樟芝（Antrodia cinnamomea）為台灣知名的傳統藥用真菌，民間長久認為其對食物中毒、腹瀉、嘔吐和農藥中毒等具有解毒作用。本研究首先以脂多醣（lypopolysaccharides）誘導小鼠急性發炎的動物模式，評估樟芝子實體抽出物之抗發炎活性，試驗結果發現樟芝乙醇抽出物具有顯著的抗發炎活性，可有效抑制小鼠體內因LPS所誘導的一氧化氮生成酵素、第二型環氧酵素和核轉錄因子-κB等酵素之表現。接著並以活性為導向的分離策略，從樟芝子實體乙醇抽出物中分離出具抗發炎活性成分antrocamphin A，續利用LPS誘導小鼠巨噬細胞（RAW 264.7）產生發炎的模式，解析antrocamphin A之抗發炎機制，結果證實antrocamphin A確可有效抑制一氧化氮自由基和前列腺素的生成，透過蛋白質表現分析得知，antrocamphin A之抗發炎活性是經由抑制一氧化氮生成酵素和第二型環氧酵素的mRNA表現，進而抑制了一氧化氮生成酵素、第二型環氧酵素和核轉錄因子-κB等酵素之表現。本研究為首次以動物模型證實樟芝抽出物可抑制動物體內之發炎反應，並分離獲得其抗發炎活性成分與釐清其作用機制，對於未來進一步的利用具有相當的參考價值。

Antrodia cinnamomea is a well-known traditional medicinal fungus in Taiwan. It has been used for centuries as detoxificant for food poisoning, diarrhea, vomiting and agrochemical poisoning. In the present study, the LPS-challenged ICR mice acute inflammation model was used to evaluate the anti-inflammatory activity of A. cinnamomea. The ethanol extract of A. cinnamomea was proved to have a significant inhibition on iNOS, COX-2, and NF-κB expression in the LPS-induced acute inflammatory mice. To understand the anti-inflammatory mechanism and active compounds of A. cinnamomea, antrocamphin A was purified from previous ethanol extraction using bioactivity-guided fractionation. As results, the antrocamphin A could significantly inhibit NO and PGE2 autacoids production in LPS-induced RAW 264.7 macrophage cells. Meanwhile, the mRNA and protein expression levels of iNOS and COX-2 were inhibited by antrocamphin A in a dose-dependent manner. Antrocamphin A also reduced the translocation of NF-κB induced by LPS, which was associated with the prevention of the degradation of I-κB, and subsequently decreased p65/p50 proteins level in the nucleus. This is the first report demostrating the A. cinnamomea could significant reduce the inflammation in animal. Moreover, the mechanism of its anti-inflammatory compound, antrocamphin A, was also revealed. which could be a valuable reference for developing the pharmaceutical or functional food of A. cinnamomea in the future.

目次.....................................................i
圖表目次................................................iv
摘要.....................................................1
Summary..................................................2
專有名詞縮寫.............................................3
一、前言.................................................4
二、文獻回顧.............................................6
（一）樟芝藥理活性與成分研究之探討.......................6
1.抗癌活性...........................................6
2.抗氧化與抗發炎活性.................................7
3.樟芝成分研究.......................................8
（二）抗發炎活性評估與細胞毒性試驗......................11
1.抗發炎活性評估....................................11
2.細胞毒性試驗......................................12
三、材料與方法..........................................14
（一）試驗材料..........................................14（二）成分萃取與分離....................................14
1.樟芝乙醇抽出物製備................................14
2.液相-液相分配（Liquid–liquid partition）萃取.....14
3.管柱層析（Column chromatography, CC）.............15
4.薄層層析（Thin layer chromatography, TLC）........15
5.高效能液相層析（High performance liquid chromatography, HPLC）與核磁共振（Nuclear magnetic resonance, NMR）光譜鑑定................................15
6.Antrocamphin A定量分析............................16
（三）急性體內抗發炎試驗................................17
1.小鼠血清內一氧化氮自由基（NO）生成抑制............17
2.小鼠肝臟蛋白萃取與蛋白表現量分析..................18（四）抗發炎活性成分之作用機制探討......................19
1.抑制LPS誘導巨噬細胞產生NO自由基之試驗.............19
2.Prostaglandin E2（PGE2）生成抑制試驗..............20
3.全細胞及核內、外蛋白萃取..........................20
4.蛋白表現量分析（西方墨點法）......................21
5.mRNA表現量分析（RT - PCR）........................22（五）統計分析..........................................22
四、結果與討論..........................................23（一）樟芝乙醇抽出物抑制LPS誘導小鼠發炎活性之探討.......23
1.乙醇抽出物抑制小鼠血清中一氧化氮生成量............23
2.乙醇抽出物抑制小鼠體內急性發炎之機制探討..........25（二）樟芝抗發炎活性成分分析............................27
1.樟芝抽出物、乙酸乙酯層與水層抑制LPS誘導小鼠巨噬細胞產生NO自由基之活性........................................27
2.乙酸乙酯層之各分離部抑制LPS誘導小鼠巨噬細胞產生NO自由基......................................................31
3.化合物Antrocamphin A之分離與鑑定..................33
4.Antrocamphin A定量分析............................36
5.Antrocamphin A抑制LPS誘導小鼠巨噬細胞產生NO自由基.38
6.Antrocamphin A抑制LPS誘導小鼠巨噬細胞產生PGE2.....41
7.Antrocamphin A抑制LPS誘導小鼠巨噬細胞產生發炎因子iNOS與COX-2的mRNA...........................................43
8.Antrocamphin A抑制LPS誘導小鼠巨噬細胞產生發炎因子iNO與COX-2蛋白表現量.......................................45
9.Antrocamphin A抑制NF-κB路徑相關蛋白表現量.........46
五、結論................................................50
六、參考文獻............................................52

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