進階搜尋


  查詢北醫館藏
系統識別號 U0007-2207201016525700
論文名稱(中文) 兩種真菌醱酵液之生物活性成分研究
論文名稱(英文) Chemical investigation of the fermented broths of Theissenia rogersii and Stilbohypoxylon elaeicola
校院名稱 臺北醫學大學
系所名稱(中) 生藥學研究所
系所名稱(英) Graduate Institute of Pharmacognosy
學年度 98
學期 2
出版年 99
研究生(中文) 張雅雯
研究生(英文) Ya-Wen Chang
學號 M303097009
學位類別 碩士
語文別 中文
口試日期 2010-06-24
論文頁數 181頁
口試委員 委員-李水盛
委員-蕭哲志
指導教授-李宗徽
中文關鍵字 真菌  誘導型一氧化氮合成酶  Theissenia rogersii  Stilbohypoxylon elaeicola  elaeicolaside  elaeicolactone 
英文關鍵字 isopimarane diterpene glycoside  elaeicolaside  elaeicolactone  Theissenia rogersii  Stilbohypoxylon elaeicola  iNOS 
學科別分類
中文摘要 利用抑制一氧化氮合成酶的活性分析平台來篩選真菌培養株,藉以篩檢出具有抗發炎活性的真菌株,發現某些真菌株的醱酵培養液或菌絲體萃取物,對於RAW264.7細胞的產生一氧化氮具有顯著的抑制作用。遂選定:Theissenia rogersii(#92031201)及Stilbohypoxylon elaeicola(#173)兩株進行其活性成分研究。在選擇以麥芽抽取物(malt extract)為培養基加以擴大培養後,針對醱酵液所含代謝產物進行分析與分離,計獲得12個化合物,分別為: 2,5-dihydroxymethylfuran(1)、pycnidione(2)、maltol(3)、tyrosol(4)、hymatoxin K(5)、hymatoxin L(6)、elaeicolactone(7)、5-hydroxymethylfurfural(8)、elaeicolasdie A(9)elaeicolaside B(10)、elaeicolaside C(11)和 elaeicolaside D(12),其中屬於倍半萜之化合物7,及屬於二萜糖苷的化合物9、10及12,皆為新化合物。在100 μM的濃度下,10對於RAW264.7細胞的產生一氧化氮具有顯著的抑制活性且不具細胞毒性。此外,2及10對於A549肺腺癌細胞具有強的毒殺作用,其GI50分別為5.25 nM及9.79 μM。
英文摘要 The ethyl acetate extracts of the fermented broths of Theissenia rogersii (#92031201) and Stilbohypoxylon elaeicola (#173) were found to exhibit significant activity against inducible nitric oxide synthase (iNOS) in our preliminary screening. Therefore, bioassay-guided fraction and separation of the active component from these two broths were carried out which resulted in the isolation of twelve compounds. Their structures were elucidated to be 2,5-dihydroxymethylfuran (1), pycnidione (2), maltol (3), tyrosol (4), hymatoxin K (5), hymatoxin L (6), elaeicolactone (7), 5-hydroxymethylfurfural (8), elaeicolasdie A (9), elaeicolaside B (10), elaeicolaside C (11) and elaeicolaside D (12). Of these compounds identified, 7, 9, 10 and 12, classified as respective a sesquiterpene and three diterpene glycosides, were novel chemical entities. Compound 10 exhibited significant inhibitory activity on NO production of RAW264.7 cells without any cytotoxicity. In addition, 2 and 10 exerted potent cytotoxicity against an A549 lung cancer cell line with GI50 values of 5.25 nM and 9.79 μM, respectively.
論文目次 摘要 I
Abstract II
總目錄 III
圖目錄 VII
縮寫表 XII
壹、緒論與研究目的 1
貳、炭角菌科真菌之天然物文獻回顧 5
参、實驗結果與討論 34
3.1.1 2,5-Dihydroxymethylfuran(1)之結構解析 39
3.1.2 Pycnidione(2)之結構解析 43
3.1.3 Maltol(3)之結構解析 54
3.1.4 Tyrosol(4)之結構解析 58
3.1.5 Hymatoxin K(5)之結構解析 61
3.1.6 Hymatoxin L(6)之結構解析 73
3.1.7 Elaeicolactone(7)之結構解析 82
3.1.8 5-Hydorxymethylfurfural(8)之結構解析 92
3.1.9 Elaeicolaside A(9)之結構解析 96
3.1.10 Elaeicolaside B(10)之結構解析 106
3.1.11 Elaeicolaside C(11)之結構解析 116
3.1.12 Elaeicolaside D(12)之結構解析 126
3.2 一氧化氮濃度及細胞毒性測定結果 135
3.3 A549細胞生長抑制測試結果 137
3.4 討論 138
肆、實驗部分 141
4.1 儀器設備與試劑 141
4.2 真菌材料 143
4.3 培養基配置 143
4.4.1 #92031201 培養液之成分分離與純化 145
4.4.2 #173培養液之成分分離與純化 148
4.5 單糖組成分析 152
4.6 單糖旋光度測定 153
4.7 一氧化氮(NO)濃度之測定:Griess reagent assay 154
4.8 細胞毒性測試(alamarBlue assay) 157
4.9 細胞生長測試(sulforhodamine B, SRB) 159
4.10 各成分之物理數據 161
參考文獻 165
附錄一 173
參考文獻 1 Nathan, C. Nitric oxide as a secretory product of mammalian cells. The FASEB Journal : official publication of the Federation of American Societies for Experimental Biology 6, 3051-3064 (1992).
2 Wink, D. A. & Mitchell, J. B. Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radical Biology & Medicine 25, 434-456 (1998).
3 Chen, Y. C., Lin S., S. Y. & Lin, J. K. Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. Journal of Cellular Physiology 177, 324-333 (1998).
4 Laroux, F. S., Pavlick, K. P., Hines, I. N., Kawachi, S., Harada, H., Bharwani, S., Hoffman, J. M. & Grisham, M. B. Role of nitric oxide in inflammation. Acta Physiologica Scandinavica 173, 113-118 (2001).
5 Knowles, R. G. & Moncada, S. Nitric oxide synthases in mammals. The Biochemical Journal 298, 249-258 (1994).
6 Sautebin, L. Prostaglandins and nitric oxide as molecular targets for anti-inflammatory therapy. Fitoterapia 71, S48-S57 (2000).
7 吳美麗,台灣真菌多樣性資源與研究現況,環境教育學刊, pp. 143-163 (2002)
8 吳聲華,菌類多樣性,行政院農委會林務局委託研究報告 (2003)
9 邵廣昭、彭鏡毅、吳文哲,台灣物種名錄,行政院農委會林務局,台北,台灣, p. iv (2008)
10 曹雅菁,台灣兩種真菌醱酵液及菌絲體之生物活性成分研究,台北醫學大學生藥學研究所碩士論文, pp. 3-4 (2009)
11 Quang, D. N., Hashimoto, T. & Asakawa, Y. Inedible mushrooms: a good source of biologically active substances. Chemical Record 6, 79-99 (2006).
12 Steglich, W., Klaar, M. & Furtner, W. (+)-Mitorubrin derivatives from Hypoxylon fragiforme. Phytochemistry 13, 2874-2875 (1974).
13 Stadler, M., Baumgartner, M., Grothe, T., Muhlbauer, A., Seip, S. & Wollweber, H. Concentricol, a taxonomically significant triterpenoid from Daldinia concentrica. Phytochemistry 56, 787-793 (2001).
14 Quang, D. N., Harinantenaina, L., Nishizawa, T., Hashimoto, T., Kohchi, C., Soma, G.-I. & Asakawa, Y. Inhibitory activity of nitric oxide production in RAW 264.7 cells of daldinals A-C from the fungus Daldinia childiae and other metabolites isolated from inedible mushrooms. Journal of Natural Medicines 60, 303-307 (2006).
15 Edwards, R. L., Maitland, D. J. & Whalley, A. J. S. Metabolites of the higher fungi. Part 24. Cytochalasin N, O, P, Q, and R. New cytochalasins from the fungus Hypoxylon terricola Mill. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 57-65 (1989).
16 Edwards, R. L., Maitland, D. J., Scowen, I. J., De Sousa, A. J. T. & Whalley, A. J. S. Metabolites of the higher fungi. Part 32. Rosnecatrone, a phytotoxic bicyclo[4.1.0]hept-3-en-2-one from the fungus Rosellinia necatrix Prill. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 537-542 (2001).
17 Stadler, M., Quang, D. N., Tomita, A., Hashimoto, T. & Asakawa, Y. Changes in secondary metabolism during stromatal ontogeny of Hypoxylon fragiforme. Mycological Research 110, 811-820 (2006).
18 Pedersen, E. J., Larsen, P. & Boll, P. M. Engleromycin, a new cytochalasan from Engleromyces goetzei Hennings. Tetrahedron Letters 21, 5079-5082 (1980).
19 Ondeyka, J., Hensens, O. D., Zink, D., Ball, R., Lingham, R. B., Bills, G., Dombrowski, A. & Goetz, M. L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. II. Isolation and structure. The Journal of Antibiotics 45, 679-685 (1992).
20 Lingham, R. B., Hsu, A., Silverman, K. C., Bills, G. F., Dombrowski, A., Goldman, M. E., Darke, P. L., Huang, L., Koch, G., Ondeyka, J. G. & et al. L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. III. Biological activity. The Journal of Antibiotics 45, 686-691 (1992).
21 Dagne, E., Gunatilaka, A. A. L., Asmellash, S., Abate, D., Kingston, D. G. I., Hofmann, G. A. & Johnson, R. K. Two new cytotoxic cytochalasins from Xylaria obovata. Tetrahedron 50, 5615-5620 (1994).
22 Chen, Y. S. Studies on the metabolic products of Rosellinia necatrix. I. Isolation and characterization of several physiologically active neutral substances. Bulletin of the Agricultural Chemical Society of Japan 24, 372-381 (1960).
23 Whalley, A. J. S. & Edwards, R. L. The Xylariaceae: A case study in biological and chemical diversity. Pure and Applied Chemistry 70, 2123-2134 (1998).
24 Chinworrungsee, M., Kittakoop, P., Isaka, M., Chanphen, R., Tanticharoen, M. & Thebtaranonth, Y. Halorosellins A and B, unique isocoumarin glucosides from the marine fungus Halorosellinia oceanica. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 2473-2476 (2002).
25 Quang, D. N., Hashimoto, T., Tanaka, M., Baumgartner, M., Stadler, M. & Asakawa, Y. Chemical constituents of the ascomycete Daldinia concentrica. Journal of Natural Products 65, 1869-1874 (2002).
26 Schlingmann, G., Milne, L. & Carter, G. T. Isolation and identification of antifungal polyesters from the marine fungus Hypoxylon oceanicum LL-15G256. Tetrahedron 58, 6825-6835 (2002).
27 Adeboya, M., Edwards, R. L., Laessoe, T., Maitland, D. J. & Whalley, A. S. J. Metabolites of the higher fungi. Part 28. Globoscinic acid and globoscin, a labile acid-lactone system from Xylaria globosa and Xylaria obovata. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 2067-2072 (1995).
28 Edwards, R. L., Maitland, D. J., Oliver, C. L., Pacey, M. S., Shields, L. & Whalley, A. J. S. Metabolites of the higher fungi. Part 31. Longianone, a C7H6O4 spiro bicyclic lactone from the fungus Xylaria longiana (Rehm.). Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 715-720 (1999).
29 Boonphong, S., Kittakoop, P., Isaka, M., Pittayakhajonwut, D., Tanticharoen, M. & Thebtaranonth, Y. Multiplolides A and B, new antifungal 10-membered lactones from Xylaria multiplex. Journal of Natural Products 64, 965-967 (2001).
30 Anderson, J. R., Edwards, R. L. & Whalley, A. J. S. Metabolites of the higher fungi. Part 19. Serpenone, 3-methoxy-4-methyl-5-prop-1-enylfuran-2(5H)-one, a new gamma -butyrolactone from the fungus Hypoxylon serpens (Barrons strain) (Persoon ex Fries) Kickx. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 215-221 (1982).
31 Edwards, R. L. & Whalley, A. J. S. Metabolites of the higher fungi. Part 18. 3-Butyl-4-methylfuran-2(5H)-one and 3-butyl-4-methylenefuran-2(5H)-one. New gamma -butyrolactones from the fungus Hypoxylon serpens (Persoon ex Fries) Kickx. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 803-806 (1979).
32 Quang, D. N., Stadler, M., Fournier, J. & Asakawa, Y. Carneic acids A and B, chemotaxonomically significant antimicrobial agents from the xylariaceous ascomycete Hypoxylon carneum. Journal of Natural Products 69, 1198-1202 (2006).
33 Whalley, A. J. S. & Edwards, R. L. Secondary metabolites and systematic arrangement within the Xylariaceae. Canadian Journal of Botany 73, S802-S810 (1995).
34 Gu, W. & Ding, H. Two new tetralone derivatives from the culture of Xylaria hypoxylon AT-028. Chinese Chemical Letters 19, 1323-1326 (2008).
35 Borgschulte, K., Rebuffat, S., Trowitzsch-Kienast, W., Schomburg, D., Pinon, J. & Bodo, B. Isolation and structure elucidation of hymatoxins B-E and other phytotoxins from Hypoxylon mammatum fungal pathogen of leuce poplars. Tetrahedron 47, 8351-8360 (1991).
36 Jossang, A., Mbeminack, B., Pinon, J. & Bodo, B. Hymatoxins K and L, novel phytotoxins from Hypoxylon mammatum, fungal pathogen of aspens. Natural Product Letters 6, 37-42 (1995).
37 Anderson, J. R., Edwards, R. L., Poyser, J. P. & Whalley, A. J. S. Metabolites of the higher fungi. Part 23. The punctaporonins. Novel bi-, tri-, and tetracyclic sesquiterpenes related to caryophyllene, from the fungus Poronia punctata (Linnaeus:Fries) Fries. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 823-831 (1988).
38 Edwards, R. L., Maitland, D. J., Poyser, J. P. & Whalley, A. J. S. Metabolites of the higher fungi. Part 25. Punctaporonin G from the fungus Poronia punctata (Linnaeus: Fries) Fries. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 1939-1941 (1989).
39 Shiono, Y., Motoki, S., Koseki, T., Murayama, T., Tojima, M. & Kimura, K. Isopimarane diterpene glycosides, apoptosis inducers, obtained from fruiting bodies of the ascomycete Xylaria polymorpha. Phytochemistry (Elsevier) 70, 935-939 (2009).
40 McDonald, L. A., Barbieri, L. R., Bernan, V. S., Janso, J., Lassota, P. & Carter, G. T. 07H239-A, a new cytotoxic eremophilane sesquiterpene from the marine-derived xylariaceous fungus LL-07H239. Journal of Natural Products 67, 1565-1567 (2004).
41 Lee, I. K., Yun, B. S., Kim, Y. H. & Yoo, I. D. Two neuroprotective compounds from mushroom Daldinia concentrica. Journal of Microbiology and Biotechnology 12, 692-694 (2002).
42 Quang Dang, N., Bach Dang, D., Hashimoto, T. & Asakawa, Y. Chemical constituents of the Vietnamese inedible mushroom Xylaria intracolorata. Natural Product Research 20, 317-321 (2006).
43 Edwards, R. L., Maitland, D. J. & Whalley, A. J. S. Metabolites of the higher fungi. Part 26. Cubensic acid, 3,7,11,15-tetrahydroxy-18-(hydroxymethyl)-2,4,6,10,14,16,20-heptamethyldocosa-4E,8E,12E,16E-tetraenoic acid, a novel polysubstituted C22 fatty acid from the fungus Xylaria cubensis (Mont.) Fr. with substituents and substitution pattern similar to the macrolide antibiotics. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 1411-1417 (1991).
44 Edwards, R. L., Maitland, D. J., Pittayakhajonwut, P. & Whalley, A. J. S. Metabolites of the higher fungi. Part 33. Grammicin, a novel bicyclic C7H6O4 furanopyranol from the fungus Xylaria grammica (Mont.) Fr. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 1296-1299 (2001).
45 Bodo, B., Tih, R. G., Davoust, D. & Jacquemin, H. Hypoxylone, a naphthyl-naphthoquinone pigment from the fungus Hypoxylon sclerophaeum. Phytochemistry 22, 2579-2581 (1983).
46 Edwards, R. L., Fawcett, V., Maitland, D. J., Nettleton, R., Shields, L. & Whalley, A. J. S. Hypoxyxylerone. A novel green pigment from the fungus Hypoxylon fragiforme (Pers.: Fries) Kickx. Journal of the Chemical Society, Chemical Communications, 1009-1010 (1991).
47 Anderson, J. R., Edwards, R. L. & Whalley, A. J. S. Metabolites of the higher fungi. Part 21. 3-Methyl-3,4-dihydroisocoumarins and related compounds from the ascomycete family Xylariaceae. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 2185-2192 (1983).
48 Chen, Y. S. The metabolic products of Rosellinia necatrix. II. The structure of rosellinic acid. Agricultural and Biological Chemistry 28, 431-435 (1964).
49 Edwards, H. G. M., Edwards, R. L., Hartley, M. J. & Quinn, M. Diene conformation in the naturally occurring tricarboxylic acid, telfairic acid, by Raman spectroscopy. Journal of Molecular Structure 789, 24-29 (2006).
50 Gunawan, S., Steffan, B. & Steglich, W. Xylaral, a hydroxyphthalide derivative from fruiting bodies of Xylaria polymorpha (Ascomycetes). Liebigs Annalen der Chemie, 825-827 (1990).
51 Jang, Y. W., Lee, I. K., Kim, Y. S., Lee, S., Lee, H. J., Yu, S. H. & Yun, B. S. Xylarinic acids A and B, new antifungal polypropionates from the fruiting body of Xylaria polymorpha. Journal of Antibiotics 60, 696-699 (2007).
52 Lee, I. K., Jang, Y. W., Kim, Y. S., Yu, S. H., Lee, K. J., Park, S. M., Oh, B. T., Chae, J. C. & Yun, B. S. Xylarinols A and B, two new 2-benzoxepin derivatives from the fruiting bodies of Xylaria polymorpha. Journal of Antibiotics 62, 163-165 (2009).
53 Edwards, R. L., Anderson, J. R. & Whalley, A. J. S. Metabolites of the higher fungi. Part 20. Chesterseine, 4-(4-methoxyphenoxy) buta-1,2-diene: an allenic ether from the fungus Hypoxylon chestersii. Phytochemistry 21, 1721-1723 (1982).
54 Fawcett, A. H., Yau, T. F., Mulemwa, J. N. & Tan, C. E. The free-radically prepared copolymers of acrylonitrile with furfuryl alcohol and similar furan derivatives. British Polymer Journal 19, 211-221 (1987).
55 Harris, G. H., Hoogsteen, K., Silverman, K. C., Raghoobar, S. L., Bills, G. F., Lingham, R. B., Smith, J. L., Dougherty, H. W., Cascales, C. & Pelaez, F. Isolation and structure determination of pycnidione, a novel bistropolone stromelysin inhibitor from a Phoma sp. Tetrahedron 49, 2139-2144 (1993).
56 Sun, P., Ye, W., Zhao, J., Pei, Y., Wang, Z., Chen, Y., Ogihara, Y. & Takeda, T. Studies on the constituents of Epimedium koreanum. Chemical & Pharmaceutical Bulletin 43, 703-704 (1995).
57 Rasser, F., Anke, T. & Sterner, O. Secondary metabolites from a Gloeophyllum species. Phytochemistry 54, 511-516 (2000).
58 Lee, C. K., Fang, J. M. & Cheng, Y. S. Norditerpenes from Juniperus chinensis. Phytochemistry 39, 391-394 (1995).
59 Chen, Y. L., Tan, C. H., Tan, J. J., Zhao, X. M., Jiang, S. H. & Zhu, D. Y. Two new diterpenoid glucosides from Clerodendranthus spicatus. Helvetica Chimica Acta 92, 2802-2807 (2009).
60 Pittayakhajonwut, P., Theerasilp, M., Kongsaeree, P., Rungrod, A., Tanticharoen, M. & Thebtaranonth, Y. Pughiinin A, a sesquiterpene from the fungus Kionochaeta pughii BCC 3878. Planta Medica 68, 1017-1019 (2002).
61 Wanner, R. M., Spielmann, P., Stroka, D. M., Camenisch, G., Camenisch, I., Scheid, A., Houck, D. R., Bauer, C., Gassmann, M. & Wenger, R. H. Epolones induce erythropoietin expression via hypoxia-inducible factor-1alpha activation. Blood 96, 1558-1565 (2000).
62 Baldwin, J. E., Mayweg, A. V. W., Neumann, K. & Pritchard, G. J. Studies toward the biomimetic synthesis of tropolone natural products via a hetero Diels-Alder reaction. Organic Letters 1, 1933-1935 (1999).
63 Ju, Y. M., Hsieh, H. M., Ho, M. C., Szu, D. H. & Fang, M. J. Theissenia rogersii sp. nov. and phylogenetic position of Theissenia. Mycologia 99, 612-621 (2007).
64 Hsieh, H. M., Lin, C. R., Fang, M. J., Rogers, J. D., Fournier, J., Lechat, C. & Ju, Y. M. Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily. Molecular Phylogenetics and Evolution 54, 957-969 (2010).
65 Cho, S. Y., Park, S. J., Kwon, M. J., Jeong, T. S., Bok, S. H., Choi, W. Y., Jeong, W. I., Ryu, S. Y., Do, S. H., Lee, C. S., Song, J. C. & Jeong, K. S. Quercetin suppresses proinflammatory cytokines production through MAP kinases and NF-kB pathway in lipopolysaccharide-stimulated macrophage. Molecular and Cellular Biochemistry 243, 153-160 (2003).
66 O'Brien, J., Wilson, I., Orton, T. & Pognan, F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. European Journal of Biochemistry 267, 5421-5426 (2000).
67 Vichai, V. & Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature Protocols 1, 1112-1116 (2006).
論文全文使用權限
  • 同意授權瀏覽/列印電子全文服務,於2013-08-12起公開。


  • 若您有任何疑問,請與我們聯絡!
    臺北醫學大學 圖書館 簡莉婷
    E-mail:etds@tmu.edu.tw
    Tel:(02) 2736-1661 ext.2519
    Fax:(02) 2737-5446