Isolation, Characterization, and Anticancer Evaluation of Alkaloids from Eumachia montana (Rubiaceae)

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