2.3.2. Acetylcholinesterase (AChE) Inhibition

The inhibition of AChE was carried out in 96-well microplates, measuring the absorbance at 450 nm 11 times using a Synergy H1 Hybrid Multi-Mode Reader [22]. Each well contained 25 μL of ATCI (15 mM) in MilliQ water, 50 μL of buffer B (50 mM Tris-HCl, pH = 8, 0.1% bovine serum), 125 μL of DTNB (3 mM) in buffer C (50 mM Tris-HCl, pH = 8, 100 mM NaCl, 20 mM MgCl2), and 25 μL of urolithin A at different concentrations in buffer A (50 mM Tris-HCl, pH = 8). Finally, 25 μL of the enzyme (0.22 U/L) was added to the control and samples to complete the reaction. Blanks contained 25 μL of buffer A instead of the enzyme. Galantamine (Sigma-Aldrich, St. Louis, MO, USA), a drug used for Alzheimer's disease, was assayed for comparative purposes as a reference inhibitor. Percentages of AChE inhibition were calculated with the following formula: % Inhibition = [1 − ((Inhibitory Slope)/(Control Slope))] × 100
