Untargeted metabolomic approach to assess the acute metabolism and urinary excretion of olive leaf bioactive compounds in humans

Olive (Olea europaea) leaves are a valuable source of bioactive compounds with numerous associated health benefits. These beneficial effects may arise from metabolites generated in vivo rather than from the native constituents of the extract. However, the human metabolism of these compounds remains underexplored. This study profiles urinary metabolites of olive leaves in humans, demonstrating extensive metabolism and identifying key derivatives. Urine samples from seventeen healthy volunteers were collected pre- and post-supplementation with an olive leaf extract and analyzed using an untargeted metabolomics workflow based on HPLC-ESI-qTOF-MS. Over 18,500 molecular features were detected, which were subsequently filtered and examined using a combination of R-based data processing packages and both supervised and unsupervised statistical analyses. Results revealed 513 significant features related to olive leaf extract intake, 176 of which were successfully annotated. Key metabolites included 28 oleuropein conjugates (e.g., hydroxyoleuropein) and 48 compounds derived from oleuropein degradation and attributed to hydroxytyrosol and elenolic acid metabolism (e.g., homovanillic acid and oleoside), along with several flavonoids and phenolic acids. Both phase I and phase II modifications dominated, while intact parent compounds were scarcely detected, highlighting extensive biotransformation and rapid urinary excretion, predominantly within the first 6 h. Moreover, 19 metabolites overlapped with those previously identified in plasma from the same volunteers, underscoring coordinated systemic processing. These findings emphasize the power of untargeted metabolomics to uncover novel bioactive structures and metabolic pathways, and provide a basis for future research on the health potential of olive-derived metabolites, which remains limitedly studied.