- 學位論文
抗癌中藥材之研究第八報抗血癌之天然物及其作用機制
Antitumor Activity of Chinese Medicines VIII：The Antitumor Activity of Natural Products on Leukemia
摘要
血癌位居國內癌症死亡原因之第十四位，臨床上以化學藥物治療為主。而臨床上使用之抗癌藥物常有抗藥性及副作用的問題，故抗癌新藥之開發在癌症治療上乃刻不容緩之課題。 生藥為藥物開發的來源之一，taxol就是一個很成功的例子。然而本研究乃擬找尋對癌細胞具有毒性、對正常細胞無毒性之抗癌藥物，因此本實驗以血癌細胞株 (HL-60 cell line)為標的，應用trypan blue及MTT細胞染色法，建立血癌藥物體外試驗模式。血癌細胞株在96孔培養盤之最適生長濃度為1.5×104 cells/ml，在加藥後第二天為較佳觀察時間。首先進行含有多酚類之十五種省產植物70%丙酮抽取物及十六種天然物之細胞毒性測定，結果發現桃金孃科 (Myrtaceae) 之香果 (Syzygium jamos L. Alston) ，xantoangelol及4-hydroxyderricin具有明顯之抑制作用(香果抽取物濃度100μg/ml 有87.9%之抑制率，xanthoangelol濃度40μg/ml 有100%之抑制率，4-hydroxyderricin濃度40μg/ml 有94.5%之抑制率)。因此本研究乃大量採集香果葉，配合活性分析追蹤法進行成份分離。分離結果得到兩個水解型單寧，以NMR及Mass等儀器分析，經圖譜判斷解析其化學結構為1-O-galloyl castalagin及casuarinin，且對血癌細胞處理48小時後，IC50分別為10.8μM及12.5μM；xanthoangelol及4-hydroxyderricin的IC50分別為12.9μM及45.7μM。故繼而探討四個具有細胞毒性之活性天然物引起血癌細胞死亡之機制。 首先針對人類正常淋巴球細胞，發現1-O-galloyl castalagin與casuarinin的毒性低(天然物濃度10μg/ml有7.5%的抑制率)；體外細胞毒性機轉試驗以分子生物學和細胞形態學兩方面來進行探討。利用流式細胞儀(Flow cytometry)分析1-O-galloyl castalagin，casuarinin，xanthoangelol及4-hydroxyderricin對細胞週期的影響，結果發現兩者皆有Sub-G1的現象出現。另外，利用去氧核糖核酸斷裂片分析法(DNA fragmentation)，結果發現有DNA ladder的產生。在細胞型態學研究方面，利用光學顯微鏡觀察HL-60 細胞之型態變化，結果在光學顯微鏡下發現細胞有凋亡小體。 綜合以上之結果，推論1-O-galloyl castalagin，casuarinin，xanthoangelol及4-hydroxyderricin對HL-60細胞具有細胞毒性，是經由誘導細胞凋亡的路徑，且1-O-galloyl castalagin與casuarinin對正常淋巴球細胞的毒性低，總之，1-O-galloyl castalagin與casuarinin具有開發成為抗癌藥的潛力。
並列摘要
Leukemia is one of the most common and worldwide malignant tumors, and could be treated effectively by chemotherapy. Several previous studies demonstrated chemotherapy for leukemia caused resistance and side effects on patients, therefore, development of effective therapeutic agents on leukemia is an important and urgent topic in the future. Chinese medicines and natural products are the important sources for developing new antitumor drugs and several well-known effective antitumor compounds are derived from them such as taxol. Based on this paper, 15 kinds of natural plants from Chinese herbal medicines full of polyphenols were used to screen their cytotoxic effects on human leukemia cells (HL-60) by trypan blue and MTT. The most suitable cell concentration which HL-60 cells in 96-well plate is 1.5 × 104 cells/ml. The doubling time of HL-60 cells is 83.9 hours. The test time for observation is two days after treating with the tested compounds. We found that the most effective plant is Syzygium jambos L. Alston (Myrtaceae). The cytotoxicity percentage of the extract of S. jambos (100μg/ml) is 92%. Therefore, the components of S. jambos were surveyed. In the present investigation on the tannins of S. jambos, we have isolated two hydrolyzable tannins. They are 1-O-galloyl castalagin and casuarinin, respectively. The IC50 of 1-O-galloyl castalagin and casuarinin were 10.8 and 12.5μM, respectively. Therefore, we studied the mechanisms of 1-O-galloyl castalagin and casuarinin. Meanwhile, 16 kinds of compounds were also submitted to screen their cytotoxic effects against HL-60 cells. We found that xanthoangelol and 4-hydroxyderricin were effective and the IC50 were 12.9 and 45.7μM, respectively. The distribution of DNA content in the cell population was evaluated by flow cytometry, and found that the presence of apoptotic cells with sub-G1. Light-microscopic study was applied to find those apoptotic bodies. The apoptosis induced by these compounds also was demonstrated by DNA fragmentation assay and microscopic observation. These results suggest that cytotoxic mechanism of 1-O-galloyl castalagin, casuarinin, xanthoangelol and 4-hydroxyderricin might induce apoptosis in HL-60 cells. On the analysis of cytotoxic activities of these compounds on normal blood cells, the results demonstrated that the cytotoxic activities of 1-O-galloyl castalagin and casuarinin were far less than those of xanthoangelol and 4-hydroxyderricin. These data provided more beneficial evidences to indicate that 1-O-galloyl castalagin and casuarinin showed the promising potential to develop as anticancer agents.
參考文獻
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