Activity-Guided Isolation of Cytotoxic Non-Glycosidic Ester Iridoids from Valeriana alliariifolia Adams and Unravelling Their Cell Death Mechanisms

The aim of this study was to isolate the cytotoxic compounds from V. alliariifolia via activity-guided isolation and to determine the mechanism of actions of the most potent ones. The crude EtOH extract as well as CHCl₃ and AcOEt subextracts demonstrated remarkable cytotoxic activities against A549, MCF7, HGC27 and PC3 cancer cells. Sequential chromatographic separations on active subextracts yielded 14 secondary metabolites, including 11 iridoids (1-11) most of which belong to non-glycosidic ester iridoids, two phenylpropanoids (12 and 13) and one lignan (14). The chemical structures of purified compounds were elucidated by NMR and MS analysis. Among the isolates, 7-deisovaleroylvaltrate (3) was isolated for the first time as a natural product. According to the cytotoxic assay compounds, 2, 4-6 and 8 were found to be the potent cytotoxic compounds (IC₅₀ <10 μM) against at least one of the tested cancer cell lines. Thus, 2, 4-6 and 8 were investigated for their effects on apoptotic, necrotic and autophagic pathways as well as cell cycle progression. They exerted anticancer activities by inducing different cell death mechanisms depending on the cancer cells. The results demonstrated that 2, 4-6 and 8 could be potential anticancer drug leads that deserve further in vivo and clinical studies on the way to discover novel natural compounds with anticancer properties.