Spinacia oleracea L. Baby Leaves as a Source of Bioactive Principles: The Chemical Profiling of Eco-Sustainable Extracts by Using LC-ESI/HRMS- and 1H NMR-Based Metabolomics

<i>Spinacia oleracea</i> L. cultivar platypus leaves are identified as a functional food due to their nutrient composition which promotes health beyond basic nutrition. Considering the increasing use of food supplements, <i>S. oleracea</i> baby leaves have been extracted by maceration, solid-liquid dynamic extraction (SLDE)-Naviglio, and ultrasound-assisted extraction (UAE) using EtOH and EtOH:H₂O mixtures. The analysis of the extracts by using LC-ESI/HRMSMS revealed 42 compounds (flavonoids, polar lipid derivatives, and 20-hydroxyecdysone), along with primary metabolites, detected by NMR analysis. A principal component analysis (PCA) of LC-ESI/HRMS and NMR data was performed, revealing how 20-hydroxyecdysone and flavonoids, the specialized metabolites mainly responsible for the biological activity of <i>S. oleracea</i> leaves, occurred in the highest amount in the EtOH and EtOH:H₂O (70:30, <i>v</i>/<i>v</i>) extracts obtained by SLDE-Naviglio extraction. 20-hydroxyecdysone was also quantified in all the extracts via LC-ESI/QTrap/MS/MS using the Multiple Reaction Monitoring (MRM) method. The EtOH extracts obtained by SLDE-Naviglio and maceration showed the highest content (82.16 and 81.27 mg/g extract, respectively). The total phenolic content (118.35-206.60 mg GAE/g), the flavonoid content (10.90-41.05 mg rutin/g), and the Trolox Equivalent Antioxidant Capacity (TEAC) (1.63-2.05 mM) of the extracts were determined. The EtOH:H₂O (70:30, <i>v</i>/<i>v</i>) extract analyzed by using SLDE-Naviglio showed the highest phenolic and flavonoid content and radical scavenging activity.