臺灣博碩士論文加值系統

本研究目的為尋找Aspergillus terreus 中具有生物活性的成分，並且研究過去文獻中未曾被發現的新結構。從此株真菌中共分離得到18個化合物：包括6個固醇類化合物、6個異香豆素化合物、3個土麴霉酮類化合物、1個丁內酯類化合物、1個戊二酐類化合物以及1個亞硝基苯類化合物。其中，5個為新化合物：12S,15S,25,28-tetrahydroxy-ergosta-4,6,8(14),22-tetraene-3-one (2), 12S,15R,25,28-tetrahydroxy-ergosta-4,6,8(14),22-tetraene-3-one (3), 12S,15S,25,26-tetrahydroxy-ergosta-4,6,8(14),22-tetraen-3-one (4), 3-(1’-propenyl)-glutaric anhydride (17) and nitrobenzene A (18)；7個化合物為首次從此株真菌中分離得到。所有化合物結構皆由各種光譜資料 (紫外光譜、紅外光譜、圓二色光譜、核磁共振光譜、質譜)以及化學方法 (Mosher’s method)加以證明確定。
除了 ergosta-4,6,8(14),22-tetraen-3-one (1)、4B-(1’-propenyl)-1B,2B
-cyclopentanediol (14) 和 4a-(1’-propenyl)-1B,2B-cyclopentanediol (15) 之外的化合物皆進行抗癌和抗發炎活性測試。當中(+)-terrein (13) 和 5a,8a-epidioxyergosta-6,22-dien-3B-ol (5) 對OECM-1細胞株具有細胞毒殺作用，其IC50 分別為38.5 μM和36.4 μM。抗發炎活性方面，固醇類化合物則比其他類化合物相對來的佳。

The aim of this project is to isolate the bioactive compounds from Aspergillus terreus as an attempt to identify novel compounds which potentially may be useful for pharmaceutical research or never been reported in literatures.
Eighteen compounds, including six steroids, six isocoumarins, three terreins, one butyrolactone, one glutaric anhydride and one nitrobenzene, were isolated from Aspergillus terreus. Among them, five compounds, 12S,15S,25,28
-tetrahydroxy-ergosta-4,6,8(14),22-tetraene-3-one (2), 12S,15R,25,28-tetrahydroxy-ergosta-4,6,8(14),22-tetraene-3-one (3), 12S,15S,25,26-tetrahydroxy-ergosta
-4,6,8(14),22-tetraen-3-one (4), 3-(1’-propenyl)-glutaric anhydride (17) and nitrobenzene A (18), have never been reported previously and seven compounds were isolated from this fungus for the first time. All structures have been characterized by spectroscopic methods (UV, IR, CD, NMR, HR-ESIMS) and chemical assay (Mosher’s method).
All compounds, except ergosta-4,6,8(14),22-tetraen-3-one (1), 4B-(1’-propenyl)
-1B,2B-cyclopentanediol (14) and 4a-(1’-propenyl)-1B,2B-cyclopentanediol (15), were tested for their anti-cancer and anti-inflammatory activities. (+)-terrein (13) and 5a,8a-epidioxyergosta-6,22-dien-3B-ol (5) possessed anti-oral cancer (OECM-1) activity, and the IC50 were 38.5 μM and 36.4 μM respectively. Moreover, steroid compounds showed superior anti-inflammatory activity compared to other compounds, by inhibiting IL-6 and TNF-a expression in LPS-stimulated macrophages.

摘　要 I
ABSTRACT III
致謝 V
CONTENT VI
LIST OF TABLES VIII
LIST OF FIGURES IX
Chapter 1 INTRODUCTION 1
1.1 Thermophilic Fungi 1
1.2 Aspergillus terreus 2
1.3 Literature Review of Aspeergillus terreus 4
1.4 Objective 15
Chapter 2 MATERIALS AND METHODS 16
2.1 Materials 16
2.1.1 Fungi Strains 16
2.1.2 Medium for Aspergillus terreus 16
2.1.3 Instruments 16
2.2 Methods 18
2.2.1 Flow Chart 18
2.2.2 Extraction, Isolation and Purification 18
2.2.3 Modified Mosher’s Method 23
2.2.4 Cell Cultures 23
2.2.5 Anti-Cancer Activity Assays 23
2.2.6 Anti-inflammatory Activity Assays 24
Chapter 3 RESULTS 25
Section 3.1 Ergosta-4,6,8(14),22-tetraen-3-one (1) 25
Section 3.2 12S,15S,25,28-Tetrahydroxy-ergosta-4,6,8(14),22-tetraene-3 -one (2)* 30
Section 3.3 12S,15R,25,28-Tetrahydroxy-ergosta-4,6,8(14),22-tetraene-3 -one (3)* 41
Section 3.4 12S,15S,25,26-tetrahydroxy-ergosta-4,6,8(14),22-tetraen-3 -one (4)* 49
Section 3.5 5

1.Chen, G.Y., Taxonomical study of thermophilic and thermotolerant fungi in Taiwan, National Taiwan University, Taipei, 1992.
2.Cooney, D.G., Thermophilic fungi: an account of their biology, activities and classification, Freeman & Co. San francisco and London, 1964.
3.Denning, D.W., Invasive aspergillosis, Clinical Infectious Diseases, 1998, 26, 781-803.
4.Balajee, S.A., Aspergillus terreus complex, Medical Mycology, 2009, 47, 42-46.
5.Bonnarme, P., Itaconate biosynthesis in Aspergillus terreus, Journal of Bacteriology, 1995, 177, 3573-3578.
6.Dwiarti, L., Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1, Bioresource Technology, 2007, 98, 3329-3337.
7.Alberts, A.W., Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent, Proceedings of the National Academy of Sciences of the United States of America Biological Sciences, 1980, 77, 3957-3961.
8.Chen, J.W.; Ling, K.H., Territrems: naturally occurring specific irreversible inhibitors of acetylcholinesterase, Journal of Biomedical Science, 1996, 3, 54-58.
9.Ronnest, M.H., (+)-Geodin from Aspergillus terreus, Acta Crystallographica Section C Crystal Structure Communications, 2011, 67, O125-O128.
10.Nomoto, K., Chemical structure of a new metabolite isolated from the mycelium of Aspergillus terreus var. aureus, Chemical & Pharmaceutical Bulletin, 1984, 32, 4213-4216.
11.Hassall, C.H.; T.C. Mcmorris, The constitution of geodoxin, a metabolic product of Aspergillus terreus Thom, Journal of the Chemical Society, 1959, 2831-2834.
12.Inamori, Y., Studies on metabolites produced by Aspergillus terreus var. aureus. I. chemical structures and antimicrobial activities of metabolites isolated from culture broth, Chemical & Pharmaceutical Bulletin, 1983, 31, 4543-4548.
13.Elsohly, H.N., Metabolites of Aspergillus cervinus massee (Moniliaceae), Journal of Pharmaceutical Sciences, 1974, 63, 1632-1633.
14.Kiriyama, N., Studies on metabolic products of Aspergillus terreus. II. structure and biosynthesis of metabolites of strain ATCC 12238, Chemical & Pharmaceutical Bulletin, 1977, 25, 1265-1272.
15.Wijeratne, E.M.K., Cytotoxic constituents of Aspergillus terreus from the Rhizosphere of Opuntia versicolor of the Sonoran Desert, Journal of Natural Products, 2003, 66, 1567-1573.
16.Lee, I.K., Betulinans A and B, two benzoquinone compounds from Lenzites betulina. Journal of Natural Products, 1996, 59, 1090-1092.
17.Wu, S.H., A new spiroketal from Aspergillus terreus, an endophytic fungus in Opuntia ficusindica Mill, Journal of Basic Microbiology, 2008, 48, 140-142.
18.He, J., Cytotoxic and other metabolites of Aspergillus inhabiting the rhizosphere of Sonoran desert plants, Journal of Natural Products, 2004, 67, 1985-1991.
19.Li, G.Y., Sesterterpenoids, terretonins A-D, and an alkaloid, asterrelenin, from Aspergillus terreus, Journal of Natural Products, 2005, 68, 1243-1246.
20.Gordon W.K., Structure and synthesis of cis-3,6-dibenzyl-3,6-bis(methyIthio) -piperazine-2,5-dione, a new metabolite of Aspergillus terreus, Journal of the Chemical Society, Chemical Communications, 1983, 812-813.
21.Linschitz, H.; Connolly, J.S., Aranotin and Related Metabolites from Avachniofus Aureus. I. determination of structure, Journal of the American Chemical Society, 1968, 90, 2980.
22.Lin, Z., 1H and 13C NMR assignments of two new indolic enamide diastereomers from a mangrove endophytic fungus Aspergillus sp. Magnetic Resonance in Chemistry, 2008. 46, 1212-1216.
23.Garson, M.J., Isolation of some new 3,6-Dialkyl-1,4-Dihydroxypiperazine -2,5-Diones from Aspergillus terreus, Journal of the Chemical Society-Perkin Transactions 1, 1986, 901-903.
24.Kuriyama, T., Receptor assay-guided isolation of anti-GABAergic insecticidal alkaloids from a fungal culture, Journal of Agriculture and Food Chemistry, 2004, 52, 3884-3887.
25.Kagamizono, T., Terpeptin, a novel mammalian cell cycle inhibitor, produced by Aspergillus terreus 95F-1, Tetrahedron Letters, 1997, 38, 1223-1226.
26.Lee, S.M., Terreusinone, a novel UV-A protecting dipyrroloquinone from the marine algicolous fungus Aspergillus terreus, Tetrahedron Letters, 2003, 44, 7707-7710.
27.Arai, K., Metabolic products of Aspergillus terreus. IV. metabolites of the strain IFO-8835.(2). The isolation and chemical structure of indolyl benzoquinone pigments, Chemical & Pharmaceutical Bulletin, 1981, 29, 961-969.
28.Arai, K.; S. shake; Y. Yuzuru, Metabolic products of Aspergillus terreus. VI. Metabolites of the strain IFO 8835. (3). The isolation and chemical structure of colorless metabolites. Chemical & Pharmaceutical Bulletin, 1981, 29, 1005-1012.
29.Parvatkar, R.R., Aspernolides A and B, butenolides from a marine-derived fungus Aspergillus terreus. Phytochemistry, 2009, 70, 128-132.
30.Niu, X., Butyrolactone I derivatives from Aspergillus terreus carrying an unusual sulfate moiety. Journal of Natural Products, 2008, 71, 689-692.
31.Rao, K.V., Butyrolactones from Aspergillus terreus, Chemical & Pharmaceutical Bulletin (Tokyo), 2000, 48, 559-562.
32.Ojima, N., Structures of pulvinone derivatives from Aspergillus terreus, Phytochemistry, 1975, 14, 573-576.
33.Kiriyama, N., Studies on metabolic products of Aspergillus terreus. III. Metabolites of strain IFO 8835. (1), Chemical & Pharmaceutical Bulletin, 1977, 25, 2593-2601.
34.Vertesy, L., Kodaistatins, novel inhibitors of glucose-6-phosphate translocase T1 from Aspergillus terreus thom DSM 11247. Isolation and structural elucidation, The Journal of Antibiotics (Tokyo), 2000, 53, 677-686.
35.Golding, B.T., New metabolites of Aspergillus terreus: 3- Hydroxy-2,5- bis-(p- hydroxyphenyl)penta-2,4-d ien-4-olide and Derivatives, Journal of the Chemical Society, Perkin Transactions 1, 1975, 19, 1961-1963.
36.Nakagawa, M., New sesquiterpenes from Aspergillus terreus Thom, Agricultural and Biological Chemistry, 1984, 48, 2279-2283.
37.Yoo, I.D., Isoterreulactone A, a novel meroterpenoid with anti-acetylcholinesterase activity produced by Aspergillus terreus, Bioorganic & Medicinal Chemistry Letters, 2005. 15, 353-356.
38.Ling, K.H.; Yang, C.K.; Peng, F.T., Territrems, tremorgenic mycotoxins of Aspergillus terreus, Applied and Environmental Microbiology, 1979, 37, 355-357.
39.Franck, B.; Gehrken, H.P., Citreoviridine aus Aspergillus terreus, Angewandte Chemie, 1980, 92, 484-486.
40.Nishihara, Y., Takase, S., New anti-influenza agents, FR198248 and its derivatives II. characterization of FR198248, its related compounds and some derivatives, Journal of Antibiotics, 2001, 54, 297-303.
41.Naito, S., Two new phenolic reductones from Aspergillus terreus, Tetrahedron Letters, 1969, 10, 4675-4678.
42.Stefanelli, S., Inhibitors of type-I interleukin-1 receptor from microbial metabolites, Journal of Antibiotics, 1997, 50, 484-489.
43.Fernando, C.M., Terreinol–a novel metabolite from Aspergillus terreus: structure and 13C labeling, Tetrahedron Letters, 2004, 45, 53-55.
44.Ballanti, J.A.; Hassall, C.H.; Jones, B.D., The biosynthesis of phenols XVII. Some phenolic metabolites of mutant strains of Aspergillus regulosus, Phytochemistry, 1968, 7, 1529-1534.
45.Yoshikawa, K., Novel abietane diterpenoids and aromatic compounds from Cladonia rangiferina and their antimicrobial activity against antibiotics resistant bacteria, Chemical & Pharmaceutical Bulletin, 2008, 56, 89-92.
46.Nitao, J.K., Isolation of flavipin, a fungus compound antagonistic to plant-parasitic nematodes, Nematology, 2002, 4, 55-63.
47.Arai, K., Metabolic products of Aspergillus terreus. VIII. Astepyrone: a novel metabolite of the strain IFO 4100, Chemical & Pharmaceutical Bulletin, 1983, 31, 925-933.
48.Sankawa, U., Biosynthesis of citrinin in Aspergillus terreus: incorporation studies with [2-13C,2-2H3], [1-13C,18O2] and [1-13C,17O]-acetate, Tetrahedron, 1983, 39, 3583-3591.
49.Puel, O., Biosynthesis and toxicological effects of patulin, Toxins, 2010, 2, 613-631.
50.Curtis, R.F., The biosynthesis of Phenols. part II. Asterric acid, a metabolic product of Aspergillus terreus Thom, Journal of the Chemical Society, 1960, 4838-4842.
51.Weng, Y., Ganodermasides A and B, two novel anti-aging ergosterols from spores of a medicinal mushroom Ganoderma lucidum on yeast via UTH1 gene, Bioorganic & Medicinal Chemistry Letters, 2010, 18, 999-1002.
52.Tanaka, N., Photochemical reaction of ergosta-4,6,8(14),22-tetraen-3-one, Chemical & Pharmaceutical Bulletin, 1996, 44, 843-846.
53.Ohtani, I.; Kusumi, T., High-field FT NMR application of mosher''s method. The absolute configurations of marine terpenoids, Journal of the American chemical Society, 1991, 113, 4092-4096.
54.Rosecke, J.; Konig, W.A., Constituents of the fungi Daedalea quercina and Daedaleopsis confragosa var. tricolor, Phytochemistry, 2000, 54, 757-762.
55.Takaishi, Y., Glycosides of ergosterol derivatives from Hericum erinacens, Phytochemistry, 1991, 30, 4117-4120.
56.Nes, W.D.; Heupel, R.C.; Le, P.H., Biosynthesis of ergosta-6(7),8(14),22(23)-trien-3-ol by Gibberella fujikuroi: its importance to ergosteril''s metabolic pathway. Journal of the Chemical Society, Chemical Communications, 1985, 1431-1433.
57.Krohn, K., Dihydroisocoumarins from fungi: isolation, structure elucidation, circular dichroism and biological activity, Phytochemistry, 1997, 45, 313-320.
58.Schlingmann, G.; Milne, L.; Carter, G.T., Isolation and identification of antifungal polyesters from the marine fungus Hypoxylon oceanicum LL-15G256, Tetrahedron, 2002, 58, 6825-6835.
59.Hutchiso, R.D.; Steyn, P.S., Isolation and structure of 4-hydroxyochratoxin and 7-carboxy-3,4-dihydro-8-hydroxy-3-methylisocoumarin from Penicillium viridicatum, Tetrahedron Letters, 1971, 4033
60.Assante, G., Screening of the genus Cercospora for secondary metabolites. Phytochemistry, 1977, 16, 243-247.
61.Shimada, A., 4-hydroxykigelin and 6-demethylkigelin, root growth promoters, produced by Aspergillus terreus, Zeitschrift Fur Naturforschung C, 2004, 59, 218-222.
62.Dethoup, T., Merodrimanes and other constituents from Talaromyces thailandiasis, Journal of Natural Products, 2007, 70, 1200-1202.
63.Chinworrungsee, M., Isolation and structure elucidation of a novel antimalarial macrocyclic polylactone, menisporopsin A, from the fungus Menisporopsis theobromae, Journal of Natural Products, 2004, 67, 689-692.
64.Govindachari, T.R., isolation and structure of two new dihydroisocoumarins from kigelia pinnata, Phytochemistry, 1971, 10, 1603-1606.
65.Kawakubo, J., Production of phenolic compounds by Aspergillus S-4 in sake cake medium and identification of terrein, Bioscience Biotechnology and Biochemistry, 1993, 57, 1208-1209.
66.Wakana, D., Isolation of isoterrein from Neosartorya fischeri, Mycotoxins 2006, 56, 3-6.
67.Ghisalberti, E.L.; Narbey, M.J.; Rowland C.Y., Metabolites of Aspergillus terreus antagonistic towards the take-all fungus, Journal of Natural Products, 1990, 53, 520-522.
68.Rao, K.V., Butyrolactones from Aspergillus terreus, Chemical & Pharmaceutical Bulletin, 2000, 48, 559-562.
69.Chao, L.K., Anti-inflammatory bioactivities of honokiol through inhibition of protein kinase C, mitogen-activated protein kinase, and the NF-kB Pathway to reduce LPS-induced TNF-a and NO expression, Journal of Agricultural and Food Chemistry, 2010, 58, 3472-3478.