- 學位論文
蛇床子之抗血癌成分研究
The Antitumor Principle Constituents of Cnidium monnieri on Leukemia
摘要
蛇床子為繖形科(Umbelliferae)植物蛇床Cnidium monnieri (L.) Cusson的乾燥成熟果實，屬於補益藥。本研究以乙醇萃取蛇床子並檢測其對血癌(HL-60)、子宮頸癌(HeLa)、直腸癌(CoLo 205)等癌細胞之毒性，發現對HL-60具有明顯的抑制效果，再將蛇床子乙醇萃取物經由矽膠管柱層析及再結晶等方法，分離純化得到蛇床子素、前胡內酯、佛手柑內酯、異虎耳草素及花椒毒素等五種香豆素，和花生酸及山萮酸兩種脂肪酸。其中蛇床子素及前胡內酯對HL-60有較強的細胞毒性，處理二十四小時後其百分之五十生長抑制濃度（IC50）分別是14.9 mg/ml (61.1 mM) 及18.8 mg/ml (69.6 mM)。由光學顯微鏡觀察以蛇床子素及前胡內酯處理後之HL-60細胞，可見到凋亡小體；利用瓊膠電泳法及流式細胞儀，發現蛇床子素及前胡內酯能誘導HL-60細胞產生有規則性的去氧核糖核酸斷鏈；西方印蹟法的分析可見不活化型的PARP被切割成活化型之片段，上述結果證明蛇床子素及前胡內酯能誘導HL-60走向細胞凋亡。進而以此兩種香豆素作為蛇床子中抗癌活性指標成分，建立了簡單、方便、準確之高效液相層析分析品質管制法，並檢測六種市售品及一種台灣原生種蛇床果實，發現市售品的蛇床子素及前胡內酯的含量分別為0.288~1.211 mg/g和0.104~0.303 mg/g，且台灣蛇床果實蛇床子素含量甚少，因此本分析系統可提供篩選中藥材中含有蛇床子素及前胡內酯的簡易方法，對未來蛇床子品管及抗癌活性成分的篩選都有很大的幫助。
並列摘要
Cnidii fructus, the dried mature fruits of Cnidium monnieri (L.) Cusson (Umbelliferae), is used as a tonic agent in traditional Chinese medicine. The cytotoxicity effects of the ethanol extracted-Cnidii fructus were stronger on human leukemia (HL-60) cells than cervical carcinoma (HeLa) and colorectal carcinoma (CoLo 205) cells. The ethanol extract of Cnidii fructus was subjected to silica gel column chromatography and recrystallization to give five coumarins: osthol, imperatorin, bergapten, isopimpinellin, xanthotoxin and two fatty acids: arachidic acid and behenic acid. Among these compounds, osthol and imperatorin showed the strongest cytotoxicity activity on HL-60 cells with the IC50 values of 14.9 mg/ml (61.1 mM) and 18.8 mg/ml (69.6 mM), respectively for 24h. Osthol and imperatorin caused apoptotic bodies, DNA fragmentation and enhanced PARP degradation on HL-60 cells by biochemical analysis. These results indicated that osthol and imperatorin could induce apoptosis in HL-60 cells. Therefore, osthol and imperatorin were as antitumor marker substances in Cnidii fructus. The quality control of Cnidii fructus was established an easy, quick, economic and reproductive HPLC analysis method. The quantity of osthol and imperatorin in Cnidii frutus from markets were 0.288~1.211 mg/g and 0.104~0.303 mg/g, respectively. However, osthol was poor content in C. formosanum fructus. Therefore, we can use the HPLC analysis system to search for the antitumor principles of Chinese herbs and control the quality of Cnidii frutus.