臺灣博碩士論文加值系統

本研究以16種栽培品系之山苦瓜為材料，分別以其水抽取物或甲醇抽取物進行抗氧化、細胞毒性、抗菌活性及SSAO酵素抑制活性之篩選，並針對細胞毒性物質進行初步分離。
結果發現，依據品系的不同，在各個活性之表現亦有相當大的差異，在抗氧化活性方面，對於DPPH自由基清除作用最強之品種，其IC50可達181 g/mL，相當於每克水粗抽取物內約有25.500 g之水溶性維他命E。在對超氧自由基的清除方面，少數的水抽取物與幾乎所有的甲醇抽取物均具有活性，且以甲醇抽取物之清除能力較佳。在低密度脂蛋白氧化試驗方面，無論以瓊膠電泳染色觀察或是以TBARS assay測定MDA之含量，均顯示水或甲醇粗抽取物具有抑制Cu2+促進LDL過氧化之活性。而以電子自旋共振儀測定粗抽物清除氫氧自由基能力之試驗中，我們也可以看到山苦瓜之水或甲醇抽取物，均能有效的清除氫氧自由基。
於細胞毒性方面，僅有少數品系之水以及甲醇粗抽取物對於HT 1080細胞株具有細胞毒性。進一步以不同濃度之D品系水與甲醇抽取物處理HT 1080細胞株後，可以得到其IC50分別為0.368 mg/mL及0.550 mg/mL。不過在以正常纖維母細胞進行細胞毒性的試驗當中，大多數對於HT 1080細胞株具有細胞毒性之樣品，也同樣的具有細胞毒性，故其對癌細胞與正常細胞之毒殺濃度，還有待進一步之釐清。
抗菌方面，則依據菌種的不同，有抑菌活性之品系也不同，數種品系之水抽物以及甲醇抽取物對於大腸桿菌和沙門氏桿菌有抑制之作用，且樣品經過加熱後，仍具有抑菌活性。但所有的品系對於抗藥性之金黃色葡萄球菌以及綠膿桿菌均無抑菌之活性。
在SSAO酵素抑制活性方面，大多數品系之山苦瓜甲醇粗抽取物，在1 mg/mL之濃度下均對於SSAO之活性有不錯的抑制作用，而在水粗抽物方面，僅有兩品系在1 mg/mL能使抑制率達到50%。而在甲醇粗抽取物中，抑制率在90%左右之品系為D、E、F、H、K、N與P，其IC50分別為0.594、0.653、0.637、0.673、0.397、0.386與0.750 mg/mL。

The water or methanol extracts from sixteen species of Momordica charantia L. var. abbreviata Seringe (MCs) were used for anti-oxidation, cytotoxicity, anti-bacteria and SSAO inhibition screening assays.
It was found that the variations among the species of each biological activity were found. For screening of the anti-oxidation activity, the IC50 for the most potent species on DPPH scavenging activity assay was 181 g/mL equivalent to 25.500 g of Trolox® per gram water extracts. For screening of superoxide radical scavenging activity using autoxidation of pyrogallol assay system, it was found that less number of the water extracts and almost all the methanol extracts exhibited the scavenging activity against superoxide radicals. In the anti-LDL oxidation assay, determined by both the electrophoretic mobility and TBARS assays both extracts of MCs showed positively inhibitory activity on Cu2+-induced LDL peroxidation. Furthermore, the both extracts of MCs showed good scavenging activity on the hydroxyl radical generated by Fenton reaction using the ESR spectrometric method.
On the screening of anti-cancer activity, some water and methanol extracts exhibited cytotoxicity on HT 1080 cells. For the D species of MCs, the IC50 of the water and methanol crude extracts were 0.368 and 0.550 mg/mL, respectively, and the cytotoxic activity increased after Sephadex LH-20 purifications. However, the potent extracts with cytotoxicity on HT 1080 cells were also found to have cytotoxic activity on HDF (primary culture cells). Further investigations will be done in the future.
On the screening of anti-bacteria activity, all extracts had no activities against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, however, some extracts did have the inhibitory activity on the growth of Escherichia coli and Salmonella enterica. The inhibitory activity remained even after heating at 100°C for 5 minutes.
On the screening of SSAO inhibitory activity, the methanol extracts of MCs showed potent inhibitory activity against SSAO; while only two species of MCs’ water extracts reached the 50% inhibition. The species of the methanol extracts reached the 90% inhibition including D, E, F, H, K, N and P, which the IC50 were 0.594, 0.653, 0.637, 0.673, 0.397, 0.386 and 0.750 mg/mL, respectively.

目錄 •••••••••••••••••••••••••••••••••••••••••••••••••••• I
圖目錄 •••••••••••••••••••••••••••••••••••••••••••••••••• V
表目錄 ••••••••••••••••••••••••••••••••••••••••••••••••• IX
中文摘要 •••••••••••••••••••••••••••••••••••••••••••••••• X
英文摘要 •••••••••••••••••••••••••••••••••••••••••••••• XII
縮寫表 •••••••••••••••••••••••••••••••••••••••••••••••• XIV

第一章 序論
第一節、前言 •••••••••••••••••••••••••••••••••••••••••••• 1
第二節、文獻回顧 •••••••••••••••••••••••••••••••••••••••• 1
一、苦瓜之介紹 •••••••••••••••••••••••••••••••••••••••••• 1
（一）命名 •••••••••••••••••••••••••••••••••••••••••••••• 1
（二）生產與分佈 •••••••••••••••••••••••••••••••••••••••• 2
（三）性狀 •••••••••••••••••••••••••••••••••••••••••••••• 2
（四）栽種環境 •••••••••••••••••••••••••••••••••••••••••• 3
（五）品種 •••••••••••••••••••••••••••••••••••••••••••••• 4
（六）典籍記載 •••••••••••••••••••••••••••••••••••••••••• 4
（七）植物化學 •••••••••••••••••••••••••••••••••••••••••• 5
（八）藥理活性 ••••••••••••••••••••••••••••••••••••••••• 12
（九）山苦瓜之介紹 ••••••••••••••••••••••••••••••••••••• 23
二、自由基與抗氧化物之介紹 ••••••••••••••••••••••••••••• 24
三、Semicarbazide-Sensitive Amine Oxidase (SSAO)之介紹 ••••• 28
第三節、實驗動機與目的 ••••••••••••••••••••••••••••••••• 31

第二章 材料與方法
第一節、實驗材料 ••••••••••••••••••••••••••••••••••••••• 32
一、山苦瓜 ••••••••••••••••••••••••••••••••••••••••••••• 32
二、細胞株 ••••••••••••••••••••••••••••••••••••••••••••• 35
三、菌種 ••••••••••••••••••••••••••••••••••••••••••••••• 39
第二節、實驗方法 ••••••••••••••••••••••••••••••••••••••• 42
一、抗氧化活性之測定 ••••••••••••••••••••••••••••••••••• 42
（一）DPPH自由基清除試驗 ••••••••••••••••••••••••••••• 42
（二）超氧自由基清除試驗 ••••••••••••••••••••••••••••••• 42
（三）抗低密度脂蛋白氧化能力試驗 ••••••••••••••••••••••• 43
（四）TBARS assay •••••••••••••••••••••••••••••••••••••• 45
（五）電子自旋共振儀測定氫氧自由基清除試驗 ••••••••••••• 45
二、抗癌活性之測定 ••••••••••••••••••••••••••••••••••••• 46
（一）癌細胞之存活率試驗 ••••••••••••••••••••••••••••••• 46
（二）正常細胞之存活率試驗 ••••••••••••••••••••••••••••• 47
三、抗癌活性成分之分離 ••••••••••••••••••••••••••••••••• 49
（一）DGH之活性成分分離 •••••••••••••••••••••••••••••• 49
（二）DGM之活性成分分離 •••••••••••••••••••••••••••••• 49
四、抗菌活性之測定 ••••••••••••••••••••••••••••••••••••• 51
（一）生長抑制情況測定 ••••••••••••••••••••••••••••••••• 51
五、抑制SSAO活性之試驗 ••••••••••••••••••••••••••••••• 52
（一）抑制SSAO活性試驗 ••••••••••••••••••••••••••••••• 52

第三章 結果與討論
一、山苦瓜粗抽物之製備 ••••••••••••••••••••••••••••••••• 53
二、抗氧化活性探討 ••••••••••••••••••••••••••••••••••••• 55
（一）DPPH自由基清除試驗 ••••••••••••••••••••••••••••• 55
（二）超氧自由基清除試驗 ••••••••••••••••••••••••••••••• 60
（三）抗低密度脂蛋白氧化能力試驗 ••••••••••••••••••••••• 64
（四）TBARS assay •••••••••••••••••••••••••••••••••••••• 67
（五）電子自旋共振儀測定氫氧自由基清除試驗 ••••••••••••• 70
三、抗癌活性之測定 ••••••••••••••••••••••••••••••••••••• 75
（一）癌細胞之存活率試驗 ••••••••••••••••••••••••••••••• 76
（二）正常細胞之存活率試驗 ••••••••••••••••••••••••••••• 82
四、抗癌活性成分之分離 ••••••••••••••••••••••••••••••••• 85
（一）Sephadex LH-20區分之細胞毒性試驗 ••••••••••••••••• 85
五、抗菌活性之測定 ••••••••••••••••••••••••••••••••••••• 89
（一）生長抑制情況測定 ••••••••••••••••••••••••••••••••• 89
六、抑制SSAO活性之試驗 •••••••••••••••••••••••••••••• 101
（一）抑制SSAO活性試驗 •••••••••••••••••••••••••••••• 101

第四章 結論 ••••••••••••••••••••••••••••••••••••••••••• 105

第五章 參考文獻 ••••••••••••••••••••••••••••••••••••••• 109

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