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研究生:蔡玉鈴
研究生(外文):Tsai, Yuling
論文名稱:紫錐菊花部活性成分之萃取及抗大腸癌細胞生長作用機制之探討
論文名稱(外文):Studies On The Extraction Of Active Components Of Echinacea Purpurea Flower And Its Anti-tumor Mechanism In Human Colon Cancer Cells
指導教授:詹恭巨詹恭巨引用關係
指導教授(外文):Chan, Kungchi
口試委員:張珍田楊肇基林聖敦謝長奇
口試委員(外文):Chang, ChentienYang, JawjiLin, ShengdunHsieh, Changchi
口試日期:2012-06-04
學位類別:博士
校院名稱:靜宜大學
系所名稱:食品營養學系
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:117
中文關鍵詞:紫錐菊咖啡酸衍生物菊苣酸細胞凋亡端粒酶凋亡蛋白酶大腸癌
外文關鍵詞:Echinacea purpurwaCaffeic acid derivativeCichoric acidApoptosisTelomeraseCaspasecolon cancer
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紫錐菊(Echinacea purpurea)原產於北美洲中西部,具有增強及刺激人體免疫系統的效果。這些保健功效與紫錐菊所含之藥理活性成分有關,如咖啡酸衍生物、烷醯胺及多醣體等,特別是菊苣酸(cichoric acid)。研究證實,菊苣酸是紫錐菊最具有藥理活性的主要成分,也是紫錐菊產品品質的主要參考指標成分。紫錐菊花中菊苣酸的含量最高,目前國外的研究,多著重在紫錐菊的根部萃取及其生理活性之探討,對於花中有效物質的萃取及生理活性之探討,則待研究。而誘導癌細胞細胞週期停滯及細胞凋亡是研究藥物或天然物抗癌作用的兩大重要指標。因此,本研究第一部份,以乙醇為萃取溶劑,探討不同乙醇濃度及不同萃取溫度,對紫錐菊花部萃取物活性成分之影響,並了解其抗氧化性及抗致突變性。第二部份則是以人類大腸癌細胞Caco-2和HCT-116為對象,探討紫錐菊花部乙醇萃取物及菊苣酸對癌細胞生長之影響,並探討其作用機轉。
實驗結果顯示,50%乙醇及65℃為凍乾紫錐菊花部活性成分的最適萃取條件,其50%乙醇萃取物具有清除DPPH自由基能力、還原力、螯合亞鐵離子能力且不具毒性,並具有抗致突變性。以 (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT)分析細胞生長時發現,紫錐菊花部50%乙醇萃取物及菊苣酸可抑制大腸癌細胞的生長,且具有時間與劑量依存的關係。另外,添加菊苣酸能抑制人類端粒酶反轉錄酶(human telomerae reverse transcriptase; hTERT)mRNA的表現,亦能抑制端粒酶活性。在誘導細胞凋亡的部份,菊苣酸會導致DNA片段化、增加p53的表現、caspase-7, -9的活化,poly-ADP-ribose polymerase (PARP)及β-catenin的降解,而抗凋亡因子Bcl-2的表現也明顯受到菊苣酸的抑制。在MAPK訊號傳遞途徑方面,高濃度的菊苣酸會誘導P38及JNK的活化,抑制ERK的表現並且造成細胞週期G1期停滯。。
綜合以上之結果,紫錐菊花部乙醇萃取物及菊苣酸具有抗氧化及細胞毒殺功能。另外,菊苣酸具有抑制大腸癌細胞生長及誘導細胞凋亡之功能,這些效應可能經由抑制hTERT和Bcl-2的表現、抑制端粒酶的活性、導致細胞週期停滯、活化caspase及DNA片段化,同時菊苣酸也可經由活化P38及JNK,最後誘導細胞進行粒腺體- casapse凋亡路徑,走向細胞凋亡。由此可知,紫錐菊花部乙醇萃取物及菊苣酸可應用於大腸癌之輔助治療。
Echinacea purpurea L. is one of the top selling medicinal plants widely used to stimulate immune system. The immuno-stimulating properties are attributed to the bio-active phytochemicals including caffeic acid derivatives, alkamides, polysaccharides, and glycoproteins. Among these caffeic acid derivatives, cichoric acid is the major component that contains biological activities. This study evaluated the effects of ethanol concentration and extraction temperature on the quality of extracts obtained from freeze-dried E. purpurea flowers. The antioxidant and antimutagenic effects of flower extract were also examined. Moreover, the inhibitory effects of flower extract and cichoric acid on human colonic cancer cells growth were investigated.
The optimal extraction conditions for freeze-dried flowers were 50% aqueous ethanol and 65°C extraction temperature, with extraction yield of 37.4%. The 50% ethanolic flower extract did not show toxicity and mutagenicity toward Salmonella typhimurium TA98 and TA100 with or without S9 mix. The ethanolic extract at 0.25–5 mg/plate exhibits a dose-dependent inhibitory effect against the mutagenicity of 2-aminoanthracene. Thus, freeze-dried E. purpurea flower ethanolic extract exhibits high antioxidant and antimutagenic activities.
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that the growth of human colon cancer cells was inhibited by flower extract and cichoric acid in dose- and time-dependent manners. Cichoric acid treatment inhibited the expression of human telomerase reverse transcriptase (hTERT) mRNA and telomerase activity, but activated p53、caspase-7, -9 and downregulated the expression of Bcl-2、poly-ADP-ribose polymerase and β-catenin. Cytometric analysis showed that cichoric acid induced G1-phase arrest within 24-48 h. In addition, cichoric acid may induce mitochondria-dependent apoptosis through the activation of p38 and JNK. Thus, the 50% ethanol extract of E. purpurea flowers and cichoric acid could be used as a potential therapeutic agent against colon cancer.
表目錄 I
圖目錄 II
縮寫檢索表 III
中文摘要 1
英文摘要 3
第壹章、前言 5
第貳章、文獻回顧 7
第一節、紫錐菊 7
(一)、菊苣酸(Cichoric acid) 8
(二)、萃取方法 12
第二節、大腸癌 13
第三節、端粒(Telomere)的長度及端粒酶(Telomerase)的活性 14
(一)、端粒 14
(一)、端粒酶 15
第四節、細胞凋亡(Apoptosis) 19
第五節、 MAPK訊號傳遞路徑 26
第六節、 研究目的 29
第参章、紫錐花中有效成份之萃取及其抗氧化與抗致突變性之探討 30
第一節、前言 31
第二節、材料與方法 33
一、試驗材料及化學藥品 33
二、紫錐菊花部乙醇萃取物之製備 34
(一)、原料前處理 34
(二)、紫錐菊花部乙醇萃取物之製備方法 34
1、不同濃度乙醇之影響 34
2、不同萃取溫度之影響 34
(三)、品質分析 35
1、總酚類含量測定 35
2、咖啡酸衍生物含量測定 36
(四)、抗氧化能力分析 37
1、DPPH自由基清除能力測定 37
2、還原力測定 37
3、螯合亞鐵離子能力測定 38
(五)、安姆氏試驗 39
1、Solmonella菌種 39
2、毒性試驗(Toxicity test) 39
3、致突變性試驗(Mutagenicity test) 40
4、抗致突變性試驗(Antimutagenicity assay) 41
(六)、統計分析 42
第三節、結果與討論 43
一、不同乙醇濃度對萃取率、總酚類及咖啡酸衍生物含量之影響 43
二、不同萃取溫度對萃取率、總酚類及咖啡酸衍生物含量之影響 44
三、抗氧化性 45
(一)、抗氧化力 45
(二)、還原力 46
(三)、亞鐵離子螯合能力 46
(四)、毒性、致突變性及抗致突變性 47
第肆章、紫錐菊花部乙醇萃取物和菊苣酸對人類大腸癌細胞生長之影響及作用機轉之探討 57
第一節、前言 58
第二節、材料與方法 60
一、細胞與化學藥品 60
二、紫錐菊花部乙醇萃取物之製備方法 61
三、細胞培養 61
四、細胞存活率試驗(MTT法) 62
五、端粒酶活性試驗 63
六、細胞片段化試驗(DNA fragmentation assay) 64
七、 RT-PCR 65
八、西方墨點法 68
九、細胞週期分析 70
十、統計分析 70
第三節、結果與討論 71
一、紫錐菊花部50%乙醇萃取物和cichoric acid可抑制大腸癌細胞之生長...71
二、菊苣酸抑制hTERT mRNA的表現及降低細胞內端粒酶的活性 72
三、菊苣酸誘導大腸癌細胞凋亡機制之探討 74
第伍章 結論 81
第陸章 參考文獻 78
附錄一、Caffeic acid derivatives, total phenols, antioxidant and antigenic activities of Echinacea purpurea flower extracts 116
*http://herbalhealing.tumblr.com/post/15249406253/echinacea-echinacea- purpure a-e-angustifolia.
*http://articles.mercola.com/sites/articles/archive/2010/02/23/science-finally- reveals-how-you-can-actually-revese-aging.aspx
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