Acetylcholinesterase from electrophorus electricus

Acetylcholinesterase (from Electrophorus electricus) was obtained from Sigma-Aldrich. The effects of benzimidazolium salt 1, palladium-based complexes (2–6) and reference compounds including tacrine and donepezil on AChE activity were performed according to Ellman's method [58 (link)] as given previously [59 (link)]. The hydrolysis of acetylcholine iodide (AChI) by AChE was recorded at 412. Briefly, an aliquot (0.1 mL) of Tris/HCl buffer (1.0 M, pH 8.0) and different concentration of the synthesized benzimidazolium salt 1 and palladium-based complexes (2–6) were added to 50 μL of AChE enzyme solution (5.32 × 10⁻³ EU) and incubated for 10 min at 25 °C. Then, 50 μL of 5,5′-dithio-bis(2-nitro-benzoic) acid (DTNB) (0.5 mM) was added. The reaction was allowed to be initiated upon addition of 50 mL of AChI. The hydrolysis of AChI was monitored spectrophotometrically by the formation of the yellow 5-thio-2-nitrobenzoate anion, as a result of the reaction of DTNB with thiocholine, which released by enzymatic hydrolysis of AChI with maximum absorption at 412 nm [60 ].

Studied substances: N-allylcytisine-12-carbamide (A-63), cytisine-12-carbamide (A-36), N-1-adamantylcytisine-12-thiocarbamide (U-12), and 1-hydroxyquinopimaric acid (U-201) were synthetized in Ufa Institute of Chemistry of Russian Academy of Science. Acetylthiocholine iodide, acetylcholinesterase (from Electrophorus electricus), and 5,5′-dithiobis(2-nitrobenzoicacid) were purchased from Sigma- Aldrich Chemical Company.