Butyrylcholinesterase

acetylcholinesterase/butyrylcholinesterase (AChE/BuChE) inhibitory activity of the isolated compound (1) was assayed by the spectrophotometric method developed by Ellman’s method with slight modification [46 (link)]. S-Acetylthiocholine iodide, S-butyrylthiocholineiodide, 5,5′-dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman’s reagent), acetylcholinesterase and butyrylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical (St. Louis, MO, USA). The compounds were dissolved in DMSO. The reaction mixture (totally 200 μL) containing phosphate buffer (pH 8.0), test compound (50 μM), and acetyl cholinesterase (0.02 U/mL) or butyrylcholinesterase (0.016 U/mL), was incubated for 20 min (37 °C). Then, the reaction was initiated by the addition of 40 μL of the solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) or butyrylthiocholine iodide (0.625 mM) for AChE or BuChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholineorbutyrylthiocholine was monitored at 405 nm every 30 s for one hour. Tacrine was used as a positive control with a final concentration of 0.333 μM. All of the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E − S)/E × 100 (E is the activity of the enzyme without test compound, and S is the activity of the enzyme with test compound).

All the chemicals and reagent used in this work were of laboratory and analytical grade. Diphenyl-1-picrylhydrazyl (DPPH), 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 3,5-dinitrosalicylic acid (DNSA), α-amylase, acarbose, 4-nitrophenyl butyrate (p-NPB), lipase, orlistat, l-3,4-dihydroxyphenylalanine (L-DOPA), mushroom tyrosinase, kojic acid, N-succinyl-Ala-Ala-p-nitroanilide (AAAPVN), porcine pancreatic elastase, quercetin, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), acetylthiocholine iodide, butyrylcholine iodide, 5,5′-dithiobis-(2-nitrobenzoic acid) (DNTB), and galantamine were purchased from Sigma-Aldrich, USA.

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).