台灣株Theissenia cinerea醱酵液之生物活性成分研究

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Title:	台灣株Theissenia cinerea醱酵液之生物活性成分研究
Authors:	李亮欣
Contributors:	生藥學研究所
Date:	2010
Issue Date:	2010-10-21 10:37:49 (UTC+8)
Abstract:	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。 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.
Relation:	87頁
Description:	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 1. 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