Donepezil hydrochloride

Sulforaphene (SF, purity ≥ 95%) was obtained from Chengdu Biopurify Pharmaceutical Co. Ltd. (Chengdu, Sichuan, China), and its identity was confirmed by comparing its ¹H NMR and ¹³C NMR spectra with those published in the literature [17 (link)]. Donepezil hydrochloride, lipopolysaccharide, LY294002, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). STZ was purchased from Santa Cruz Biotechnology (Dallas, USA). All other reagents and chemicals used in this study were of analytical grade.

Acetylcholinesterase (AChE, E.C. 3.1.1.7, obtained from electric eel) was purchased from Sigma-Aldrich (St. Louis, MO, USA, product code: C3389-2KU). Butyrylcholinesterase (BuChE, E.C. 3.1.1.8, obtained from equine serum) was purchased from Aladdin (Shanghai, China, product code: B128570-2KU). 5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent DTNB), acetylthiocholine chloride (ATC), butylthiocholine chloride (BTC), donepezil hydrochloride and tacrine hydrochloride were purchased from Sigma-Aldrich. The compounds were dissolved in a minimum volume of DMSO (1%) and then diluted in 0.1 M KH₂PO₄/K₂HPO₄ buffer (pH 8.0) to provide a final concentration range.
All the methods were under 0.1 M KH₂PO₄/K₂HPO₄ buffer (pH 8.0), using a Shimadzu UV-2450 Spectrophotometer. Enzyme solutions were prepared to give 2.0 units/ mL in 2 mL aliquots. The assay medium contained phosphate buffer, pH 8.0 (1 mL), 50 μL of 0.01 M DTNB, 10 μL of enzyme, and 50 μL of 0.01 M substrate (ATC). The substrate was added to the assay medium containing enzyme, buffer, and DTNB with inhibitor after 15 min of incubation time. The activity was determined by measuring the increase in absorbance at 412 nm at 1 min intervals at 37 °C. In vitro BuChE assay used the similar method described above [31 (link)]. Each concentration was assayed in triplicate.

The H3R antagonist DL77 [1-(3-(4-tert-pentylphenoxy) propyl)piperidine] was designed and synthesized in the Department of Technology and Biotechnology of Drugs Krakow, Poland, according to a previously described procedure^{23 (link)}. Sodium valproate (VPA) (500 mg/kg, i.p.), donepezil hydrochloride (DOZ) (1 mg/kg, i.p.) (reference drug), the H3R agonist RAMH (10 mg/kg, i.p.), the brain-penetrant H1R antagonist PYR (10 mg/kg, i.p.), the brain-penetrant H2R antagonist ZOL (10 mg/kg, i.p.), LPS (from E. coli serotype 0111:B4), and the assay kit for GSH were all purchased from Sigma-Aldrich (St. Louis, MO, USA). The lipid peroxidation assay kit for estimation of malondialdehyde (MDA) was obtained from North West Life Science (Vancouver, WA, USA). For estimation of the proinflammatory cytokines IL-1β, IL-6 and TNF-α mediated by lipopolysaccharide (LPS), commercially available ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). All the reagents used in the study were of analytical grade. All drugs were dissolved in isotonic saline solution and injected intraperitoneally (i.p.) at a volume of 10 ml/kg adjusted to body weight, and all doses are expressed in terms of the free base.

Behavioral and neurogenic effects of S 38093 (Servier, France) were compared to fluoxetine hydrochloride (Anawa Trading, Zurich, Switzerland) or Donepezil hydrochloride (Sigma-Aldrich, St Quentin Fallavier, France).

Rivastigmine tartrate (R), donepezil hydrochloride (D) and memantine hydrochloride (M) were procured from Sigma Aldrich, St Louis, MO, USA and the stocks were prepared [110 (link),111 (link),112 (link),113 (link)]. Rivastigmine, donepezil and memantine were used at 2 µM, 4 µM and 3 µM, respectively, for the subsequent experiments.

Donepezil hydrochloride (DNP, ≥98%, HPLC), cholesterol (Chol, ≥99%), α-tocopherol (TOC), and thioflavin S were procured from Sigma-Aldrich (St. Louis, MO, USA). Soybean L-α-phosphatidylcholine (PC, 95%) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) was used as the main component of liposomes. Tetradecyltriphenylphosphonium bromide (TPPB-14) was synthesized according to the published method [74 (link)]. Chloroform and ethanol (HPLC) were purchased from JSC “№1 BASE Chemical reagents” (Staraya Kupavna, Russia). Rhodamine B (RhB) (Acros Organics, Morris Plains, NJ, USA) and fluorescent lipid DOPE-RhB (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Avanti Polar Lipids, Inc., Alabaster, AL, USA) were used to visualize liposomes inside cells. Liposomal dispersions were prepared using ultrapure Milli-Q water purified by the Simplicity^® UV system (Millipore SAS, Molsheim, France). For visualization of the immunoexpression of synaptophysin, primary rabbit monoclonal antibody to synaptophysin (ab 32127) and secondary donkey anti-rabbit (Alexa Fluor^® 488) antibody (ab 150073) were purchased from Abcam (Cambridge, UK). Mayer’s hematoxylin was purchased from Biovitrum (Saint Petersburg, Russia).