Acetylcholinesterase

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.

The kenaf stems (KS) were collected from the Futian farm in Ji'an, Jiangxi Province. Graphite powder (99.95% and 325 mesh) and paraffin were acquired from Aladdin. DMFc, 1,3,5-triformylbenzene, acetylthiocholine chloride (ATCl), 1,4-diaminobenzene, and Acetylcholinesterase (1,000 U/mg), were obtained from Sigma-Aldrich (USA). Trichlorfon was bought from Kanghe Yinong Biotechnology Co., Ltd. Other reagents utilized were of analytical grades and procured from Shanghai Guoyao Group Chemical Reagent (China). Distilled water (18.2 MΩ cm) was employed for making all the solutions and purged by nitrogen prior to experiments. PBS was freshly made using dihydrogen phosphate and sodium disodium hydrogen phosphate.

The samples of some parts used, such as leaves, heartwood, and trunk bark of ELC, were collected in Yok Don National Park, Dak Lak Province, Vietnam, in 2022. The dried herbal parts were packed in PE bags and stored at −30 °C before extraction. Acetylcholinesterase and berberine chloride were acquired from Sigma Aldrich (St. Louis, MO, USA). The highest-grade solvents and common chemical agents available were used in this study.

2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydryl (DPPH), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), acetylcholinesterase (AChE), acetylthiocholine (AcSCh), butylated hydroxytoluene (BHT), dichloromethane (DMC), dimethyl sulfoxide (DMSO), donepezil, methanol (MeOH), magnesium chloride hexahydrate, phosphate buffered saline (PBS), tris hydrochloride (Tris-HCl) and sodium sulfate anhydrous were purchased from Sigma-Aldrich (San Luis, MO, USA). Mueller Himton broth, Mueller Hinton II broth and fluid thioglycollate medium were purchased from DIPCO (Quito, Ecuador). The standard aliphatic hydrocarbons were purchased from ChemService (West Chester, PA, USA). Helium was purchased from INDURA (Quito, Ecuador). All chemicals were of analytical grade and used without further purification.

Potassium hexacyanoferrate (III), potassium hexacyanoferrate (II) trihydrate, 25% GA, acetylcholine iodide (ATCh), acetylcholinesterase (AChE) from electric eel, nitric acid, and phosphate buffer saline (PBS) were purchased from Sigma‐Aldrich. Phosmet was obtained from J&K Scientific (Hong Kong) Ltd. Nb₂AlC (>99%) was obtained from Laizhou Kai Ceramic material Co., Ltd. Carbon fiber cloths (CFCs) were purchased from CeTech (W0S1002) with a thickness of 0.33 mm. The electrochemical characterizations were performed by using Solartron Electrochemical workstation with standard three electrode system in 1 m PBS (aq). All these chemicals were used as received without further purification.