4.5.1. Determination of Anti-Elastase Activity

To assess the inhibition potential of matrix metalloproteinase, a fluorometric kit (Abcam, ab118971, Cambridge, MA, USA) was used to determine the activity of neutrophil elastase (NE). The analysis was based on the instructions provided by the manufacturer and the procedure previously described by Zagórska-Dziok et al. [3]. Fluorometric analyses were performed in a 96-well plate with black walls and clear bottom. The ability to inhibit the activity of elastase was determined for the FEE at the concentrations of 100, 500 and 1000 µg/mL. Initially, NE enzyme solutions, NE substrate, and inhibitor control (succinyl-alanyl-alanyl-prolyl-valine chloromethylketone; SPCK) were prepared according to the manufacturer’s guidelines. The NE solution was then added to all wells. The test samples, the inhibitor control, and the enzyme control (assay buffer) were added to the subsequent wells, and the samples were mixed thoroughly. All samples were analyzed in duplicate. The prepared plate was incubated in the dark at 37 °C for 5 min. Meanwhile, a reaction mixture was prepared by mixing assay buffer and NE substrate and added to each well. The fluorescence was then immediately measured at excitation wavelength λ = 400 nm and emission λ = 505 nm using a microplate reader (FilterMax F5, Thermo Fisher Scientific, Waltham, MA, USA). During the measurements, the kinetic mode was used (30 min at 37 °C). The influence of the tested samples on the NE activities was calculated from the equation: