2.10. Human Neutrophil Elastase (HNE) Inhibition Assay

Essential oils and individual compounds were dissolved in 100% DMSO at 5 mM stock concentrations. The final concentration of DMSO in the reactions was 1%, and this level of DMSO had no effect on enzyme activity. Sivelestat, a known HNE inhibitor, was used as a positive control. The inhibition assay was performed, as described previously [29]. Briefly, a solution containing 200 mM Tris-HCl (pH 7.5), 0.01% bovine serum albumin, 0.05% Tween-20, and 20 mU/mL of human neutrophil elastase was added to black, flat-bottom 96-well microtiter plates containing different concentrations of test compounds. Reactions were initiated by addition of 25 µM elastase substrate N-methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin in a final reaction volume of 100 µL/well. Kinetic measurements were obtained every 30 s for 10 min at 25 °C using a Fluoroskan Ascent FL fluorescence microplate reader (Thermo Electron, MA, USA) with excitation and emission wavelengths at 355 and 460 nm, respectively. The concentration of compound that caused 50% inhibition of the enzymatic reaction (IC₅₀) was calculated by plotting % inhibition versus logarithm of inhibitor concentration.