Metabolic profile of eupatorin (10 μM) metabolism by CYP1 family enzymes and identification of cirsiliol as the primary metabolite. (a) Typical high-pressure liquid chromatography (HPLC) traces of 20-minute incubation of CYP1 enzymes with eupatorin. (b) Expansion of (a) showing metabolites E2 and E3. (c) Co-elution studies of eupatorin with cirsiliol. A 20-minute CYP1B1 incubate of eupatorin was spiked with cirsiliol (0.2 μM). Reaction mixtures contained eupatorin, NADPH (nicotinamide adenine dinucleotide phosphate), and recombinant microsomes purchased from Gentest Corporation (now part of BD Biosciences). Reactions were terminated by the addition of 1% acetic acid in methanol. (d) HPLC trace of metabolism of eupatorin in MDA-MB-468 cells. Samples were analysed by HPLC using a UV detector at 350 nm. Experiments were performed in triplicate. A350: absorption of light at wavelength 350 nm. AU: arbitrary units.