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dc.description.abstract台灣肖楠(Calocedrus formosana Florn)為台灣珍貴的針葉樹種,且台灣肖楠葉子抽出成分已被證實具有細胞毒性、抗氧化及抗真菌等生物活性,實具有開發為天然抗癌製劑之潛力。因此,本論文將首次針對台灣肖楠葉子抽出成分及其活性化合物進行體內及體外抗腫瘤活性評估。所得結果發現,台灣肖楠葉子抽出成分中,以正己烷可溶部具有最佳細胞毒性。進一步以管柱層析方式將正己烷可溶部細分為10個次分離部,並針對各分離部進行細胞毒性評估後發現,各次分離部中以次分離部1及4具有較強的細胞毒性。 基於生物活性導向之分離策略,本研究續利用HPLC系統對次分離部1及4進行分離及純化。經HPLC分析後,由次分離部1中得到順式瓔柏酸(trans-Communic acid)及Isoabienol共2個化合物。而由次分離部4中則係得到松柏酸(Pinusolidic acid)及(-)-反式-3-(3,4-亞甲基二氧基芐基)-2-(3,4,5-三甲氧基芐基)丁內酯(Yatein)共2個化合物。各化合物經細胞毒性篩選後,發現以Yatein對A549及CL1-5細胞具有最強細胞毒殺效果,二者中又以對CL1-5細胞之效果較佳。由流式細胞儀分析結果顯示,Yatein可誘導A549及CL1-5細胞之細胞週期停滯於G2/M時期,同時亦可造成肺癌細胞株產生細胞凋亡現象。此外,經Yatein處理後之肺癌細胞株,其Cyclin B1及磷酸化Cdc 2等G2/M時期相關調節蛋白表現量會有明顯增加情形。然而,經Yatein處理後之A549及CL1-5細胞,其Cdc 2及Cdc 25c等調節蛋白之表現量並無明顯改變。而本論文之試驗結果亦發現,Yatein之抗肺癌作用機轉與上游之ATM/ATR及p53等調控路徑活化有密切的關連性。另一方面, Yatein可扮演微管去穩定劑之角色,進而干擾A549及CL1-5細胞之微管聚合動力學,此為Yatein誘導肺癌細胞株產生G2/M時期停滯可能原因之一。 此外,由體內試驗結果可以發現,將Yatein投予腫瘤異體移殖小鼠後,可有效降低腫瘤之體積以及重量,且對於小鼠體重及其日常攝食量則無明顯影響。基於上述結果,本論文證實Yatein在體外及體內模式中,具有良好的細胞毒性及抗腫瘤功效,顯示Yatein實具有進一步開發為抗非小細胞肺癌植物製劑之發展潛能。zh_TW
dc.description.abstractCalocedrus formosana Florin is a well-known softwood tree species in Taiwan. The leaves extract of C. formosana had been reported with excellent bioactivities such as cytotoxic, antioxidant and antifungi effects. According to these studies, the leaves extract of C. formosana may be a potential candidate for novel natural anticancer agent. In the present study, the anti-lung cancer efficacy of C. formosana leaves extract and its derived active phytocompounds, were evaluated using in vitro cell-based models and in vivo xenograft mice model for the first time. Among the crude extract and its derived fractions from leaves of C. formosana, the n-hexane fraction exhibited the best cytotoxicity potential. Following by column chromatography, 10 subfractions were obtained from the n-hexane fraction, and the cytotoxic effects of each subfraction were also determined in this study. The results revealed that the sunfractions 1 and 4 exhibited better cytotoxic potential than other subfractions. Based on the bioactivity-guided isolation principle, the subfractions 1 and 4 were then separated using HPLC system. After HPLC analyzed, trans-Communic acid and isoabienol were isolated from subfraction 1; pinusolidic acid and yatein were isolated from subfraction 4. Among these phytocompounds, yatein exhibited the best cytotoxicity in A549 and CL1-5 cells. In addition, the CL1-5 cells were more sensitive than A549 cells after yatein treatment. According to flow cytometry analysis, the result revealed that yatein induced G2/M arrest and apoptosis in these two lung cancer cell lines. Furthermore, the expression of G2/M arrest related regulatory proteins such as Cyclin B1 and p-Cdc 2 were increased after treatment with yatein in A549 and CL1-5 cells. However, the Cdc 2 and Cdc 25c expressions of A549 and CL1-5 cells were no significant difference between vehicle control group and yatein treatment group. The upstream pathways including ATM/ATR pathway and p53 pathway were also involved in the anti-ling cancer mechanism of yatein. In this study, we also found that yatein act as a microtubule destabilizer that interfered microtubule dynamic of A549 and CL1-5 cells, which is another possible reason for yatein induced significantly cell populations staying in G2/M arrest. On the other hand, the in vivo anti-lung cancer activity of yatein was also evaluated in this study using xenofraft mice model. The tumor size and weight in xenograft mice were decreased after yatein treatment. Accordingly, the daily diet intake and body weight of xenograft mice have no significant difference between the vehicle control group and the yatein treatment group. Based on these findings, it provides insight into the in vitro and in vivo anti-lung tumor efficacy of yatein, rendering this phytocompound a potential anticancer drug for NSCLC.en_US
dc.description.tableofcontents目錄 i 表目次 iii 圖目次 v 摘要 1 Abstract 3 第一章 前言 5 第二章 文獻回顧 8 一、肖楠屬抽出成分及相關活性介紹 8 二、常見細胞死亡方式之介紹 15 三、天然物應用於抑制微管動態平衡之相關研究 27 四、木酚素細胞毒性相關研究然 31 第三章 材料與方法 34 一、試驗材料 34 二、台灣肖楠指標成分之定性與定量 34 三、體外細胞試驗 35 四、體內抗腫瘤活性試驗 42 五、統計分析 44 第四章 結果與討論 45 一、台灣肖楠葉子甲醇粗萃物及其各可溶部之細胞毒性評估 45 二、以生物活性為導向對正己烷可溶部進行初步篩選及分離 50 三、次分離部4對A549及CL1-5細胞細胞週期之影響 51 四、台灣肖楠葉子正己烷可溶部細胞毒性化合物之分離與鑑定 52 五、台灣肖楠葉子成分對人類肺癌細胞株之生長抑制效果 74 六、Yatein對肺癌細胞株細胞週期之影響 77 七、Yatein對肺癌細胞株產生細胞凋亡之影響 81 八、Yatein對肺癌細胞株細胞週期調控相關蛋白之影響 87 九、Yatein對肺癌細胞株細胞凋亡相關蛋白之影響 91 十、Yatein造成肺癌細胞DNA損壞並導致ATM/ATR路徑活化 94 十一、Yatein對肺癌細胞株微管聚合動力學之影響 101 十二、Yatein對肺癌細胞株產生ROS之影響 106 十三、Yatein與其他抗癌藥物之相互作用 109 十四、Yatein於A549細胞之異體移殖小鼠體內抗腫瘤功效 113 十五、Yatein抗非小細胞肺癌細胞生長之作用機轉 120 第五章 結論 122 參考文獻 124zh_TW
dc.subjectCalocedrus formosana Flornen_US
dc.subjectlung canceren_US
dc.subjectcell cycle arresten_US
dc.titleYatein, Antitumor Active Compound from Calocedrus formosana Florin Leave and Its Mechanismen_US
dc.typeThesis and Dissertationen_US
item.openairetypeThesis and Dissertation-
item.fulltextwith fulltext-
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