Abstract
Olive leaves are rich in phenolic compounds. This study explored the chemical profiles and contents of free phenolics (FPs) and bound phenolics (BPs) in olive leaves, and further investigated and compared the antioxidant properties of FPs and BPs using chemical assays, cellular antioxidant evaluation systems, and in vivo mouse models. The results showed that FPs and BPs have different phenolic profiles; 24 free and 14 bound phenolics were identified in FPs and BPs, respectively. Higher levels of phenolic acid (i.e., sinapinic acid, 4-coumaric acid, ferulic acid, and caffeic acid) and hydroxytyrosol were detected in the BPs, while flavonoids, triterpenoid acids, and iridoids were more concentrated in the free form. FPs showed a significantly higher total flavonoid content (TFC), total phenolic content (TPC), and chemical antioxidant properties than those of BPs (p < 0.05). Within the range of doses (20-250 μg/mL), both FPs and BPs protected HepG2 cells from H2O2-induced oxidative stress injury, and there was no significant difference in cellular antioxidant activity between FPs and BPs. The in vivo experiments suggested that FP and BP treatment inhibited malondialdehyde (MDA) levels in a D-galactose-induced oxidation model in mice, and significantly increased antioxidant enzyme activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and the total antioxidant capacity (T-AOC). Mechanistically, FPs and BPs exert their antioxidant activity in distinct ways; FPs ameliorated D-galactose-induced oxidative stress injury partly via the activation of nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway, while the BP mechanisms need further study.