O phosphoric acid

All experiments have been performed using analytical-grade chemicals. Folin-Ciocalteu solution, gallic acid, sodium carbonate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid 114 (Trolox), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and all HPLC Standards of gallic acid (3,4,5-Trihydroxybenzoic acid), p-Hydroxybenzoic acid, (+)-catechin hydrate, protocatechuic, rutin hydrate, quercetin (3,3’,4’,5,7-Pentahydroxyflavone), chlorogenic acid, p-coumaric acid, caffeic acid (3,4-Dihydroxycinnamic acid), trans-cinnamic acid, ellagic acid, resveratrol, and sinapic acid (3,5-Dimethoxy-4-hydroxycinnamic acid) were obtained from Sigma-Aldrich (Darmstadt, Germany). All assay kits for FRAP, CUPRAC, and proanthocyanin were purchased from Bioquochem (Asturias, Spain). As for the solvents, aqua and O-Phosphoric Acid 85% extra pure were provided by Fisher Scientific (Janssen Pharmaceuticalaan, Belgium), and Acetonitrile was provided by LiChrosolv^® (Darmstadt, Germany). Polyoxyethylene-20 (Tween 20) was obtained from Biotech (Markham, ON, Canada). A basic cream formula and two commercial exfoliating scrubs were purchased from a local store (Beirut, Lebanon) and used as positive controls, one containing 5% sugar and the other containing 5% raspberry seed powder and Argania spinosa shell powder.

Sodium arsenite, sodium arsenate dibasic heptahydrate (≥98%), methanol (Optima^TM for HPLC), tert-butanol (t-BuOH), acetonitrile (HPLC grade), o-phosphoric acid (85%, ACS grade), formic acid (88%), ammonium hydroxide (29.2%), and ferric chloride hexahydrate (FeCl₃·6H₂O, 98.8%) were purchased from Fisher Scientific (Waltham, MA, USA). Ferrous chloride tetrahydrate (FeCl₂·4H₂O, ≥99%), humic acid sodium salt (Lot#STBC5468V), coumarin (COU) (≥99%), potassium sorbate (≥99%), and 7-hydroxycoumarin (7-HC) (99%) were obtained from Sigma Aldrich (St. Louis, MO, USA). Furfuryl alcohol (FFA) and the tetrabutylammonium hydroxide (TBAH) (40 wt.%) were purchased from Acros Organic (Geel, Antwerp, Belgium). The 2,4,6-trimethylphenol (98%), was obtained from TCI (Portland, OR, USA). The malonic acid (reagent grade, 99.5%) was purchased from Alfa Aesar (Haverhill, MA, USA). Millipore water (MilliQ water, resistivity~18.0 MΩ cm⁻¹ at 25 °C) was used for sample and standard preparation unless otherwise indicated.

Standards for gas chromatography (oleic acid, ≥ 99%), high performance liquid chromatography (nonadecanoic acid methyl ester, 99%), and thin layer chromatography (glyceryl trioleate, ≥ 99%; dioleoylglycerol, ≥ 99%; 1-oleoyl-rac-glycerol, ≥ 99%; and oleic acid, ≥ 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). For gas chromatography, derivatization of all fatty acids (both esterified and free fatty acids) was achieved using acetyl chloride (≥ 99%, Sigma-Aldrich (St. Louis, MO, USA)), while derivitiation of free fatty acids alone was accomplished using diazomethane prepared from diazald (TLC Pharmaceutical Standards Ltd., Aurora, ON, Canada) and a Diazald® kit from Sigma-Aldrich (St. Louis, MO, USA). For thin layer chromatography, iodine (≥ 99.99%) and diethyl ether (≥ 98%) were also purchased from Sigma-Aldrich (St. Louis, MO, USA). The solvents used for analysis, methanol (HPLC grade, > 99.9%), toluene (HPLC grade, > 99.9%), hexane (HPLC grade, > 99.9%), acetic acid (> 99.85%), along with the o-phosphoric acid (85%) used for acidification, were all obtained from Fisher Scientific (Fairlawn, NJ, USA). Nitrogen gas (99.998%) was obtained from Praxair (Mississauga, ON, Canada).

To confirm the increase in level of total Aβ protein expression in injected eyes, retinal tissues were collected from each experimental group 8 week after subretinal injection and ELISA assays were performed. The retinal tissues were homogenized in Diethyl Amine (DEA) buffer using a hand held disposable micro tissue grinder (Pellet Pestle, Fisher Scientific, Waltham, MA). The DEA buffer contained 0.2% DEA in 50 mM NaCl solution and Complete Protease Inhibitor (Roche Applied Science, Indianapolis, IN). Homogenized tissue was centrifuged at 100,000 g for 30 minutes and supernatant collected. The supernatant was neutralized with 10% (v/v) 0.5 M Tris-HCl (pH = 6.8) immediately. Total Aβ levels were determined from the neutralized samples using a sandwich ELISA strategy: Aβ capture with mAb5 (human Aβ 1–5, T.E. Golde) and detection with HRP-conjugated 4G8 (human Aβ17–24; Covance, Princeton, NJ). The colorimetric reaction was developed with 3,3′,5,5′-Tetramethylbenzidine substrate and halted with 6.7% o-Phosphoric Acid (Fisher Scientific, Waltham, MA). The plates were analyzed using Molecular Devices spectrometer and Softmax Pro 6 software (Molecular Devices, Sunnyvale, CA). All measurements were performed in duplicate and the data represents the mean of at least two assay results.

4-chloroPhenol (C₆H₅ClO, 99+ %) and palladium catalyst (Pd/Al₂O₃, 1% Pd on alumina powder, with a specific surface area of 150 m²g⁻¹) were purchased from Acros and Alfa Aesar, respectively. Phenol (C₆H₅OH, 89.6%), sodium sulfate anhydrous (Na₂SO₄, 99%), sulfuric acid (H₂SO₄, 98%), sodium bicarbonate (NaHCO₃, 99–100%), acetonitrile (99.8+ %), sodium hydroxide (NaOH, 96%), benzoic acid (C₇H₆O₂, 99.9+%), 4-hydroxylbenzoic acid (C₇H₆O3, 99.9+%), o- Phosphoric acid (H₃PO₄, 85%), and Acetic Acid (Glacial, HPLC grade) were acquired from Fisher Scientific. Ferrous sulfate (FeSO₄.7H₂O, pro analysis) was obtained from J.T. Baker Analyzed. palladium catalyst (1% on carbon 4 to 8 mesh), Potassium-hydrogen phthalate (C₈H₅KO₄), HPLC grade water, and methanol were bought from Sigma-Aldrich. The syringe filters with 0.22 μm and 0.45 μm pore sizes were purchased from Millex. Titanium sulfate (TiSO₄, 65%) was obtained from GFS Chemicals. All solutions were prepared in de-ionized water (18.2 mΩ.cm), obtained from a Millipore Milli-Q system.