Butyrylthiocholine

AChE from Electrophorus electricus (CAS: 9000-81-1) was obtained from Sigma Aldrich, St. Loius, MO, United States, while BChE was derived from equine serum (9001-08-5) and was purchased from Sigma–Aldrich GmbH, Germany. Acetylthiocholine iodide (CAS1866-15-5) and butyrylthiocholine iodide (CAS 2494-56-6) were purchased from Sigma–Aldrich United Kingdom and Sigma–Aldrich Switzerland respectively. 5,5-Dithio-bis-nitrobenzoic acid (DTNB) (CAS 69-78-3) (Sigma–Aldrich GmbH, Germany) and galanthamine HBr Lycoris Sp. (CAS: 1953-04-4) (Sigma–Aldrich, France) were used in enzymes studies. Antioxidant reagents including DPPH (CAS: 1898-66-4) ABTS (CAS: 30931-67-0) were purchased from Sigma Aldrich St. Loius, MO, United States. H₂O₂ (batch no: A040) was obtained from Rehmat pharma Lahore, Pakistan. Potassium peroxodisulfate (LOT NO: 51240) was obtained from Labor chemikalien GmbH & Co KGD-30926 Seelze. For genotyping of transgenic animals, GF-1 tissue DNA extraction kit (Cat:GF-TD-100, Vivantis), agarose (Invitrogen CAT:75510-011, Carlsbad, CA, United States), boric acid (Serva CAT 15165, Germany), DNA Ladder (Serva CAT:15165, Germany), EDTA (Invitrogen CAT:75576-028, Carlsbad, CA, United States), ethanol (Merck CAT:26225745, Germany), ethidium bromide (Sigma CAT:E7637, United States), MgCl₂ (Invitrogen CAT:AM9530G, Carlsbad, CA, United States), DNTPs (Promega CAT:U1515, United States), Taq polymerase (Thermo Scientific CAT: EP0402, United States), PCR primers (Thermo Scientific CAT:OIMR3610 F, OIMR3611 R), PCR grade distilled water (Thermo Scientific CAT: R0581), sucrose (Invitrogen CAT: 15503-022, Carlsbad, CA, United States), Tris EDTA solution (50X), 2XPCR Master mix (Fermentas CAT: K0171, EU), NaCl (Invitrogen CAT: 24740-011, Carlsbad, CA, United States) and tris (Invitrogen CAT: 15504-020, Carlsbad, CA, United States) were purchased from authorized dealers in Pakistan. Solvents and buffer salts used were of extra pure quality.

Two recombinant hBChE enzymes were used for crystallography. The first (BChE_CHO), a truncated monomer containing residues 1-529 whose tetramerization domain and four N-glycosylation sites were deleted, was expressed in Chinese hamster ovary (CHO) cells [58 (link)]. The second (BChE_S2) was a truncated monomer containing residues 1-529 expressed in Drosophila S2 cells [38 (link)]. Both BChE_CHO and BChE_S2 were secreted into serum-free culture medium and purified by affinity and exclusion chromatography as previously described [38 (link)]. BChE_CHO crystals are preferred over BChE_S2 crystals because they are generally of better quality. BChE_S2 was used as a backup in case of failure to obtain the structure of the complexes with BChE_CHO.
For enzyme kinetics experiments, purified recombinant hAChE (~1500 units as determined by the supplier) was purchased from Sigma Aldrich (St. Louis, MO, USA). The AChE concentration was calculated using the assumption of 450 units/nmol (4.8 ∆A412/(min × nM)). Purified human plasma wild-type BChE was a gift from Dr. Oksana Lockridge (University of Nebraska Medical Center, Omaha, NE, USA). BChE concentration was calculated assuming 62.5 unit/nmol (0.94 ∆A412/(min × nM)) [59 (link)]. As defined previously, 0.1 unit is the amount of BChE that gives 1.0 ∆A/min in the presence of 1.6 × 10⁻⁴ M butyrylthiocholine in a 1.5 mL assay [60 (link)].
For isothermal titration calorimetry, BChE_CHO and AChE_CHO were used. AChE_CHO is a truncated monomer of hAChE containing residues 1-543 (no tetramerization domain) expressed in CHO cells [61 (link)].
ThT chloride, decamethonium bromide, ethopropazine hydrochloride, propidium iodide, and edrophonium chloride were purchased from Sigma (Saint-Quentin-Fallavier, France), and huprine 19 was provided by ChemForAse (Rouen, France). ThT was recrystallized, as described previously [12 (link)].

For antioxidant assays, 1,1-diphenyl, 2-picrylhydrazyl (DPPH) and 2, 2-azinobis [3-ethylbenzthiazoline]-6-sulfonic acid (ABTS) (CAS 1898–66-4 and CAS 30931–67-0 Sigma Aldrich, USA,K₂S₂O₄ (Riedel-de Haen Germany) were purchased from authorized dealers in Pakistan. For anticholinesterase and α-glucosidase studies, AChE from Electric eel (type-VI-S, CAS 9000–81-1), BChE from equine serum lyophilized (CAS 9001–08-5) and α-glucosidase from Saccharomyces cerevisiae CAS 9001–42-7 were purchased from Sigma-Aldrich GmbH USA. The substrates including acetylthiocholine iodide (CAS1866–15-5), butyrylthiocholine iodide (CAS 2494–56-6), p-nitrophenyl-α-D-glucopyranoside glucopyranoside (CAS 2492–87-7), were purchased from Sigma-Aldrich UK and Sigma-Aldrich, Switzerland, respectively. Similarly, the indicator substance, 5,5-dithio-bis-nitrobenzoic acid (DTNB) CAS 69–78-3 was purchased from Sigma-Aldrich Germany. The standard drugs, galantamine hydrobromide, Lycoris Sp. (CAS 1953–04-4), acarbose alfa aesar (CAS 56180–94-0) and ascorbic acid were purchased from Sigma-Aldrich France. The buffer system including (K₂HPO₄), (KH2PO₄), KOH and solvents used were of extra pure quality.

Acetylcholinesterase from electric eel type VI-S, butyrylcholinesterase from equine serum, acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), 5,5′-dithiobis[2-nitrobenzoic acid] (DTNB), bovine serum albumin (BSA), tris buffer, and galantamine were purchased from Sigma–Aldrich. The organic solvents (methanol and ethanol) and reagents used in the analysis were of analytical grades.

The in vitro inhibitory potential of PEE from C. gilliesii, M. acuminata, and T. multiflora were evaluated by Ellman’s method. PEE of each sample (50 µL, 2 mg/mL) was mixed with 120 µL of 5,5-dithio-bis (2-nitrobenzoic) acid (DTNB) 0.3 mM, AChE (0.26 U/mL, from Electric eel), or BChE (0.26 U/mL, from horse serum) solution (25 µL) in Tris-HCl buffer 50 mM (pH = 8.0) in a 96-well microplate and incubated for 20 min at 37 °C. The reaction was initiated by the addition of 25 µL of acetylthiocholine iodide (ATCI) 1.5 mM or butyrylthiocholine chloride (BTCl) 1.5 mM. The absorbances were recorded three times at 405 nm at 37 °C using a microplate reader (Multiskan EX, Thermo). Galantamine hydrobromide was used as a positive control. The cholinesterase inhibitory activity was expressed as IC₅₀ (µg mL⁻¹, concentration range 15 to 500 µg mL⁻¹). All data were recorded in triplicate.

Alkane standard solution (C8–C20, ~40 mg/L each, in hexane), 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 5,5-dithio-bis(2-nitrobenzoic) acid, acarbose, acetonitrile, ammonium acetate, ammonium molybdate, amylase (EC. 3.2.1.1, from porcine pancreas), α-bisabolol, butyrylthiocholine chloride, caffeic acid, β-caryophyllene, cupric chloride, 1,1-diphenyl-2-picrylhydrazyl (DPPH), eel acetylcholinesterase (AChE, type: VI-S, EC 3.1.1.7), ethanol, ferric chloride, ferrous sulfate hexahydrate, ferrozine, Folin–Ciocalteu reagent, formic acid, galantamine, acetylthiocholine iodide, gallic acid, glucose, glucosidase (EC. 3.2.1.20, from Saccharomyces cerevisiae), α-humulene, horse serum butyrylcholinesterase (BChE, EC 3.1.1.8), hydrochloric acid, hydroxybenzoic acid, kojic acid, limonene, β-myrcene, Mueller–Hinton (MH) broth, rosmarinic acid, rutin, sodium carbonate, sodium hydroxide, sodium molybdate, sodium nitrate, 2,4,6-tris(2-pyridyl)-s-triazine, Trolox, ethylenediaminetetraacetate (EDTA), and tyrosinase (EC1.14.18.1, mushroom) were from Merck KGaA (Darmstadt, Germany). β-Caryophyllene oxide was from Thermo Scientific (Olching, Germany), α-humulene was from Biomol (Hamburg, Germany), and liquid CO₂ (≥99.7%) from Westfalen AG (Münster, Germany). Methanol was bought from VWR Chemicals (Ismaning, Germany). Acetonitrile and formic acid were from Avantor (Gliwice, Poland).