本論文分兩個部分探討佛甲草對肝癌細胞 Hep 3B cells的影響,並尋找其有效成分物質。首先,利用細胞實驗探討佛甲草水相萃取物及經液相層析儀純化之混合物 對Hep 3B cells細胞存活狀況的影響。藉由 MTT 分析法與Trypan blue 分析法中發現均可提高細胞的死亡率,根據MTT 分析法所得之數據計算出水相萃取物 IC50 濃度為0.76 mg/ml,液相層析儀純化之混合物 IC50 濃度為0.231 mg/ml、0.391 mg/ml。但是其產量太低,因此將改變其萃取方式來提高產率並進一步降低IC50 濃度。 第二部分探討乙醇相萃取物之靜置時間對有效成分是否有影響,結果發現浸泡十九天的產物, IC50 濃度低於浸泡三天與七天萃取物之IC50 濃度。比較水相混合物 與乙醇相萃取物的IC50 濃度後發現,乙醇相萃取物的IC50 濃度較低,為 0.199mg/ml。故本實驗以乙醇相萃取物作進一步的探討,並利用TLC 片進行兩次純化後再分別用MTT 分析法與Trypan blue 分析法探討細胞的死亡率。第一次TLC純化(EA:MeOH = 5:4)分別得到混合物 1、2及3,藉由細胞實驗測試發現混合物 1 對於提高細胞死亡率的效果最好,其 IC50 濃度為0.037 mg/ml。將混合物1 進行第二次 TLC 純化(EA:Hexane = 3:4)分別得到混合物 1-1、1-2、1-3、1-4及1-5,並利用MTT 分析法與 Trypan blue 分析法探討五個混合物 對於細胞存活狀況的影響。進一步對混合物 1-3 及 1-4 作細胞核染色實驗,得知具有提高細胞死亡率之效果,且發現肝癌細胞之細胞核出現凋亡小體型態。於化學顯色反應中得知混合物 1-3 含有黃酮類化合物 。 綜合以上實驗結果發現混合物 1-3及1-4 可能藉由誘發細胞凋亡來達到提高細胞死亡率,其中TLCⅡ之混合物 1-3 可能含有黃酮類化合物因此本論文推測可能與提高細胞死亡率有關。
Abstract This study focuses on the effect of Sedum extracts extracted using two different solvents, ethanol and water, on hepatoma (Hep 3B) cells in vitro. First, active components were extracted from Sedum using water and were separated by HPLC prior to use in cell assay. MTT and Trypan blue assays confirmed that the crude Sedum extract can reduce cell activity. Based on the MTT assay, the IC50 concentration of the crude extract was 0.76 mg/ml whereas HPLC-separated mixtures obtained at different time points had IC50 concentrations of 0.231 mg/ml (16 – 35 mins) and 0.391 mg/ml (36 – 55 mins). However, water extraction yielded insufficient amounts of the extract. Upon using ethanol as solvent, the crude extract obtained after 19 days of soaking yielded a larger amount and lower IC50 concentration (0.199 mg/ml) than extracts obtained after 3 (0.253 mg/ml) and 7 days (0.334 mg/ml). TLC purification (EA: MeOH = 5:4) of the extract with the lowest IC50 yielded mixtures 1, 2, and 3, with mixture 1 conferring the highest decrease in cell viability with an IC50 of 0.037 mg/ml. Further purification (EA: Hexane = 3:4) of mixture 1 generated mixtures 1-1, 1-2, 1-3, 1-4, and 1-5, which were used for MTT and Trypan blue assays to assess their effect on cell activity. Immunofluorescence experiments using both mixtures revealed the formation of apoptotic bodies among treated Hep3B cancer cells. Chemical characterization in order to partially identify the class of the mixture suggests that 1-3 may possibly contain a flavanone.