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目的分离纯化槲寄生抗肺癌A549细胞增殖活性的化学成分,对获得的活性成分促A549细胞凋亡的作用进行评价。方法采用硅胶柱层析、Sephadex LH-20凝胶柱层析等方法对槲寄生提取物的乙酸乙酯萃取部分进行分离纯化;~1H NM R和~(13)C NM R波谱法确定化合物结构;M TT法检测化合物对细胞增殖的影响;流式细胞术检测细胞周期、凋亡及胞内活性氧(ROS)的变化;蛋白免疫印迹法检测蛋白表达的变化。结果获得了10个酚类化合物;其中化合物1为1,7-二(4-羟基苯基)-4E,6E-庚二烯-3-酮,对A549细胞表现出剂量依赖的增殖抑制作用(F=196.768,P=0.001),可引起G2/M期细胞增多(P<0.05),S期细胞减少(P<0.05);化合物1可引起胞内ROS水平显著升高(P<0.05),促进细胞凋亡,并引起细胞Caspase-9,Caspase-3和PARP蛋白表达水平的改变(F值分别为19.340、4.388、194.571,P值分别为0.001、0.042、0.001)。结论槲寄生中的酚类成分主要是二苯基庚烷类和黄酮类,二苯基庚烷类化合物是该药材抗肺癌细胞增殖的主要活性成分。
OBJECTIVE: To isolate and purify the chemical components of the anti-lung cancer cell line A549 and to evaluate the effect of the active ingredient on the apoptosis of A549 cells. Methods Silica gel column chromatography and Sephadex LH-20 gel column chromatography were used to separate and purify the ethyl acetate fraction of the mistletoe extract. The structures of the compounds were determined by ~ 1H NM R and ~ (13) C NM R spectroscopy The effect of compound on cell proliferation was detected by MTT assay. The cell cycle, apoptosis and intracellular reactive oxygen species (ROS) were detected by flow cytometry. The protein expression was detected by Western blotting. As a result, 10 phenolic compounds were obtained. Compound 1 was 1,7-bis (4-hydroxyphenyl) -4E, 6E-heptadien-3-one and showed a dose- F = 196.768, P = 0.001), the cells in G2 / M stage increased (P <0.05) and the cells in S stage decreased (P <0.05). Compound 1 increased the intracellular ROS levels significantly (P < (P = 19.340,4.388,194.571, P = 0.001,0.042,0.001 respectively), which could promote the apoptosis of cells and induce the expression of Caspase-9, Caspase-3 and PARP. Conclusion The main phenolic compounds in the mistletoe are diphenylheptane and flavonoids. Diphenylheptane compounds are the main active components of the anti-proliferation of lung cancer cells.