臺灣博碩士論文加值系統

根據衛生署近20 年來的十大死因分析報告顯示，癌症仍屬十大死因的榜首，臨床進行癌症治療的方法很多，其中以中草藥成分物質進行癌症治療日益重要。本論文選定臺灣特有種臺灣蛇莓進行研究，臺灣蛇莓(Duchsnea chrysantha)具止血、解熱、除毒之用途以及治療疔瘡及胃病等，而本研究的目的則著眼於台灣蛇莓的主成分萃取、分析與細胞層級的各項活性評估等。
本論文共分五章，第一章介紹研究的目的，第二章敘述文獻回顧，第三章為實驗的材料與方法，第四章為結果與討論，第五章為結論。依本論文研究顯示：1.因季節不同，其本身所含的有效成分含量亦有所不同，在夏季的臺灣蛇莓對於癌細胞具抑制生長效果，而冬季
的臺灣蛇莓不具有細胞抑制生長的效果；2.從抗氧化能力探討對DPPH 自由基的清除能力，其清除DPPH 自由基的能力可由原本的27%提升至60 %；3.細胞內的抗氧化能力知臺灣蛇莓對細胞具有保護能力的，可使細胞存活率由原本的53%提升至74%，而臺灣蛇莓對細
胞沒有明顯修復的能力；4.在抗輻射能力評估上，雖加入320μg/mL的臺灣蛇莓後，其細胞致死率減少63%，但由於採用低劑量Cs-137照射，其抗輻射殺傷力較不具意義。
本論文顯示臺灣蛇莓確實有抑制癌細胞增生、抗自由基傷害及抗氧化的功效，針對其成分進行化學結構鑑定與各種生物活性測試，可作為藥物毒殺細胞等進行深入探討，以為基礎臨床研究作貢獻。

According to the report of the Department of Health for the past 20
years, cancer is still the first disease of the ten leading death cause. There are many effective methods for clinical cancer treatment, among them, herbs substances for cancer treatment has become increasingly important.
This paper selects unique plants in Taiwan, Duchsnea chrysantha, to proceed the major components extraction, substance characterization, cell level evaluation, etc. The pharmacology of Duchsnea chrysantha exhibits the function of hemostasis, antipyretic, disinfection and the treatment of boils and stomach problems, etc.
The paper is divided into five chapters, the first chapter describes the purpose of the study. In the second chapter, we reviewed the recent researches. The third chapter shows the experimental materials and methods, and the results and discussion are in the forth chapter. The conclusions of the paper are in the fifth chapter. Some phenomenons were observed in the paper and concluded briefly as following: 1. Depending on the season of harvest, the effective components of Duchsnea chrysantha show little difference, the summer Duchsnea chrysantha proves the suppression effect of cells growth, while the winter Duchsnea chrysantha exhibits no suppression effect. 2. Explore the DPPH radical scavenging capacity of Duchsnea chrysantha from the antioxidant capacity, DPPH radical scavenging capacity from the original 27% to 60%. 3. In the intracellular antioxidant capacity, Duchsnea chrysantha can protect cells, and the survival rate increase from the original 53% to 74%. However, Duchsnea chrysantha confirms no cells repair ability. 4. Although the spiked concentration of Duchsnea chrysantha was as high
as 320 μg /mL in the cell culture medium, cell death rate, caused by radiation exposure, was also decreased to 63%. However, this results, due to low dose of Cs-137 exposure, exhibit less meaningful.
The paper shows that Duchsnea chrysantha is indeed inhibit cancer
cell proliferation, anti-free radical damage and anti-oxidation. Studies such as characterization chemical components of Duchsnea chrysantha, structure identification and biological activity test can be used to clinical evaluation, further studies will be proceed in the future.

目錄
致謝 I
中文摘要 II
英文摘要 III
目錄 V
圗目錄 VIII
表目錄 X
第一章 前言 1
1.1 前言 1
1.2研究目的 1
1.3 天然物的使用 2
1.3.1 天然物的各種特性 3
1.3.2 臺灣蛇莓 3
1.3.3 臺灣蛇莓之成分與藥理作用之研究 5
第二章 文獻回顧 6
2.1藥用植物的重要性 6
2.2中草藥的萃取和初步分離方法 8
2.3高效液相層析(HPLC)分析條件選擇的介紹 10
2.4癌細胞的介紹 14
2.4.1 細胞週期調控之異常與癌細胞生長 14
2.4.2 癌細胞生長與細胞凋亡 15
2.5 抗氧化劑的介紹 16
2.5.1自由基之介紹 16
2.5.2自由基對生物體分子之氧化性傷害 25
2.5.3自由基與疾病之相關性 26
2.6生物體內抗氧化防禦系統 26
第三章 儀器與分析方法 31
3.1 實驗儀器 31
3.2 實驗材料 32
3.3 試劑 32
3.4 蛇莓成分物萃取 33
3.4.1臺灣蛇莓萃取方法 33
3.4.2傳統熱水萃取法(HW) 34
3.4.3 超高壓水萃取法(HPW) 34
3.4.4製備級純化方式 35
3.5細胞培養 36
3.5.1細胞來源 36
3.5.2細胞培養液之配製 38
3.5.3 老鼠正常肝細胞(Clone – 9)的培養方法 39
3.5.4 前列腺癌細胞(Du -145)的培養方法 39
3.5.5 細胞數目計算 40
3.6 細胞株解凍與保存 41
3.6.1 細胞株解凍 41
3.6.2細胞株保存 41
3.7 細胞實驗方法 42
3.7.1 細胞存活率MTT assay原理 42
3.7.2方法步驟 44
3.8清除DPPH自由基實驗 44
3.9細胞核染色 45
第四章 結果與討論 47
4.1蛇莓萃取物最佳化條件之液相層析圖譜 47
4.1.1熱水及高壓冷水蛇莓萃取物之HPLC圖譜比較 49
4.1.2 不同季節蛇莓萃取物之HPLC圖譜比較 50
4.2臺灣蛇莓對於癌細胞的抑制能力 51
4.2.1 熱水及高壓冷水蛇莓萃取物之抑制不同癌細胞能力 51
4.2.2不同季節蛇莓萃取物抑制不同癌細胞能力 52
4.3 DAPI細胞核染色 55
4.4蛇莓之抗氧化能力評估 55
4.4.1蛇莓萃取物細胞外(In vitro)之抗氧化力之評估 55
4.4.2蛇莓萃取物細胞內(In vivo)之抗氧化力之評估 58
4.4.3 細胞內抗氧化保護實驗 59
4.4.4細胞內抗氧化修復實驗 63
4.5蛇莓之抗輻射能力評估 64
第五章 結論與展望 66
參考文獻 68

圗目錄
圗1-1 臺灣蛇莓的產地分布 4
圗1-2蛇莓--植株 4
圗1-3蛇莓--果實 4
圖2-1氧分子的氧化還原及激發狀態 22
圖2-2氧自由基及含氮自由基之生成途徑 22
圖3-1臺灣蛇莓全株乾燥後的植株 33
圖3-2臺灣蛇莓全株研磨後粉末 33
圖3-3 高壓冷水萃取系統 34
圖3-4高壓幫浦設備 35
圖3-5不銹鋼萃取槽 35
圖3-6冷凍乾燥機 35
圖3-7臺灣蛇莓高壓水萃取粉末 36
圖3-8臺灣蛇莓熱水萃取粉末 36
圖3-9老鼠正常肝細胞株(CLONE-9) 36
圖3-10 攝護腺癌細胞(DU-145) 37
圖3-11 人類肝癌細胞(HUH– 7) 37
圖3-12 人類血癌細胞(K562) 38
圖3-13 細胞計數盤與細胞計數區域放大簡圖 40
圖3-14 血球計數器 41
圖3-15 血球計數器放大圖 41
圖3-16 MTT反應中MTT黃色分子轉變為藍紫色FORMAZAN 42
圖3-17 96-WELL待測樣品 43
圖3-18 HUH-7 CELL 43
圖3-19 HUH-7 CELL 43
圖3-20 酵素吸附免疫分析儀(ELISA READER ) 44
圖3-21 DPPH(Α,Α-DIPHENYL-Β-PICRYL- HYDRAZYL) 自由基反應 45
圖3-22 DAPI(4',6-DIAMIDINO-2-PHENYLINDOLE)的結構式 45
圖4-1 臺灣蛇莓之分析級HPLC圖譜 48
圖4-2臺灣蛇莓之分析級HPLC圖譜 48
圖4-3臺灣蛇莓之製備級HPLC圖譜 49
圖4-4 臺灣蛇莓之分析級HPLC圖譜 50
圖4-5 臺灣蛇莓之分析級HPLC圖譜 51
圖4-6臺灣蛇莓高壓水與熱水萃取液對人類肝癌細胞(HUH-7)的影響 52
圖4-7 臺灣蛇莓夏季採收之萃取液對老鼠正常肝細胞(CLONE-9)的抑制作用 53
圖4-8 臺灣蛇莓夏季採收之萃取液對人類攝護腺癌細胞(DU-145)的抑制作用 54
圖4-9 臺灣蛇莓冬季採收之萃取液對老鼠正常肝細胞(CLONE-9)的抑制作用 54
圖4-10 臺灣蛇莓冬季採收之萃取液對人類攝護腺癌細胞(DU-145)的抑制作用 55
圖4-11 DAPI細胞核染色技術於螢光顯微鏡觀察細胞凋亡的情形 56
圖4-12 清除DPPH自由基之測定 57
圖4-13 鼠類正常肝細胞(CLONE-9)對於氧化劑H2O2之耐受性探討 60
圖4-14 鼠類正常肝細胞(CLONE-9)對於氧化劑H2O2之耐受性探討 60
圖4-15 不同濃度之蛇莓應用於老鼠肝細胞(CLONE-9)之保護作用 62
圖4-16 不同濃度之蛇莓應用於老鼠肝細胞(CLONE-9)之修護作用 63
圖4-17 蛇莓之抗輻射能力評估(本實驗為MTT ASSAY之結果) 65

表目錄
表2-1 細胞凋亡與壞死的比較 17
表2-2 自由基及活性氧的種類 19
表2-3 活性氧物質的來源 21
表4-1清除DPPH自由基之測定方法 57
表4-2 鼠類正常肝細胞(CLONE-9)對於氧化劑H2O2之耐受性探討 61
表4-3 不同濃度之蛇莓應用於老鼠肝細胞(CLONE-9)之保護作用 62
表4-4 不同濃度之蛇莓應用於老鼠肝細胞(CLONE-9)之修護作用 64

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