Phytochemical Profiling of Extracts

The phytochemical profile of EE and HGE was elucidated by LC-DAD-ESI-MS analysis. Separation was carried out by a Luna Omega PS C18 column (150 mm × 2.1 mm, 5 µm; Phenomenex, Torrance, CA, USA) at 25 °C by using 0.1% formic acid (Solvent A) and acetonitrile (Solvent B) as a mobile phase according to the elution program reported in Danna et al. [71 (link)]. Five microliters of each extract were injected, and the UV–Vis spectra of analytes were recorded in the range of 190 to 600 nm. Chromatograms were acquired at 260, 292, 330, 370, and 520 nm, to detect all polyphenol classes, whereas the ion trap (model 6320, Agilent Technologies, Santa Clara, CA, USA) was carried out in full-scan mode (90–1000 m/z) following both positive and negative electrospray ionizaton (ESI) according to Danna et al. [71 (link)]. Identification was carried out by comparing the retention times and UV–Vis and MS spectra of each analyte with those of commercially available HPLC-grade standards (see Table 2), as well as with literature data and UV–Vis and mass spectra databases. Quantification was carried out by using external calibration curves of the authentic reference standard, when commercially available, or structurally similar compounds (see Table 2 footnotes). Results were expressed as mg of each compound/100 mL of LE ± standard deviation of three independent analyses in triplicate (n = 3).