Isolation, Characterization, and Anticancer Evaluation of Alkaloids from <i>Eumachia montana</i> (Rubiaceae)

The dimeric (-)-calycanthine (<b>1</b>), the hexameric (+)-oleoidine (<b>2</b>), the heptameric (+)-caledonine (<b>3</b>), the octameric (+)-eumatanine (<b>4</b>), and a tricyclic pyrroloquinoline alkaloid, eumatricine (<b>5</b>), are alkaloids isolated from the leaves of <i>Eumachia montana</i>; compounds <b>4</b> and <b>5</b> are novel, previously uncharacterized alkaloids. Preliminary anticancer assays, including MTT, cell count, and clonogenic assays, revealed potent inhibition of cell growth and colony formation by compounds <b>2-4</b> against seven carcinoma cell lines (including breast, colorectal, lung, and glioblastoma multiforme cell lines). The GI₅₀ values of compounds <b>2-4</b> ranged from 0.1 to 2.7 μM, and potent cytotoxic activity against HCT-116 and MCF-7 was observed (<b>3</b> > <b>4</b> > <b>2</b>). Cell cycle analysis and apoptosis assays revealed that compound <b>4</b> evokes profound apoptosis without cell cycle phase-specific arrest, accompanied by caspase activation, corroborating apoptosis-induction. Detection of ROS induced by <b>4</b> infers a role for oxidative stress in anticancer activity. γH2AX detection indicates the presence of DNA double-strand breaks in cells treated with compound <b>4</b>. Visualization of <b>4</b>-treated cells by fluorescence microscopy implies multiple cell death pathways are simultaneously triggered (e.g., apoptosis, autophagy, necrosis, and paraptosis).