Olive solid wastes: UHPLC-MS/MS-based biochemometric approach for investigating the effect of conventional versus modern extraction methods on in vitro antioxidant, α-glucosidase, and lipase actions

The olive oil industry produces huge amounts of solid by-products (e.g., pomace, leaf, and stone), representing a rich source of polyphenols. Different extraction techniques were compared, including conventional (e.g., maceration) versus modern (e.g., microwave-, ultrasound-, and enzyme-assisted extraction), to yield a total of 17 extracts to include 2, 7, and 8 leaf, pomace, and stone extracts, respectively. Ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) combined with unsupervised and supervised modeling were employed for assessing extracts’ heterogeneity and markers identification, respectively. Verbascoside and γ-tocopherol were potentially associated with microwave- (MAE) and supercritical fluid CO₂-assisted (SFE) extracts of olive pomace, respectively. Besides, hydroxylated product of the decarboxylated form of hydroxy elenolic acid (HDHEA), hydroxytyrosol, and verbascoside were correlated with antioxidant activity based on partial least squares regression (PLS) (R² = 0.8) for pomace extracts, whereas verbascoside appeared as the strongest antioxidant marker in stone extracts. Furthermore, total phenolics (TPC) and flavonoids (TFC) were determined showing that MAE-prepared extracts exhibited the highest TPC at 72.0 and 53.6 mg GAE.g⁻¹ dw from aqueous extracts of pomace and stone, respectively. This study identifies MAE, particularly aqueous extracts, as the most effective method for obtaining phenolic and antioxidant-rich extracts from olive by-products. The MAE not only maximizes the yield of beneficial compounds but also enhances their bioactivity. Future research should focus on conducting both in vivo assays, which shall provide deeper insights into the potential applications of olive by-products and validate the efficacy of the extraction methods employed.