Bioassay-guided isolation of cytotoxic metabolites from Daphne mucronata and evaluate of apoptosis inductive effect on potent compound

Daphne mucronata Royle. shrub is known as a medicinal plant in several parts of Asia. Extract of aerial parts of this plant has been used in Iranian Folk Medicine to treat breast tumors and various cancers. In the present study, cytotoxic and apoptosis-inducing activities of the aerial parts methanolic extract of the plant, fractions and active purified components were investigated. First, the cytotoxic activity of methanolic extract of D. mucronata was evaluated against various cell lines (MCF-7, Hela, K562, and HepG2) by MTT assay. It was revealed that methanolic extract had the highest cytotoxic activity against breast cancer cell line (MCF-7). Then, the methanolic extract was suspended in water and sequentially partitioned with n-hexane, ethyl acetate and n-butanol. The resulting fractions were tested against MCF-7 cells, of which the n-hexane fraction demonstrated the most promising activity with an IC50 value of 75.15 µg/mL. Activity-guided fractionation of the n-hexane extract led to the isolation of five compounds including three coumarins (daphkoreanin, daphnin, demethyldaphnoretin-7-O-glucoside), one triterpenoid (lupeol), and one steroid (daucosterol). The structures of the compounds were established using high-field NMR spectroscopy and comparison of the data with those reported in the literature. Among the isolated compounds, daphkoreanin (1), demethyldaphnoretin-7-O-glucoside (3), and daucosterol (5) were isolated from D. mucronata for the first time. The cytotoxicity of the isolated compounds was evaluated using the MTT assay. Among the isolated compounds, daphkoreanin (1) depicted remarkable activity (IC50 = 58.77 µM). Further investigations using Flow Cytometry, Western Blotting, DAPI staining, and AO staining assays showed that daphkoreanin induces apoptosis on MCF-7 cells efficiently. The results suggested that daphkoreanin from D. mucronata could efficiently induce MCF-7 cells apoptosis through mitochondria-dependent pathway and activation of the caspase cascade. Molecular docking study showed more interaction of daphkoreanin with Bax than Bcl-2 proteins, which confirms its ability to induce apoptosis in MCF-7 cells.