- 學位論文
抗癌中藥材之研究 第十報 牽牛子之抗直腸癌活性研究
Antitumor Activity of Chinese Medicines Ⅹ: The Antitumor Activity of Pharbitis nil Choisy on Colorectal Carcinoma
摘要
直腸癌為台灣地區好發率高的癌症，而臨床上使用之抗癌藥物常有抗藥性及副作用的問題，故將由中藥材中開發新的抗癌藥物。本研究收集坊間常用之26種抗癌中藥材，分別對直腸癌細胞株(CoLo 205, HT-29)進行毒性試驗篩選，結果以黑牽牛子之50%乙醇萃取物對直腸癌細胞具有最強的抑制作用，並在相同濃度時對人類正常肝細胞毒性較小，且會誘導直腸癌細胞凋亡。繼而利用細胞毒性追蹤活性成份之分離黑牽牛子方式，經過一連串的管柱層析分離步驟，經70% Acetone沖提Diaion HP-20管柱之沖提物(D-70A)，對CoLo 205細胞毒性最強，其在48小時IC50 為17.38 μg/ml，進一步利用去氧核糖核酸斷片分析法、流式細胞儀與Giemsa染色法及西方印蹟法得知D-70A會誘導CoLo 205細胞DNA斷鏈及造成PARP被切割產生85kDa的片段，證明其會誘導CoLo 205細胞死亡模式為凋亡。繼而D-70A再經由RP-18層析管柱得到gibberellin, 其在48小時IC50 為20.36 μg/ml, 抑制癌細胞作用不及D-70A強，但作用48小時後，仍可引起DNA斷鏈而誘導凋亡。Gibberellin誘導之細胞毒性會被catalase所抑制，推測gibberellin誘導CoLo 205細胞之毒性作用，可能與自由基產生有關。黑牽牛子經一系列之活性追蹤管柱層析後，得到D-70A為最具活性之劃分部並分離得到gibberellin之天然物，故未來可利用gibberellin做為D-70A抗癌活性指標成份進行品質管制。
並列摘要
Colorectal carcinoma is one of the leading causes of cancer deaths in Taiwan. Several previous studies demonstrated chemotherapy for colorectal carcinoma caused resistance and side effects on patients. Therefore, the new anticolorectal tumor drugs are going to develop from Chinese herbs. In this study, the cytotoxicity effects of the 50% ethanol extracts of Chinese herbs were screened by MTT assay in human colon adenocarcinoma cell lines (CoLo 205, HT-29). The cytotoxicity effects of 50% EtOH extracted-Pharbitidis Semen Atrum were exerted stronger in CoLo 205 cells than Chang normal liver cell (CNL). The cytotoxic principles of semen were isolated by bioassay-guided fractionation. The semen were extracted with methanol and chromatographed over Diaion HP-20 column with 70% aqueous acetone Moreover, the cytotoxicity effect of 70% acetone fraction (D-70A) showed stronger than the other fractions in CoLo 205 cells and IC50 was 17.38 μg/ml for 48h. Furthermore, the cytotoxic mechanisms of D-70A were measured by agarose gel electrophoresis, fluorescence flow- cytometry, Giemsa stain, Western blot analysis. The results showed that D-70A could induce apoptosis in CoLo 205 dells. Gibberellin was isolated from D-70A with RP-18 column chromatography. The IC50 value of gibberellin was 20.36 μg/ml and induced apoptosis in CoLo205 cells for 48h. Moreover, treatment with catalase prevented gibberellin-induced cytotoxicity. In according to the results, we suggested that gibberellin might produce free radicals and induce apoptosis in CoLo 205 cells. However, the cytotoxic effect of bibberellin was lesser than D-70A in CoLo 205 cells. Therefore, we suggested the D-70A fraction extract is good for antitumor effects. In the future, gibberellin will be a biosubstance to control the quality of D-70A.