臺灣博碩士論文加值系統

牛樟芝(Antrodia cinnamomea)和其宿主牛樟樹 (Cinnamomum kanehirae )為台灣本土的特有種。牛樟芝為一種昂貴的藥用真菌，經實驗研究證實牛樟芝富含多醣體和三萜類，對於抗癌、抗發炎、降血壓、抗氧化具有功效。本研究特別針對更為稀有的白牛樟芝，作一系列的研究，藉由白牛樟芝與紅牛樟芝的化合物做分析比較，以及誘導紅牛樟芝白化，進一步探討紅牛樟芝與白牛樟芝的相對關係。本研究利用不同的光照處理，觀察牛樟芝是否因不同刺激產生不同的生長變化。實驗結果，成功以藍光誘導牛樟芝菌絲的白化，同時經由實驗也發現，紅光可以造成牛樟芝不穩定生成子實體的現象。從薄層分析與高效能液相層析圖譜中比較不同的時期的萃取物，可以看出白牛樟芝與紅牛樟芝化合物的差異。而經本實驗誘導白化的牛樟芝在分析圖譜中也較接近白牛樟芝而又保有紅牛樟芝的某些特徵。分析牛樟芝幾個指標性三萜類化合物的改變，得知經誘導白化的牛樟芝，不但三萜類組成接近白牛樟芝，同時也還保留紅牛樟芝的化合物特徵。利用細胞融合技術，本研究進一步探討白牛樟芝是否為基因受損而變異。觀察白色牛樟芝經細胞融合後所挑出菌株的生長狀況，其菌種菌絲呈現紅色，其液態培養基萃取物有別於一般牛樟芝的二次代謝物，但固態培養後期卻又出現白色菌絲的菌落，其詳細機制仍待進一步的分析研究。

Antrodia cinnamomea and its host Cinnamomum kanehirae, are both endemic species unique to Taiwan. A. cinnamomea is an expensive medicinal fungus. Many studies have confirmed that A. cinnamomea is rich in polysaccharides and triterpenoids that have medicinal effects in anti-cancer, anti-inflammation, antihypertensive and anti-oxidation. This study, makes a series of observations of white A. cinnamomea which occurs rarely in nature. Chemical variations among white A. cinnamomea, regular A. cinnamomea and induced white A. cinnamomea were compared. This study, illustrates the relative relationship between red A. cinnamomea and white A. cinnamomea which occur naturally or through radiation induction. Different light radiation was used to stimulate A. cinnamomea. Blue light was found to induce A. cinnamomea mycelia whitening and red light was able to cause irregular formation of A. cinnamomea fruiting bodies. The chemical profiles of A. cinnamomea extracts at different growth stages was compared using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The TLC and HPLC profiles indicate that specific triterpenoids were varied among different A. cinnamomea. The compounds profiles were different between white A. cinnamomea and red A. cinnamomea. Moreover, the compounds of induced white A. cinnamomea were similar to those of naturally occurring white A. cinnamomea but retained specific characteristics of red A. cinnamomea. Another issue explored in this study is whether white A. cinnamomea is genetic modification causes its different growth conditions. Some protoplast fusion strains were made from white A. cinnamomea. Interestingly, the mycelia of the cell-fusion strain was red color. The extraction from liquid culture media showed their secondary metabolites were different between cell-fusion strains and regular A. cinnamomea. Further analysis is needed to verify these variations.

1. 前言 1
1.1 牛樟芝背景及重要性簡介 1
1.2 牛樟芝在分類上的地位 2
1.3 牛樟芝的形態及生長環境 3
1.4 牛樟芝功效及其應用價值 3
1.4.1 抗癌的活性 4
1.4.2 消炎、免疫的調節作用 5
1.4.3 抗B型肝炎 5
1.4.4 抗氧化活性 5
1.4.5 保肝活性 6
1.4.6 肝纖維化的預防 7
1.4.7 降血壓的作用 7
1.5 牛樟芝成分 8
1.5.1 牛樟芝之多醣體 9
1.5.2 牛樟芝之三萜類 9
1.6 其他有效成分 10
1.7 光照在真菌學上的應用 10
2. 研究動機 13
3. 材料方法 15
3.1 牛樟芝菌種記錄與保存 15
3.2 牛樟芝的繼代培養 15
3.3 實驗菌種來源 16
3.4 牛樟芝的光照處理 17
3.5 牛樟芝光照處理後的再生 17
3.6 萃取紅牛樟芝、白牛樟芝、誘導白化牛樟芝之不同時期菌絲 17
3.7 萃取牛樟芝液態培養基之二次代謝物 18
3.8 牛樟芝原生質體的製備 18
3.9 細胞融合 19
3.10 牛樟芝細胞核螢光染色 19
3.11 薄層色層分析 20
3.12 高效能液相層析儀的分析 21
3.13 質譜儀的分析 22
4. 結果 23
4.1 牛樟芝的繼代培養 23
4.2 牛樟芝的光照處理 23
4.3 牛樟芝光照處理後的再生 24
4.4 牛樟芝原生質體的製備 25
4.5 牛樟芝細胞核螢光染色 25
4.6 細胞融合 25
4.7 薄層色層分析 26
4.8 高效能液相層析儀分析 27
4.9 質譜儀分析 29
5. 討論 31
6. 引用文獻 37
7. 圖表 47
8. 附錄 83

朱祐頡. 2008. 人工培養基栽培之樟芝的分子定性. 國立東華大學生物技術研究所碩士論文.
楊瑞玫. 2011. 牛樟芝在不同固態培養基上之生長差異研究.
宋祖瑩. 2003. 樟芝深層培養液抗氧化及抗腫瘤特性之研究. 國立中興大學食品科學系博士論文.
李名訓. 2006. 樟芝栽培之研究. 國立嘉義大學農學院林業暨自然資源研究所碩士論文.
李順來. 2009. 台灣國寶:牛樟芝.
林志遠. 2005. 牛樟芝子實體形成之探討. 國立東華大學生物技術研究所碩士論文.
張東柱. 2010. 牛樟芝的神奇療效:保肝抗癌的森林奇蹟.
郭淑卿. 2009. 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.
陳勁初, 林文鑫, 陳清農, 許勝傑, 黃仕政 and 陳炎鍊. 2001. 台灣特有真菌 - 樟芝菌絲體芝開發. Fung. Sci. 16 7-22.
黃鈴娟. 2000. 樟芝與姬松茸之抗氧化性質及其多醣組成分析. 國立中興大學食品科學系碩士論文.
Ambra, R., B. Grimaldi, S. Zamboni, P. Filetici, G. Macino and P. Ballario. 2004. Photomorphogenesis in the hypogeous fungus Tuber borchii: isolation and characterization of Tbwc-1, the homologue of the blue-light photoreceptor of Neurospora crassa. Fungal Genet Biol 41:688-697.
Ao, Z. H., Z. H. Xu, Z. M. Lu, H. Y. Xu, X. M. Zhang and W. F. Dou. 2009. Niuchangchih (Antrodia camphorata) and its potential in treating liver diseases. J Ethnopharmacol 121:194-212.
Bayram, O., S. Krappmann, M. Ni, J. W. Bok, K. Helmstaedt, O. Valerius, S. Braus-Stromeyer, N. J. Kwon, N. P. Keller, J. H. Yu and G. H. Braus. 2008. VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 320:1504-1506.
Bayram, O., F. Sari, G. H. Braus and S. Irniger. 2009. The protein kinase ImeB is required for light-mediated inhibition of sexual development and for mycotoxin production in Aspergillus nidulans. Mol Microbiol 71:1278-1295.
Cerda-Olmedo, E. 2001. Phycomyces and the biology of light and color. FEMS Microbiol Rev 25:503-512.
Chang, C. Y., C. L. Lee and T. M. Pan. 2006. Statistical optimization of medium components for the production of Antrodia cinnamomea AC0623 in submerged cultures. Appl Microbiol Biotechnol 72:654-661.
Chang, H. L., G. R. Chao, C. C. Chen and J. L. Mau. 2001. Non-volatile taste components of Agaricus blazei, Antrodia camphorata and Cordyceps militaris mycelia. Food Chem Toxicol 74:203-207.
Chang, T. T. and W. N. Chou. 2004. Antrodia cinnamomea reconsidered and A-salmonea sp nov on Cunninghamia konishii in Taiwan. Bot Bull Acad Sini 45:347-352.
Chen, C. and M. B. Dickman. 2002. Colletotrichum trifolii TB3 kinase, a COT1 homolog, is light inducible and becomes localized in the nucleus during hyphal elongation. Eukaryot Cell 1:626-633.
Chen, C. H., S. W. Yang and Y. C. Shen. 1995. New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum. J Nat Prod 58:1655-1661.
Chen, T. I., C. C. Chen, T. W. Lin, Y. T. Tsai and M. K. Nam. 2010. A 90-day subchronic toxicological assessment of Antrodia cinnamomea in Sprague-Dawley rats. Food Chem Toxicol 49:429-433.
Chen, Y. S., J. H. Pan, B. H. Chiang, F. J. Lu and L. Y. Sheen. 2008. Ethanolic extracts of Antrodia cinnamomea mycelia fermented at varied times and scales have differential effects on hepatoma cells and normal primary hepatocytes. J Food Sci 73:179-185.
Cheng, J. J., N. K. Huang, T. T. Chang, D. L. Wang and M. K. Lu. 2005a. Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells. Life Sci 76:3029-3042.
Cheng, J. J., C. J. Yang, C. H. Cheng, Y. T. Wang, N. K. Huang and M. K. Lu. 2005b. Characterization and functional study of Antrodia camphorata lipopolysaccharide. J Agric Food Chem 53:469-474.
Cherng, H., D. P. Wu and H. C. Chiang. 1996. Triterpenoids From Antrodia Cinnamomea. Phytochemistry 41:263-267.
Chiang, H. C., D. P. Wu, I. H. Cherng and C. H. Ueng. 1995. A sesquiterpene lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. Phytochem 39:613-616.
Chou, J. C., T. Z. Wu and J. Y. Lin. 2006. In vitro induction of fruiting body in Antrodia cinnamomea –a medicinally important fungus. Bot Stud 47:267-272.
Chu, Y. C., R. M. Yang, T. T. Chang and J. C. Chou. 2010. Fructification of Antrodia cinnamomea Was Strain Dependent in Malt Extract Media and Involved Specific Gene Expression. J Agric Food Chem 58:257-261.
Dai, Y. Y., C. H. Chuang and C. C. Tsai. 2003. The Protection of Anthrodia Camphorata Against Acute Hepatotoxicity of Alcohol in Rats. J Food Drug Anal 11:177-185.
De Fabo, E. C., R. W. Harding and W. Shropshire. 1976. Action Spectrum between 260 and 800 Nanometers for the Photoinduction of Carotenoid Biosynthesis in Neurospora crassa. Plant Physiol 57:440-445.
Du, Y. C., T. Y. Wu, F. R. Chang, W. Y. Lin, Y. M. Hsu, F. T. Cheng, C. Y. Lu, M. H. Yen, Y. T. Tsui, H. L. Chen, M. F. Hou, M. C. Lu and Y. C. Wu. 2012. Chemical profiling of the cytotoxic triterpenoid-concentrating fraction and characterization of ergostane stereo-isomer ingredients from Antrodia camphorata. J Pharm Biomed Anal 58:182-192.
Ehmann, A. 1977. The van urk-Salkowski reagent--a sensitive and specific chromogenic reagent for silica gel thin-layer chromatographic detection and identification of indole derivatives. J Chromatogr 132:267-276.
Fox, E. M. and B. J. Howlett. 2008. Secondary metabolism: regulation and role in fungal biology. Curr Opin Microbiol 11:481-487.
Haggblom, P. and T. Unestam. 1979. Blue light inhibits mycotoxin production and increases total lipids and pigmentation in Alternaria alternata. Appl Environ Microbiol 38:1074-1077.
Herrera-Estrella, A. and B. A. Horwitz. 2007. Looking through the eyes of fungi: molecular genetics of photoreception. Mol Microbiol 64:5-15.
Hill, E. P. 1976. Effect of light on growth and sporulation of Aspergillus ornatus. J Gen Microbiol 95:39-44.
Hseu, Y. C., W. C. Chang, Y. T. Hseu, C. Y. Lee, Y. J. Yech, P. C. Chen, J. Y. Chen and H. L. Yang. 2002. Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life Sciences 71:469-482.
Hseu, Y. C., F. Y. Wu, J. J. Wu, J. Y. Chen, W. H. Chang, F. J. Lu, Y. C. Lai and H. L. Yang. 2005. Anti-inflammatory potential of Antrodia Camphorata through inhibition of iNOS, COX-2 and cytokines via the NF-kappaB pathway. Int Immunopharmacol 5:1914-1925.
Hsiao, G., M. Y. Shen, K. H. Lin, M. H. Lan, L. Y. Wu, D. S. Chou, C. H. Lin, C. H. Su and J. R. Sheu. 2003. Antioxidative and hepatoprotective effects of Antrodia camphorata extract. J Agric Food Chem 51:3302-3308.
Hsu, Y. L., P. L. Kuo, C. Y. Cho, W. C. Ni, T. F. Tzeng, L. T. Ng, Y. H. Kuo and C. C. Lin. 2007. Antrodia cinnamomea fruiting bodies extract suppresses the invasive potential of human liver cancer cell line PLC/PRF/5 through inhibition of nuclear factor kappaB pathway. Food Chem Toxicol 45:1249-1257.
Hsu, Y. L., Y. C. Kuo, P. L. Kuo, L. T. Ng, Y. H. Kuo and C. C. Lin. 2005. Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines. Cancer Lett 221:77-89.
Huang, K. F., W. M. Huang and H. C. Chiang. 2001. Phenyl compounds from Antrodia cinnamomea. Chin Pharmaceut. 53:327-331.
Huang, S. J. and J. L. Mau. 2007. Antioxidant Properties of Antrodia camphorata in Submerged Culture. J. Agric. Food Chem.105:1702-1710
Huang, R. L., Q. Huang, C. F. Chen, T. T. Chang and C. J. Chou. 2003. Anti-viral effects of active compounds from Antrodia camphorata on wild-type and lamivudine-resistant mutant HBV. J Chin Pharmaceu Sci. 55:371-379.
Huanga, M. Z., S. C. Chenga, S. S. Jhanga, C. C. Choua, C. N. Chenga, J. Shieaa, I. A. P. and E. N. Nikolaevb. 2012. Ambient molecular imaging of dry fungus surface by electrospray laser desorption ionization mass spectrometry. Int J Mass Spectrom.325-327:172-182
Idnurm, A. and J. Heitman. 2005. Light controls growth and development via a conserved pathway in the fungal kingdom. PLoS Biol 3:e95.
Kuo, P. L., Y. L. Hsu, C. Y. Cho, L. T. Ng, Y. H. Kuo and C. C. Lin. 2006. Apoptotic effects of Antrodia cinnamomea fruiting bodies extract are mediated through calcium and calpain-dependent pathways in Hep 3B cells. Food Chem Toxicol 44:1316-1326.
Lee, I. H., R. L. Huang, C. T. Chen, H. C. Chen, W. C. Hsu and M. K. Lu. 2002. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiol Lett 209:63-67.
Lin, W. C., S. C. Kuo, W. L. Lin, H. L. Fang and B. C. Wang. 2006. Filtrate of fermented mycelia from Antrodia camphorata reduces liver fibrosis induced by carbon tetrachloride in rats. World J Gastroenterol 12:2369-2374.
Lu, Z. M., W. Y. Tao, X. L. Zou, H. Z. Fu and Z. H. Ao. 2007. Protective effects of mycelia of Antrodia camphorata and Armillariella tabescens in submerged culture against ethanol-induced hepatic toxicity in rats. J Ethnopharmacol 110:160-164.
Miyake, T., A. Mori, T. Kii, T. Okuno, Y. Usui, F. Sato, H. Sammoto, A. Watanabe and M. Kariyama. 2005. Light effects on cell development and secondary metabolism in Monascus. J Ind Microbiol Biotechnol 32:103-108.
Purschwitz, J., S. Muller and R. Fischer. 2009. Mapping the interaction sites of Aspergillus nidulans phytochrome FphA with the global regulator VeA and the White Collar protein LreB. Mol Genet Genom 281:35-42.
Purschwitz, J., S. Muller, C. Kastner and R. Fischer. 2006. Seeing the rainbow: light sensing in fungi. Curr Opin Microbiol 9:566-571.
Ranga, V. and G. K. Saini. 2011. Biochemical and molecular characterization of wild-type and fused protoplasts of Beauveria bassiana and Metarhizium anisopliae. Folia Microbiol (Praha) 56:289-295.
Schmoll, M., L. Franchi and C. P. Kubicek. 2005. Envoy, a PAS/LOV domain protein of Hypocrea jecorina (Anamorph Trichoderma reesei), modulates cellulase gene transcription in response to light. Eukaryot Cell 4:1998-2007.
Schuster, A., C. P. Kubicek, M. A. Friedl, I. S. Druzhinina and M. Schmoll. 2007. Impact of light on Hypocrea jecorina and the multiple cellular roles of ENVOY in this process. BMC Genomics 8:449.
Shen, Y. C., Y. H. Wang, Y. C. Chou, C. F. Chen, L. C. Lin, T. T. Chang, J. H. Tien and C. J. Chou. 2004. Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata. Planta Med 70:310-314.
Shen, Y. C., S. W. Yang, C. S. Lin, C. H. Chen, Y. H. Kuo and C. F. Chen. 1997. Zhankuic acid F: a new metabolite from a formosan fungus Antrodia cinnamomea. Planta Med 63:86-88.
Song, T. Y. and G. C. Yen. 2002. Antioxidant properties of Antrodia camphorata in submerged culture. J Agric Food Chem 50:3322-3327.
Song, T. Y. and G. C. Yen. 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.
Tai, T., A. Akahori and T. Shingu. 1993. Triterpenes of Poria cocos. Phytochem 32:1239-1244.
Tisch, D. and M. Schmoll. 2010. Light regulation of metabolic pathways in fungi. Appl Microbiol Biotechnol 85:1259-1277.
Wang, G. J., H. W. Tseng, C. J. Chou, T. H. Tsai, C. T. Chen and M. K. Lu. 2003. The vasorelaxation of Antrodia camphorata mycelia: involvement of endothelial Ca(2+)-NO-cGMP pathway. Life Sci 73:2769-2783.
Wang, W. N., R. Y. Wu and W. H. Ko. 2005. Variation and segregation following nuclear transplantation in Antrodia cinnamomea. Bot Bull Acad Sini 46:217-222.
Wu, D. P. and H. C. Chiang. 1995. Constituents of Antrodia cinnamomea. J Chin Chem Soc. 42:797-800.
Yang, S. W., Y. C. Shen and C. H. Chen. 1996. Steroids and triterpenoids of Antrodia cinnamomea-A fungus parasitic on Cinnamomum micranthum. Phytochem 41:1389-1392.
Young, D. S., H. C. Chiang and L. K. Liu. 1998. Identification of bioactive components in Antrodia cinnamomea by MS/MS via EI ionization. J Chin Chem Soc. 45:123-129.
Yu, Y. L., I. H. Chen, K. Y. Shen, R. Y. Huang, W. R. Wang, C. J. Chou, T. T. Chang and C. L. Chu. 2009. A triterpenoid methyl antcinate K isolated from Antrodia cinnamomea promotes dendritic cell activation and Th2 differentiation. Eur J Immunol 39:2482-2491.
Zhu, J. C. and X. J. Wang. 2005. Effect of blue light on conidiation development and glucoamylase enhancement in Aspergillus niger. Acta microbiol Sin 45:275-278.