2.3.8. Determination of Individual Phenolic Compounds

The phenolic acids (rosmarinic acid, *p*-coumaric acid, chlorogenic acid, vanillic acid, caffeic acid, *p*-hydroxybenzoic acid, protocatechiuc acid, gallic acid) and flavonoids (kaempferol, quercetin, luteolin and myricetin) were determined from the methanolic extract using a high performance liquid chromatograph Shimadzu (Kyoto, Japan) with diode array detector. The separation was carried out on a Zorbax SP-C18 column, with 150 mm length, 4.6 mm i.d. 5 μm-diameter particle was used for the separation [35]. The separation of the compounds were realized on a system with 0.1% acetic acid (mobile phase A) and acetonitrile (mobile phase B) based on the elution range described by Palacios et al. [36] as: min 0—A 100%, min 6.66—B 5%, min 66.66—B 40% and min 74—B 80%. The flow rate was set at 1 mL·min−1, and the injection volume was 10 <sup>μ</sup>L. Gallic acid, vanillic acid, protocatechuic acid and *p*-hydroxybenzoic acid were determined at 280 nm, and chlorogenic acid, *p*-coumaric acid, caffeic acid, rosmarinic acid, myricetin, quercetin, luteolin and kaempherol were determined at 320 nm, respectively. The phenolics compounds were expressed as mg/kg dry mater. All the measurements were carried out in triplicate.
