Determination of acetylcholinesterase (AChE) inhibition

AChE inhibitory activity was measured by using Ellman's colorimetric method [23]. Briefly, in 96-well plates, 25 μL of 15 mM ATCI, 75 μL of 3 mM DTNB and 50 μL of 50 mM Tris–HCl, pH 8.0, containing 0.1% bovine serum albumin (BSA), and 25 μL of the tested phytochemicals were added. The absorbance was measured at 405 nm after a 5-min incubation at room temperature. Then, 25 μL of 0.22 U.ml^-1 of AChE was added and incubated for 5 min at room temperature, and the absorbance was measured at 412 nm. Acetylcholinesterase (5–1,000 μM) was used as a reference standard. The percentage inhibition was calculated using the following equation: Inhibition (%) = 1 – (A_sample/A_control) × 100, where A_sample is the absorbance of the sample extracts, and A_control is the absorbance of the blank (50% aqueous methanol in buffer).

In addition to the in vitro assay of AChE mentioned earlier, we also determined AChE activity in the hippocampal homogenate. In brief, the hippocampus was isolated and homogenized in ice-cold 0.1 M phosphate-buffered saline (pH 8.0). The homogenate was centrifuged at 1,000 g for 10 min at 4 °C, and the supernatant was used as the source of the enzyme in the AChE assay. AChE activity in hippocampus was evaluated using Ellman's method with slight modifications [24].