靈芝 (Ganoderma) 是一種普遍的藥用蕈類，為亞洲國家使用超過了兩千年的傳統中草藥。先前的研究顯示靈芝主要抗癌的成份為多醣體與三萜類化合物。但不同種類的靈芝可能因活性成份的不同導致醫藥活性差異大，因此本論文探討松杉靈芝 (Ganoderma tsugae) 的萃取物是否可抑制人類大腸癌及卵巢癌細胞的生長，以及是何種萃取物成份抑制了這些癌細胞的生長。本論文以乙醇萃取靈芝子實體的粉末，得到靈芝乙醇萃取物；此靈芝乙醇萃取物溶於50%乙醇後再分別以乙酸乙酯 (EA) 與正丁醇進行液相分配 (partitioning)。分別得到EA層、 正丁醇層與水層。將EA層、正丁醇層與水層分別減壓濃縮得三種萃取物。再根據MTT assay結果選擇EA層做柱狀色層分析，得10種fractions，再分別做MTT assays，其中Fraction G對大腸癌與卵巢癌細胞有較佳的生長抑制效果。最後選取Fraction G以高效能液相層析法、液相層析-串聯質譜及核磁共振法來確認化合物種。結論為松杉靈芝萃取物可抑制人類卵巢癌及大腸癌細胞的生長，但其中的 ganoderic acid C2成份是否為有效成份，仍有待進一步的實驗證實。

Ganoderma (Lingzhi) is a popular medicinal mushroom used as a traditional Chinese medicine (TCM) in Asian countries over the past two millennia. Previous studies have shown that the main anticancer constituents of Ganoderma are polysaccharides and triterpenoids. However, medicinal activities of Ganoderma may vary significantly from one species to another, mainly due to differences in the active ingredients. Therefore, this thesis investigated whether the ethanol extract of Ganoderma tsugae (GTE) could cause the growth inhibition of human colorectal and ovarian cancer cells followed by unraveling its active ingredients inside? The GTE was dissolved in 50% ethanol and then partitioned with ethyl acetate (EA), n-butanol (n-BuOH) in turn to obtain EA, n-BuOH and water fractions, respectively. Based on the results of MTT assay, the active EA fraction was collected and further separated into 10 fractions by column chromatography. The biological activity of each fraction was determined by MTT assay. Among the 10 fractions, fraction G has the best growth inhibition effect on human colorectal and ovarian cancer cells. Finally, HPLC (high performance liquid chromatography), LC-MS/MS (liquid chromatography-mass spectrometry / mass spectrometry) and NMR (nuclear magnetic resonance) were used to confirm the chemical structure of the peak ingredient. In conclusion, the GTE can cause growth inhibition of human ovarian and colorectal cancer cells. However, the inhibiting effect of the GTE ingredient ganoderic acid C2 remains to be confirmed by further investigation.

中文摘要 i
英文摘要 ii
誌謝 iii
表目錄 viii
圖目錄 ix
縮寫表 xi
第一章：緒論 12

第一節、研究緣起與目的 12

第二節、靈芝 13

壹、靈芝概述及分類 14

貳、靈芝之化學組成及生理功能 15

第二章：材料與方法 17
第一節、實驗材料 17

壹、細胞株 17

貳、松杉靈芝 17

參、主要儀器 17

肆、化學試劑藥品 18

第二節、實驗方法 19

壹、 松杉靈芝溶劑的萃取與液相分配 (partition) 19

貳、 細胞株培養 19

參、測量松杉靈芝萃取物對細胞株的半數抑制濃度 (IC50) 20

肆、松杉靈芝乙酸乙酯層之管柱色層分離試驗(column chromatography) 21

伍、測量松杉靈芝B到G層對細胞株的半數抑制濃度 (IC50) 21

陸、高效能液相層析分析 (High Performance Liquid chromatography, HPLC) 22
柒、核磁共振分析 (Nuclear Magnetic Resonance, NMR) 22

捌、液相層析-質譜/質譜串聯 (liquid chromatography – mass spectrometry/mass spectrometry, LC-MS/MS) 22
第三章：結果 23
第一節、松杉靈芝萃取物對癌細胞株的半數抑制濃度 (IC50) 23

壹、松杉靈芝乙醇萃取物、乙酸乙酯層萃取物、正丁醇層萃取

物及水層萃取物對癌細胞的半數抑制濃度 (IC50) 23

貳、松杉靈芝乙酸乙酯萃取物B到G層對癌細胞株的半數抑制濃度 (IC50) 23

第二節、松杉靈芝G層的萃取物對卵巢癌與大腸癌細胞株最有效成份

的結構研究 24

第三節、驗證是否ganoderic acid C2對卵巢癌與大腸癌細胞株為有效的抑制物 26

第四章：討論 27
第一節、松杉靈芝萃取物對癌細胞株的半數抑制濃度 (IC50)實驗的誤差
27


第二節、液相層析法 (Liquid Chromatography, LC) 的原理與分離乙酸乙酯萃取物時所造成的誤差 27



第三節、逆向高效能液相層析法的原理 (Reversed-Phase High

Performance Liquid Chromatography, R-P HPLC) 28

第四節、液相層析-質譜/質譜串聯 (liquid chromatography - mass spectrometry / mass spectrometry, LC-MS/MS) 30

第五節、為何ganoderic acid C2對卵巢癌细胞株無法有效抑制之探討 31
第六節、HPLC光譜圖上的化合物以UV偵測並收集有其瓶頸 32
第五章：結論 33
第六章：參考文獻 34
第七章：圖表 37

第一節、表 37

第二節、圖 39
附錄 58
附錄ㄧ、台灣地區常見野生靈芝一覽表 (許瑞祥教授發表) 58

附錄二、靈芝的主要成份及療效 59

附錄三、細胞株培養方法 60

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