臺灣博碩士論文加值系統

內生真菌是生存在植物寄主體內，不會造成明顯病徵的一種真菌，其生長期間有的會產生具有藥物活性的二次代謝物質，因此，植物內生真菌被認為是新藥開發的來源。由中研院取得的50株內生真菌經PDA培養到第七天，95%萃取物之抑菌測試後得到五株具有明顯抑制Bacillus cereus、Escherichia coli、Salmonella typhimurium、Staphylococcus aureus或Serratia liquefyacieus ，也可以抑制病原性真菌Aspergillus niger、Botrytis cinerea或Rhizopus oryzae。其中以038及039兩株抑菌效果較佳，經分子生物鑑定的結果，038為Coniothyrium sp.，039為Eupenicillium brefeldianum。使用M broth液態培養兩株真菌五天後用乙酸乙酯萃取的物質對人類肺癌細胞A549及肝癌細胞Huh7有毒殺的效果，其中以對Huh7的效果較強；對於正常新生大鼠心臟內皮細胞則具有較少的毒害性。分析038萃取物質，純化出單一化合物Compound 1，根據流式細胞儀的結果發現具有更強的細胞毒性且會誘導癌症細胞行細胞凋亡途徑，目前已進行初步化學結構式的分析。

Endophytic fungi are referred to those fungi that live within the host plants without causing noticeable symptoms of disease. Some of these fungi produce bioactive material with pharmaceutical efficacy. Actually, endophytic fungi have been considered as a source for new medicines. In this study, 50 endemic endophytic fungal strains were screened for the purpose to find new bioactive agents. Our results show that the cultures of strain #038 (Coniothyrium sp.) and strain #039 (Eupenicillium brefeldianum) contain antimicrobial substances with inhibitory effect on the proliferation of bacteria (containing Bacillus cereus, Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Serratia liquefyacieus) and the fungi (including Aspergillus niger, Botrytis cinerea, Rhizopus oryzae). The ethyl acetate extracts of the cultures from #038 and #039 have showed cytotoxicity in A549 and Huh7 cancer cell lines. Data from flow cytometry indicate that the extracts might contain bioactive agent which can induce apoptotic cell death in these cancer cell lines. The bioactive agent has been isolated, and the structure of this bioactive agent has been analyzed preliminarily.

目 錄
中文摘要--------------------------------------------------------------------------------------------І
英文摘要--------------------------------------------------------------------------------------------Ⅱ
壹、前言------------------------------------------------------------------------------------------1
貳、文獻回顧----------------------------------------------------------------------------------------2
一、內生真菌簡介-----------------------------------------------------------------------------------2
（一）內生真菌定義及研究歷史--------------------------------------------------------------------2
（二）內生真菌特性----------------------------------------------------------------------------------3
1. 草本植物內生真菌-----------------------------------------------------------------------------3
2. 木本植物內生真菌-----------------------------------------------------------------------------3
二、內生真菌特異代謝物質及其應用-------------------------------------------------------5
（一）抗病原細菌二次代謝物----------------------------------------------------------5
（二）抗病原真菌二次代謝物----------------------------------------------------------6
（三）抑制癌細胞方面-----------------------------------------------------------------10
（四）其種活性--------------------------------------------------------------------------14
三、內生真菌於食品科技上的應用--------------------------------------------------------16
（一）抗黴菌活性-----------------------------------------------------------------------16
（二）抗氧化活性-----------------------------------------------------------------------16
（三）促進植物根的發育--------------------------------------------------------------17
（四）香氣物質--------------------------------------------------------------------------17
四、內生真菌近年來的發展趨勢-----------------------------------------------------------18
參、材料與方法--------------------------------------------------------------------------------20
一、試驗材料-----------------------------------------------------------------------------------20
（一）內生真菌--------------------------------------------------------------------------------20
（二）受試病原菌-----------------------------------------------------------------------------20
（三）受試細胞---------------------------------------------------------------------------------20
（四）培養基與試藥---------------------------------------------------------------------------20
二、實驗儀器------------------------------------------------------------------------------------22
三、實驗方法------------------------------------------------------------------------------------22
（一）病原菌純化及培養基的製備---------------------------------------------------------22
1、病原菌純化--------------------------------------------------------------------------22
2、含菌培養基的製備-----------------------------------------------------------------22
（二）抑菌物質產生之內生真菌篩選-------------------------------------------------------23
1、抑菌物質測試-----------------------------------------------------------------------23
2、抑菌物質之萃取--------------------------------------------------------------------23
（三）內生真菌之分子生物鑑定-------------------------------------------------------------24
1、實驗方法-----------------------------------------------------------------------------24
（四）產生抑菌物質之內生真菌外觀形態及生長----------------------------------------29
1. 固態培養基上的型態及狀態------------------------------------------------------29
2. 顯微鏡下菌絲形態------------------------------------------------------------------29
（五）液態培養內生真菌抑菌物質之萃取-------------------------------------------------29
1、菌元製備-----------------------------------------------------------------------------29
2、液態培養條件-----------------------------------------------------------------------29
3、有機溶劑萃取-----------------------------------------------------------------------29
（六）抑菌物質之細胞毒性測試-------------------------------------------------------------30
1. PDA培養及液態培養之細胞毒性測試------------------------------------------30
2. 液態培養乙酸乙酯萃取物之細胞毒性測試------------------------------------30
（七）液態培養乙酸乙酯萃取物之抑菌測試----------------------------------------------30
（八）內生真菌活性物質之初步分析-------------------------------------------------------31
1. 全光譜測定---------------------------------------------------------------------------31
2. 第一次HPLC分離------------------------------------------------------------------31
（九）HPLC產物之活性測試---------------------------------------------------------------31
1. 抑菌測試---------------------------------------------------------------------------31
2. 細胞毒性測試---------------------------------------------------------------------32
3. 第二次HPLC分離及製備有效成分------------------------------------------32
4. 細胞固定染色---------------------------------------------------------------------32
4.1 原理--------------------------------------------------------------------------32
4.2 實驗方法--------------------------------------------------------------------33
5. 流式細胞儀 (Flow cytometer) 之細胞停滯階段測試--------------------33
5.1 原理--------------------------------------------------------------------------33
5.2 實驗方法--------------------------------------------------------------------34
5.3 PI染色------------------------------------------------------------------------35
肆、結果與討論--------------------------------------------------------------------------------36
一、含抑菌物質之內生真菌篩選-----------------------------------------------------------36
（一）抑制病原細菌--------------------------------------------------------------------------36
（二）抗生素濃度對於病原菌抑菌比較--------------------------------------------------40
（三）抑制病原真菌--------------------------------------------------------------------------40
二、五株內生真菌分子鑑定結果-----------------------------------------------------------43
三、內生真菌生長及形態--------------------------------------------------------------------45
四、抑菌物質之細胞毒性測試--------------------------------------------------------------51
（一）細胞存活率-----------------------------------------------------------------------------51
（二）細胞形態之變化-----------------------------------------------------------------------56
五、內生真菌液態培養之乙酸乙酯萃取物抑菌測試-----------------------------------59
六、細胞毒性有效成分之分離--------------------------------------------------------------59
（一）全光譜分析-----------------------------------------------------------------------------59
（二）HPLC第一次分析及細胞毒性測試-----------------------------------------------61
（三）Fraction 3經HPLC分離（第二次HPLC分離）------------------------------61
（四）Compound 1之細胞毒性測試------------------------------------------------------67
（五）Compound 1之DAPI染色結果---------------------------------------------------67
（六）Compound 1之細胞週期停滯階段------------------------------------------------73
七、內生真菌038活性物質之結構式鑑定---------------------------------------------------76
（一）全光譜分析-----------------------------------------------------------------------------76
（二）IR 吸收光譜分析 (Infrared Absorption Spectrum)------------------------------76
（三）NMR-1H譜------------------------------------------------------------------------------76
伍、結論----------------------------------------------------------------------------------------79
陸、參考文獻----------------------------------------------------------------------------------80
附錄一、實驗儀器----------------------------------------------------------------------------85
附錄二、不同濃度的Ampicillin對四種病原性細菌之抑菌環大小-----------------86
附錄三、不同濃度的Tetracycline對四種病原性細菌之抑菌環大小---------------87

圖 次
附圖一、Phoma medicaginis E01071內生真菌形態-----------------------------------------4
附圖二、Phomosichalasin 結構式---------------------------------------------------------------5
附圖三、(1) helvolic acid (2) monomethylsulochrin (3) ergosterol 和
(4) 3b-hydroxy-5a,8a-epidioxy-ergosta-6,22-diene 的結構式--------------------6
附圖四、Cryptocandin 結構式--------------------------------------------------------------------7
附圖五、Ambuic acid 結構式---------------------------------------------------------------------9
附圖六、Macrosphelide A結構式-----------------------------------------------------------------9
附圖七、Taxol結構式------------------------------------------------------------------------------11
附圖八、Brefeldin A結構式----------------------------------------------------------------------12
附圖九、Radicicol結構式-------------------------------------------------------------------------13
附圖十、 Jesterone 及 Hydroxy-jesterone 結構式------------------------------------------16
附圖十一、Graphislactone A 結構式-----------------------------------------------------------17
附圖十二、Peniprequinolone 結構式-----------------------------------------------------------17
附圖十三、Irone 及 Nookatone 結構式------------------------------------------------------18
附圖十四、真菌rDNA基因之基本結構及PCR兩個引子位置圖-----------------------19
附圖十六、抑菌環測量方式---------------------------------------------------------------------23
附圖十七、yT&A 載體基因圖譜---------------------------------------------------------------28
附圖十八、DAPI染色-----------------------------------------------------------------------------33
圖一、內生真菌PDA培養第七天之乙醇萃取物對G (+) 的抑菌環大小--------------37
圖二、內生真菌固態培養第七天之乙醇萃取物對G (-)菌抑菌環大小------------------37
圖三、PDA培養之內生真菌乙醇萃取物對G (+)的抑菌作用之時間效應----------------------38
圖四、PDA培養之內生真菌乙醇萃取物對G (-)的抑菌作用之時間效應----------------------39
圖五、內生真菌PDA培養第十一天之乙醇萃取物對三種病原真菌之抑菌環大小-----------------41
圖六、內生真菌Conio. #038在PDA培養基上2-9天之生長情形-------------------------48
圖七、內生真菌Eu. #039在PDA培養基上2-9天之生長情形-----------------------------49
圖八、內生真菌在顯微鏡下的菌絲形態。------------------------------------------------------50
圖九、內生真菌四種萃取物對A549細胞毒性測試------------------------------------------52
圖十、內生真菌Conio. #038乙酸乙酯萃取物質對四種細胞之毒性測試-----------------------54
圖十一、內生真菌Eu. #039乙酸乙酯萃取物質對四種細胞之毒性測試------------------------55
圖十二、內生真菌Conio. #038萃取物質加入A549細胞24及48小時之細胞型態圖---------------57
圖十三、內生真菌Eu. #039萃取物質加入A549細胞24及48小時之細胞型態圖------------------58
圖十四、內生真菌Conio. #038及Eu. #039液態培養五天乙酸乙酯萃取物復溶於甲醇之紫外光-可見光光譜圖---60
圖十五、內生真菌Conio. #038液態培養五天乙酸乙酯萃取物之HPLC分析----------62
圖十六、內生真菌Eu. #039液態培養五天乙酸乙酯萃取物之HPLC分析--------------63
圖十七、Brefeldin A (BFA) 標準品1 mg/ml之HPLC分析圖-----------------------------64
圖十八、內生真菌Conio. #038液態培養五天乙酸乙酯萃取物的第三個區域之HPLC分析----------66
圖十九、Compound 1對人類肺癌細胞A549之細胞毒性作用-----------------------------68
圖二十、Compound 1對人類肝癌細胞Huh 7之細胞毒性作用--------------------------68
圖二十一、Compound 1加入A549中反應24小時之DAPI染色圖--------------------69
圖二十二、Compound 1加入A549中反應48小時之DAPI染色圖-----------------------70
圖二十三、Compound 1加入Huh 7中反應24小時之DAPI染色圖--------------------71
圖二十四、Compound 1加入Huh 7中反應48小時之DAPI染色圖--------------------72
圖二十五、Compound 1對A549反應24及48小時後之細胞週期測定------------------74
圖二十六、Compound 1對Huh7反應24及48小時後之細胞週期測定------------------75
圖二十七、Compound 1之紫外光-可見光光譜圖--------------------------------------------76
圖二十八、Compound 1 之IR光譜圖----------------------------------------------------------77
圖二十九、Compound 1之1H-NMR光譜圖---------------------------------------------------78

表 次
附表一、 純化的cryptocandin與其他抗真菌劑對多種人類病原真菌之最小抑制濃度--------------8
附表二、細胞凋亡與細胞壞死之比較----------------------------------------------------------10
附表三、內生真菌所產生之天然活性物質----------------------------------------------------14
表一、六內生真菌培養7、9及11天對三種病原真菌之抑菌環大小比較----------------42
表二、五株內生真菌之菌種鑑定結果----------------------------------------------------------44
表三、內生真菌Conio. #038的生長紀錄------------------------------------------------------46
表四、內生真菌Eu. #039的生長紀錄----------------------------------------------------------47
表五、內生真菌Conio. #038乙酸乙酯萃取物其HPLC初步產物之細胞毒性測試----------------------65

文才藝，吳元華，田秀玲。2004。植物內生真菌研究進展及其存在問題。生態學雜誌。23，86 – 91。

任安芝，高玉葆。2001，植物內生真菌-一類應用前景廣闊的資源微生物。微生物學通報。28，90 – 93。

李飛，李春杰。2006。內生真菌對禾草類植物抗旱性的影響。草葉科學。23，57 – 62。

宋朝霞，邢苗，曾憲錄。2000。細胞鬆弛素B對多頭絨泡菌有絲分裂的影響。遺傳
學報。1，83 – 89。

邱德有，黃美娟，方曉華等。1994。一種雲南紅豆杉內生真菌的分離。真菌學報。37，417 – 424。

柯勇。1992。Botryosphaeria dothidea引起的梨輪紋病。植保會刊34： 342。

馬天有，董兆麟。1999。植物分離產紫杉醇的內生真菌的研究。西北大學學報。29，47 – 49。

袁國芳。1997。新生物技術在真菌分類鑑定之應用。食品工業 微生物分類鑑定專輯。

曾松榮，王海坤，徐成東。2001。抗癌藥用植物內生真菌的潛在應用價值。韶關學院學報。22，123 – 126。

張玲琪，孫曉鵬，樓家鳳。1997，對鳶尾新鮮根狀莖發酵生香的微生物研究。雲南大學學報。19，362 – 365。

黃勝忠，蔡奇助，許謙信。2001。利用PCR選殖菊花rDNA之內轉錄間隔區。台中區農業改良場研究彙報。71，57 – 68。

Agee C, Hill N, 1994. Ergovaline variability in Acremonium-infected tall fescue due to environment and plant genotype. Crop Sci. 34, 221 – 226.

Carrol GC. 1986. The biology of endophytism in plants with particular reference to
woody perennials. In Fokkema N.J.and van den Heuvel J. eds .Microbiology of the Phyllosphere.Cambridge Cambridge University Press 205 – 222.

Chen G, Lin Y, Wen L, Vrijmoed LLP, Gareth-Jones EB, 2003. Two new metabolites of a marine endophytic fungus (No. 1893) from an estuarine mangrove on the South China Sea coast. Tetrahedron. 59, 4907 – 4909.

Chen Y. J, 2001. Studies on antimicrobial substances of the fungus THL. M.S. thesis, Tunghai University, Taichung, Taiwan (in Chinese with English abstract).

Clay K, 1989.Clavicipitaceous endophytes of grasses; their potential as biocontrol agents. Mycol.Res. 92, 1 – 12.

Dai JR, Carte’ B, Sidebottom P, Sek AL, Ng S, Huang Y, Butler M, 2001. Circumdatin G, a new alkaloid from the fungus Aspergillus ochraceus. J. Net. Prod. 64, 125 – 126.

Furusawa M, Hashimoto T, Noma Y, Asakawa Y, 2005. Highly efficient production of nootkaton, the grapefruit aroma from valencene by biotransformation. Chem. Pharm. Bull. 53, 1515 – 1514.

Gunatilaka AAL., 2006. Natural products form plant-associated micro-organisms: distribution, structural diversity, bioactivity, and implications of their occurrence. J. Nat. Prod. 60, 509 – 526.

Harri E, Loeffler W, Sigg HP, Tamm H, 1963. Uber die Isolierung neuer
Stoffwechselproduckte aus Penicillium brefeldianum DODGE. Helv. Chim. Acta. 46, 1235–1243.

Haldar S, Chintalli J, Croce CM, 1996. Taxol induces bcl-2 phosphorylation and death of prostate cancer cell. Cancer Res. 56, 1253 – 1255.

Hensens OD, Ondeyka JG, Dombrowski AW, Ostlind DA, Zink DL, 1999. Isolation and structure of Nodulisporic acid A1 and A2, novel insecticides from a Nodulisporium sp. Tetrahedron Lett. 40, 5455 – 5458.

Hofmann H, Choi SM, Wilsmann-Theis D, Horre R, de Hoog GS, Bieber T, 2005. Invasive chromoblastomycosis and sinusitis due to Phialophora verrucosa in a child from northern Africa. Mycoses. 48, 456 – 461.

Horn WS, Simmonds MSJ, Schwartz RE, Blaney WM, 1995. Phomopsichalasin, a novel antimicrobial agent from an endophytic Phomopsis sp. Terahedron. 51, 3969 – 3978.

KimuraY, Kusano M, Koshino H, Uzawa J, Fujioka S, Tani K, 1996. Penigequinolones A and B, pollen growth-inhibitors produced by Penicillium sp. No. 410. Tetrahedron Letters. 37, 4961 – 4964.

Kuldau GA, Yates IE, 2000. Evidence for Fusarium endophytes in cultivated and wild plants. In Microbial Endophytes (Bacon, C. W., White, J. F., eds), 85 – 117. Mareel Dekker. New York.

Lewis FJ, 1924. An endotrophic fungus in the Coniferae. Natrue. 114, 860.

Li JY, Harper JK, Grant DM, Tombe BO, Bashyal B, Hess WM, Strobel GA, 2001. Ambuic acid, a highly functionalized cyclohexenone with antifungal activity from Pestalotiopsis spp. and Monochaetia sp. Phytochemistry. 54, 463 – 468.

Li JY, Strobel GA, 2001. Jesterone and hydroxyl-jesterone antioomycete cyclohexenone epoxides from the endophytic fungus Pestalotiopsis jesteri. Phytochem. 57, 261 – 265.

Li Y, Song YC, Liu JY, Ma YM, Tan RX, 2005. Anti-Helicobacter pylori substances from endophytic fungal cultures. World J. Microbiol. Biotech. 28, 553 – 558.

Lin SW, 2005. Studies on antimicrobial substance of endemic endophytic fungi. M.S. thesis, Tunghai University, Taichang, Taiwan (in Chinese with English abstract).

McCloud TG, Burns MP, Majadly FD, Muschik GM, Miller DA, Poole KK, Roach JM, Ross JT, Lebherz WB, 1995. Production of brefeldin-A. J Ind Microbiol. 15, 5 – 9.

Nebenfuhr A, Ritzenthaler C, Robinson DG, 2002. Brefeldin A: deciphering an enigmatic inhibitor of secretion. Plant Physiol. 130, 1102 – 1108.

Ondeyka JG, Helms GL, Hensens OD, Goetz MA, Zink DL, Tsipouras A, Shoop WL, Slayton L, Dombrowski AW, Polishook JD, Ostlind DA, Tsou NN, Ball RG, Singh SB, 1997. Nodulisporic Acid A, a novel and potent insecticide from a Nodulisporium sp. isolation, structure determination, and chemical transformations. 119, 8809 – 8816.

Robertshaw H, Higgins E, 2004. Cutaneous infection with Alternaria tenuissima in an immunocompromised patient. Br J Dermatol. 153, 1047 – 1049.

Schmeda-Hirschmann G, Hormazabal E, Astudillo L, Rodriguez J, Theoduloz C, 2005. Secondary metabolites from endophytic fungi isolated from the Chilean gymnosperm Prumnopitys andina (Lleuque). World J. Microbiol. Biotech. 21, 27 – 32.

Song YC, Huang WY, Sun C, Wang FW, Tan RX, 2005. Characterization of graphislactone A as the antioxidant and free radical-scavenging substance from the culture of Cephalosporium sp. IFB-R001,an endophytic fungus in Trachelospermum jasmineides. Biol. Pharm. Bull. 28, 506 – 509.

Stierle AA, Stierle DB, Bugni T, 2001. Sequoiatones C-F, constituents of the redwood endophyte Asperfillus parasiticus. J. Nat. Prod. 64, 1350 – 1353.

Stierle A, Strobel G, Stierle D, 1993. Taxol and taxane production by Taxomyces andreanae anendophytic fungus of Pacific yew. Science. 260, 214 – 216.

Stierle DB, Stierle AA, Bugni T, 2003. Sequoiamonascins A-D: Novel anticancer metabolites isolated from a redwood endophyte. J. Org. Chem. 68, 4966 – 4969.

Strobel GA, Miller RV, Miller CM, Condron MM, Teplow DB, Hess WM, 1999. Cryptocandin, a potent antimycotic from the endophytic fungi Cryptosporiopsis cf. quercina. Microbiol. 145, 1919 – 1926.

Strobel G, Yang XS, Sears J, Kramer R, Sidhu RS, Hess WM, 1996. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiol. 142, 435 – 440.

Turbyville T J, Kithsiri-Wijeratne EM, Liu MX, Burns AM, Seliga CJ, Luevano LA, David CL, Faeth SH, Whitesell L, Leslie-Gunatilaka AA, 2006. Search for Hsp90 inhibitors with potential anticancer activity: isolation and SAR studies of radicicol and monocillin І from two plant-associated fungi of the sonoran desert. J. Nat. Prod. 69, 178 – 184.

de Vrije T, Antoine N, Buitelaar RM, Bruckner S, Dissevelt M, Durand A, Gerlagh M, Jones EE, Luth P, Oostra J, Ravensberg WJ, Renaud R, Rinzema A, Weber FJ, Whipps JM, 2001. The fungal biocontrol agent Coniothyrium minitans: production by solid-state fermentation, application and marketing. Appl Microbiol Biotechnol. 56, 58 – 68.

Wang J, Huang Y, Fang M, 2002. Brefeldin A, a cytotoxin produced by Paecilomyces sp. and Aspergillus clavatus isolated from Taxus mairei and Torreya grandis. FEMS Immunology and Medical Microbiology 34, 51 – 57.

Weber D, Sterner O, Anke T, Gorzalczancy S, Martino V, Acevedo C, 2004. Phomol, a new anti-inflammatory metabolite from an endophyte of the medicinal plant Etrythrina crista-galli. J. Antibio. 57, 559 – 563.

Weber RWS, Stenger E, Meffert A, Hahn M, 2004. Brefeldin A production by Phoma medicaginis in dead pre-colonized plant tissue: a strategy for habitat conquest? Mycol. Res. 108, 662 – 671.