Valorization of Underutilized Radish Leaves Through Extraction-Method-Dependent Antioxidant, NO-Inhibitory Activity, and Metabolite Profiles

Radish leaves, often discarded during radish (Raphanus sativus var. longipinnatus) cultivation, are rich in bioactive compounds but underutilized compared to roots. Despite extensive research on radish roots, the functional benefits of radish leaves remain underexplored. This study evaluated the effects of different extraction methods on the antioxidant and nitric oxide (NO)-inhibitory activities of radish leaf extracts and characterized their metabolites using UPLC Q-Exactive Orbitrap MS/MS-based non-targeted profiling. Among the tested methods, Pectinex-assisted ethanol extraction (PAE) showed the highest total phenolic content, DPPH radical-scavenging activity, and TEAC values, reaching 17.91 ± 0.53 mg GAE/g dry weight (d.w.), 43.40 ± 0.78 μM TE/g d.w., and 57.19 ± 1.14 μM TE/g d.w., respectively. Ultrasound-assisted extraction (UAE) yielded the highest total flavonoid content at 6.17 ± 0.30 mg CE/g d.w. In LPS-stimulated RAW 264.7 cells, PAE most effectively reduced NO production, decreasing it from 17.76 ± 0.21 to 5.68 ± 0.32 μM. Metabolite profiling yielded putative annotations for 54 compounds, including flavonoids, flavonoid glycosides, phenolic acids, organic acids, and amino acids. These findings demonstrate that PAE and UAE enhance the recovery of bioactive metabolites from radish leaves, supporting their valorization as functional food ingredients and sustainable agricultural byproducts.