- 學位論文
有機溶劑紅藻萃取物之抗氧化與抗癌症影響
Antioxidant and anticancer effects of red algal organic solvent extracts
摘要
最近氧化壓力被認為是導致選擇性殺死癌細胞的指標之一。數種海藻萃取物也被發現有這項氧化壓力調節功能。我的研究目的是製備與檢測乙酸乙酯萃取紅藻萃取物(GTEA)是否改變癌細胞之氧化壓力，進而造成選擇性毒殺癌症的效果。在本論文中，主要有3大目標，包括: 1)我首先確保GTEA萃取物在不同季節與來源的品質；2)分析GTEA抗氧化相關之生化活性；3)進行細胞實驗確定，GTEA是否對不同癌細胞有選擇性毒殺效果。在品質控制方面，我蒐集並萃取四個季節所產的紅藻，發現它們的核磁共振(nuclear magnetic resonance; NMR)圖譜相近。在抗氧化生化分析方面，我使用ABTS•+ 自由基捕捉能力(ABTS•+ radical scavenging activity; ABTS)、亞鐵離子螯合能力(ferrous ions chelating activity)、1,1-diphenyl-2-picrylhydrazyl自由基捕捉能力(DPPH radical scavenging activity)和總類黃酮能力(total flavonoids capacity; TFC)分析GTEA，都發現GTEA具有抗氧化相關活性。有關選擇性殺死癌症細胞的評估中，我進行細胞存活率、細胞週期、細胞凋亡、氧化壓力和DNA受損等分析。由細胞存活率實驗發現，GTEA可以選擇性殺死口腔細胞株(CAL 27與Ca9-22)、肺部細胞株(H1299)和乳房細胞株(SKBR3)，但是對於其對應的正常組織的細胞株，則傷害較小(分別是HGF-1、HEL299與 M10細胞)。而選擇性殺死機制探討，我主要著重在口腔癌細胞。其中細胞週期變動與細胞凋亡方面，顯示GTEA會影響口腔癌細胞的所subG1期細胞累積，造成G2/M細胞週期停滯，並增加annexin V-positive (%)的細胞比例。利用流式細胞儀進行氧化壓力分析，發現GTEA會誘發口腔癌細胞ROS升高。同時GTEA也會造成口腔癌細胞的DNA受損。結論是，本研究開發出GTEA，可能對口腔癌、肺癌與乳癌細胞有選擇性殺死的作用。
並列摘要
Reactive oxygen species (ROS)-modulation is a potential treatment for selective killing against cancer cells. Several crude extracts of seaweeds have been reported to regulate ROS level in cancer cells. The purpose of my thesis is to evaluate the possible selective killing effects of cancer cells of ethyl acetate extracts of red seaweed (GTEA). The strategy of this study has three aims, including: 1) to prepare GTEA under quality control by considering the materials from different seasons and sources, 2) to perform the antioxidant biochemical analyses of GTEA, and 3) to investigate the possible selective killing responses of GTEA against several cancer cells. For quality control, the red seaweeds from four seasons had been collected and I found that they had similar patterns of nuclear magnetic resonance (NMR) spectroscopy for chemical fingerprints. For antioxidant biochemical analyses, GTEA displayed several antioxidant abilities in terms of ABTS•+ radical scavenging activity (ABTS), ferrous ions chelating activity, and total flavonoids capacity (TFC) antioxidant tests. The selective killing of cancer cells were investigated in terms of cell viability, cell cycle, apoptosis, oxidative stress, and DNA damage analyses. In MTS survival assay, GTEA selectively killed the oral (CAL 27 and Ca9-22), lung (H1299), and breast cancer cells (SKBR3) but less harmful to its corresponding normal cells (HGF-1, HEL299, and M10, respectively). The other detailed selective killing mechanisms were mainly focused on the oral cancer cells (CAL 27 and Ca9-22). For cell cycle disturbance and apoptosis, GTEA induced subG1 accumulation and G2/M arrest as well as the increase of annexin V-positive (%). GTEA also induced ROS generation and γH2AX-based DNA damage of oral cancer cells in terms of flow cytometry. In conclusion, the contribution of this study is to identify the GTEA for potential selective killing to oral, lung, and breast cancer cells.