Acetylthiocholine iodide

Glyphosate (PESTANAL^®, analytical standard; CAS N. 1071-83-6; MW: 169.07), Imidacloprid (PESTANAL^®, analytical standard; CAS N. 138261-41-3; MW: 255.66), Imazalil (PESTANAL^®, analytical standard; CAS N. 35554-44-0; MW: 297.18), lamba-cyhalothrin (PESTANAL^®, analytical standard; CAS N. 91465-08-6: MW: 449.8), acetylcholinesterase from Electrophorus electricus (CAS N. 9000-81-1; E.C. 3.1.1.7), butyrylcholinesterase from equine serum (CAS N. 9001-08-5; E.C. 3.1.1.8), tyrosinase from mushroom (CAS N. 902-10-2; E.C. 1.14.18.1), acetylthiocholine iodide (CAS N. 1866-15-5; MW: 289.18), butyrylthiocholine iodide (CAS N. 1866-16-6; MW: 317.23), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; CAS N. 69-78-3; MW: 396.35), 3,4-dihydroxy-L-phenylalanine (CAS N. 59-92-7; MW: 197.19) and kojic acid (CAS N. 501-30-4; MW: 142.11) were purchased from Merck (Darmstadt, Germany). All other reagents and salts were of analytical grade and were also obtained from Merck (Darmstadt, Germany).

All reagents and chemicals were analytical grades. Trizma base, hydrochloric acid, 5,5′-dithiobis-(2-nitrobenzoic acid), physostigmine, acetylthiocholine iodide, and acetylcholinesterase were purchased from Merck (Darmstadt, Germany). The COX-2 Inhibitor Screening Kit was purchased from Abcam (Cambridge, UK). LC-MS grade acetonitrile, LC-MS grade methanol, ethanol, dimethyl sulfoxide, catechin, sodium nitrite, aluminum chloride, sodium hydroxide, gallic acid, 2,2-difenil-1-picrilhidrazil (DPPH), iron (III) chloride hexahydrate, and formic acid used were purchased from Sigma-Aldrich (St. Louis, MO, USA). Folin-Ciocalteu reagent, sodium acetate solution, sodium carbonate, and 2,4,6-tripyridyl-s-triazine (TPTZ) were purchased from Thermo Fisher (Waltham, MA, USA). Finally, 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) was purchased from Roche (Basel, Switzerland), and the water used was Milli-Q type obtained from Millipore purification equipment (Billerica, MA, USA).

Dimethyl sulfoxide (DMSO), acetylcholinesterase (AChE) from Electrophorus electricus and butyrylcholinesterase (BChE) from equine serum, acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), galanthamine hydrobromide, and all the buffer components (TWEEN20, bovine serum albumin, Trizma base, Trizma HCl, MgCl₂·6H₂O), cyclohexane, nonane, petroleum ether, ethyl acetate, 1,8-cineole, linalool, linalyl acetate, 4-terpineol, and α-terpineol were purchased from Merck Life Science S.r.l. (Milan, Italy).
Six batches of Elettaria cardamomum (L.) Maton EO were obtained via steam distillation from botanically identified plant material (dried fruits, geographical origin Guatemala) and supplied by Erboristeria Magentina (Poirino, Torino, Italy). The hydrodistilled EO was obtained by submitting 100 g of dried fruits (same geographical origin) purchased from a local herbal shop to hydrodistillation for 4 h in Clevenger apparatus (a pic of the plant used to obtain the hydrodistilled EO is reported in the Supplementary Material Figure S2). The EO was collected and stored in the dark at +4 °C until use.

GC/MS-grade heptane, boron trifluoride-methanol solution 14% in methanol, and hexane, in addition to LC/MS-grade methanol, water, acetylthiocholine iodide, acetonitrile, folic acid, thiamine, riboflavin, ultrapure water, pyridoxine, niacin, Acetylcholinesterase (from Electrophorus electricus), and 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl, were purchased from Merck (Darmstadt, Germany). Fatty acid methyl esters (unsaturated Kit) and sodium chloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade and were purchased from Chempur (Piekary Slaskie, Poland).
Freeze-dried buffalo worms (Alphitobius diaperinus Panzer), house crickets (Gryllus campestris), mealworms (Tenebrio molitor), and grasshoppers (Chorthippus biguttulus) were purchased from a local commercial source (Delibugs, Lelystad, Netherlands). Before analysis, the insects were shredded.
Insects (10 g) previously ground in a mortar were added to virgin olive oil (1 L). Olive oil with insects was left for 48 h in a cool, dark place.

A standardised extract of WS was kindly provided by Natural Remedies Private Limited, Bangalore, India. WS extract was certified to contain 2·6 % withanolides including withanosides IV (0·87 %) and V (0·65 %), withaferin A (0·56 %), withanolide A (0·20 %) and B (0·06 %), and 12-deoxy withastramonolide (0·26 %) as determined by HPLC. Dulbecco's minimum essential medium (DMEM), fetal bovine serum, penicillin/streptomycin, acrolein, acetylthiocholine iodide, neostigmine bromide, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), H₂O₂ and TOX-2 (XTT based) were obtained from Sigma-Aldrich Inc. The cytotoxicity detection kit based on the lactate dehydrogenase (LDH) assay was from Roche Diagnostics and 2′,7′-dichlorofluorescein diacetate (DCF-DA) was from Invitrogen. β-Amyloid peptide_(25–35) was purchased from American Peptide.

All chemicals used in the present study are of analytical grade. Sucrose (saccharose); Trizma^®hydrochloride–Tris [hydroxymethyl] aminomethan hydrochloride, L-glutamic acid, Kainic acid monohydrate (2-Carboxy-3-carboxymethyl-4-isopropenyl-pyrrolidine.H₂O), Na₂HPO₄.2H₂O (sodium phosphate dibasic dihydrate), NaH₂PO₄.H₂O (sodium phosphate monobasic monohydrate), acetyl-thiocholine iodide, DTNB (5,5′-Dithiobis (2-nitrobenzoic acid), Ezerin Salicylate, TCA (Trichloroacetic acid), EDTA (Ethylenediaminetetraacetic acid, EDTA disodium salt), Ouabain, NaCl, KCl, MgCl₂ are purchased from Sigma-Aldrich (St. Louis, MO, USA). ATP (adenosine-5′-triphosphate, Na₂ salt) is purchased from Serva Electrophoresis GmbH (Heidelberg, Germany). The synthetic monounsaturated phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti Polar Lipids Inc., Alabaster, AL 35007, USA) is used. Bidistilled water from quartz distiller is used for the preparation of aqueous solution.