- 學位論文
台灣蔬果及藥材之自由基清除與乳癌生長之活性探討
Free radical-scavenging capability and human breast cancer cells proliferative activities of selected Taiwanese vegetable, fruit and botany
摘要
台灣處於亞熱帶，四季物種豐富且多樣，尤其水果和天然植物更是台灣不可或缺的資產，已有許多文獻指出，天然植物當中普遍含有大量多酚類化合物， 其中多酚類化合物更被研究發現具有抗老化、抗氧化、預防心血管疾病與清除自由基功能。自由基為帶有不成對電子的原子、分子或離子，生物體常見的自由基為氧自由基 ( Oxygen free radicals )，種類包含氫氧自由基 ( Hydrogen radical ) 等，易導致致癌基因 ( Oncogene ) 活化或抑癌基因 ( Tumor suppressor gene ) 失去功能，進而引發癌症產生。癌症即惡性腫瘤，由於近年來罹患人數與死亡人數持續高攀，已成為世界衛生組織與醫療研究機構關注的焦點，而乳癌目前是台灣女性發生率最高的癌症，其死亡率高居第四位，且發生率與死亡率仍逐年上升，與歐美國家相比，更有年輕化的現象。 本論文收集台灣本土蔬菜9種、水果2種、藥材7種、香料與其他植物3種，總計21種天然植物，並分自由基清除能力與乳癌細胞之生長活性能力測試兩部分加以探討研究： < 第一部份 >： 21種蔬果及藥材自由基清除之能力 多酚含量測定結果包含：總多酚含量（Gallic acid equivalent，GAE）( μg Gallic acid / mg）以：咖啡 ( GAE = 160.75 ± 4.36 )、百里香 ( GAE = 194.47 ± 4.99 )、紫蘇 ( GAE = 192.41 ± 4.88 )、蔓澤蘭 ( GAE = 227.20 ± 9.33 )、桂花( GAE = 254.74 ± 5.87 )、龍眼花( GAE = 718.78 ± 7.37 ) 和YC-27 ( GAE = 631.28 ± 8.12 ) 之多酚含量較高。總類黃酮含量（Rutin equivalent，RE）( μg Rutin / mg）則以：咖啡 ( RE = 12.75 ± 0.23 )、百里香 ( RE = 24.12 ± 0.98 )、紫蘇 ( RE = 22.98 ± 1.20 )、蔓澤蘭 ( RE = 30.72 ± 1.32 )、龍眼花 ( RE = 17.47 ± 0.52 ) 和YC-27 ( RE = 45.29 ± 2.23 ) 之總類黃酮含量較高。總黃酮醇含量 （ Rutin equivalent，RE ） ( μg Rutin / mg ） 則以：咖啡 ( RE = 30.63 ± 0.57 )、百里香 ( RE = 29.64 ± 0.83 )、山藥 ( RE = 21.99 ± 0.87 )、紫蘇 ( RE = 31.36 ± 1.49 )、蔓澤蘭 ( RE = 26.03 ± 1.19 )、龍眼花 ( RE = 29.40 ± 0.68 ) 和YC-27 ( RE = 86.08 ± 3.52 ) 之總黃酮醇含量較高。 經過一系列抗氧化測試後，發現其中良好抗氧化之萃取物包括：咖啡、百里香、地瓜、原生種山藥、洛神花、紫蘇、蔓澤蘭、桂花、龍眼花和YC-27有顯著清除DPPH自由基能力，咖啡、百里香、紫蘇、蔓澤蘭、桂花和龍眼花有顯著FRAP還原能力，虎斑瓠皮和火龍果皮有顯著螯合亞鐵離子能力，黑胡椒有顯著抑制超氧陰離子能力，苦瓜、洋蔥、百里香、龍眼花和YC-27顯著清除超氧陰離子能力，咖啡、百里香、地瓜、原生種山藥、山藥、洛神花、紫蘇、蔓澤蘭、桂花、龍眼花和YC-27具有顯著抑制肝粒線體脂質過氧化之能力。為了進一步了解植物性化合物與癌症的關聯，本論文第二部份分別以兩株人類乳癌細胞-MCF-7與MDA-MB-231，評估21種台灣蔬果及藥材的抗乳癌能力，並篩選具抗氧化與抗癌活性潛力之天然植物：YC-27，探討其抗兩株乳癌細胞之活性機轉。 < 第二部份 >：分別探討 ( 1 ) 21種台灣蔬果及藥材抗人類乳癌細胞之能力與 ( 2 ) YC-27抗乳癌活性機轉。 ( 1 ) 21種台灣蔬果及藥材抗人類乳癌細胞之能力 利用MTT試劑測試天然植物分別對雌激素受體陽性乳癌細胞株MCF-7，與雌激素受體陰性乳癌細胞株MDA-MB-231兩株乳癌細胞之活性機轉。MTT實驗結果顯示在200 μg / ml濃度下，與MCF-7共同培養24小時之抑制細胞生長能力 ( % ) 黑胡椒 = 66.72 ± 5.91和百里香 = 58.95 ± 6.21，以及對MDA-MB-231細胞生長活性能力：YC-27 = 62.16 ± 15.20，皆對乳癌細胞產生細胞活性影響，表現抗乳癌之潛力，並且黑胡椒和百里香 / YC-27於濃度100 μg / ml下，皆使MCF-7 / MDA-MB-231有凋亡小體 ( apoptotic body ) 如細胞萎縮與染色體濃縮及空泡之產生，顯示經由凋亡路徑抑制MCF-7 / MDA-MB-231細胞生長活性。其中YC-27對較惡性之雌激素受體陰性乳癌細胞MDA-MB-231表現抗癌活性，並有良好抗氧化能力，因此進一步探討其抗癌活性機轉。 ( 2 ) YC-27之抗乳癌活性及機轉 將YC-27以不同極性溶劑：正己烷 ( Hexane )、乙酸乙酯 ( Ethyl acetate，EA )、丙酮 ( Acetone )、70 % 甲醇 ( 70 % MeOH ) 和甲醇 ( MeOH ) 萃取後，再分別以不同極性溶劑劃分萃取，得到不同極性之濃縮萃取化合物。MTT試驗之細胞活性結果：YC-27萃取物與劃分萃取物於濃度200 μg / ml下，對MCF-7細胞生長抑制率 ( % ) 為：YC-27-E = 76.77 ± 2.94，YC-27-EF1 = 79.29 ± 11.85，YC-27-EF2 = 93.35 ± 3.28，YC-27-A = 54.66 ± 10.54，YC-27-AF1 = 54.47 ± 5.59，YC-27-MF1 = 63.53 ± 6.99，YC-27-MF2 = 91.70 ± 2.59；對MDA-MB-231細胞生長抑制率 ( % ) 為：YC-27-E = 93.80 ± 8.68，YC-27-EF1 = 61.24 ± 5.47，YC-27-A = 97.68 ± 3.18，YC-27-AF1 = 77.40 ± 7.27，YC-27-AF2 = 82.54 ± 4.86，YC-27-M = 62.16 ± 5.20，YC-27-MF2 = 87.08 ± 6.20，YC-27-MF3 = 71.37 ± 3.12，YC-27-70 % M = 65.64 ± 1.40。其中YC-27-AF2、YC-27-M、YC-27-MF3、YC-27-70 % M對兩株乳癌細胞有選擇性抑制能力，具有藥物研發潛力，因此進一步進行細胞活性機轉實驗：以 ( a ) 照相螢光顯微鏡觀察經過染色之細胞型態：YC-27-AF2、YC-27-M、YC-27-MF3、YC-27-70 % M於濃度100 μg / ml 下，皆使MDA-MB-231有細胞萎縮與染色質濃縮及空泡之產生，顯示YC-27經由凋亡路徑抑制MDA-MB-231細胞生長活性。另進一步進行YC-27萃取物與劃分萃取物，與兩株乳癌細胞作用後所產生之 ( b ) 細胞核內去氧核醣核酸斷裂片 ( DNA fragmentation ) 情形： YC-27-MF3產生DNA斷裂 ( c ) 西方墨點轉漬法 ( Western blotting ) 偵測細胞中蛋白質表現變化顯示：YC-27-M、YC-27-MF3與YC-27-70%M於濃度100 μg / ml下，MDA-MB-231皆有Caspase 3蛋白質表現。 綜觀以上結果YC-27具有良好之抗氧化能力，並能抑制兩株不同雌激素受體表現型之乳癌細胞，因此進一步探討YC-27抑制乳癌細胞活性的詳細機轉，並作為抗癌新藥之開發。
並列摘要
Free radicals are correlated with many diseases such as brain and vessel disease、coronary heart disease、diabetes, aging, etc. Polyphenols are the plant-derived compounds which associated with antioxidative and chemopreventive benefits by free-radical reduction. Breast cancer was the top ten death cancer which frequently occurred in past decades. The cases of breast cancer and death rate are still on the rise nowadays. Today, breast cancer is the most common cancer in Taiwan and be associated with women suffering. Therefore, 21 selected Taiwan vegetable,fruit and botany ethanol extractions were investigated their antioxidative potential and anti-cancer proliferative property on two breast cancer cell lines：estrogen-receptor ( ER )-positive breast cancer cell line—MCF-7,and ER-negative breast cancer cell line—MDA-MB-231,to disscuse the diet of breast cancer patient’s in different subtypes. This paper contained the following 2 parts： < Part I > Free radical scavenging activities and polyphenolic content of selective Taiwanese vegetable,fruit and botany The polyphenolic contents were evaluated by Total phenol/flavonoid/flavonol quantitative analysis. The results exhibited that the polyphenol-rich plant were： Coffea canephora / C. arabicaJ ( YC-12 )、Thymus vulgaris L. ( YC-13 )、Perilla frutescens L. Britton ( YC-21 )、Mikania cordata Burm. f. B. L. Rob ( YC-24 )、Osmanthus fragrans L. ( YC-25 )、Dimocarpus longan L. ( YC-26 ) and YC-27. And several free-radical-associated assays were used and exhibiting the significant effects with EC50 ( μg / ml ) that show as the following：( 1 ) DPPH free radical scavenging ability：YC-12 = 19.78 ± 0.59、YC-13 = 15.94 ± 1.49、YC-21 = 13.89 ± 1.63、YC-24 = 8.90 ± 2.53、YC-25 = 11.01 ± 0.74、YC-26 = 2.83 ± 0.25 and YC-27 = 19.58 ± 0.36, ( 2 ) Fe2+ chelating ability：Hylocereus costaricensis ( Weber ) Britt. & Rose peel ( YC-14 ) = 75.89 ± 1.93, ( 3 ) XO inhibition：YC-11 = 30.15 ± 6.97, ( 4 ) O2•- radical scavenging ability：YC-26 = 2.27 ± 0.42 and YC-27 = 33.33 ± 7.59, ( 5 ) LPO inhibition：YC-12 = 47.44 ± 8.09、YC-13 = 7.03 ± 0.44、YC-21 = 98.32 ± 14.24、YC-24 = 81.24 ± 9.30、YC-26 = 12.66 ± 0.70 and YC-27 = 32.91 ± 1.19. And, in concentration of 200 μg / ml,the significant ( 6 ) FRAP ability in trolox equivalent antioxidant capacity ( TEAC ) ( μg / ml ) were：YC-12 = 85.62 ± 1.50、YC-13 = 90.37 ± 1.29、YC-21 = 104.77 ± 1.57、YC-24 = 89.72 ± 1.99、YC-25 = 108.40 ± 2.78 and YC-26 = 98.37 ± 0.42. < Part II > Human breast cancer cells proliferative activities of selected Taiwanese vegetable, fruit and botany A. Proliferative activities of selective Taiwanese vegetable,fruit and botany to human breast cancer cell lines—MCF-7 and MDA-MB-231 YC-11 and YC-13 exhibited the inhibitory effect on MCF-7 breast cancer cell proliferation activities with IC50 ( μg / ml ) = 136.67 ± 5.50 and 191.59 ± 0.78. On the other hand, YC-27 inhibited the MDA-MB-231 breast cancer cell proliferation activities with IC50 ( μg / ml ) = 112.79 ± 16.96. Moreover,in cell morphology assay,apoptotic body including cell shrinkage and chromatin condensation were found in 100 μg / ml-YC-11- and 100 μg / ml-YC-13-added MCF-7 cell culture,as well as in 100 μg / ml-YC-27-added MDA-MB-231 cell culture. Liebermann-Burchard test was used for steroid determination and results showed that：steroid-contained samples were YC-11、YC-13、YC-14、YC-20、YC-21、YC-24、YC-25 and YC-26. B. In vitro anti-tumor genetic effect of YC-27. Since results of MTT test exhibited YC-27 extraction from methanol ( MeOH ) were significantly inhibit MDA-MB-231 cell line which associated with malignant and metastatic abilities,the botany YC-27 were extracted and fractionated with solvents in different polarity such as hexane,ethyl acetate ( EA ), acetone and MeOH to begin the further exploration. In MTT assay, EA / Acetone / MeOH extraction/fraction at concentration of 200 μg / ml exhibited significantly inhibitory effect ( % ) on ( 1 ) MCF-7 cell：YC-27-E = 76.77 ± 2.94, YC-27-EF1 = 79.29 ± 11.85, YC-27-EF2 = 93.35 ± 3.28 and YC-27-MF2 = 91.70 ± 2.59, and on ( 2 ) MDA-MB-231 cell：YC-27-E = 93.80 ± 8.68, YC-27-EF1 = 61.24 ± 5.47, YC-27-A = 97.68 ± 3.18, YC-27-AF1 = 77.40 ± 7.27, YC-27-AF2 = 82.54 ± 4.86, YC-27-M = 62.16 ± 5.20, YC-27-MF2 = 87.08 ± 6.20, YC-27-MF3 = 71.37 ± 3.12, YC-27-70 % M = 65.64 ± 1.40. Furthermore, in cell morphology assay,apoptotic body including cell shrinkage and chromatin condensation were found in 100 μg / ml-extraction / fraction-added MCF-7 cell culture, as well as in 100 μg / ml-extraction / fraction-added MDA-MB-231 cell culture. In MTT assay, the selected anti-cancer ability between MCF-7 and MDA-MB-231 cell of extractions / fractions－YC-27-AF2、YC-27-M、YC-27-MF3、YC-27-70 % M showed potential of drug development. DNA fragmentation and western blotting showed that YC-27-MF3 exhibited DNA fragment in DNA fragmentation assay and expresses apoptotic protein－caspase 3 in western blotting assay. In view of antioxidant and anti-breast cancer properties of botany YC-27, the investigation of the potential carcinogenesis and other diseases prevention is feasible and anticipative in the future.
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