In vitro anti-inflammatory activity

For evaluating anti-inflammatory activity, the inhibitory activity against cyclooxygenases was determined using the previously described method by Langhansova et al.,⁴⁰ with slight modifications. COX-2 (0.125 units/reaction) was added to 180 µL of incubation mixture consisting of 100 mM Tris buffer (pH 8.0), 5 µM hematin porcine, 50 µM Na₂EDTA, and 18 mM L-epinephrine. The essential oil samples were dissolved in DMSO and 10 μL was added to incubation mixture in the 96-well microplate with 5 μL of COX enzyme. After adding 10 µM arachidonic acid the reaction was initiated. After 20 min of incubation at 37 °C, the reaction was ceased by adding 10 μL of 10% formic acid. The PGE₂ concentration was determined by PGE₂ ELISA kit according to the manufacturer’s instructions and the final solutions were diluted at 1:15 in assay buffer. The absorbance was measured with a microplate reader (Tecan Infinite M200) at 405 nm and the inhibitory activity was calculated as the percentage inhibition of PGE₂ production compared to blank. (S)-(+)-ibuprofen was used as a reference inhibitor and DMSO as the blank. The experiment was repeated at least two times with at least two technical replicates in each experiment.