傷寒草地上部對口腔癌細胞致死作用之探討
The Lethal Effect of the Aerial Part of Vernonia Cinerea to CT-5'&apos
中文摘要
本實驗主要目的是探討傷寒草對口腔癌細胞的致死作用。論文中以口腔癌細胞 (CT-5＇)為實驗對象，探討傷寒草萃取物對口腔癌細胞的影響。利用各種的生 物活性試驗方法，尋找傷寒草殺死口腔癌細胞(CT-5＇)的可能機制。傷寒草 (Vernonia cinerea)，又名「一支香」，屬於菊科(composiate)植物，花朵為 略帶紫色的野草，分布於熱帶亞洲。台灣坊間將傷寒草以酒水蒸煮後食用，發現 對治療喉癌，口腔癌症有一定的療效。
傷寒草經過50%酒精萃取濃縮後，進行細胞實驗，實驗結果中發現，將傷寒草
全草濃縮液處理口腔癌細胞(CT-5＇)24 hrs 後，其 LD50 約為 375 μg/cc。
若將傷寒草莖葉經50% ETOH 萃取濃縮後再以氯仿 partition 分層取有機層濃 縮液，進行細胞測試，其24 hrs 的 LD50 約為 100μg/cc。另做纖維母細胞 (fibroblast)為正常細胞對照組，進行 MTT 毒性測試，發現傷寒草莖葉氯仿萃 取液之濃度對纖維母細胞(fibroblast)及口腔癌細胞(CT-5＇)的毒性皆與濃度 成正相關，但對纖維母細胞具較低的毒性。將不同濃度之傷寒草莖葉氯仿萃取液 處理細胞24 小時後，以 D2O/PBS 中的 D 置換細胞內的 H，利用 NMR 儀器分 析，觀察細胞內CH2 及 CH3 於 H＇-NMR 圖譜上之 peak 高度變化，peak 的
高度，可分別表示細胞CH2 及 CH3 的濃度。發現隨著細胞處理藥物的濃度的
升高，H＇-NMR 圖譜上 CH2/CH3 高度比值隨之減低，顯示細胞內 CH2/CH3 內的濃度比值下降。由此實驗初步證明傷寒草莖葉氯仿萃取液引起口腔癌細胞 (CT-5＇)的死亡機制可能經由細胞程式死亡(apoptosis)。另將不同濃度傷寒草 莖葉氯仿萃取液處理細胞24 小時，以流式細胞儀(flow cytometry)進行分析，
發現細胞隨著處理之傷寒草莖葉氯仿萃取液濃度增加，Sub-G1 peak 越高，細 胞內的DNA 斷裂越多，此現象可能表示細胞 apoptosis 的現象越明顯。另外，
利用acrydine orange 將細胞內的染色體染色，觀察細胞加入藥物後的型態變 化，發現未加藥物之細胞，細胞的型態完整。隨著藥物濃度的增加，細胞染色質 濃縮現象越明顯，亦產生越多的凋亡細胞(apoptotic body)，且具
dose-dependent 的現象。由上證明傷寒草莖葉氯仿萃取液殺死口腔癌細胞 (CT-5＇)之途徑是經由細胞自殺性死亡(apoptosis)。
英文摘要
The main purpose of the experiment is to explore the lethal effect of Vernonia cinerea to oral cancer cell line. We want to explore the lethal effect of the extracts from Vernoniaa cinerea to oral cancer cell line (CT-5’) in the essay. We also try to fine the
possible mechanism of Vernonia cinerea to oral cancer cell line (CT-5’) with any kinda bio-assay methods. Vernonia cinerea, as known as composiate, a Chinese herb with purpose bloom, distributed in tropic Asia. People in Taiwan boiled it with rice wine and water to take and find that has its anticancer effect for both oral cancer and larynx cancer.
We extracted the Vernonia cinerea with 50% alcohol and concentrated by
rotavapor .The LD50 in extracts of the whole plant of Vernonia cinerea to CT-5’ cell after 24hrs treatment was 375μg/cc.However,the chloroform phase extract from the aerial part of Vernonia cinerea after 50% alcohol extraction the LD50 was 100 μg/cc after 24hrs treatment. And keep the test of cytotoxicity by MTT assay as fibroblast as control. The chloroform phase extracts from the aerial part of Vernonia cinerea to CT-5’ and fibroblast was dose-dependent manner. But it had less cytotoxicity to fibroblast. We replace the H of CT-5'' with D from D2O/PBS after treating CT-5'' with different concentrations after 24hrs.According to the NMR result ,We find the ratio of the peak high in -CH2 and -CH3 on the H’-NMR spectrum that was dose-dependent with the ratio of the two peaks. With the increasing of the concentrations of the chloroform phase extracts from the aerial part of Vernonia cinerea ,the ratio of CH2/CH3 decrease in CT-5’ cell. What we mention above prove me the pathway might be by apoptosis in the effect of CT-5’ induced by chloroform phase extracts of the aerial part of Vernonia cinerea.The result show from cytometry after treating CT-5’ with different chloroform phase extracts .The Sub-G1 peak is does-dependent with the concentrations of the extracts that show the cleavage of the DNA in the cell is getting more and more .The phenomenon above may show the signal of apoptosis in the CT-5’ cell. In the other hand, we stained the chromosome of the cell with acrydine orange after treating cell with different concentrations of the chloroform phase extracts from the aerial part of Vernonia cinerea .We observe that morphology of the untreated cell is intact .The phenomenon of chromosome
condensation and the forming of apoptotic bodies became more and more when we increased the concentrations of the extract to the cell. What phenomenon we observe above provide the possible pathway that the chloroform phase extracts from the aerial part of Vernonia cinerea in CT-5’ lethal effect might be by apoptosis.
