The experiment was carried out in a 384-well black plate
(BD Falcon), using the instructions provided by the supplier of
the assay kit as follows: 2 μL of each of the sample solutions (in
DMSO) and 10 μL of a freshly prepared 1 in 50 dilution (made in
assay buffers) of the enzyme substrate were put in a separate
well. The procedure was repeated using DMSO and embelin in
two additional separate wells as negative and positive controls respectively. Thiswas followed by the addition of 8 μL of freshly
prepared 0.5 μg/mL dilution of the enzyme into each well and
subsequent incubation at 37 °C for 30 min. The fluorescence
activity of the contents of each well was then measured at
excitation/emission wavelengths of 490 nm/520 nm respectively
by a Thermo Scientific Varioskan Flashmicroplate reader.
Percentage inhibition was calculated as follows: % Inhibition=
100 × (Fvehicle − Fsample) / Fvehicle. The experiment was carried
out in triplicate for each sample and the results analysed by
average % inhibition versus sample concentration plots, from
which IC50, i.e., concentration producing 50% inhibition, was
determined.