Helleborus cyclophyllus Boiss is a rhizomatous plant species, with strong allelochemical properties, that has been used since ancient times for its therapeutic properties. In the present study we investigated the ability of an aqueous-soluble fraction of the methanol extract of H. cyclophyllus Boiss leaves, to induce apoptotic cell death on A549 human bronchial epithelial adenocarcinoma cells. A primary human lung fibroblasts’ cell line was used as a model of normal-healthy cells for comparison. Cell morphology was examined after appropriate staining, cytotoxic activity of the extract was determined by the MTT assay, the type of cell death was analyzed by flow cytometry, confirmation of apoptosis was evaluated with the analysis of caspase-3, PARP1 by western blotting, while the chemical composition was assessed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). H. cyclophyllus Boiss extract was selectively active on A549 cells inducing significant morphological changes, even at low concentrations. Characteristic morphological alterations included the release of vesicular formations from A549 cell membranes (ectosomes), detachment of cells from their substrate, generation of a large vesicle into the cytoplasm (thanatosome) and the formation of apoptotic bodies. The selective apoptotic action on treated cells was also confirmed by biochemical criteria. Low concentrations, however, did not affect normal cells. The phytochemical analysis of the extract revealed the presence of cardiac glucosides, bufadienolides and phytoecdysteroids. To the best of our knowledge, the above-mentioned sequences of events leading selectively cancer cells to apoptosis, has not been reported before.

铁葱(Helleborus cyclophyllus Boiss) 是一种根茎植物，具有很强的化感作用，自古以来就因其治疗特性而被使用。在本研究中，我们研究了H. cyclophyllus Boiss 叶的甲醇提取物的水溶性部分诱导 A549 人支气管上皮腺癌细胞凋亡的能力。使用原代人肺成纤维细胞系作为正常健康细胞的模型进行比较。适当染色后检查细胞形态，MTT法测定提取物的细胞毒活性，流式细胞仪分析细胞死亡类型，蛋白质印迹法分析caspase-3、PARP1评估细胞凋亡的确认，同时通过液相色谱-串联质谱（LC-MS/MS）评估化学成分。 H. cyclophyllus Boiss 提取物对 A549 细胞具有选择性活性，即使在低浓度下也能诱导显着的形态变化。特征性形态学改变包括从 A549 细胞膜（胞外体）释放囊泡结构、细胞与其基质分离、在细胞质中产生大囊泡（致死体）以及凋亡小体的形成。对处理细胞的选择性凋亡作用也通过生化标准得到证实。然而，低浓度不会影响正常细胞。提取物的植物化学分析表明存在强心苷、蟾蜍二烯内酯和植物蜕皮类固醇。据我们所知，上述选择性导致癌细胞凋亡的事件序列以前从未有过报道。