臺灣博碩士論文加值系統

本研究為釐清文蛤之有效抗癌生理活性物質本體，及文蛤熱水萃取物效果不穩定之可能原因，將文蛤區分為肌肉及消化道處理，分別以乙酸乙酯萃取後可得 HCEAM 和 HCEAA。藉由 DNA 片段化、Annexin V-FITC/PI 雙染、粒線體膜電位改變以及 sub-G1 DNA distribution 進行凋亡機制分析；以 MTT assay 測定細胞存活率；以 TLC 和 HPLC 分析可能活性成份；以 DCFH-DA染色分析其對胞內 ROS 之影響；並以測量清除 DPPH 之能力評估體外抗氧化活性。結果顯示HCEAA 和 HCEAM可顯著抑制人類肝癌細胞株 HepG2及Hep3B 之增生，HCEAA分別對 HepG2 和 Hep3B 處理後，IC50 分別為 397.95 及 553.98 μg/mL。HCEAA (IC50 = 397.95 μg/mL) 相較於 HCEAM (IC50 = 770.88 μg/mL) 對 HepG2 的細胞毒性有兩倍以上；產率方面，HCEAA 其產量為 4.77% 較 HCEAM 1.33% 高；且HCEAA 與 HCEAM 均檢出 epidioxysterol 成份，HCEAA 可以提高細胞內的ROS 含量，在體外抗氧化實驗具有清除 DPPH 自由基的能力。由以上結果得知，乙酸乙酯萃取所得有效抗癌成份主要分佈於文蛤消化道。文蛤乙酸乙酯粗萃物 (IC50 = 397.95 μg/mL) 較文蛤熱水粗萃物 (IC50 = 607.39 μg/mL) 有較低的半致死劑量，但其效果並無 WG30-50 (IC50 = 100 μg/mL) 顯著。

The goal of this study was to explain the uncertained effect of hard clam hot water extraction and identify the distribution of bioactive compounds. We used ethyl acetate to extract steroids from alimentary tract and muscle in hard clam, which named as HCEAA and HCEAM. The cell viability is determined by MTT assay. The molecular mechanisms of apoptosis inducing by HCEAA was determined by DNA fragmentation, Annexin V-FITC/PI double stain, analysis of mitochondrial transmembrane potential and sub-G1 DNA distribution. The sterol compounds were analysis by TLC and HPLC. Determination of ROS generation by DCFH-DA stained. Evaluate the antioxidization activity by DPPH scavenging activity. The activity of epidioxysterols analogs compound of extract from hard clam, HCEAA and HCEAM, were significantly decreased Hep3B and HepG2 cell viability. HCEAA (Yield: 4.77%) have higher yield compared to HCEAM (Yield: 1.33%), and showed more strong effect on cell cytotoxicity by 2 folds. By using TLC and HPLC, epidioxysterol was presented in HCEAA and HCEAM. HCEAA can increase the intracellular ROS concentration. In vitro, it has DPPH scavenging activity. Therefore, this study suggested that, extract by ethyl acetate can get bioactive compounds and distribute in alimentary tract. But compared with WG30-50 (IC50 = 100 μg/mL), HCEAA’s cytotoxicity (IC50 = 397.95 μg/mL) is not outstanding. The bioactive compound of hard clam is not necessarily epidioxysterol.

名詞縮寫 i
目錄 ii
圖目錄 viii
表目錄 xi
附錄目錄 xii
摘要 xiii
Abstract xiv
壹、前言 1
貳、文獻整理 3
ㄧ、肝癌 3
1. 肝癌的形成 3
2. 肝癌的治療方法 5
二、文蛤 (MERETRIX LUSORIA) 的簡介 6
1. 文蛤名稱及分類 7
2. 文蛤台灣養殖情況 7
3. 文蛤的成分分析與季節變化 8
4. 文蛤中的固醇類化合物 9
三、細胞凋亡 ( APOPTOSIS ) 10
1. 細胞死亡模式分析 10
2. 細胞週期 (cell cycle) 12
3. Apoptosis 13
3.1 細胞凋亡的執行者— caspase 14
3.2 死亡受體訊息傳遞 (Signal transduction induced death receptor pathway ) 15
3.3 粒線體調控途徑 (Mitochondrial associated pathway) 17
3.3.1 Bcl-2 家族 17
3.3.2 Mitochondrial permeability transition pore 18
3.4 內質網壓力途徑 19
3.5 活性氧 (Reactive Oxygen Species; ROS) 的氧化傷害 21
?、實驗設計 22
肆、實驗材料與方法 23
一、實驗材料 23
1. 細胞株 23
2. 文蛤來源 23
3. 藥品 23
3.1 文蛤萃取物的製備 23
3.1.1. 熱水萃取 23
3.1.2. 乙酸乙酯萃取 23
3.2 細胞培養 23
3.3. 文蛤之抗腫瘤有效成分分析 24
3.3.1 TLC 之 sulfuric acid 呈色反應 24
3.3.2 HPLC 分析 24
3.4 細胞存活率測定 (MTT assay) 24
3.5 DNA 片段化試驗（DNA Fragmentation） 25
3.6 cell cycle 分析 26
3.7 DNA 片段化百分比 26
3.8 粒線體膜電位ΔΨm之測定 26
3.9偵測磷脂醯絲胺酸外翻 26
3.10細胞內 ROS 產生量的分析 26
4. 儀器設備 26
二、實驗方法 28
1. 文蛤萃取物的製備 28
1.1 文蛤熱水萃取物的製備 28
1.2 文蛤乙酸乙酯萃取物的製備 29
2. 文蛤之抗腫瘤有效成分分析 29
2.1 TLC 之 sulfuric acid 呈色反應 29
2.2 HPLC 分析 30
3. 細胞的培養與凍結 30
3.1 懸浮性細胞 30
3.2 黏著性細胞培養 30
3.3 細胞凍結 31
4. 細胞存活率試驗 (MTT assay) 32
5. 細胞凋亡之測定 32
5.1 DNA 片段化之測定 (DNA Fragmentation Test) 32
5.2 細胞週期分析 33
5.3 DNA 片段化百分比 34
5.4 螢光染色法 35
6. 流式細胞計數儀測試 35
6.1試劑配製 35
6.2 細胞週期與細胞 DNA 損壞情形 35
6.3 實驗步驟 36
7. 偵測磷脂醯絲胺酸外翻 37
7.1 原理 37
7.2 實驗步驟 37
8. 細胞內粒線體膜電位 ΔΨm 的分析 38
8.1原理 38
8.2 實驗步驟 38
9. 細胞內 ROS 產生量的分析 39
9.1 原理 39
9.2 實驗步驟 39
10. 清除DPPH自由基之能力測定 39
11. 統計分析 40
伍、結果與討論 41
一、文蛤抗腫瘤活性因子的萃取 41
1. 文蛤熱水萃取經硫酸銨沉澱蛋白質對人類肝癌細胞生長之影響 41
2. 文蛤不同部位經乙酸乙酯進行萃取後對人類肝癌細胞生長之影響 42
3. 文蛤消化道乙酸乙酯萃取物 HCEAA 對不同癌細胞存活率的影響 42
4. Epidioxysterol 對人類肝癌細胞生長之影響 43
二、DNA 片段化測定 43
三、HCEAA 引發肝癌細胞的形態改變 44
四、HCEAA 引發染色質的凝集現象 45
五、ANNEXIN V-FITC/PI 雙染法測定 45
六、細胞週期分析 47
七、HCEAA 引發人類肝癌細胞HEPG2 粒線體膜電位 ΔΨM 與 ROS 之改變 47
八、分析文蛤萃取物中是否含有可以有效誘導細胞凋亡的化合物 EPIDIOXYSTEROLS 49
陸、結論 52
柒、參考文獻 54
捌、圖表 62
玖、附錄 97

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