牛樟芝為台灣特有種真菌，近年來有越來越多不同的活性物質被發現，最受矚目的是三萜類化合物被發現具有抗腫瘤能力，所以有許多研究將牛樟芝放在對癌症的影響上。已發表的研究多直接以牛樟芝之菌絲體、子實體、發酵液或發酵液乾燥物等為實驗材料，然而對不同極性區分萃取物生理活性之探討相當有限。本論文利用索式萃取法、管柱層析法與半製備HPLC分離法將子實體粗萃取物中不同極性的成分進一步精萃出FK1~FK6等六個劃分 ，並測試對肝癌細胞株HepG2抑制細胞存活率的方式篩選出最有效區段為FK6。再將FK6進一步以半製備HPLC分離出兩個純度高於 95 % 的單一波峰FK6-1與FK6-2，測試後確定對HepG2有抑制作用的有效成分為FK6-1並計算其IC50約為26 μg/ml。以FK6-1處理HepG2細胞後以流式細胞儀偵測細胞週期與annexinV&PI雙染，結果HepG2細胞隨濃度上升趨向凋亡的比例也隨之上升。接著利用western blot證實FK6-1會活化HepG2細胞膜表面的Fas/FasL蛋白複合體並啟動其細胞凋亡機制及系列路徑。總結如下，本研究證實FK6-1首先活化Fas/FasL蛋白複合體而促使caspase-8表現，接著促使Bid活化成tBid，造成BAX表現上升、而Bcl-2表現下降，導致cytochrome c釋放到粒腺體外，活化caspase-9，並進一步活化下游的caspase-3，casepase-3被活化後引起ICAD失去活性，造成DNA斷裂成片斷，HepG2細胞逐漸走向凋亡。基於以上結果FK6-1應有機會進一步發展成新的抗肝癌藥物。

Taiwanofungus is a kind of unique fungi species which only grow in the inner cavity of the endemic species Cinnamomum kanehirae . The fruiting body of Taiwanofungus had been identified that contained several different physiological active compounds, especially the triterpenoids, which were found having anticarcinoma and antiangiogenesis abilities. Recently, more and more studies focused on the functions of using Taiwanofungus mycelium, fermented broth, or fruiting body’s extracts as experimental reagents in cancer cell lines study. Few of them focused on the fractionated extracts or purified components to elucidate the effectiveness on cancer cell lines . This study focused on the various partilly purified fractions, FK1~FK6, prepared from series extraction and purification steps by using Soxlet, column chromatography and Semi-preparative HPLC method. The primary results showed FK6 fraction was the most effective one on hepatic cancer cell line, HepG2. Subsequenly, FK6 was further separated into two single peaks with purity more then 95% subfractions samples, FK6-1 and FK6-2, by using Semi-preparative HPLC. The viabilites of HepG2 cancer cell line was inhibited by FK6-1 with IC50 values about 26 μg/ml. Then apoptosis induced by FK6-1 in HepG2 cells was characterized by flow cytometry cell cycle analysis and Annexin V/PI double staining in HepG2 cells. The result of western blot showed that FK6-1 activated the Fas/FasL apoptosis pathway in HepG2 cell line. The activation of Fas/FasL by FK6-1 induced caspase-8 activation. Caspase-8 activated tBid, and then tBid further stimulated with the increase of Bax and decrease of Bcl-2. As Bcl-2 family inactivated, cytochrome c released from mitochondria to cytoplasm and activated caspase-9. Finally, caspase-9 activated caspase-3 and inhibited the function of ICAD, breaking DNA into pieces. According to the results of this study, FK6-1 should have a great potential to develop into a new anticancer drug.

中文摘要 Ⅰ
Abstract Ⅱ
目錄 Ⅲ
表目錄 Ⅳ
圖目錄 Ⅴ
第一章 前言 1
第二章 文獻回顧 3
一、牛樟芝簡介 3
（一）、生物分類學中牛樟芝的分類 3
（二）、生長特徵 5
二、牛樟芝成份分析 6
（一）一般成份 6
（二）生理活性成份 6
1. 多醣（polysaccharides） 7
2. 三萜類化合物（triterpenoids） 8
（二）菌絲體與子實體成份比較 14
1. 一般成份 14
2. 金屬離子 16
3. 胺基酸 17
4. 固醇與脂肪酸 17
三、牛樟芝的功效 18
四、肝癌簡介 21
(一) 肝臟 21
(二) 肝硬化 21
(三) 肝癌 22
(四) 肝癌的治療方式 24
第三章 研究目的 25
第四章、實驗材料與儀器 27
一、 實驗材料 27
細胞株 (Cell lines) : 27
材料 : 27
藥品 : 27
抗體 (antibody) : 30
二、實驗耗材 31
三、實驗儀器 31
第五章、實驗方法 33
一、牛樟芝菌絲體萃取 33
超音波震盪萃取 33
二、牛樟芝子實體粉末萃取 33
索氏萃取法 33
管柱層析法 34
半製備HPLC分離法 35
三、HPLC分析 36
四、人類肝癌細胞株HepG2培養 36
DMEM細胞培養液配置 37
冷凍細胞解凍及活化 37
細胞繼代及分盤 37
細胞凍管保存 38
細胞存活率測試 ( MTT assay ) 38
細胞凋亡測試 39
顯微鏡鏡檢螢光染色細胞 39
細胞週期測試 40
細胞 annexinV&PI雙染測試 40
DNA萃取 41
DNA片斷化測試 42
五、西方點墨法 42
蛋白質萃取 42
蛋白質定量 43
蛋白質電泳及轉漬 43
蛋白質免疫呈色 43
六、統計分析 44
第六章、實驗結果 45
一、牛樟芝樣品分離 45
(一) 牛樟芝菌絲體與子實體乙醇萃取 45
(二) 牛樟芝子實體管柱層析、索氏萃取與半製備HPLC分離 45
(三) FK6半製備HPLC純化分離 45
二、細胞型態與存活率測試 ( MTT assay ) 46
三、細胞週期測試 (Flow Cytometry ) 48
四、DNA片斷化測試 ( DNA fragmentation ) 49
五、西方轉印法(Western blot) 50
第七章、結果討論 53
第八章、結論 87
第九章、未來展望 89
第十章 參考文獻 91


表目錄

第二章 文獻回顧
表2- 1 牛樟芝一般成份分析 15
表2- 2 牛樟芝微量金屬含量分析 16
表2- 3 牛樟芝胺基酸組成分析 17
表2- 4 牛樟芝與抗癌相關的研究摘錄 19
表3-1民國99年度十大癌症死因統計表 23


圖目錄


第二章 文獻回顧
圖2-1 牛樟芝生物分類學系統樹 5
圖2- 2 以β-1,3鍵合的D-葡聚醣骨架呈現螺旋型結構 8
圖2- 3 比較牛樟芝特有24(28)-en結構與靈芝三帖類差異 9
圖2- 4 三種以ergostane為骨架的三萜類化合物 10
圖2- 5發現三種新的三萜類化合物 11
圖2- 6 四種新的三萜類化合物 12
圖2- 7 以ergostane為骨架的新化合物 13
圖2- 8 具有神經保護功能的三萜類化合物 14
圖3- 1 肝炎、肝硬化與肝癌的關係 23
第五章、實驗方法
圖一、 索氏萃取裝置 34
圖二、管柱層析萃取裝置 35
圖三、半製備HPLC樣品分離裝置 36
第六章、實驗結果
圖1-1A菌絲體HPLC分析圖譜 61
圖1-1B子實體HPLC分析圖譜 62
圖1-2子實體HPLC分析圖譜分離區段選擇 63
圖1-3A FK1 HPLC分析圖譜 64
圖1-3B FK2 HPLC分析圖譜 65
圖1-3C FK3 HPLC分析圖譜 66
圖1-3D FK4 HPLC分析圖譜 67
圖1-3E FK5 HPLC分析圖譜 68
圖1-3F FK6 HPLC分析圖譜 69
圖1-4A FK6-1 HPLC分析圖譜 70
圖1-4B FK6-2 HPLC分析圖譜 71
圖2-1乙醇對HepG2細胞株存活率的影響 72
圖2-2菌絲體與子實體乙醇萃取物對HepG2細胞株存活率的影響 72
圖2-3牛樟芝分離物FK1~FK6對HepG2細胞株存活率的影響 73
圖2-4分離物FK6-1對HepG2細胞株存活率的影響 73
圖2-5分離物FK6-2對HepG2細胞株存活率的影響 74
圖2-6分析比較FK6中包含之分離物對HepG2細胞株存活率的影響 74
圖2-7比較FK6-1對HepG2與CCD-966SK細胞株存活率的影響 75
圖2-8倒立式顯微鏡鏡檢CCD-966SK與HepG2細胞 76
圖3-1在FK6-1作用下HepG2的細胞週期變化 77
圖3-2倒立式螢光顯微鏡鏡檢FK6-1作用後的HepG2細胞 78
圖3-3 HepG2螢光染色細胞凋亡比例 79
圖4-1 HepG2在FK6-1作用下產生DNA片斷化 80
圖5-1 DFF45/ICAD與P53表現量變化 81
圖5-2 Proaspase-3與Caspase-3表現量變化。 82
圖5-3 Procaspase-9與Caspase-9表現量變化 83
圖5-4 Cytochrome c表現量變化 84
圖5-5 Bcl-2、tBid與BAX表現量變化 85
圖5-6 Fas lignd ( FasL )與 Proaspase-8表現量變化 86
第八章、結論
FK6-1誘導HepG2引發凋亡的路徑圖 88

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