Phosphoric acid

For an estimation of the brain tissue monoamine levels and NC activity assays, the brains of rats were harvested and brain areas were dissected bilaterally: ventral striatum (VS), dorsal striatum (DS), dorsal hippocampus (DH), ventral mesencephalon (VM), and hypothalamus (Hyp). Tissue of one hemisphere was used for monoamine analysis. The tissue was homogenized in 0.5-M perchloric acid, centrifuged, filtered, and stored at − 80 °C until further analysis. All samples were analyzed using HPLC with electrochemical detection to measure dopamine (DA), serotonin (5-HT) and norepinephrine (NA) levels. The column was an ET 125/2, Nucleosil 120-5, C-18 reversed-phase column (Macherey & Nagel). The mobile phase consisted of 75-mM NaH2PO4, 4-mM KCl, 20-µM EDTA, 1.5-mM sodium dodecylsulfate, 100-µl/L diethylamine, 12% alcohol and 12% acetonitrile adjusted to pH 6.0 using phosphoric acid (Carl Roth GmbH). The electrochemical detector (Intro) was set at 500 mV vs. an ISAAC reference electrode (Antec) at 30 °C. This setup allows simultaneous measurement of DA, 5-HT, and NA [32 (link), 33 (link)].

HT, Ole, HVA, and DOPAC (all purity ≥ 98%) were from Cayman Chemical (Ann Arbor, MI, USA). Pure HT-3-S (98%) was supplied by ChemCruz (Huissen, The Netherlands); Tyr (purity ≥ 99.5%) was from Sigma-Aldrich (Taufkirchen, Germany). HT-3-G (purity ≥ 97%) was from Biozol (Eching, Germany); citric acid, phosphoric acid, and L(+)-ascorbic acid were from Roth (Karlsruhe, Germany). Water, acetonitrile, methanol, acetic acid, and formic acid (all LC-MS-grade) were purchased from VWR Chemicals (Darmstadt, Germany).
The liquid food supplements Oliphenolia bitter and Oliphenolia, and the EVOO were provided by Fattoria La Vialla S.A.S (Castiglion Fibocchi, Arezzo, Italy). HTEssence Hydroxytyrosol Liquid was kindly provided by Wacker Chemie AG (Munich, Germany).

Lipid extracts were prepared according to the protocol published by Bligh and Dyer [35 (link)]. Alkaline methanolysis of glycerolipids was performed as described by Bodennec et al. [36 (link)]. Lipids were dried in a SpeedVac vacuum concentrator, dissolved in chloroform/methanol (1:1), and loaded onto HPTLC plates. Lipid standards were loaded onto adjacent lanes of the same HPTLC plate. Chromatograms were developed in the solvent mixture chloroform/methanol/water (70:30:4; v/v/v) or twice in the solvent mixture chloroform/methanol/water (144:25:2.8; v/v/v) using a horizontal developing chamber (CAMAG, Muttenz, Switzerland). Lipids were visualized by spraying plates with 625 mM cupric sulfate (cat# C1297, Sigma-Aldrich) in 8% (v/v) phosphoric acid (cat# 9079, Carl Roth, Karlsruhe, Germany) followed by heating to 180 °C for 5 min [37 (link)]. Immediately after stained plates had been cooled to room temperature, they were scanned using a flatbed scanner (Epson Perfection V700 Photo) and the intensity of lipid bands was quantified using the program AIDA (Elysia-Raytest, Straubenhardt, Germany).

Acetonitrile, chloroform, methanol, acetic acid, phosphoric acid, and hydrochloric acid were purchased from Carl Roth GmbH & Co KG (Karlsruhe, Germany). Ammonium acetate, copper sulfate, and potassium chloride were purchased from Sigma-Aldrich GmbH (Munich, Germany). The standard substances SQDG 816 (2-O-hexadecanoyl-1-O-(9Z,12Z,15Z-octadecatrienoyl)glycerol 3-(6-deoxy-6-sulfo-α-d-glucopyranoside)) and 3-O-sulfo-d-galactosyl-β1-1′-N-heptadecanoyl-d-erythro-sphingosine as internal standard (ISD) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). All aqueous solutions were prepared with deionized water, generated by a Purelab flex water purification system (Veolia Water Technologies Deutschland GmbH, Celle, Germany). NH₂ cartridges (6 mL, 500 mg) were purchased from Macherey-Nagel GmbH & Co. KG (Düren, Germany).

All solvents were at least HPLC grade. Water, 2-propanol, and phosphoric acid were purchased from Roth (Karlsruhe, GER), methanol from J.T.Baker (Austin, TX, USA), formic acid, TAG 45:0 and TAG 51:0 from Sigma (Vienna, AUT), and ammonium acetate from Merck (Darmstadt, GER). Glass beads (0.45–0.50 mm) were from SiLi (Warmensteinach, GER).