臺灣博碩士論文加值系統

摘要
肝病發生率在台灣一直居高不下，而肝癌更是名列台灣十大死因之中，令人聞之色變。五爪金英屬於菊科植物，別名〞王爺葵〞，〞小向日葵〞，〞樹菊〞等，藥用方面，其全草有利尿解熱、清肝解毒之效，為台灣民間用於治療肝病之傳統用藥。
本研究將中草藥五爪金英以兩種不同方式萃取所得的萃取物，經由細胞實驗應用於抗癌作用及抗氧化作用分析上，其萃取方式分別為高壓水萃取法及甲醇冷浸萃取法。在實驗中的細胞包括人類攝護腺癌細胞DU-145和人類肝癌細胞Huh-7。實驗結果顯示，針對人類攝護腺癌細胞株，加入萃取物24小時後結果發現其半致死劑量約為3µg/mL，加入萃取物後的48小時和72小時其實驗結果和加入萃取物後24小時其半致死劑量是相同的。而針對人類肝癌細胞株，加入萃取物後24小時其半致死劑量約為4.5µg/mL，加入萃取物後的48小時和72小時其半致死劑量約為3µg/mL。在高壓水萃取物方面雖然增加萃取物濃度，但細胞存活率仍維持在100%，故高壓水萃取物對於此兩種癌細胞株並無明顯抑制效果。
針對具有抗癌效果之甲醇萃取物，利用製備級HPLC進行分離與純化，純化後的物質重複以上細胞實驗，確認所純化的物質為抗癌有效成份，接著以核磁共振光譜(NMR)以及液相層析質譜儀(LC-MS)做結構鑑定，結果發現我們的有效成份為Tagitinin C和Desacyltagitinin C-8-O-(2-methylbutyrate)兩者的混合物。
此外針對不具有毒殺作用的高壓水萃取物進行細胞抗氧化實驗，結果發現高壓水萃取物可以保護細胞免於受到H2O2的氧化傷害，進而證實五爪金英高壓水萃取物具有增進細胞抗氧化能力的效果。

Abstract
The liver cancer is one of the ten greatest deaths in Taiwan. Tithonia diversifolia, the Composite plant, is the folk medicine to cure the liver cancer, sarcoma and skin cancer in Taiwan.
In this study, two kinds of extracts (extracted by high-pressure water and methanol extraction) were tested the properties anti-cancer property in prostate cancer cells (DU-145) and hepatoma cells (Huh-7) and anti-oxidation properties in Mouse's normal liver cells (clone-9). The preliminary results show that the methanol extracts have anti-cancer effects in DU-145 and Huh-7 cells. After 24, 48 and 72 hours treatments with the extracts for DU-145, the cells’ growths were dramatically inhibited, and the IC50 was evaluated about 2.5μg/ml. For Huh-7 cells, the IC50 was evaluated about 4.5μg/ml after 24 hours treatment and 3μg/ml after 48 and 72 hours treatments. In the other way, the preliminary result shows that the high-pressure water extracts don’t have the anti-cancer effects.
In the anti-oxidant experiments, the cells were treated with the exteacts of Tithonia diversifolia to prevent the H2O2-induced damage in Mouse’s normal liver cells (clone-9). The results show that high-pressure water extracts can protect cells to resistant H2O2-induced oxidation damage.

目錄
中文摘要………………………………………………………………1
英文摘要………………………………………………………………2
第一章 緒論
1.1 前言………………………………………………………………3
1.2 文獻回顧…………………………………………………………4
1.3 研究動機…………………………………………………………5
1.4 五爪金英構造之觀察……………………………………………6
1.4.1 生藥性狀………………………………………………….6
1.4.2 內部構造………………………………………………….6
1.5 癌細胞的介紹…………………………………………………..8
1.5.1細胞週期調控之異常與癌細胞生長……………………...8
1.5.2 癌細胞生長與細胞凋亡………………………………….9
1.6氧化與抗氧化的介紹……………………………………………11
1.6.1 自由基的定義…………………………………………….11
1.6.2 自由基的形成原由和導致之細胞傷害………………….11
1.6.3 活性氧分子的種類和造成之毒性……………………….12
1.6.4 與人類疾病相關的自由基約可分為五大類…………….12
1.6.5 體內抗氧化機制………………………………………….13

第二章 實驗材料
2.1中草藥五爪金英和實驗細胞株………………………………….15
2.2 萃取與分離純化實驗……………………………………………15
2.3 細胞實驗…………………………………………………………16

第三章 實驗方法
3.1萃取方法………………………………………………………….17
3.2高效能液相層析圖譜的最佳條件……………………………….18
3.3細胞培養………………………………………………………….19
3.3.1 MTT Assay 原理………………………………………….21
3.4 細胞毒性實驗…………………………………………………..23
3.4.1 萃取物對人類攝護腺癌細胞株(DU 145)的毒性試驗….23
3.4.2 萃取物對人類肝癌細胞株(Huh-7)的毒性試驗…………23
3.5細胞外抗氧化實驗……………………………………………….24
3.6細胞內抗氧化實驗……………………………………………….25
3.6.1細胞內抗氧化實驗最佳條件探討………………………..25
3.6.2 細胞內抗氧化保護實驗………………………………….26

第四章 結果與討論
4.1高壓水最佳萃取壓力的選擇…………………………………….28
4.2萃取物之分離與分析…………………………………………….29
4.3 五爪金英對細胞生長的抑制能力……………………………….30
4.3.1人類攝護腺癌細胞株(DU 145)……………………………30
4.3.2人類肝癌細胞株(Huh-7)……………………………………31
4.4細胞外抗氧化能力評估………………………………………....33
4.5細胞內抗氧化保護實驗條件探討………………………………..34
4.6細胞內抗氧化保護實驗……………………………………………36
4.7五爪金英純化物對細胞生長的抑制能力………………………..38
4.7.1人類攝護腺癌細胞株(DU 145)…………………………….38
4.7.2人類肝癌細胞株(Huh-7)…………………………………..39
4.8五爪金英純化物結構鑑定………………………………………..41

第五章 結論………………………………………………………….47
參考文獻……………………………………………………………….49

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