Butyrylthiocholine iodide

Purified horse BChE was from Worthington Biochemical Corporation, dithio-bis-di-nitro benzoic acid and butyrylthiocholine iodide (BSCh) were from Sigma, tetramethylammonium iodide (TMA), tetraethylammonium iodide (TEA), edrophonium chloride (EDR), and methanesulfonylfluoride (MSF) were from BDH; decamethonium bromide (DME) was from Koch Light Laboratories; sodium fluoride and d-Tubocurarine were from Fluka AG. N-((1-benzylpiperidin-3-yl)methyl)-N-methylnaphthalene-2-sulfonamide (942) was synthesized in the Faculty of Pharmacy, University of Ljubljana (Ljubljana, Slovenia). All other reagents used were of analytical grade. The experiments were carried out at 25 °C in 25 mM phosphate buffer, pH 7.0.

A modified protocol of Ellman’s spectrophotometric assay [54 (link)] adapted to a 96-well plate procedure was followed as previously described [39 (link)]. Incubation samples of AChE from electric eel or BChE from equine serum (eeAChE, 463 U/mg, and esBChE, 13 U/mg, Sigma-Aldrich, Milan, Italy) were set in phosphate buffer (pH 8.0) containing 0.5 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; Sigma-Aldrich, Milan, Italy) as the chromophoric reagent alone or in the presence of the inhibitor (10 μM). Incubations were carried out in clear flat-bottomed, 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in duplicate. For most active compounds (inhibition > 60%), IC₅₀ was determined from seven solutions (ranging from 30 μM to 0.03 μM as the final concentrations) of inhibitor and prepared by diluting a stock DMSO solution 1000 μM with a work buffer. After incubation for 20 min at 25 °C, 0.5 mM acetyl- or butyrylthiocholine iodide (Sigma-Aldrich, Milan, Italy) were added as the substrates and AChE-catalyzed hydrolysis was followed by measuring the increase of absorbance at 412 nm for 5 min at 25 °C in a Tecan Infinite M1000 Pro multiplate reader (Tecan, Cernusco S.N., Italy). Inhibition values and IC₅₀s were calculated with a GraphPad Prism as the mean of three independent experiments and are expressed as mean ± SEM.

Acetylcholinesterase (AChE) (EC 3.1.1.7) from electric eel, Butyrylcholinesterase (BuChE) (EC 3.1.18), from equine serum, acetylthiocholine iodide (ATC), butyrylthiocholine iodide (BTC), 5:5-dithiobis-2-nitrobenzoic acid (DTNB), sodium phosphate (mono and dibasic), sodium bicarbonate, Galanthamine hydrobromide from Lycoris sp, (-)-epicatechin (EC), catechin, (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG) were purchased from Sigma-Aldrich (UK).

The AChE and BuChE inhibition assay was performed spectrophotometrically for methanolic extracts and its derived fractions using acetylthiocholine iodide and butyrylthiocholine iodide (Sigma-Aldrich, USA), respectively as substrate following the method of Ellman et al., with minor changes [20 (link)]. In a 96–well plate, 10 μL of enzyme from stock solution (AChE, 2U/mL and BuChE, 2U/mL) was taken and 10 μL of plant extract or fraction (15–150 μg/mL) and 100 μL Phosphate buffer were added to them. To this mixture, 50 μL DTNB solution (3.96 mg DTNB and 1.5 mg sodium bicarbonate dissolved in 10 mL of 200 mM phosphate buffer pH 7.7) was added and incubated for 5 minutes at 25°C. Substrates with the volume of 15 μL (10.85 mg acetylthiocholine iodide or butyrylthiocholine iodide in 5mL phosphate buffer) were added and incubated for further 5 minutes at 25°C. The colour developed due to the formation of 5-thio-2-nitrobenzoate anion was measured at 412 nm. Galantamine, at 0.12, 0.23, 0.46, 0.92, 1.84, 3.68 and 7.37 μg/mL, and the reaction mixture excluding plant sample were taken as positive and negative controls, respectively. The percent of inhibition and IC₅₀ values were determined.

Acetylthiocholine iodide (ATCh), butyrylthiocholine iodide (BuTCh), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), and galantamine were purchased from Sigma Chemicals Co., Ltd. (Shanghai, China). Phenylhydrazine-HCl and α-ketoglutaric acid were purchased from Aladdin Industrial Inc. (Shanghai, China). Other reagents and solvents were obtained locally and of analytical grade or purified according to standard methods before use. The water used was redistilled and ion-free.