The polyphenolic fraction present in olive leaf was extracted using methanol. Particularly, 10 mg of olive leaf powder were added to 3.0 mL of methanol, then mixed for 20 min through sonication, and lastly the supernatant was separated by centrifugation at 4700 g. The methanolic extract was filtered at 0.45 μm and was diluted 1:10 with methanol.

HPLC/ESI–MS analyses of olive leaf extracts were carried out in negative mode by a MicroTOF-Q II mass spectrometer (Bruker Daltonics, Macerata, Italy) in the range 50–1000 m/z, equipped with an ESI ion source with nitrogen as nebulizing gas (4 atm) and drying gas (10 L/min, 180 °C); capillary voltage at 3500 V and end plate offset at − 500 V.

Mass accuracy was verified by infusing a solution of Na-formate made up of 10 mL of 98% formic acid, 10 μL of sodium hydroxide (1.0 M), 490 μL of i-propanol and 490 μL of deionized water. Extracts were introduced (20 μL) in the HPLC (Agilent 1200 Series), using an autosampler in a RP column Synergi 4 µm Fusion-RP 80 Å, 100 mm × 3.0 mm. Flow rate was set to 0.5 mL/min using deionized water (resistivity: 18 MΩ cm, eluent A) and acetonitrile (LC/MS grade, eluent B) with 0.1% of formic acid, in gradient: 1% of B in the first minute and shifting to 100% B within the following 15 min. The composition was held 2 min, returning to 1% B in 2 min and keeping this condition for additional 3 min to achieve the column stabilization before next run (total run time was 18 min). The raw data were collected as continuum mass spectra at regular time interval (spectra rate of 1 spectrum/s); mass spectra were processed using Data Analysis 4.0 (Bruker Daltonik GmbH, Bremen, Germany).

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