摘要

海洋微生物在生物活性化學支架被視作最有潛力的使用品之ㄧ。為找尋來自海洋環境的抗微生物次生代謝產物，本研究從海綿 ( genus : Haliclona ) 中分離出ㄧ種海洋真菌 Aspergillus terreus (MB14-HBr)。於2014年五月，收集自台灣東沙環礁群島。藉由乙酸乙酯 ( EtOAc )，由實驗室自行培養出的真菌，萃取出代謝物。由抗微生物檢測初步結果表示，以EtOAc萃取出的MB14-HBr能分別地抑制細菌病原體鮑氏不動桿菌 (Acinetobacter baumannii ) 和白色念珠菌 (Candida albicans)。有趣的是，本研究還發現以EtOAc粗萃取時，還有其它的純物質, 包含 : dihydrogeodin (A-01), butryolactone II (A-02) ， butryolactone I (A-06), Terrelumamide A (A- 11),另, Methyl 3,4,5-trimethoxy-2-(2-nicotinamido)benzamido) benzoate (A-15) 和鐵螯合於色天青S (Chrome-azurol S, CAS)檢測裡。由於鐵載體有抓住天然鐵的特性，讓其可應用於癌症化療。鮑氏不動桿菌 (Acinetobacter baumannii ) ，一種伺機式感染人類的病原體，容易受到臨床經常使用的抗生素而突變，也常為院內感染的元凶。而且，三種化合物，butryolactone II (A-02), butryolactone I (A-06) 和 methyl 3, 5 dichloro-asterric acid (A-03) 在螢光素脢活性分析，表明這些化合物可能阻止了(TGF)-β誘導的轉錄反應。本研究的目的是在分叢菌株 Aspergillus terreus 中分離出生物活性物。為了分離的生物活性成分，將EtOAc萃取物進行Sephadex LH-20 管柱層析法和逆向高效液相層析法。其結果是，共15種已知化合物從此真菌提取物中分離，包含 dihydrogeodin (A -01), butryolactone (A-02), methyl 3, 5 dichloro-asterric acid (A-03), 3R-methyl-6-hydroxy-8-methoxy isocoumarin (A-04), terretonin C (A-05), butryolactone I (A-06), geodin hydrate (A-07), 6,7-dimethoxy-4-hydroxymellein (A-08), butryolactone V (A-09), asterric acid (A-10), terrein (A-12) asterrelenin (A-13), terretonin (A-14) and methyl 3,4,5-trimethoxy-2-(2-nicotinamido)benzamido)benzoate (A-15). 此外，這些被分離出的結構，透過1D和2D NMR被闡述在本研究中。

關鍵字 : 土麴黴 (Aspegillus terreus)，抗微生物活性，鮑氏不動桿菌，螢光素脢活性分析，MTS細胞生存能力檢測，螯合鐵檢測

Abstract

Marine microbes are regarded as one of the worthiest treasures for bioactive chemical Scaffolds. To search for anti-microbial secondary metabolites from marine environment, we isolated a marine-derived fungus Aspergillus terreus (MB14-HBr) from the sponge Haliclona species, Collected from Dongsha Atoll, Taiwan in May 2014. The isolated fungus was further cultured in laboratory and fungal metabolites were extracted by ethyl acetate (EtOAc). Preliminary anti-microbial assay indicated that EtOAc extract of MB14-HBr could inhibit the bacterial pathogen Acinetobacter baumannii and the fungal one Candida albicans, respectively. Besides interestingly, we also discovered that the crude EtOAc extract, fraction as well as pure compounds including dihydrogeodin (A-01), butryolactone II (A-02), Butryolactone I (A-06), Terrelumamide A (A-11) and Methyl 3,4,5-trimethoxy-2-(2-nicotinamido)benzamido)benzoate (A-15) exhibited siderophore-like (iron-chelating) effects in Chrome-azurol S (CAS) assay. Due to natural iron clutching properties of sidherophores, they have impending applications in cancer chemotherapy. Acinetobacter baumannii is an opportunistic human pathogen which is easy mutant to be several clinical antibiotics and very famous due to cause of nosocomial infection in human. Moreover, three compounds, butryolactone II (A-02), Butryolactone I (A-06) and methyl 3, 5 dichloro-asterric acid (A-03) showed activity in luciferase assay, suggested that these components might block Transforming Growth Factor (TGF)-β-induced transcriptional responses. The objective of this study is to isolate the bioactive components from the strain Aspergillus terreus. In order to isolate bioactive components, the EtOAc extract was subjected to Sephadex LH-20 column chromatography and reversed phase high performance liquid chromatography. As a result, a total of fifteen known compounds have been isolated from this fungal extract, including dihydrogeodin (A-01) butryolactoneII (A-02), methyl 3,5 dichloro-asterrric acid (A-03), 3R-methyl-6-hydroxy-8-methoxy isocoumarin (A-04), terretonin C (A-05), butryolactone I (A-06), geodin hydrate (A-07), 6,7-dimethoxy-4-hydroxymellein (A-08), butryolactone V (A-09), asterric acid (A-10), terrein (A-12) asterrelenin (A-13), terretonin (A-14) and methyl 3,4,5-trimethoxy-2-(2-nicotinamido)benzamido)benzoate (A-15). Moreover, we isolated one are peptide named terrelumamide-A (A-11). The structures of these isolates were elucidated on the basis of 1D and 2D NMR, Mass spectrometry, as well as literature data.
Key words: Aspegillus terreus, Anti-microbial activity, Acinetobacter baumannii, luciferase assay activity, MTS cell viability effect, Iron chelating effects.

Content
Varification letter i
Acknowledgement ii
Abstract iii
List of figures ix
List of tables xii
List of schemes xii
List of abbreviations and symbols xiii
Chapter - 1 1
Introduction 1
1.1 Natural products in history 1
1.2 Historically important natural products 2
1.3 Natural products from fungi 4
1.4 Secondary metabolites and antibiotics 5
1.4-1 Antibiotics history 6
1.4-2 Antibiotics definition 6
1.4-3 Antibiotics function 7
1-5. Litreture review of Aspergillus terreus 8
1-5-1. Butryolactone types compounds from Aspergillus terreus 9
1-5-3. Statin types compounds from Aspergillus terreus 11
1-5-4. Sesterterpenoid types compounds from Aspergillus terreus 11
1-5-4. Asterric Acid types compounds from Aspergillus terreus 12
1-5-5. Quinone types compounds from Aspergillus terreus 12
1-5-6. Asterriquinone types compounds from Aspergillus terreus 13
1-5-8. Alkaloid types compounds from Aspergillus terreus 15
1-5-11. Meroterpenoid types compounds from Aspergillus terreus 17
1-5-12. Lumazine peptides 18
1-5-13. Others 19
1-6. Biological activities – 19
Chapter - 2 24
Materials and Methods 24
2.1 Experimental methods 24
2.2 Marine fungus 26
2.2.1. Collection and purification 26
2.3 Initial purification of sponge (Haliclona species) 29
2.4 Extraction, isolation and purification 30
2.5 Marine agar 34
2.8 Other media 36
2.8-1 Salts seawater 36
2.8-2 MSWYE (PYE) 37
2.8-3 Potato dextrose agar (PDA) 37
2.8-4 CAMHB 37
2.9 Instruments 38
2.9-1 Optical rotation spectrometer: 38
2.9-2 UV-visible spectrometer (UV-Vis): 38
2.9-3 Infrared spectrometer (IR) 38
2.9-4 Nuclear magnetic resonance spectrometer (NMR) 38
2.9-5. Mass spectrometer (MS) 39
2.9-6 Column chromatography 39
2.9-7 Solvents 39
2.10 Bioassay methods 39
2.10-1 Paper disc agar diffusion assay 39
2.10-2 Punching-plate assay 40
2.10-3 Chrome azurol S (CAS) 40
2.10-4 Minimum inhibitory concentrations assay 41
Chapter – 3 47
Results and Discussion 47
3.1 Dihydrogeodin (A-01) 47
3.2 Butryolactone II (A-02) 54
3.3 Methyl 3, 5 dichloro-asterrric acid (A-03) 60
3.4 3R-methyl-6-hydroxy-8-methoxy isocoumarin (A-04) 66
3.5 Terretonin C (A-05) 71
3.6 Butryolactone I (A-06) 76
3.7 Geodin hydrate (A-07) 82
3.8 6,7-dimethoxy-4-hydroxymellein (A-08) 88
3.9 Butryolactone V (A-09) 95
3.10 Asterric acid (A-10) 101
3.11 Terrelumamide-A (A-11) 106
3.12 Terrein (A-12) 113
3.13 Asterrelenin (A-13) 118
3.14 Terretonin (A-14) 124
3.15 Methyl 3, 4, 5-trimethoxy-2-(2-nicotinamido)benzamido)benzoate (A-15) 130
Chapter 4 136
Biological activities 136
4.1 Disc diffusion assay 136
4.2 Chrome azurol S (CAS) assay 137
4.3 Luciferase activity assay 140
4.4 MTS cell viability assay 140
Chapter – 5 142
Conclusion 143
Chapter-6 144
References 144

Chapter-6
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