Optimization of the Extraction of Bioactive Compounds from Walnut (Juglans major 209 x Juglans regia) Leaves: Antioxidant Capacity and Phenolic Profile

This work studies the extraction of phenolic compounds from walnut leaves of the hybrid Juglans major 209 x Juglans regia based on extract antioxidant capacity. Once the solid/liquid ratio was selected (1/10 g/mL), by means of a Box-Benkhen experimental design, the influence of temperature (25-75 °C), time (30-120 min), and aqueous ethanol concentration (10-90%) on extraction yield and ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) antioxidant activities were analyzed. In all cases, the quadratic effect of % EtOH was the most significant, followed by the linear effect of temperature and, for most of the responses, the effect of time was almost negligible. Response surface analysis allowed to select the optimal extraction conditions: 75 °C, 120 min and 50% ethanol, which led to the following extract properties: extraction yield, 30.17%; FRAP, 1468 nmol ascorbic acid equivalents (AAE)/mg extract d.b.; DPPH, 1.318 mmol Trolox equivalents (TRE)/g extract d.b.; DPPH EC₅₀, 0.11 mg/mL; ABTS, 1.256 mmol TRE/g extract (on dry basis) and ABTS EC₅₀, 0.985 mg/mL. Quercetin 3-β-D-glucoside, neochlorogenic acid, and chlorogenic acid, in this order, were the main compounds identified in this extract by ultra-performance liquid chromatography coupled with electrospray ionization and time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS), with various potential applications that support this valorization alternative for walnut leaves.