In vitro aromatase inhibition assay

In vitro aromatase inhibition assay was performed for quercetin, naringenin and naringin. Aromatase inhibition was quantified by measuring the fluorescent intensity of fluorescein, the hydrolysis product of dibenzylfluorescein, by aromatase, as previously described²⁹. In brief, the test substance (10 µL) was pre-incubated with the NADPH regenerating system (90 µL of 2.6 mM NADP⁺, 7.6 mM glucose 6-phosphate, 0.8 U/mL glucose 6-phosphate dehydrogenase, 13.9 mM MgCl₂, and 1 mg/mL albumin in 50 mM potassium phosphate, pH 7.4) for 10 min at 37 °C before 100 µL of the enzyme and substrate mixture [4 pmol/well enzyme (CYP19, BD Biosciences, San Jose, CA), 0.4 µM dibenzylfluorescein, and 4 mg/mL albumin in 50 mM potassium phosphate, pH 7.4] were added. Then, the reaction mixture was incubated for 30 min at 37 °C to allow aromatase to generate the product and quenched with 75 µL of 2 N NaOH. After the reaction was terminated, shaking was done for 5 min followed by incubation for 2 h at 37 °C to enhance the noise/background ratio, then fluorescence was measured at 485 nm (excitation) and 530 nm (emission). Three independent experiments were performed in duplicates, and the average values were used to construct the dose–response curves. At least four concentrations of each test substance were used, and the IC₅₀ values were calculated and compared to ketoconazole as reference standard at a concentration of 0.1 µg/mL.