臺灣博碩士論文加值系統

三葉青 (Tetrastigma hemsleyanum) 為葡萄科 (Vitaceae) 植物，其根部在亞洲被使用作為消炎、鎮痛和解熱等特性的民間藥物。主要的活性成分，如蘆丁 (Rutin) 和山萘酚 (Kaempferol)，具有抗氧化、抗發炎和增強抑制癌細胞之作用。然而，來自三葉青萃取物和活性化合物的抗癌作用機制，至今仍未完全釐清。肺癌 (Lung carcinoma)是一種惡性肺腫瘤，其特徵在於肺組織中不受控制的細胞生長，並透過轉移到附近組織或身體其他部位。肺癌是台灣十大癌症排名之首，也是全球死亡的主要原因。在這些死亡病例中，近60％的患者已進展為晚期之轉移。因此，若能找到更有效的抑制肺癌方式，將有助於提高治療效果。本研究目的探討從三葉青中利用H2O和EtOH提取萃取物，並測定萃取物的抗氧化活性、總酚和類黃酮含量，以及活性化合物抑制肺癌細胞的評估。結果顯示，透過1,1-diphenyl-2-picrylhydrazyl (DPPH) 方法測定，三葉青的EtOH萃取物具有較好的抗氧化活性，而H2O萃取物具有較高的酚類和黃酮類含量，顯示三葉青中所含的成分極多，作用活性也不盡相同，故進一步分析已知的活性化合物Rutin和Kaempferol。利用3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) 方法測試，顯示Kaempferol可有效抑制A549人類肺癌細胞和LLC1小鼠肺癌細胞之存活率。然而，Rutin在50 ～ 400 µM濃度測試下，對A549和LLC1細胞之生長並沒有顯著的抑制作用。此外，Kaempferol與紫杉醇 (Paclitaxel)的合併處理時，增強了對兩種細胞株的抑制作用。在Kaempferol 50 µM合併Paclitaxel 50 µM 時，A549細胞之抑制比單獨使用Paclitaxel處理增加約11%的細胞抑制作用，以及LLC1細胞增加約30%的抑制細胞生長作用。在細胞週期分析上，Kaempferol會使得A549細胞週期停滯於S期，Paclitaxel (50 µM)會誘導sub-G1期上升並停滯在S期，兩者合併處理可明顯提高S期和G2/M期比例。LLC1細胞經過Paclitaxel、Kaempferol或合併處理後，對LLC1細胞週期之sub-G1期皆無明顯變化，Kaempferol單獨和合併處理有促進上升G2/M期，顯示Kaempferol使其細胞週期停滯於G2/M期，而Paclitaxel對LLC1細胞的作用不敏感，仍需進一步探討。總結，本研究證明，三葉青的水萃和酒萃萃取物具有不同的抗氧化活性、總酚和類黃酮含量，而活性成分Kaempferol對肺癌細胞具有比Rutin較高的生長抑制作用，可能是透過細胞週期之停滯，且結合Paclitaxel在抗癌活性中具有增強抑制癌細胞生長之效果。

Tetrastigma hemsleyanum is a genus of plants in the grape family, Vitaceae. The roots of T. hemsleyanum are used as a folk medicine in Asia for the anti-inflammatory, analgesic, and antipyretic properties. The major active ingredients, rutin and kaempferol, were reported with the activities of anti-oxidation, anti-inflammatory, and enhancing the inhibition of cancer cells. However, the exact anti-cancer activities of the crude extracts and active compounds from T. hemsleyanum are still unclear. Lung cancer, also known as lung carcinoma, is a malignant lung tumor characterized by uncontrolled cell growth in tissues of the lung. The growth can spread beyond the lung by the process of metastasis into nearby tissue or other parts of human body. Lung cancer is the top ten cancer rankings in Taiwan and the leading cause of death worldwide. Of these deaths, nearly 60% of patients progress into advanced stages with metastasis. Therefore, new strategies for enhancing the treatment efficiency of lung cancer are necessary. The aim of this study explored the extraction by H2O and EtOH from T. hemsleyanum, and assayed the antioxidant activity, total phenolic and flavonoid content from the extracts, as well as investigation of the active integrates on anti-cancer effects. The results demonstrated that EtOH extracts from T. hemsleyanum have better antioxidant activities by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, while H2O extracts have higher phenolic and flavonoid contents, indicating that there are many components contained in the T. hemsleyanum with various activities. The known active compounds rutin and kaempferol were further analyzed. Cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that kaempferol was effective in inhibition of both human A549 and murine LLC1 lung cancer cell lines. However, rutin did not significantly inhibit the growth of A549 and LLC1 cells at the concentrations ranging from 50 to 400 μM. In addition, the combination of kaempferol and paclitaxel enhanced the inhibition of both cell lines. At the concentration of 50 μM, kaempferol combined with paclitaxel (50 μM) increased the inhibition of cell growth by 11% and 30% in A549 and LLC1 cells, respectively, as compared to paclitaxel (50 μM) alone. In cell cycle analysis, kaempferol induced the cell cycle arrest at the S phase in A549 cells, paclitaxel increased the sub-G1 and S phases, and the combined treatment significantly upregulated the S and G2/M arrest. After treatment with paclitaxel, kaempferol alone, or combinational treatment in LLC1 cells, all of them showed no significant change of cell cycle. Kaempferol alone and combinational treatment promoted the G2/M phase in LLC1 cells, indicating that kaempferol arrested the cell cycle at G2/M phase. The reason that paclitaxel effect on LLC1 cells is not sensitive still needed to be further clarification. In summary, the present study demonstrated that the H2O and EtOH extracts from T. hemsleyanum have different antioxidant activities, total phenolic, and flavonoid content. The active ingredient kaempferol has a higher growth inhibitory effect on lung cancer cells than rutin. The combination of kaempferol with paclitaxel has an enhancing effect on anti-lung cancer activity

目錄 1
中文摘要 3
英文摘要 6
I. 前言 9
1.1 三葉青 9
1.2 山萘酚(Kaempferol) 10
1.3蘆丁(Rutin) 11
1.4 紫杉醇(Paclitaxel) 12
1.5肺癌 13
1.6細胞週期 (Cell cycle) 18
II. 研究目的與動機 20
III. 材料與方法 21
3.1 實驗材料 21
3.2實驗方法 25
IV. 結果 35
4.1三葉青的水萃取物和酒精萃取物進行DPPH清除自由基活力測試 35
4.2三葉青的水萃取物和酒精萃取物進行總酚含量測試 35
4.3三葉青的水萃取物和酒精萃取物進行總類黃酮含量測試 36
4.4 A549細胞在Rutin和Kaempferol處理48小時下的細胞存活率影響 36
4.5 LLC1細胞在Rutin和Kaempferol處理48小時下細胞存活率影響 37
4.6 A549細胞在Kaempferol和Paclitaxel處理24小時下細胞存活率影響 38
4.7 LLC1細胞在Kaempferol和Paclitaxel處理24小時下細胞存活率影響 39
4.8 A549細胞在Kaempferol和Paclitaxel處理24小時下細胞型態影響 40
4.9 LLC1細胞在Kaempferol和Paclitaxel處理24小時下細胞型態影響 40
4.10 A549細胞在Kaempferol和Paclitaxel處理24小時下細胞週期影響 41
4.11 LLC1細胞在Kaempferol和Paclitaxel處理24小時細胞週期影響 42
V. 討論 44
5.1三葉青的水萃取物和酒精萃取物進行DPPH自由基清除能力、總酚、總類黃酮含量測試 44
5.2探討三葉青中活性成分Rutin和Kaempferol對A549和LLC1細胞存活率之影響 45
5.3探討Kampferol結合抗癌藥物Paclitaxel，對A549和LLC1細胞存活率之影響 46
5.4探討Kampferol結合抗癌藥物Paclitaxel對A549和LLC1細胞型態之影響 47
5.5探討Kampferol結合抗癌藥物Paclitaxel對A549和LLC1細胞週期之影響 48
VI. 總結 50
VII. 參考文獻 51
VIII. 圖表 71
IX. 附錄 88

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