臺灣博碩士論文加值系統

本研究評估並建立可生產具有抑制人類肝癌細胞活性與護肝功能特性的樟芝（Antrodia cinnamomea, AC)深層發酵條件，結果發現搖瓶發酵8週的樟芝菌絲體乙醇萃取物具有較佳抑制Hep3B生長的活性，其中以glucose為碳源發酵8週的樟芝菌絲體乙醇萃取物（AC8-Em-glu）對Hep3B生長抑制的IC50最低，為76.2

The objective of this research was to establish proper fermentation conditions for submerged cultivation of Antrodia cinnamomea (AC) to produce a fermentation product with antihepatoma activity. AC was fermented for 1, 2, 4, 6 and 8 weeks with galactose, lactose and glucose as carbon sources of the submerged cultivation medium in 250 ml flask. The cell viability of Hep3B was more inhibited with the treatment of ethanolic extract of AC mycelia fermented for 8 weeks than those fermented for 1, 2, 4 and 6 weeks. The IC50 of ethanolic extract of AC mycelia fermented for 8 weeks with glucose as a carbon source (AC8-Em-glu) was 76.2

中文摘要 1
英文摘要 3
前言 5
研究架構 7
文獻整理 8
一、樟芝相關之研究 8
（一）化學成分鑑定方面 8
（二）生理活性方面 18
第一章 不同發酵樟芝菌絲體乙醇萃取物對人類肝癌細胞之抑制活性 26
一、摘要 26
二、研究背景及緣由 27
三、材料與方法 28
（一）樟芝深層發酵 28
（二）樟芝樣品之前處理 29
（三）樟芝發酵產物相關性質之分析 30
（四）不同發酵條件發酵樟芝乙醇萃取物抑制肝癌活性之分析 31
（五）探討樟芝菌絲體乙醇萃取物對Hep3B及HepG2 細胞週期之影響 34
（六）探討樟芝菌絲體乙醇萃取物對Hep3B及HepG2細胞內caspase-3活性之影響 35
四、結果與討論 36
（一）不同250 mL搖瓶發酵條件的樟芝菌絲體 36
（二）不同250 mL搖瓶發酵條件的樟芝菌絲體乙醇萃取物對人類肝癌細胞之影響 36
（三）5 L發酵槽深層發酵之樟芝菌絲體 37
（四）不同發酵時間5 L發酵槽深層發酵之樟芝菌絲體對人類肝癌細胞之影響 38
（五）500 L發酵槽深層發酵 39
（六）不同發酵時間500 L發酵槽深層發酵樟芝菌絲體乙醇萃取物對人類肝癌細胞之影響 39
（七）可能有效成分之分析 39
（八）樟芝菌絲體乙醇萃取物對HepG2及Hep3B cell-cycle之影響 40
（九）探討樟芝菌絲體乙醇萃取物對Hep3B及HepG2細胞內caspase-3活性之影響 41
五、結論 42
第二章 樟芝菌絲體乙醇萃取物對大白鼠初代肝細胞之影響 64
一、摘要 64
二、研究背景及緣由 65
三、材料與方法 66
（一）探討樟芝發酵菌絲體乙醇萃取物對大白鼠正常初代肝細胞之影響 66
（二）樟芝發酵菌絲體乙醇萃取物對四氯化碳誘導大白鼠初代肝細胞損傷之影響 71
四、結果與討論 72
（一）樟芝發酵菌絲體乙醇萃取物對正常大白鼠初代肝細胞之影響 72
（二）樟芝發酵菌絲體乙醇萃取物對四氯化碳大白鼠誘導初代肝細胞損傷之影響 74
五、 結論 76
第三章 樟芝發酵菌絲體乙醇萃取物對四氯化碳誘導大白鼠肝損傷之影響 90
一、摘要 90
二、研究背景及目的 91
三、材料與方法 92
四、結果與討論 94
五、結論 96
第四章 樟芝發酵菌絲體乙醇萃取物之化學成分分析 99
一、摘要 99
二、研究背景及目的 100
三、材料與方法 101
四、結果與討論 102
五、結論 103
參考文獻 114
附錄 122

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.
林苡芬. 2004. 不同發酵碳源之牛樟芝菌絲體發酵過濾液對人類肝癌細胞株之影響. 台灣大學食品科技研究所碩士論文.
洪淑敏. 2000. 靈芝促進人類肝癌細胞凋亡之研究. 國立台灣大學食品科技研究所碩士論文.
蔡敬民 & 劉翠玲. 2002. 樟芝對於血醣及血脂質之影響-2. 行政院國家科學委員會91年度保健食品研究開發計畫報告. p. 23-27.
蔡敬民 & 嚴貴榮. 2002. 樟芝對於血醣及血脂質之影響-1. 行政院國家科學委員會九十一年度保健食品研究開發計畫報告.
顏國欽. 2002. 樟芝菌絲體液體培養液萃取物抗氧化性及抗腫瘤特性之探討. 91年行政院國科會專題研究計畫. p. 8-12.
Bellomo G, Mirabelli F, Dimonte D, Richelmi P, Thor H, Orrenius C & Orrenius S. 1987. Formation and Reduction of Glutathione-Protein Mixed Disulfides during Oxidative Stress - a Study with Isolated Hepatocytes and Menadione (2-Methyl-1,4-Naphthoquinone). Biochemical Pharmacology 36(8):1313-1320.
Chang CY, Huang ZN, Yu HH, Chang LH, Li SL, Chen YP, Lee KY & Chuu JJ. 2008. The adjuvant effects of Antrodia Camphorata extracts combined with anti-tumor agents on multidrug resistant human hepatoma cells. J Ethnopharmacol 118(3):387-395.
Chang CY, Lee CL & Pan TM. 2006. Statistical optimization of medium components for the production of Antrodia cinnamomea AC0623 in submerged cultures. Applied microbiology and biotechnology 72(4):654-661.
Chang HL, Chao GR, Chen CC & Mau JL. 2001. Non-volatile taste components of Agaricus blazei, Antrodia camphorata and Cordyceps militaris mycelia. Food Chem 74(2):203-207.
Chang TT & Chou WN. 1995. Antrodia-Cinnamomea Sp-Nov on Cinnamomum-Kanehirai in Taiwan. Mycol Res 99:756-758.
Chang TT & Chou WN. 2004. Antrodia cinnamomea reconsidered and A-salmonea sp nov on Cunninghamia konishii in Taiwan. Botanical Bulletin of Academia Sinica 45(4):347-352.
Chang TT & Wang WR. 2005. Basidiomatal formation of Antrodia cinnamomea on artificial agar media. Botanical Bulletin of Academia Sinica 46(2):151-154.
Chen CC, Chyau CC & Hseu TH. 2007a. Production of a COX-2 inhibitor, 2,4,5-trimethoxybenzaldehyde, with submerged cultured Antrodia camphorata. Letters in applied microbiology 44(4):387-392.
Chen CC, Shiao YJ, Lin RD, Shao YY, Lai MN, Lin CC, Ng LT & Kuo YH. 2006. Neuroprotective diterpenes from the fruiting body of Antrodia camphorata. J Nat Prod 69(4):689-691.
Chen CH, Yang SW & Shen YC. 1995. New steroid acids from Antrodia cinnamomea, a fungal parasite of Cinnamomum micranthum. J Nat Prod 58(11):1655-1661.
Chen JJ, Lin WJ, Liao CH & Shieh PC. 2007b. Anti-inflammatory benzenoids from Antrodia camphorata. J Nat Prod 70(6):989-992.
Chen KC, Peng CC, Peng RY, Su CH, Chiang HS, Yan JH & Hsieh-Li HM. 2007c. Unique formosan mushroom Antrodia camphorata differentially inhibits androgen-responsive LNCaP and -independent PC-3 prostate cancer cells. Nutr Cancer 57(1):111-121.
Chen SC, Lu MK, Cheng JJ & Wang DL. 2005. Antiangiogenic activities of polysaccharides isolated from medicinal fungi. FEMS Microbiol Lett 249(2):247-254.
Chen YJ, Cheng PC, Lin CN, Liao HF, Chen YY, Chen CC & Lee KM. 2008. Polysaccharides from Antrodia camphorata mycelia extracts possess immunomodulatory activity and inhibits infection of Schistosoma mansoni. Int Immunopharmacol 8(3):458-467.
Cheng JJ, Huang NK, Chang TT, Wang DL & Lu MK. 2005a. Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells. Life Sci 76(26):3029-3042.
Cheng JJ, Yang CJ, Cheng CH, Wang YT, Huang NK & Lu MK. 2005b. Characterization and functional study of Antrodia camphorata lipopolysaccharide. J Agric Food Chem 53(2):469-474.
Cheng PC, Hsu CY, Chen CC & Lee KM. 2008. In vivo immunomodulatory effects of Antrodia camphorata polysaccharides in a T1/T2 doubly transgenic mouse model for inhibiting infection of Schistosoma mansoni. Toxicol Appl Pharmacol 227(2):291-298.
Cherng IH, Chiang HC, Cheng MC & Wang Y. 1995. 3 New Triterpenoids from Antrodia-Cinnamomea. J Nat Prod 58(3):365-371.
Cherng IH, Wu DP & Chiang HC. 1996. Triterpenoids from Antrodia cinnamomea. Phytochemistry 41(1):263-267.
Chiang HC, Wu DP, Cherng IW & Ueng CH. 1995. A Sesquiterpene Lactone, Phenyl and Biphenyl Compounds from Antrodia-Cinnamomea. Phytochemistry 39(3):613-616.
Chiang SY, Hsieh CL, Chang LL, Pei SY, Lin MY & Kao ST. 2004. The safety and anti-genotoxic effects of Antrodia camphorata in vitro, in pregnant mice and their fetuses. Toxicology and Applied Pharmacology 197(3):350-351.
Chien CC, Tsai ML, Chen CC, Chang SJ & Tseng CH. 2008. Effects on Tyrosinase Activity by the Extracts of Ganoderma lucidum and Related Mushrooms. Mycopathologia.
Dai YY, Chuang CH, Tsai CC, Sio HM, Huang SC, Chen JC & Hu ML. 2003. The protection of Antrodia camphorata against acute hepatotoxicity of alcohol in rats. Journal of Food and Drug Analysis 11(3):177-185.
Habig WH, Pabst MJ & Jakoby WB. 1974. Glutathione S-Transferases - First Enzymatic Step in Mercapturic Acid Formation. Journal of Biological Chemistry 249(22):7130-7139.
Han HF, Nakamura N, Zuo F, Hirakawa A, Yokozawa T & Hattori M. 2006. Protective effects of a neutral polysaccharide isolated from the mycelium of Antrodia cinnamomea on Propionibacterium acnes and lipopolysaccharide induced hepatic injury in mice. Chemical & pharmaceutical bulletin 54(4):496-500.
Hseu YC, Chang WC, Hseu YT, Lee CY, Yech YJ, Chen PC, Chen JY & Yang HL. 2002. Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes. Life Sci 71(4):469-482.
Hseu YC, Chen SC, Tsai PC, Chen CS, Lu FJ, Chang NW & Yang HL. 2007. Inhibition of cyclooxygenase-2 and induction of apoptosis in estrogen-nonresponsive breast cancer cells by Antrodia camphorata. Food Chem Toxicol 45(7):1107-1115.
Hseu YC, Chen SC, Yech YJ, Wang L & Yang HL. 2008. Antioxidant activity of Antrodia camphorata on free radical-induced endothelial cell damage. J Ethnopharmacol 118(2):237-245.
Hseu YC, Yang HL, Lai YC, Lin JG, Chen GW & Chang YH. 2004. Induction of apoptosis by Antrodia camphorata in human premyelocytic leukemia HL-60 cells. Nutr Cancer 48(2):189-197.
Hsiao G, Shen MY, Lin KH, Lan MH, Wu LY, Chou DS, Lin CH, Su CH & Sheu JR. 2003. Antioxidative and hepatoprotective effects of Antrodia camphorata extract. J Agric Food Chem 51(11):3302-3308.
Hsu FL, Chou CJ, Chang YC, Chang TT & Lu MK. 2006. Promotion of hyphal growth and underlying chemical changes in Antrodia camphorata by host factors from Cinnamomum camphora. Int J Food Microbiol 106(1):32-38.
Hsu YL, Kuo YC, Kuo PL, Ng LT, Kuo YH & Lin CC. 2005. Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines. Cancer Lett 221(1):77-89.
Huang HY, Chen CC & Chen RC. 2005a. The immunologic response of oral administration Antrodia camphorate with BALB/c mice. Faseb Journal 19(5):A1480-a1480.
Huang NK, Cheng JJ, Lai WL & Lu MK. 2005b. Antrodia camphorata prevents rat pheochromocytoma cells from serum deprivation-induced apoptosis. FEMS Microbiol Lett 244(1):213-219.
Huang RC, Chen JC & Wang BC, inventors. 2002. Isolate of Antrodia camphorata, process for producing a culture of the same and product obtained thereby. US Patent 6395271.
Huang RL, Huang Q, Chen CF, Chang TT & Chou CJ. 2003. Anti-viral effects of active compounds from Antrodia camphorara on wild-type and lamivudine-resistant mutant HBV. The Chinese Pharmacyeutical Journal 55(5):371-379.
Josnhston DE & Kroening C. 1988. Pharmacol. Toxicol.
Kobori M, Yoshida M, Ohnishi-Kameyama M & Shinmoto H. 2007. Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cells. Br J Pharmacol 150(2):209-219.
Kreamer BL, Staecker JL, Sawada N, Sattler GL, Hsia MTS & Pitot HC. 1986. Use of a Low-Speed, Iso-Density Percoll Centrifugation Method to Increase the Viability of Isolated Rat Hepatocyte Preparations. In Vitro Cell Dev Biol 22(4):201-211.
Kuo PL, Hsu YL, Cho CY, Ng LT, Kuo YH & Lin CC. 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(8):1316-1326.
Lawrence RA & Burk RF. 1976. Glutathione Peroxidase-Activity in Selenium-Deficient Rat-Liver. Biochemical and Biophysical Research Communications 71(4):952-958.
Lee IH, Huang RL, Chen CT, Chen HC, Hsu WC & Lu MK. 2002. Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects. FEMS Microbiol Lett 209(1):63-67.
Lee TH, Lee CK, Tsou WL, Liu SY, Kuo MT & Wen WC. 2007. A new cytotoxic agent from solid-state fermented mycelium of Antrodia camphorata. Planta Med 73(13):1412-1415.
Liau YJ, Wen L, Shaw JF & Lin CT. 2007. A highly stable cambialistic-superoxide dismutase from Antrodia camphorata: expression in yeast and enzyme properties. J Biotechnol 131(1):84-91.
Lin ES & Chen YH. 2007. Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media. Bioresour technol 98(13):2511-2517.
Lin ES & Ko HC. 2005. Glucose stimulates production of the alkaline-thermostable lipase of the edible Basidiomycete Antrodia cinnamomea. Enzyme and Microbial Technology 37(2):261-265.
Lin ES & Sung SC. 2006. Cultivating conditions influence exopolysaccharide production by the edible Basidiomycete Antrodia cinnamomea in submerged culture. Int J Food Microbiol 108(2):182-187.
Lin WC, Kuo SC, Lin WL, Fang HL & Wang BC. 2006. Filtrate of fermented mycelia from Antrodia camphorata reduces liver fibrosis induced by carbon tetrachloride in rats. World J Gastroenterol 12(15):2369-2374.
Liu DZ, Liang HJ, Chen CH, Su CH, Lee TH, Huang CT, Hou WC, Lin SY, Zhong WB, Lin PJ, Hung LF & Liang YC. 2007a. Comparative anti-inflammatory characterization of wild fruiting body, liquid-state fermentation, and solid-state culture of Taiwanofungus camphoratus in microglia and the mechanism of its action. J Ethnopharmacol 113(1):45-53.
Liu DZ, Liang YC, Lin SY, Lin YS, Wu WC, Hou WC & Su CH. 2007b. Antihypertensive activities of a solid-state culture of Taiwanofungus camphoratus (Chang-chih) in spontaneously hypertensive rats. Bioscience, biotechnology, and biochemistry 71(1):23-30.
Liu JJ, Huang TS, Hsu ML, Chen CC, Lin WS, Lu FJ & Chang WH. 2004. Antitumor effects of the partially purified polysaccharides from Antrodia camphorata and the mechanism of its action. Toxicol Appl Pharmacol 201(2):186-193.
Lowry OH, Rosebrough NJ, Farr AL & Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265-275.
Lu MK, Cheng JJ, Lai WL, Lin YJ & Huang NK. 2008. Fermented Antrodia cinnamomea extract protects rat PC12 cells from serum deprivation-induced apoptosis: the role of the MAPK family. J Agric Food Chem 56(3):865-874.
Lu MK, Cheng JJ, Lai WL, Lin YR & Huang NK. 2006. Adenosine as an active component of Antrodia cinnamomea that prevents rat PC12 cells from serum deprivation-induced apoptosis through the activation of adenosine A(2A) receptors. Life Sci 79(3):252-258.
Lu ZM, Tao WY, Zou XL, Fu HZ & Ao ZH. 2007. Protective effects of mycelia of Antrodia camphorata and Armillariella tabescens in submerged culture against ethanol-induced hepatic toxicity in rats. J Ethnopharmacol 110(1):160-164.
Mau JL, Huang PN, Huang SJ & Chen CC. 2003. Time course for antioxidants production by Antrodia camphorata in submerged culture. Fungal Science 18(3, 4):59-71.
Mau JL, Huang PN, Huang SJ & Chen CC. 2004. Antioxidant properties of methanolic extracts from two kinds of Antrodia camphorata mycelia. Food Chem 86(1):25-31.
Moldeus P, Hogberg J & Orrenius S. 1978. Isolation and use of liver cells. Methods Enzymol 52:60-71.
Nakamura N, Hirakawa A, Gao JJ, Kakuda H, Shiro M, Komatsu Y, Sheu CC & Hattori M. 2004. Five new maleic and succinic acid derivatives from the mycelium of Antrodia camphorata and their cytotoxic effects on LLC tumor cell line. J Nat Prod 67(1):46-48.
Peng CC, Chen KC, Peng RY, Su CH & Hsieh-Li HM. 2006. Human urinary bladder cancer T24 cells are susceptible to the Antrodia camphorata extracts. Cancer Lett 243(1):109-119.
Rao YK, Fang SH & Tzeng YM. 2007. Evaluation of the anti-inflammatory and anti-proliferation tumoral cells activities of Antrodia camphorata, Cordyceps sinensis, and Cinnamomum osmophloeum bark extracts. J Ethnopharmacol 114(1):78-85.
Reed DJ, Babson JR, Beatty PW, Brodie AE, Ellis WW & Potter DW. 1980. High-Performance Liquid-Chromatography Analysis of Nanomole Levels of Glutathione, Glutathione Disulfide, and Related Thiols and Disulfides. Analytical Biochemistry 106(1):55-62.
Seung HK, Young PJ, Sang HS, Chang JK, Jin WK & Young CK. 2003. Hepatoprotective Dibenzylbutyrolactone Lignans of Torreya nucifera against CCl4-Induced Toxicity in Primary Cultured Rat Hepatocytes. Biol. Pharm. Bull. 26(8):1202-1205.
Shen YC, Chou CJ, Wang YH, Chen CF, Chou YC & Lu MK. 2004a. 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(1):137-143.
Shen YC, Wang YH, Chou YC, Chen CF, Lin LC, Chang TT, Tien JH & Chou CJ. 2004b. Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata. Planta Med 70(4):310-314.
Shen YC, Yang SW, Lin CS, Chen CH, Kuo YH & Chen CF. 1997. Zhankuic acid F: A new metabolite from a formosan fungus Antrodia cinnamomea. Planta Med 63(1):86-88.
Shu CH & Lung MY. 2004. Effect of pH on the production and molecular weight distribution of exopolysaccharide by Antrodia camphorata in batch cultures. Process Biochemistry 39(8):931-937.
Shu CH, Lung MY & Xu CJ. 2005. Effects of supplementation of succinic acid on the production and molecular weight distribution of exopolysaccharides by Antrodia camphorata in batch cultures. Journal of Chemical Technology and Biotechnology 80(2):216-222.
Song TY, Hsu SL, Yeh CT & Yen GC. 2005a. Mycelia from Antrodia camphorata in Submerged culture induce apoptosis of human hepatoma HepG2 cells possibly through regulation of Fas pathway. J Agric Food Chem 53(14):5559-5564.
Song TY, Hsu SL & Yen GC. 2005b. Induction of apoptosis in human hepatoma cells by mycelia of Antrodia camphorata in submerged culture. J Ethnopharmacol 100(1-2):158-167.
Song TY & Yen GC. 2002. Antioxidant properties of Antrodia camphorata in submerged culture. J Agric Food Chem 50(11):3322-3327.
Song TY & Yen GC. 2003. Protective effects of fermented filtrate from Antrodia camphorata in submerged culture against CCl4-induced hepatic toxicity in rats. J Agric Food Chem 51(6):1571-1577.
Wang BC & Huang RC. 2002. The Development and Marketing of Ganoderma lucidum and Antrodia camphorata. Food Industries 34(5):3-14.
Wu DP & Chiang HC. 1995. Constituents of Antrodia-Cinnamomea. Journal of the Chinese Chemical Society 42(5):797-800.
Wu H, Pan CL, Yao YC, Chang SS, Li SL & Wu TF. 2006. Proteomic analysis of the effect of Antrodia camphorata extract on human lung cancer A549 cell. Proteomics 6(3):826-835.
Wu MD, Cheng MJ, Wang BC, Yech YJ, Lai JT, Kuo YH, Yuan GF & Chen IS. 2008. Maleimide and Maleic Anhydride Derivatives from the Mycelia of Antrodia cinnamomea and Their Nitric Oxide Inhibitory Activities in Macrophages. J Nat Prod 71(7):1258-1261.
Wu YY, Chen CC, Chyau CC, Chung SY & Liu YW. 2007. Modulation of inflammation-related genes of polysaccharides fractionated from mycelia of medicinal basidiomycete Antrodia camphorata. Acta pharmacologica Sinica 28(2):258-267.
Yang FC, Huang HC & Yang MJ. 2003. The influence of environmental conditions on the mycelial growth of Antrodia cinnamomea in submerged cultures. Enzyme Microb Technol 33(4):395-402.
Yang HL, Chen CS, Chang WH, Lu FJ, Lai YC, Chen CC, Hseu TH, Kuo CT & Hseu YC. 2006a. Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata. Cancer Lett 231(2):215-227.
Yang HL, Hseu YC, Chen JY, Yech YJ, Lu FJ, Wang HH, Lin PS & Wang BC. 2006b. Antrodia camphorata in submerged culture protects low density lipoproteins against oxidative modification. The American journal of Chinese medicine 34(2):217-231.
Yang SW, Shen YC & Chen CH. 1996. Steroids and triterpenoids of Antrodia cinnamomea - A fungus parasitic on Cinnamomum micranthum. Phytochemistry 41(5):1389-1392.
Young DS, Chiang HC & Liu LK. 1998. Identification of bioactive components in Antrodia cinnamomea by MS/MS via El ionization. Journal of the Chinese Chemical Society 45(1):123-129.