Galantamine

The measurements of the activities were carried out using a 96-well microplate reader (PerkinElmer Multimode Plate Reader EnSpire) at the National Center for Biotechnology Research. The chemical products and reagents used were: Folin–Ciocalteu reagent (FCR), 1,1-diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxylanisole (BHA), butylated hydroxyltoluene (BHT), quercetin, α-Tocopherol, ascorbic acid, neocuproine, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)-diammoniumsalt(ABTS), trichloroacetic acid (TCA), potassium ferricyanide (C₆N₆FeK₃), phenanthroline, silver nitrate (AgNO3), trisodium citrate (Na₃C₆H₅O₇), acetylcholinesterase from electric eel (AChE, Type-VI-S, EC 3.1.1.7, 827.84 U/mg, Sigma), butyrlcholinesterase from horse serum (BChE, EC 3.1.1.8, 7.8 U/mg, Sigma), acetylthiocholine iodide, S-Butyrylthiocholine iodide, 5,5′-Dithiobis(2-nitrobenzoic) acid (DTNB), and galantamine, which were obtained from Sigma-Aldrich; and sodium carbonate, aluminum nitrate (Al(NO₃)₃, 9H₂O), iron (III) chloride (FeCl₃), sodium bicarbonate (NaHCO₃), copper (II) chloride (CuCl₂), potassium persulfate (K₂S₂O₈), and potassium acetate (CH₃CO₂K), which were obtained from Biochem Chemopharma. All other chemicals and solvents were of analytical grade.

The 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2, 4, 6-Tris(2-pyridyl)-s-triazine (TPTZ), 2, 6-di-tert-butyl-4-methylphenol (BHT), vitamin C (Vc), were obtained from Solarbio Life sciences (Beijing, China). All chemical reagents including methanol, ethyl acetate (EtOAc), petroleum ether (PE), Dichloromethane (DCM), and n-butanol (n-BuOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Chromatographic acetonitrile was purchased from Merck (Darmstadt, Germany). Folin-Ciocalteu, pyrogallol, and other analytical grade chemicals were purchased from Aladdin (Shanghai, China). Myricetin, dihydroMyricetin, quercetin, resveratrol, apigenin, baicalein, hesperitin, gallic acid, rutin, luteolin, epicatechin, epicatechin gallate, and other standards for HPLC were purchased from Yuanye Bio-Technology (Shanghai, China). Cellulase (3, 000 μ/g) and pectinase (40, 000 μ/g) were purchased from Xiya reagent (Shandong, China). α-Glucosidase (G5003) and acetylcholinesterase (AChE, C3389) were purchased from Sigma-Aldrich (St. Louis, USA). p-Nitrophenol nitrophenol α-D-glycopyranoside (pNPG), acarbose, galantamine, and cisplatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HepG2, MCF-7, Raw264.7, and A549 cells were purchased from Conservation genetics CAS Kunming cell bank.

Fluorescence and optical density were measured by Bio-TEK ELISA fluorescence reader FLx 800 and Bio-TEK ELx 808, respectively (Winooski, VT, USA). Eckol (>95%), dieckol (>95%), and 8,8′-bieckol (>95%), were bought from National Development Institute of Korean Medicine (Gyeongsangbuk-do, Korea). The BACE1 assay kit was purchased from Invitrogen (Pan Vera, Madison, WI, USA). TACE and substrate were bought from R&D Systems (Minneapolis, MN, USA). AChE from Electrophorus electricus (electric eel), 5,5′-dithiobis-(2 nitrobenzoic acid) (DTNB), resveratrol, galantamine, trypsin, chymotrypsin, elastase, and their substrates, including N-benzoyl-l-Arg-pNA, N-benzoyl-l-Tyr-pNA, and N-succinyl-Ala-Ala-Ala-p NA, were from Sigma-Aldrich (St. Louis, MO, USA).

The irisin expression construct (pET15b-His-3C-irisin) was procured from Addgene (122612). For the expression of irisin, the C41-DE3 strain of E. coli was used. Ampicillin, memantine, galantamine, and fluoxetine were obtained from Sigma-Aldrich Co. (Saint Louis, MO, Missuori, USA). For buffer preparations, analytical -grade chemicals were used.

In both studies, galantamine (4 mg/kg, Sigma Aldrich, #1287755) was administered by intraperitoneal injection (i.p.). In the prevention study (study 1), galantamine or saline (vehicle) was administered 30 min prior to either ethanol or water gavage on every gavage treatment day of the AIE paradigm. In contrast, in the restoration study (study 2), galantamine or vehicle was administered every day consecutively for 2 weeks starting on P57, after the end of the ethanol/water gavage.
Rats were euthanized at P70 (prevention, study 1) or P73 (restoration, study 2) with sodium pentobarbital and perfused with 4% paraformaldehyde/0.1 M PBS. Following, their brains were harvested, sunk in 30% sucrose/0.1 M PBS, and frozen for subsequent histological analyses.

HPLC grade solvents (ethanol, formic acid, and acetonitrile) were obtained from Merck (Santiago, Chile). Ultrapure water was obtained from a water system of purification (Milli-Q Merck Millipore, Chile). Flavonol standards (catechin, isoflavones, and flavonoids, with a high purity: 95% by HPLC) were acquired from ChromaDex (Santa Ana, CA, USA), Sigma-Aldrich (Saint Louis, Mo, USA), or Extrasynthèse (Genay, France). Folin-Ciocalteu reagent, NaOH, Na₂CO₃, AlCl₃, FeCl₃, HCl, NaNO₂, trichloroacetic acid, quercetin, Trolox sodium acetate, gallic acid, 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), nitroblue tetrazolium, xanthine oxidase, and DPPH (1,1-diphenyl-2-picrylhydrazyl radical), acetylcholinesterase (AChE) from electric eel (Torpedo californica), butylcholinesterase (BChE) from horse serum, 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB), acetylthiocholine iodide, butyrylthiocholine chloride, and galantamine were purchased from Sigma-Aldrich Chemical Company (Santiago, Chile). G. sphacelata fruits were collected in November 2016 in Valdivia, XIV Region de Los Ríos, Chile. The fruits were identified by Jorge Macaya (Universidad de Chile) and a voucher specimen were kept at the Institute of Pharmacy of the Universidad Austral de Chile under number GS20161115.

Inhibition of AChE activity of BEO was measured following the method of Ellmann with slight modifications [46 (link)]. Briefly, aliquots of BEO diluted in methanol were mixed with 50 μL of 50 mM phosphate buffer pH 8, 125 μL of dithionitrobenzoic acid (DTNB, 3 mM in 50 mM phosphate buffer pH 8) and 25 μL of AChE from electric eel, 3 U/mL in 50 mM phosphate buffer pH 8) and incubated for 15 min at 25 °C. Then, 25 μL of the substrate acetylthiocholine iodide (ACTI, 15 mM in 50 mM phosphate buffer pH 7.0) were added and AChE activity was calculated by measuring the absorbance at 412 nm (at 25 °C for 3.0 min) with a Fluostar Omega (BMG-Labtech, Ortenberg, Germany) plate reader. Galantamine (Sigma, Milan, Italy) was used as a reference substance. Results were expressed as Galantamine-equivalent inhibition capacity (GEIC), indicating the mg of Galantamine equivalents (GE) per g of BEO. Each experiment was replicated three times.