Determination of cholinesterase inhibitory activity

The AChE inhibitory assay was performed according to the colorimetric method by Ellman et al. [31] with acetylthiocholine iodide as a substrate. For the enzyme source, rat brains were homogenized by a homogenizer with five volumes of ice-cold homogenization buffer (10 mM Tris-HCl (pH 7.2), which contained 1 M NaCl, 50 mM MgCl₂ and 1% Triton X-100) and centrifuged at 10000 g for 30 min. The resulting supernatant was used as an enzyme source. All of the extraction steps were carried out at 4 °C. Protein concentration was determined by using a bicinchoninic acid kit (Sigma Co., St. Louis, MO, USA) with bovine serum albumin as a protein standard. The rates of hydrolysis by AChE were monitored spectrophotometrically. The EPL-M80 or standard (500 μL) was mixed with an enzyme solution (500 μL) and incubated at 37 °C for 15 min. Absorbance was taken at 405 nm immediately after adding Ellman’s reaction mixture (3.5-mL 0.5 mM acetylthiocholine, 1 mM 5, 5′-dithio-bis (2-nitro benzoic acid)) in a 50 mM sodium phosphate buffer (pH 8.0) to the above reaction mixture. Readings were repeated for 10 min at 2 min intervals to verify that the reaction occurred linearly. The blank reaction was measured by substituting saline for the enzyme. Donepezil was used as a positive control. The percentage inhibition of AChE activity was calculated using the following formula:

Assessment of BChE inhibition was performed as described above except that the enzyme solution was 50 μL and acetylthiocholine iodide was replaced by butyrylthiocholine iodide. Galantamine was used as positive control. The percentage inhibition of BChE activity was calculated using the same formula as mentioned above for AChE activity.