Olive Leaf Extract Characterization

The olive leaves (cv. Coratina) were picked off the trees in an olive grove in the province of Bari (40°51'16.2"N 16°47'23.6"E), stored at 4°C and processed in less than 24 h. After washing with tap water at room temperature, the olive leaves were dried at 120°C for 8 min in a ventilated oven (Argolab, Carpi, Italy) to reach a moisture content <1% and then milled in a blender (Waring-Commercial, Torrington, CT, USA). The extraction from leaves was performed in an ultrasound bath (CEIA, Viciomaggio, Italy) adding water (Eth-0) or hydroalcoholic solutions to30% (Eth-30) and 70% ethanol (Eth-70) in a 1/20 ratio (w/v). Three washes were done, each one for 30 min at 35±5°C. Finally, the extracts were filtered through Whatman (GE Healthcare, Milan, Italy) filter paper (67 g m^-2), lyophilized and stored at -20°C. The total phenol content, the antioxidant activity and the identification of single phenolic compounds were performed according to Difonzo et al. [16 ]. The Eth-0 OLE showed a content of polyphenols, determined by Folin-Ciocalteu, equal to 180 mg g^-1 gallic acid equivalents (GAE) and an antioxidant activity, determined by ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt), accounting for 750 μmol TE (Trolox equivalents) g^-1. The main phenolic compounds found in OLE were oleuropein, hydroxytyrosol glucoside, luteolin-glucosides, verbascoside, ligstroside, secologanoside and other minor compounds, detected by UHPLC-ESI-MS/MS as described in Difonzo et al. [16 ]. Cadmium detection was carried out by means of inductively coupled plasma–atomic emission spectrometry (ICP-OES) (IKAP 6500, Thermo Scientific, USA). The detailed ICP-OES analytical conditions were the following: from room temperature to 190°C in 15 minutes followed by 10 min hold at the same temperature. For this purpose, an aliquot of 250 mg of olive leaf extract was digested with 8 mL HNO₃ (69.0%) and 1 mL H₂O₂ (30%) using a microwave digestion system (Discovery-SP, CEM, USA).