Methanol

The analytical standard of FF (Sigma-Aldrich, Steinheim, Germany) and internal standard CAP (Dr Ehrenstorfer, Augsburg, Germany) were used. HPLC-grade acetonitrile, methanol and water were provided by Merck (Darmstadt, Germany). Phosphoric acid (85 wt% in water) of analytical grade was purchased from Kemika (Zagreb, Croatia).
The methanol stock solution of FF was diluted to 20.0, 15.0, 10.0, 7.5, 5.0, 2.5, 1.0, 0.5, 0.1 and 0.05 µg/mL by adding component A of the mobile phase for obtaining the calibration curve. The appropriate volumes for each dilution were also added to blank urine to prepare urine FF standards ranging from 0.05 to 20.0 µg/mL. The concentration of internal standard CAP in all calibrators was 10.0 µg/mL.
Frozen urine samples were first thawed at room temperature and then centrifuged (Centric 322A, Tehtnica Železniki d.o.o., Železniki, Slovenia) at 4500 rpm for 15 min before sample treatments. The 500 µL of a urine sample was spiked with 0.1 mL of the internal standard stock solution and diluted with component A of the mobile phase. Samples were then vortexed (Assistent Reamix 2789, Karl Hecht GmbH, Fritzens, Germany) for 1 min and filtered using a regenerated cellulose filter (pore size 0.45 μm, Agilent Technologies, Waldbronn, Germany).

Tart cherries were purchased at a local market, pits were removed, and flesh was used for the preparation of jams. The following chemicals were used: Potassium chloride, sodium acetate, hydrochloric acid, methanol, sodium carbonate, sodium bisulfite, Folin-Ciocalteu reagent were purchased from Kemika (Zagreb, Croatia). Trolox, 2,2’-azinobis (3-ethylbenzothiazoline sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazil (DPPH) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Hydrochloric acid, acetic acid, methanol, sodium carbonate, iron chloride, ammonium acetate and Folin–Ciocalteu reagent were purchased from the manufacturer Kemika (Zagreb, Croatia). Trolox was purchased from Sigma (Darmstadt, Germany). 2,2’-azinobis (3-ethylbenzothiozolinesulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Fluka (Darmstadt, Germany). 2,4,6,-tri (2-pyridyl)-s-triazine (TPTZ), gallic acid, procyanidin B2 and myrtenol were purchased from Sigma, Germany. Neocuproine and copper chloride are products of Gram-mol Zagreb, Croatia). Sucrose was purchased from Gram-mol (Zagreb, Croatia), and maltose and trehalose were obtained from Hayashibara doo (Nagase group, Tokyo, Japan). Citrus fibers were obtained from Biesterfeld AG (Zagreb, Croatia).

Inhibition potency of novel compounds was evaluated for recombinant human AChE (prepared as described earlier²⁵ (link) and kindly donated by Prof Palmer Taylor, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, USA) and BChE isolated from human plasma (kindly donated by late Dr Douglas Cerasoli and Dr David Lenz, USAMRICD, Edgewood, MD). The inhibition mixture contained a 0.1 M phosphate buffer, pH 7.4, enzyme, tested compound, and reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 0.3 mM; Sigma Chemical Co., St. Louis, MO, USA). Enzyme activity was measured upon addition of substrate, acetylthiocholine (ATCh, 0.2 or 0.1 mM; Sigma Chemical Co., St. Louis, MO, USA) by the Ellman method²⁶ (link) at 25 °C and 412 nm, on a Tecan Infinite M200PRO plate reader (Tecan Austria, GmbH, Salzburg, Austria). Due to the low solubility, a stock solution of the tested compounds was prepared in DMSO or methanol (Kemika, Zagreb, Croatia), and a corresponding solvent was in controls as well. The IC₅₀ values were determined from at least three experiments by a nonlinear fit of the compound concentration logarithm values vs. % of enzyme activity using Prism6 software (GraphPad Prism 6 Software, San Diego, USA).

Trolox, 2,4,6,-tri (2-pyridyl)-s-triazine (TPTZ), gallic acid, procyanidin B2 and myrtenol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid, acetic acid, methanol, sodium carbonate, iron chloride, ammonium acetate and Folin-Ciocalteu reagent were purchased from Kemika (Zagreb, Croatia). 2,2′-azinobis (3-ethylbenzothiozolinesulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Fluka (Buchs, Switzerland). Neocuproine and copper chloride were obtained from Gram-mol (Zagreb, Croatia). Sucrose was purchased from Gram-mol (Zagreb, Croatia), while trehalose was obtained from Hayashibara doo (Okayama, Japan). Citrus fibers were obtained from Biesterfeld AG (Zagreb, Croatia).

All chemicals to investigate the lipidemic properties, the antioxidant capacity, and the analytics of propolis were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) except ethanol, methanol, potassium acetate, potassium hydroxide, and sodium carbonate, which were purchased from Kemika (Zagreb, Croatia) or from Merck, Darmstadt, Germany (AlCl₃ and 2,4-dinitrophenylydrazine). Perkin Elmer Lambda 25 spectrophotometer (Perkin Elmer, Waltham, MA, USA) was used for the absorbance measurements.