| 摘要: | 蒿屬是菊科中最大的家族之一，植株為分布廣泛之草本或灌木，其藥用價值自古以來即備受重視。蒿屬植物在化學，藥理學和分類學上皆為研究重點，並已用於肝炎，癌症，發炎性疾病，細菌和病毒等感染治療。植物化學研究中蒿屬已被分離和鑑定出含有單?翩B倍半?翩B香豆素和結構多樣性的類黃酮。細葉山艾屬於蒿屬，為臺灣特有種。傳統上在臺灣原住民的民俗療法已用於治療類風濕性關節炎、過敏性鼻炎、頭痛和水腫。然而，這種植物的藥用潛力和潛在作用機制仍屬未知，且其活性成分尚無任何研究。此實驗的目的是在分離和鑑定細葉山艾的化學和活性成分。細葉山艾在2011年8月於合歡山翠峰採集並經過鑑定，全草曬乾磨粉後用乙醇萃取，再經液-液分層後，針對其中乙酸乙酯層進行成分分離，採用管柱層析法進一步純化分為11個部分（AM-EA-1〜11），肺癌細胞株（A549）的MTT結果顯示AM-EA-3具有最佳活性。繼續用管柱層析法和半製備型HPLC分離AM-EA-3（5.699克），得到AM-EA-3-10-3 (capillene), AM-EA-3-10-5-14-2 (capillaridin C) , AM-EA-3-10-5-4 (capillaridin F) , AM-EA-3-11-7-3-R (friedelin)，其結構由一維/二維核磁共振進行光譜鑑定。在13C光譜上，capillene, capillaridin C, capillaridin F這三種化合物顯示甲基的特徵峰位於4 ppm，為三鍵旁之甲基特徵性訊號。這些成分只有在蒿屬和禾本科植物冰草中曾被分離出來。有文獻顯示capillaridin C和capillene有顯著的抗血小板凝集作用。我們希望從細葉山艾分離出新的化合物，同時純化AM-EA-3並測試抗肺癌（A549）的活性和機制。
經由MTT證實在以下化合物4-hydroxyacetophenone, picein, friedelin, capillene中capillene的效果最好，其他的化合物並沒有明顯活性。Annexin-V FITC染色得到的結果發現細胞有晚期凋亡現象，capillene可讓細胞造成凋亡，此外西方墨點法顯示整體細胞蛋白質的表現，隨著capillene濃度上升，cleaved-PARP, cleaved-caspase 9和cleaved-caspase 3表現量增加。此外還觀察BcL-2家族中的蛋白表現，發現BcL-2表現量下降。綜合上述結果得知capillene會造成細胞凋亡。
Artemisia is one of the largest genera of the family Compositae. Artemisia is found worldwide and attracted much attention for its highly economic values, in particular for its medicinal uses. Plants belonging to Artemisia are popular from chemical, pharmacological and taxonomic points of view and have been used for the treatment of diseases, for example, hepatitis, cancer, inflammation and infections by fungi, bacteria and viruses. Phytochemical studies to Artemisia genus revealed that monoterpenes, sesquiterpenes, coumarins, and flavonoids with great structural diversity were isolated and identified. Artemisia morrisonensis Hayata is an endemic species in Taiwan. Traditionally, this plant is used for the treatment of rheumatoid arthritis, allergic rhinitis, headache, and edema in aboriginals. However, the therapeutic potential and underlying mechanisms still remain elusive and the active components of this plant have not been systematically identified. Aims of this study are trying to isolate and identify the chemical constituents. In this study, the title plant was identified and collected at Cuifeng in Hehuan Mountain in August, 2011. The whole plant was dried, pulverized and immersed in ethanol to give extract which suspended in water and partitioned against ethyl acetate. Ethyl acetate-soluble layer was further purified by silica gel column chromatography to afford 11 sub-fractions (AM-EA-1 to 11). MTT assay for human lung carcinoma (A549) showed AM-EA-3 was the most potent sub-fraction. Successive silica gel column chromatography and semi-preparative HPLC to AM-EA-3 (5.699 g) were carried out to obtain AM-EA-3-10-3 (capillene), AM-EA-3-10-5-14-2 (capillaridin C), AM-EA-3-10-5-4 (capillaridin F), AM-EA-3-11-7-3-R (friedelin) whose structures were identified by 1D/2D-NMR spectra. Capillene, capillaridin C and capillaridin F demonstrated characteristic peaks around 4 ppm in 13C spectra ascribed to methyl groups adjacent to triple bonds. Those constituents were only isolated from Artemisia genus and Agropyron repens (Poaceae). capillaridin C and capillene show significant antiplatelet aggregation in previous study. In addition, we make endeavor to find novel compounds and delineate the mechanisms for anti-human lung carcinoma (A549).
MTT assay showed that capillene possessed the most potent effect among the four compounds: 4-hydroxyacetophenone, picein, friedelin, capillene from A. morrisonensis. Western blot showed that the expression of cleaved-PARP, cleaved-caspase-9 and cleaved-caspase 3 were up-regulated with the increase of capillene concentration in cell apoptosis detected by Annexin V FITC staining. Furthermore, the protein expression in the BcL-2 family was observed and the expression of BcL-2 was decreased. These results suggest that capillene could cause apoptosis. |
