哲和陳淑貞(1997)牛樟菇培養性狀及其三帖類成分分析之研究.在:牛樟生物學及育林技術研討會論文集.林業叢刊第72號. (1997) p.133-137.台灣省林業試驗所印行.台北,台灣.

18.藤田安二(1952)ワスノキ及ぴろの近似種の種的、成分的、分布的,進化的諸關係.Bot. Mag. Tokyo 65, 771-772.

19.Adams R.P. (1995) Identification of essential oil components by gas chromatography / mass spectrometry. Carol Stream, IL: Allured Publishing.

20.Biavati B., Franzoni S., Ghazvinizadeh H. and Piccaglia R.(1996) Antimicrobial and antioxidant properties of plant essential oils. In " Proceedings of the 27th International Symposium on Essential Oils", Sep. (1996), Vienna, Austria. Ed. Franz C., Mathe A., Buchbauer, G., p.326- 331. Allured Publishing Corporation, Carol Stream, IL .

21. Chang T. T. and Chou W. W.(1995) Antrodia cinnamomea sp. Nov.on Cinnamomum kanehirae in Taiwan. Mycol. Res. 99 756-758.

22.Chang T. T. and Chou W. W.(2004) Antrodia cinnamomea reconsidered and A.salmonea sp. Nov. on Cunninghamia konishii in Taiwan.Bot.Bull.Acad.Sin.45:347-352.

23.Chen C.C., Shiao Y. J., Lin R.D., Shao Y.Y., Lai M.N., Lin C.C., Ng L.T. and Kuo Y.H. (2006) Neuroprotective diterpenes from the fruiting body of Antrodia camphorata. J. Nat. Prod. 69 689-691.

24.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.

25.EPA (1996).Detection of gene mutations in somatic cells in culture. Health effects test guidelines, OPPTS 870.5300, EPA712-C-96-221, June 1996.

26.Filek G., Bergamini M. and Lindner W. (1995) Steam distillation-solvent extraction, a selective sample enrichment technique for gas chromatographic-electron-capture detection of organochlorine compounds in milk powder and other milk products. J. Chromatography A 712, 355-364.

27.Koki A.T., Leahy K.M. and Masferrer J.L. (1999) Potential utility of COX-2 in chemoprevention and chemotherapy. Expert Opin. Investig. Drugs 8, 1623-1638.

28.Lee I. H., Huang R. L., Chen C. T., Chen H. C., Hsu W. C. and Lu M. K. (2002) Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiol.Lett. 209, 61-67.

29.Momin R.A., De Witt D.L. and Nair M.G. (2003) Inhibition of cyclooxygenase (COX) enzymes by compounds from Daucus carota L. Seeds. Phytother. Res. 17, 976-979.

30.OECD (1997). In vitro mammalian cell gene mutation test. Guideline for the testing of Chemicals, No.476, adopted 21th July (1997).

31.Rao D.V. and Stuber F.A. (1983) An efficient synthesis of 3.4.5-trimethoxybenzaldehyde from vanillin. Synthesis 4,308.

32.Shen C.C., Yang H.C., Huang R.L., Chen J.C. and Chen C.C. (2005) Anti-HBV principle from the culture broth of Antrodia camphrata (strain # BCRC-35396 ). J. Chin. Med.16, 57-61.

33.Shen Y.C., Wang Y.H., Chou Y.C., Chen C.F., Lin L.C., Chang T.T., Tien J.H. and Chou C.J. (2004a) Evaluation of the anti-inflammatory activity of Zhankuic acid isolated from the fruiting bodies of Antrodia camphorata. Planta Med. 70, 310-314.

34.Shen, Y.C., Chou, C.J., Wang Y.H., Chen C.F., Chou Y.C. and Lu M.K. (2004b) Anti-inflammatory activity of the extracts from mycelia of Antrodia camphorata cultured with water-soluble fractions from five different Cinnamomum species. FEMS Microbiol. Lett. 231, 137-143.

35.Schomberg, G. and Dielmann, G. (1973) Identification by means of retention parameters. J. Chromatographic Sci. 11, 151–159.

36. Song T.Y. and Yen G.C. (2002) Antioxidant properties of Antrodia camphorata in submerged culture. J. Agric. Food. Chem. 50, 3322-3327.

37.Tsai P.C., Hseu Y.C. and Yang H.L. (2005) Anticancer effect in MDA-MB-231 human breast cancer cells by Antrodia camphorata. In: Proceedings and Abstract of the 20th symposium on natural products, June 24-25, Taipei, p.80.

38. Toller S. (1996) A psychologist examines the use of essential oils in aromatherapy techniques. In " Proceedings of the 27th International Symposium on Essential Oils", Sep. (1996), Vienna, Austria. Ed. Franz C., Mathe A., Buchbauer, G., pp. 1-9. Allured Publishing Corporation, Carol Stream, IL.

39.Wu S.H., Ryvarden L. and Chang T.T. (1997) Antrodia camphorate (‘niu-chang-chih’), new combination of a medicinal fungus in Taiwan. Bot. Bull. Acad. Sin. 38, 273-275.

40.Wu S.H., Yu Z.H., Dai Y.C.,Chen C.T. and Su C.H. (2004) Taiwanofungus, a polypore new genus. Fung.Sci.19,109-116.

41.Yang F.C., Huang H.C. and Yang M.J. (2003) The influence of environmental conditions on the mycelium growth of Antrodia cinnamomea in submerged culture. Enzyme Microb Technol 33, 395-402.