進階搜尋


  查詢北醫館藏
系統識別號 U0007-2307201012180300
論文名稱(中文) 台灣株Theissenia cinerea醱酵液之生物活性成分研究
論文名稱(英文) Studies on the bioactive constituents of the fermented broth of Theissenia cinerea isolated in Taiwan
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 99
學期 2
出版年 99
研究生(中文) 李亮欣
研究生(英文) Liang-Hsin Lee
學號 M303097003
學位類別 碩士
語文別 中文
口試日期 2010-06-30
論文頁數 87頁
口試委員 委員-李慶國
委員-陳逸民
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 cinerea  theissenoic acid  theissenoxide  theissenolactone A  theissenolactone B  theissenolactone C 
英文關鍵字 iNOS  Theissenia cinere  theissenoic acid  theissenoxide  theissenolactone A  theissenolactone B  theissenolactone C  cytotoxicitycytotoxicity 
學科別分類
中文摘要 在曹 (2009) 的研究中,利用抑制一氧化氮合成酶 (inducible nitric oxide synthase, iNOS)的活性平台來篩選單離自本土的真菌,藉以找尋出具有抗發炎活性的真菌株,發現Theissenia cinerea (#89091602) 的醱酵培養液,對於離體RAW264.7細胞產生一氧化氮的活性,具有顯著的抑制作用。後續本研究中選擇以麥芽抽取物 (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一系列的分析、分離、純化與結構解析,計分離出9個化合物,分別為:theissenoic acid (1)、theissenoxide (2)、theissenolactone A (3)、theissenolactone B (4)、theissenolactone C (5)、5-hydroxymethylfurfural (6)、bis-2,5-hydroxymethylfuran (7)、succinic acid (8) 和tyrosol (9),其中1 - 5是過去未曾報導過的新化合物。在細胞毒殺活性上,4和5對於A549肺腺癌細胞具有中等強度的毒殺作用,其GI50分別為14.90與47.92 μM,此外,在iNOS的抑制實驗中,在100 ?慊/mL的濃度下,同樣發現4與5對RAW264.7有100%的毒殺作用,至於其劑量與活性間的相關性有待進一步探究。
英文摘要 In our preview studies, it was found that 13 strains of 34 tested fungus species exhibited significant anti-iNOS activity. Based on this findings, Theissenia cinerea (#89091602) was selected to investigate its bioactive constituents. A series of bioassay-guided chemical examination on the fermented broth of this fungus was carried out, which resulted in the isolation of five new compounds along with four known ones. Their structures were elucidated to be theissenoic acid (1)、 theissenoxide (2)、theissenolactone A (3)、theissenolactone B (4)、theissenolactone C (5)、5-hydroxymethylfurfural (6)、bis-2,5-hydroxymethylfuran (7)、succinic acid (8) and tyrosol (9) on the basis of spectroscopic analysis. Of these compounds identified, compounds 1-5 were new chemical entities. The bioactivities of all the pure isolates were further evaluated. Compounds 4 and 5 exhibited moderate cytotoxicities against A549 lung cancer cell line with GI50 values of 14.90 and 47.92 μM, respectively.

論文目次 總目錄
謝誌…………………………………………………….......................................Ⅰ
中文摘要................................................................................................................Ⅲ
Abstract..................................................................................................................Ⅳ
總目錄....................................................................................................................Ⅴ
表目錄....................................................................................................................Ⅶ
圖目錄....................................................................................................................Ⅷ
縮寫表……………………………………………………....................................Ⅸ
壹、緒論與研究目的................................................................................................1
貳、親緣相同 (近) 真菌之天然物文獻回顧.........................................................5
参、實驗結果與討論..............................................................................................10
3.1.1 Theissenoic acid (1) 之結構解析……………………………..13
3.1.2 Theissenoxide (2) 之結構解析……………………………….24
3.1.3 Theissenolactone A (3) 之結構解析………………………….35
3.1.4 Theissenolactone B (4) 之結構解析………………………….45
3.1.5 Theissenolactone C (5) 之結構解析………………………….55
3.2 化合物純質抑制一氧化氮產生與A549肺腺癌之細胞毒性分析結果...…65
3.2.1化合物純質抑制一氧化氮產生結果…………………………….65
3.2.2化合物純質對A549肺腺癌之細胞毒性分析結果 ……………66
3.3討論…………………………………………………………………………..67
肆、實驗部份

4.1 儀器設備與試劑……………………………………………………….70
4.2 培養基配置………..…………….…………….......................................71
4.3真菌培養條件……..…………….…………….........................................71
4.4 真菌菌株之萃取與分離...........................................................................72
4.5 一氧化氮 (NO) 濃度之測定: Griess reagent assay...............................76
4.6 各成分之物理數據...................................................................................79
参考文獻................................................................................................................84













表目錄
表 1、1H- and 13C-NMR data of 1。........………………...…………......…......17
表 2、13C-NMR data of 2 2, (+)-eupenoxide and (-)-3',4'-dihydrophomoxide。
……….…………...…………………………………..…...............…27表 3、1H- and 13C-NMR data of 2。........………………...…………............…28
表 4、1H- and 13C-NMR data of 3。........………………...……………...….....38
表 5、1H- and 13C-NMR data of 4。........………………...……………..…......48
表 6、1H- and 13C-NMR data of 5。........………………...…………..……......58
表 7、 化合物純質對A549肺腺癌之細胞毒性分析結果。…….…………66
表 8、化合物3-5與文獻中化學合成類似物的結構比較。..............................69
表 9、Theissenia cinerea (#89091602) 培養液之乙酸乙酯萃出物以膠濾管柱
層析分離結果。.......................................................................................72










圖目錄
圖 1、#89091602菌株培養液之乙酸乙酯萃取物的分離流程。...................11
圖 2、化合物1之高解析電灑電離質譜。………………………………….18
圖 3、化合物1之紅外光譜。…......................................................................18
圖 4、化合物1之氫譜。………......................................................................19
圖 5、化合物1之碳譜。…………..………....................................................19
圖 6、化合物1之HSQC圖譜。…………..…………………………............20
圖 7、化合物1之HMBC圖譜。….…………..……………………………..21
圖 8、化合物1之COSY圖譜。………..……………...………………….…22
圖 9、化合物1之NOESY圖譜。.…………..…………………....................23
圖 10、化合物2之高解析電灑電離質譜。.…….…..………………………29
圖 11、化合物2之紅外光譜。…....................................................................29
圖 12、化合物2之氫譜。………....................................................................30
圖 13、化合物2之HSQC圖譜。………..………..................................…..30
圖 14、化合物2之碳譜。…………..…………………..................................31
圖 15、化合物2之HMBC圖譜。….…………..……………………………32
圖 16、化合物2之COSY圖譜。………..……………...…………………...33
圖 17、化合物2之NOESY圖譜。.…………..……………………………..34
圖 18、化合物3之高解析電灑電離質譜。…..……..………………………39
圖 19、化合物3之紅外光譜。…....................................................................39
圖 20、化合物3之氫譜。………....................................................................40
圖 21、化合物3之HSQC圖譜。…………..………......................................40
圖 22、化合物3之碳譜。…………..………………………………..............41
圖 23、化合物3之HMBC圖譜。….…………..…………………...……….42
圖 24、化合物3之COSY圖譜。………..……………...……………...……43
圖 25、化合物3之NOESY圖譜。.…………..…………………...................44
圖 26、化合物4之電灑電離質譜。.….…..……..…………………..………49
圖 27、化合物4之紅外光譜。…....................................................................49
圖 28、化合物4之氫譜。………....................................................................50
圖 29、化合物4之碳譜。…………..………..................................................50
圖 30、化合物4之HSQC圖譜。…………..………………….................... 51
圖 31、化合物4之HMBC圖譜。….…………..……………………………52
圖 32、化合物4之COSY圖譜。………..……………...…………………...53
圖 33、化合物4之NOESY圖譜。.…………..…………………..................54
圖 34、化合物5之電灑電離質譜。..….……..………….........……………..59
圖 35、化合物5之紅外光譜。…....................................................................59
圖 36、化合物5之氫譜。………....................................................................60
圖 37、化合物5之碳譜。…………..………..................................................60
圖 38、化合物5之HSQC圖譜。…………..………………………………..61
圖 39、化合物5之HMBC圖譜。….…………..……………………………62
圖 40、化合物5之COSY圖譜。………..……………...…………………...63
圖 41、化合物5之NOESY圖譜。.…………..…………………..................64
圖 42、化合物1、2、4、5抑制一氧化氮的產生及RAW 264.7細胞存活
率的關係。…………………………………………………………65
圖 43、Griess Reaction 之反應圖。………………………………………77

參考文獻 1. Sohn M. J., Hur G. M., Byun H. S., Kim W. G., Cyclo(dehydrohistidyl-L- tryptophyl) inhibits nitric oxide production by preventing the dimerization of inducible nitric oxide synthase. Biochemical pharmacology, 2008. 75, 923- 930.
2. Rosselli M., Keller P. J., Dubey R. K., Role of nitric oxide in the biology, physiology and pathophysiology of reproduction. Human Reproduction Update , 1998. 4, 3-24.
3. deRojas Walker T., Tamir S.J., Wishnok H., Tannenbaum J. S., Nitric Oxide Induces Oxidative Damage in Addition to Deamination in Macrophage DNA. Chemical Research in Toxicology, 1995. 8, 473-7.
4. Spinas G. A., Laffranchi R., Francoys I., David I., Richter C., Reinecke M.,
The early phase of glucose-stimulated insulin secretion requires nitric oxide. Diabetologia, 1998. 41, 292-299.
5. Suh N., Honda T., Finlay H. J., Barchowsky A., Williams C., Benoit N. E., Xie Q. W., Nathan C., Gribble. G. W., Sporn M. B., Novel Triterpenoids Suppress Inducible Nitric Oxide Synthase (iNOS) and Inducible Cyclooxygenase (COX-2) in Mouse Macrophages. Cancer Research, 1998. 58, 717-723.
6. Landino L. M., Crews B. C., Timmons M. D., Morrow J. D., Marnett L. J., Peroxynitrite, the coupling product of nitric oxideand superoxide, activates prostaglandin biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 1996. 93, 15069- 15074.
7. Kleinert H., Pautz A., Linker K., Schwarz P. M., Regulation of the expression of inducible nitric oxide synthase. European Journal of Pharmacology, 2004. 500, 255-266.
8. Stuehr D. J., Griffith O. W., Mammalian nitric oxide synthases. Biochimical et
Biophys Acta, Bioenergetics, 1999. 1411, 217–230.
9. Quang D. N., Harinantenaina L., Nishizawa T., Hashimoto T., Kohchi C., Soma G., Asakawa Y., Inhibition of nitric oxide production in RAW 264.7 cells by azaphilones from xylariaceous fungi. Biological & Pharmaceutical Bulletin, 2006. 29, 34-37.
10. Marletta M. A., Yoon P. S., Iyengar R., Leaf C. D., Wishnok J. S., Macrophage oxidation of L-arginine to nitrite and nitrate: nitric
oxide is an intermediate. Biochemistry, 1988. 27, 8706-8711.
11. Nguyen T., Brunson D., Crespi C. L., Penman B. W., Wishnok J. S., Tannenbaum S. R., DNA damage and mutation in human cells exposed to nitric oxide in vitro. Proceedings of the National Academy of Sciences of the United States of America, 1992. 89, 3030-3034.
12. Nathan C., Xie Q. W., Nitric oxide synthases: roles, tolls, and controls.
Cell, 1994. 78, 915-918.
13. Baue J. A., Booth B. P., Fung H. L., Nitric oxide donors: Biochemical Pharmacology and Therapeutics. Advances in Pharmacology (San Diego), 1995. 34, 361-381.
14. Carlson T. J., Billings R. E., Role of nitric oxide in the cytokine
-mediated regulation of cytochrome P-450. Molecular Pharmacology, 1996. 49, 796-801.
15. Dawson V. L., Dawson T. M., Physiological and Toxicological Actions
of Nitric oxide in the Central Nervous System. Advances in Pharmacology (San
Diego), 1995. 34, 323-342
16. Duval D. L., Miller D. R., Collier J., Billings R. E., Characterization of
hepatic nitric oxide synthase: identification as the cytokine-inducible form primarily regulated by oxidants. Molecular Pharmacology, 1996. 50, 277-284.
17. Hoang L. M., Mathers D. A., Internally applied endotoxin and the activation
of BK channels in cerebral artery smooth muscle via a nitric oxide-like pathway. British Journal of Pharmacology, 1998. 123, 5-12.
18. Ishii R., Horie M., Koyama K., Ishikawa Y., Kitanaka S., Inhibitory effects of fungal bis(naphtho-gamma -pyrone) derivatives on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-gamma. Biological & Pharmaceutical Bulletin, 2005. 28, 786-790.
19. Rukachaisirikul V., Sommart U., Phongpaichit S., Hutadilok Towatana N., Rungjindamai N., Sakayaroj J., Metabolites from the Xylariaceous Fungus PSU-A80. Chemical & Pharmaceutical Bulletin, 2007. 55, 1316-1318.
20. 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, 2006. 69, 1198-1202.
21. Rogers J. D., Ju Y.M., http://mycology.sinica.edu.tw/Xylariaceae
22. Quang D. N.,Stadler M., Fournier J., Asakawa Y., Cohaerins C–F, four azaphilones from the xylariaceous fungus Annulohypoxylon cohaerens. Tetrahedron Letters, 2006. 62, 6349-6354.
23. Lin Y., Wu X., Feng S., Jiang G., Luo J., Zhou S., Vrijmoed L. L., Jones E.B., Krohn K., Steingrover K., Zsila F., Five Unique Compounds: Xyloketals from Mangrove Fungus Xylaria sp. from the South China Sea Coast. Journal of Organic Chemistry, 2001. 66, 6252-6256.
24. Quang D. N., Hashimoto T., Tanaka M., Stadler M., Asakawa Y., Cyclic azaphilones daldinins E and F from the ascomycete fungus Hypoxylon fuscum (Xylariaceae). Phytochemistry, 2004. 65, 469-473.
25. Quang D. N., Hashimoto T., Tanaka M., Stadler M., Asakawa Y., Cyclic azaphilones daldinins E and F from the ascomycete fungus Hypoxylon fuscum (Xylariaceae). Phytochemistry, 2004. 65, 469-473.
26. Krohn K., Muhammad R., Total synthesis of (+)-xyloketal D, a secondary metabolite from themangrove fungus Xylaria sp. Tetrahedron Letters, 2004. 45, 293-294.
27. 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., L-696, 474, A novel cytochalasin as an inhibitor of HIV-1 protease III Biological activity. Journal of Antibiotics, 1992. 45, 686-691.
28. Kimura Y., Nakajima H., Hamasaki T., Structure of Rosellichalasin, a New Metabolite Produced by Rosellinia necatrix. Agricultural and Biological Chemistry, 1989. 53, 1699-1701.
29. Quang D. N., Hashimoto T., Jacques F., Stadler M., Niko R., Yoshinori A., Sassafrins A–D, new antimicrobial azaphilones from the fungus Creosphaeria sassafras. Tetrahedron, 2005. 61, 1743-1748.
30. Quang D. N., Hashimoto T., Asakawa Y., Inedible Mushrooms: A Good Source of Biologically Active Substances. Chemical Record, 2006. 6, 79-99.
31. Ma C. M., Nakamura N., Min B. S., Hattori M., Triterpenes and Lignans from Artemisia caruifolia and Their Cytotoxic Effects on Meth-A and LLC Tumor Cell
Lines. Chemical & Pharmaceutical Bulletin, 2001. 49, 183-187
32. Stadler M., Quang D. N., Tomita A., Hashimoto T., Asakawa Y., Changes in secondary metabolism during stromatal ontogeny of Hypoxylon fragiforme. Mycological Research, 2006. 110, 811 - 820.
33. Ju Y. M., Rogers J. D., Hsieh H. M., The genus Theissenia: T. pyrenocrata, T. cinerea sp. nov., and T. eurima sp. nov. Mycologia, 2003. 109-116.
34. 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, 2007. 99, 612-621.
35. Mehta G., Roy S., Davis R. A., On the stereostructures of (+)-eupenoxide and (-)-30,40-dihydrophomoxide: a caveat on the spectral comparisons of oxygenated cyclohexenoids. Tetrahedron Letters, 2008. 49, 5162-5164.
36. Gao X., Snider B. B., Syntheses of (-)-TAN-2483A, (-)-Massarilactone B, and the Fusidilactone B Ring System. Revision of the Structures of and Syntheses of (±)-Waol A (FD-211) and (±)-Waol B (FD-212). Journal of Organic Chemistry, 2004. 69, 5517-5527.
37. Gao X., Nakadai M., Snider B. B., Synthesis of (-)-TAN-2483A. Revision of the Structures and Syntheses of (±)-FD-211 (Waol A) and (±)-FD-212 (Waol B). Organic Letters, 2003. 5, 451-454.
38. Liu Z., Jensen P. R., Fenical W., A cyclic carbonate and related polyketides from a marine-derived fungus of the genus Phoma. Phytochemistry, 2003. 64, 571-574.
39. Brain J. M., Stembach D. D., Andrew T. M., Synthetic and Kinetic Studies of Substituent Effects in the Furan Intramolecular Diels-Alder Reaction. Tetrahedron, 1994. 50, 6767-82.
40. Allan H. F., Yau F. T., Jane N. M., Eng T. C., The free-radically prepared
copolymers of acrylonitrile with furfuryl alcohol and similar furan derivatives.
Journal of British Polymer, 1987. 19, 211-21.
41. Dominika B., Anna K. D., Wanda ., and Adam G. K., Investigation of a wide
spectrum of inherited metabolic disorders by 13C NMR spectroscopy. Acta
Biochimica Polonica, 2008. 55, 107-18.
42. Falk Rasser, Timm Anke, Olov Sterner. Secondary metabolites from a
Gloeophyllum species. Phytochemistry, 2000. 54, 511-516.

論文全文使用權限
  • 同意授權瀏覽/列印電子全文服務,於2012-07-29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