Abstract
Background/Objectives: The consumption of olive oil and olives has been steadily increasing, leading to growing interest in the sustainable management of by-products such as olive stones. This work aimed to contribute to the development of valorization strategies by studying the chemical composition and biological potential of olive stone extracts, using both conventional and eco-friendly extraction methods with various solvents. Methods: Several extracts were prepared and chemically characterized regarding their fatty acid and phenolic profiles by GC-FID and HPLC-DAD-MS/MS, respectively. Their antioxidant, cytotoxic and antiproliferative activities were also evaluated. Results: n-Hexane Soxhlet extract yielded higher concentrations and a broader range of fatty acids when compared to the chloroform-methanol Folch extract. Oleic, palmitic, and linoleic acids were the predominant fatty acids in the extracts. A large variety of phenolic compounds were identified in extracts obtained by microwave-assisted extraction (MAE), including several flavonoids, small phenolic compounds, secoiridoids (nuezhenide and oleuropein), and hydroxytyrosol. MAE hydroalcoholic extracts showed high total phenolic content (TPC), antioxidant activity by the oxygen radical absorption capacity (ORAC) and hydroxyl radical scavenging capacity (HOSC) assays. Moreover, the aqueous ethanol (50%) and aqueous methanol (80%) extracts displayed low cytotoxicity toward the non-malignant Caco-2 cell line (IC(50) values of 1.29 and 1.40 mg/mL, respectively), while both induced complete loss of viability in the HT-29 human colon adenocarcinoma cell line at 0.63 mg/mL. Conclusions: These findings highlight the potential of olive stone as a valuable source of bioactive compounds with antioxidant and selective antiproliferative properties. The results support their further exploration in the development of sustainable valorization strategies for olive industry by-products.