4.2.3. Esterase Activity Assay

The activity of CA was assayed in RBC cytosols by following the change in absorbance at 348 nm from the 4-nitrophenylacetate (NPA) to the 4-nitrophenylate (PNP) ion over a period of 10 min at 25 ◦C with a spectrophotometer (CHEBIOS UV–VIS), according to [36]. The enzymatic reaction was carried out in a total volume of 3.0 mL, containing 1.4 mL 0.05 M Tris–SO<sup>4</sup> buffer (pH 7.4), 1 mL 3 mM NPA, 0.5 mL H2O, and 0.1 mL diluted cytosol. A reference measurement was obtained by preparing the same cuvette with a sample solution in the absence of incubation. One unit of CA activity was defined as the amount of enzyme which catalyzes the formation of 1 pmol PNP/min in standard conditions of incubation. The following formula incorporating the extinction coefficient was used to calculate: CA units <sup>×</sup> <sup>10</sup>−3/µL packed RBC = OD <sup>×</sup> sample dilution factor/(min <sup>×</sup> 667), with an extinction coefficient of 667 [28,35]. The decrease following the licorice intake (∆CA activity %) was calculated as: (1- activity T1/ activity T0)%.
