Metabolomic Mapping of Greek Olive Leaves by Untargeted NMR-Based Profiling and LC-HRMS Dereplication

Olive tree leaves are phytochemically rich yet insufficiently valorised in terms of systematic cultivar-based characterisation and exploitation, and are an abundant olive industry side-product. This study represents the first large-scale metabolome mapping based on 340 leaf extracts from five major Greek cultivars (Amfissis, Koroneiki, Manaki, Lianolia-Kerkyra and Thasou) collected across diverse regions, agronomic practices, harvest periods and years. An integrated analytical workflow combining untargeted ¹H NMR profiling and LC-HRMS dereplication was applied for the first time at this scale. NMR provided a quantitative and highly reproducible overview of the core metabolome and served as the basis for multivariate statistical analysis, while HRMS enabled confident structural annotation of 62 metabolites, including secoiridoids, flavonoids, phenolic acids, triterpenoids and fatty acids. Multivariate data analysis (PCA and OPLS-DA) revealed that cultivar identity constitutes the dominant driver of metabolic variation, exceeding the influence of environmental, agronomic and temporal factors. Pentacyclic triterpenic acids, that are, oleanolic and maslinic acids, emerged as robust, genotype-dependent chemotaxonomic markers, outperforming the widely used oleuropein, whose abundance is highly sensitive to environmental and agronomic factors. Flavonoids (apigenin, luteolin, quercetin) and the sugar alcohol mannitol displayed cultivar-specific patterns consistent with differential stress adaptation strategies rather than strict taxonomic control. Amfissis was characterised by the highest and most stable triterpenoid levels, Koroneiki/Thasou by elevated flavonoid levels, while Koroneiki/Manaki showed the highest relative concentration of oleuropein and mannitol. Overall, this integrated NMR/LC-HRMS approach establishes a robust framework for olive leaf cultivar authentication and supports genotype-driven valorisation of this agricultural side-product, enabling the development of standardised, cultivar-specific extracts for phytotherapeutic, nutraceutical and cosmetic applications.