Acetylcholine iodide

UHPLC-MS solvents, LC-MS formic acid reagent, acetonitrile were purchased from Merck (Santiago, Chile). Ultrapure water was obtained from a Millipore purification system (Milli-Q Merck Millipore, Santiago, Chile). UHPLC standards with a purity higher than 95% for HPLC) were acquired from Sigma Aldrich (St. Louis, MO, USA). Quercetin, ascorbic acid, gallic acid, sodium trioxocarbonate (VI), sodium phosphate, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), trichloroethanoic acid (TCA), thiobarbituric acid (TBA), ferrous sulphate, ethyl acetate, methanol, ethanol, Dowex^® 50WX8 hydrogen form, acetylcholine iodide (AChI), butyrylcholine iodide (BCEI) were purchased from Sigma Aldrich (St. Louis, MO, USA). Other reagents such as Folin-Ciocalteau, potassium bromide, hydrochloric acid, used for this study were of analytical grade and products procured from Qualikems Laboratories (India) and BDH Chemicals Ltd. (Poole, England). The water used for all analysis was glass distilled.

Cannabinoids standards: CBD, CBDA, Δ⁹-THC, THCA, CBG, CBGA, CBN, and CBC were obtained from Sigma-Aldrich (Poznan, Poland). Trifluoroacetic acid and acetonitrile (high-performance liquid chromatography [HPLC] grade) were supplied by Merck (Darmstadt, Germany). High-quality pure water was prepared using a Direct-Q 3 UV purification system (Millipore, Molsheim, France; model Exil SA 67120). Iron (III) chloride hexahydrate, 5,5-dithio-bis-(2-nitrobenzoic acid), 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), neocuproine, 2,4,6-tri(2-pyridyl)-s-triazine, trolox, tyrosinase, L-DOPA, acetylcholinesterase, butyrylcholinesterase, acetylcholine iodide, butyrylcholine iodide, Trizma^® hydrochloride, and Trizma^® base were purchased from Sigma-Aldrich (Schnelldorf, Germany). Sodium chloride, sodium dihydrogen phosphate, and sodium hydrogen phosphate, were purchased from Avantor Performance Materials (Gliwice, Poland). Ammonium acetate (NH₄Ac) and methanol were supplied by Chempur (Piekary Śląskie, Poland). Cupric chloride dihydrate, acetic acid (99.5%), ethanol (96%), and sodium acetate trihydrate were supplied by POCH (Gliwice, Poland).

The AChE inhibitory activity was carried out according to Ellman et al. 1961. [28 (link)], with the modifications incorporated by López et al. 2002 [29 (link)]. A stock solution of 518U of AChE from Electrophorus electricus (Sigma, Schnelldorf, Bayern, Germany) was prepared and kept at −20 °C. Acetylcholine iodide and DTNB were also obtained from Sigma, Schnelldorf, Bayern, Germany.
To perform the analysis, 50 µL of AChE in phosphate buffer (8 mM K₂HPO₄, 2.3 mM NaHPO₄, and 0.15 M NaCl, at pH 7.5) was incorporated into 50 µL of the sample dissolved in that same buffer, in flat-bottom 96-well plates. The plates were subsequently incubated for 30 min at 21–25 °C. Immediately afterwards, 100 µL of substrate solution was added (0.1 M Na₂HPO₄, 0.5 M DTNB, and 0.6 mM ACh iodide in Millipore water at a pH of 7.5). After 5 min, the absorbance was read on an ELISA plate reader (Multiscan EX Thermo Scientific^®, Waltham, MA, USA). Galanthamine was used as a positive control (employing a 1:10 dilution bank from 10⁻³ to 10⁻⁷ M). Phosphate buffer was used as blank.
To determine the inhibitory potential of each extract, their respective IC₅₀ was established. In order to do so, a semilogarithmic linear regression study was carried out from the concentrations analyzed and the inhibitory values obtained.

Trizma-HCl, EGTA, HEPES, Pronase E, acetylcholine iodide, Cyt, and all chloride salts were purchased from Sigma (USA). Mono-iodinated (3-[¹²⁵I]iodotyrosyl⁵⁴)-αBgt (∼2000 Ci/mmol) was from GE Healthcare. nAChR-enriched membranes from the electric organs of T. californica ray were kindly provided by Prof. F. Hucho (Free University of Berlin, Germany), GH₄C₁ cells transfected with human α7 nAChR were a gift from Eli-Lilly (USA). The expressed acetylcholine binding proteins (AChBP) from L. stagnalis and Aplysia californica were kindly provided by Prof. T. Sixma (Netherlands Cancer Institute, Amsterdam, the Netherlands). Venom samples from vipers V. nikolskii and V. ursinii were obtained in 2009 by milking the snakes kept in captivity at Tula zoo (the Tula Exotarium). The permission was issued by the zoo director S.A.Ryabov. The venom of krait B. fasciatus was collected from snakes obtained from the authorized, licensed local establishment for snake breeding and venom production with permission of its owner Mr. Ha Văn Ti n (Vinh Sh n, Vinh Tu' ng, Vinh Phúc province, Vietnam). Cobra (N. kaouthia) venom was obtained as described earlier [17] (link). All efforts were undertaken to minimize suffering of the animals while venom samples were collected. Venoms collected were dried over anhydrous CaCl₂ and stored at −20°C until use.

Folin–Ciocalteau’s reagent, bovine serum albumin, ABTS, acarbose, sodium dodecyl sulfate, EDTA, sodium bicarbonate, α-glucosidase, iNOS, TNF-α, IL-6, acetylcholinesterase, lipopolysaccharide, acetylcholine iodide, DTNB, TPTZ, chlorogenic acid, DMSO, and Trolox were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glycine was purchased from J.T.Baker (Phillipsburg, NJ, USA). DPPH, phenazine methosulphate, and nitroblue tetrazolium were supplied from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Thiazolyl blue tetrazolium bromide was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Butyl hydroxytoluene, nicotinamide adenine dinucleotide, sodium acetate, and potassium acetate were supplied from Acros Organics (Geel, Belgium). Ammonium persulfate and TEMED were purchased from Bio-Rad (Hercules, CA, USA). Tween20 was purchased from Merck (Darmstadt, Germany). Acetic acid was supplied from Macron Fine Chemicals (Center Valley, PA, USA). Iron (III) chloride, aluminum chloride, as well as p-Nitro-phenyl-α-D-glucopyranoside were purchased from Alfa Aesar (Lancashire, UK). Disodium hydrogenphosphate, sodium carbonate, potassium peroxodisulfate, and potassium dihydrogenphosphate were obtained from SHOWA Chemical Co. (Sun Valley, CA, USA).

Patient-derived cultures of GBM cells, as well as U87MG cells, were grown in 96-well plates in media I and II at 37 ° C, 5% CO₂ atmosphere, 100% humidity. Before calcium imaging began, the growing medium was replaced with extracellular buffer (140 mM NaCl, 2 mM CaCl₂, 2.8 mM KCl, 4 mM MgCl₂, 20 mM HEPES, 10 mM glucose, pH 7.4), and then each well was loaded with a cell-permeant 2,5 μM Fluo-4AM solution (ex/em = 494/506 nm; Thermo Fisher Scientific) for 40 min. The fluorescent dye solution was further removed, and cells were kept in an extracellular buffer for 1 h. Ca²⁺ dynamics were recorded using an Olympus IX71 epifluorescent microscope with an appropriate filter combination and a CAM-XM10 cooled CCD camera (Olympus, Tokyo, Japan). The cells were exposed to 10 μM of nAChR agonist acetylcholine iodide (Sigma-Aldrich), 1 μM of antagonists Azemiopsin, and [A10L]PnIA or RgIA (Syneuro, Moscow, Russia), and changes in the fluorescence of calcium indicator Fluo-4 were recorded for each cell independently. Videos were recorded and processed using CellA imaging software version 3.1 build 1274 (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Data analysis was performed using open-source ImageJ Fiji software (version 1.54f), where changes in fluorescent intensity per cell before and after the nAChR ligand exposure were calculated. The response of at least 5 cells was measured.