中國橄欖具有抗菌、抗病毒、抗發炎與解毒功效。先前的研究顯示其含有豐富的多酚類物質與體外抗氧化功效成分。因此，本研究目的為探討中國橄欖抗癌功能之有效成分。本實驗利用WST-1 assay作為抗癌功能之篩選平台，結果顯示，以甲醇-乙酸乙酯區分層(OM-EtOAc)進行Diaion HP-20大孔樹脂管柱層析後，所得次區分層OM-EtOAc-C和OM-EtOAc-D的抗腫瘤效果明顯優於其它次區分層。再經由90% 的Hexane/Ethyl acetate 進行沉澱， 得到上清液與沉澱後， 發現
OM-EtOAc-C的上清液對於細胞抑制效果效果優於沉澱部分，而OM-EtOAc-D的上清液與沉澱部分皆有細胞抑制效果，透過HPLC與TLC找出適當分離條件後，最後以silica gel 進行管柱層析，所得OM-EtOAc-C2b抑制細胞增生效果明顯優於OM-EtOAc-C的上清液，然而OM-EtOAc-D的上清液的次區分層之抑制細胞增生效果並無優於OM-EtOAc-D 的上清液。最後選用OM-EtOAc-C2b 進行LC-SPE-NMR與LC-MS的化學結構鑑定，共鑑定出共9個化合物，其中有2個化合物須更進一步確認分子式為何，並進行細胞存活率分析。在動物實驗方面，利用大腸癌細胞轉殖於Balb/c 6週齡公鼠，餵食高劑量(300mg/kg bw/day) 的OM-EtOAc與低劑量(150mg/kg bw/day) 的OM-EtOAc，4週後發現腫瘤細胞與腫
瘤組相比有明顯變小，推測可能進行細胞凋亡或壞死途徑。綜合上述，中國橄欖確實具有抗腫瘤增生效果，其具有功效的成分為gallic acid, methyl gallate, ellagic acid, esculetin, scopoletin。

Chinese olive is a traditional medicine material, exhibiting beneficial functions in
anti-bacterium, anti-virus, anti-inflammation and detoxification. Previous studies have
displayed Chinese olive a phenolic compounds rich resource for providing
antioxidative activity. However, the role of Chinese olive in anti-cancer remains
unknown. Therefore, our aim is to search for compounds with anti-proliferation effect
in Chinese olive. In this study, we used WST-1 assay to examine the anti-proliferation
function of compounds within Chinese olive. Firstly, we obtained the ethyl acetate
fraction from Chinese olive methanolic extract and named it as OM-EtOAc. We then
found that the subfraction of OM-EtOAc-C and OM-EtOAc-D from OM-EtOAc
through Diaion HP-20 column chromatography exhibited better antitumor effect than
the other subfractions. These two subfractions were then precipitated by 90%
Hexane / Ethyl acetate. The supernatant of OM-EtOAc-C revealed superior anti-tumor
activity to the precipitate, whereas both the supernatant and precipitate of
OM-EtOAc-D displayed comparable effect. We then used HPLC and TLC spot to
titrate appropriate separation conditions for separating the supernatants of
OM-EtOAc-C and OM-EtOAc-D (OM-EtOAc-C2 and OM-EtOAc-D2), followed by
chromatography on silica gel. The OM-EtOAc-C2b revealed better effect thanOM-EtOAc-C2, however, no fraction exhibited higer anti-tumor activity than OM-EtOAc-D2. Finally, we choose OM-EtOAc-C2b to identify functionalcompounds using LC-SPE-NMR and LC-MS, and 12 compounds were identified.Among the compounds identified, three remained to be further analyzed to confirm
the chemical structure. In animal experiments, We also implanted mouse CT26 colon cancer cells to BALB/c mice and allowed them to form tumors. The tumor-bearing mice were treated with either high dose of OM-EtOAc (300mg/kg bw/day) or lowdose of (150mg/kg bw/day) OM-EtOAc. Compared with control mice, OM-EtOActreated mice beared significantly smaller tumor after four-week treatment. In conclusion, Chinese olive has anti-tumor proliferative effect, and the functional compounds includee gallic acid, methyl gallate, ellagic acid, esculetin, scopoletin.

表目錄……………………………………………..………………………..………………………………………..……i
圖目錄……………………………………………..………………………..………………………………………….…ii
摘要 .............................................................. 1
Abstract .......................................................... 2
第一章 前言 ....................................................... 4
第二章文獻回顧 ................................................... 5
第一節、中國橄欖................................................................................................ 5
一、中國橄欖中的成分.............................................................................. 5
二、中國橄欖的體外及體內試驗.............................................................. 6
(一) 體外試驗 ....................................................................................... 6
1.抗氧化試驗................................................................................. 6
2.抗腫瘤增生與抗發炎試驗......................................................... 6
(二) 體內試驗...................................................................................... 6
1.肝毒性試驗................................................................................ 6
第二節、其他橄欖屬(Canarium)相關研究 ......................................................... 7
(一) 體外試驗.................................................................................... 10
1.抗氧化試驗............................................................................... 10
2. 抗菌試驗................................................................................. 10
3. 抗腫瘤增生試驗..................................................................... 11
(二) 體內試驗.................................................................................... 11
1.抗糖尿病................................................................................... 11
2.抗疼痛試驗............................................................................... 11
3. 肝毒性試驗............................................................................. 11
4. 心血管疾病試驗..................................................................... 12
第三節、大腸直腸癌.......................................................................................... 12
一、大腸直腸癌與發炎反應之關係.......................................................... 13
(一) Chemokines .................................................................................. 14
(二) Toll-like receptors ......................................................................... 14
(三) TIR8 ............................................................................................. 15
(四)IL-6 ................................................................................................ 15
(五) NF-κB ........................................................................................... 15
(六) Cyclooxygenase-2 ( COX2 ) ........................................................ 16
二、大腸癌與免疫反應之關係.................................................................. 17
(一)腫瘤白血球浸潤(tumor leukocyte infiltration(TILs)).................. 17
(二) 腫瘤相關巨噬細胞Tumor-associated Macrophages(TAM) ...... 18
三、參與大腸直腸癌反應路徑.................................................................. 19
(一) Wnt signaling pathway ................................................................. 19
(二)Notch signaling ............................................................................. 20
(三)BMP signaling ............................................................................... 20
第三章實驗動機與目的 ............................................ 25
一、實驗動機............................................................................................ 25
二、實驗目的............................................................................................ 25
二、實驗架構............................................................................................ 25
第四章材料與方法............................................................................................ 26
4-1 中國橄欖萃取 ............................................................................................... 26
一、實驗材料...................................................................................................... 26
1.化學藥品與溶劑............................................................................................... 26
2.儀器設備........................................................................................... 26
二、中國橄欖以不同溶劑萃取及區分層、次區分層與細區分層的製備
...................................................................................................................... 26
1. 中國橄欖萃取大量製備(進行細胞抗增生活性成份之製備與分離
使用) .................................................................................................... 26
2. 中國橄欖甲醇萃取物乙酸乙酯區分層的管柱層析..................... 27
3. 中國橄欖甲醇萃取物乙酸乙酯次區分層的沉澱反應與管柱層析
.............................................................................................................. 28
4-2 以化學分析檢驗中國橄欖之抗增生與抗發炎的能力 ............................... 29
一、實驗材料.............................................................................................. 29
1.化學藥品與溶劑............................................................................... 29
2.儀器設備........................................................................................... 29
二、化學分析.............................................................................................. 30
1.總多酚類化合物含量測定............................................................... 30
2.總黃酮類含量測定........................................................................... 30
4-3 以細胞模式檢驗中國橄欖之抗增生與抗發炎的能力 ............................... 31
一、實驗材料.............................................................................................. 31
1.化學藥品與溶劑............................................................................... 31
2.儀器設備........................................................................................... 32
二、細胞來源與培養.................................................................................. 32
1.繼代培養........................................................................................... 32
2.細胞計數........................................................................................... 33
3.細胞冷凍保存及解凍....................................................................... 33
二、細胞分析實驗...................................................................................... 34
1.細胞存活率測定.............................................................................. 34
2.細胞發炎反應之測定...................................................................... 34
4-4 以動物模式探討中國橄欖之抗增生的生理活性 ....................................... 36
一、實驗材料.............................................................................................. 36
1.化學藥品與溶劑............................................................................... 36
2.儀器設備........................................................................................... 36
二、動物分析實驗...................................................................................... 36
1.動物飼養........................................................................................... 36
2.轉植腫瘤........................................................................................... 36
3.實驗組別.......................................................................................... 37
4.腫瘤大小測量................................................................................... 37
4-5 統計分析 ...................................................................................................... 37
第四章結果 ...................................................... 38
一、以細胞模式檢驗中國橄欖萃取物之抗增生效應...................................... 38
1. 中國橄欖以不同溶劑萃取後的萃取物對於抗增生之影響................. 38
2. 中國橄欖甲醇萃取物不同區分層對於抗增生之影響......................... 38
3. 中國橄欖甲醇乙酸乙酯區分層之次區分物對CT26 細胞株生長之影
響.................................................................................................................. 39
4. OM-EtOAc-C 與OM-EtOAc-D之沉澱反應對CT26 細胞株生長之影
響.................................................................................................................. 39
5. OM-EtOAc-C2 與OM-EtOAc-D2 細區分物對CT26 細胞株生長之影
響.................................................................................................................. 40
6. OM-EtOAc-C2b HPLC 細區分物對CT26 細胞株生長之影響 ......... 40
7. OM-EtOAc-C2b 分析之結果 ................................................................. 41
二、以化學分析中國橄欖萃取物之組成.......................................................... 66
三、以動物模式探討中國橄欖之抗增生的生理活性...................................... 68
1.體重變化與腫瘤生長大小....................................................................... 68
第五章 討論 ...................................................... 71
一、萃取方式...................................................................................................... 71
二、中國橄欖萃取物對於抑制癌症的路徑...................................................... 71
三、中國橄欖具有抗腫瘤增生的成分探討...................................................... 72
第六章 結論 ...................................................... 75
第七章參考文獻 .................................................. 76

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