*3.4. Characterization of the Optimum Walnut Leaf Extract*

Both the experimental values and those predicted by the models (Equations (2)–(5), Table 4) for the extraction yield and antioxidant properties of the walnut leaf extract selected as the optimum are presented in Table 2 (Experiment 12). Additionally, Figure 6a,b show the ABTS and DPPH radical inhibition percentages, respectively, versus extract concentration for this walnut extract and for Trolox, the synthetic antioxidant used as a reference. The corresponding EC50 values were 0.985 and 0.11 mg/mL, for ABTS and DDPH, respectively. The DPPH EC50 value obtained in this work was lower (higher antioxidant activity) than those found for extracts obtained from leaves of different *Juglans regia* cultivars using other solvent systems: Aqueous extracts (0.151–0.202 mg/mL) [1], methanolic extracts (0.199 mg/mL), and petroleum ether extract (2.921 mg/mL) [3]. However, even lower EC50 values were obtained for methanolic (65.91 μg/mL) and decoction (78.97 μg/mL) extracts from certain walnut cultivars [4]. The DPPH EC50 value obtained in this work for the optimum walnut leaf extract was also significantly lower than that for *Juglans regia* green husk extracts, 0.33 mg/mL for 50% aqueous ethanol extracts [23], and 0.41 for methanolic extracts [3], and also slightly lower than that for the methanolic extract from walnut seeds (0.14 mg/mL) [3]. With respect to extraction yield it was slightly higher than that reported by Carvalho et al. [3] for the methanolic extract, 27.7%, and significantly higher than that of the petroleum ether ones, 1.1%.

**Figure 6.** Percentage of ABTS (**a**) and DPPH (**b**) radical inhibition against extract concentration for the walnut leaf extract selected as the optimum with Trolox as reference.

Finally, to identify and quantify the compounds responsible for the antioxidant activity of the optimum *Juglans major 209 x Juglans regia* leaf extract, it was analyzed by UPLC/ESI-QTOF-MS. Table 5 and Figure 7 show the phenolic profile determined. Fifteen phenolic compounds were identified and quantified, with the flavonoid quercetin 3-β-D-glucoside being the major compound, representing 49.35% of the total phenolic composition, followed by chlorogenic acid (29.16%), and neochlorogenic acid (18.34%). Both quercetin 3-β-D-glucoside and chlorogenic acid were among the main phenolic compounds found in *Juglans regia* leaf extracts from some Spanish and Portuguese cultivars [1,4,7] that also presented significant quantities of other flavonoids such as quercetin 3-*O*-galactoside, quercetin *O*-pentoside, or quercetin *O*-xyloside [1,4,5,7] not identified in this work. However, the outstanding presence of neochlorogenic acid in the *Juglans major 209 x Juglans regia* leaf extracts, also reported by Pereira et al. [1], makes the main difference with the *Juglans regia* ones.

Concerning the interest of the main phenolic compounds identified, quercetin 3-β-D glucoside has been attracting increasing research interest. It is widely distributed in fruits, vegetables, and cereals and exhibits a broad number of chemoprotective effects both in vitro and in vivo, against oxidative stress, cancer, cardiovascular disorders, diabetes, and allergic reactions [24]. Chlorogenic acids are also present in many vegetables and fruits and some health benefits have been associated with them such as the prevention of cardiovascular diseases and types 2 diabetes [25] and also antioxidants, anticancer, anti-inflammatory, and immunomodulatory properties have been shown [26]. The importance of these phenolic compounds for various industrial applications demonstrate the interest of the valorization of *Juglans major 209 x Juglans regia* leaves.


**Table 5.** Identified compounds in the optimized walnut leaf extract by UPLC/ESI-QTOF-MS (*T* = 75 ◦C, *t* = 120 min, EtOH concentration = 50%, S/L ratio = 1/10 g/mL).

**Figure 7.** UPLC chromatogram of the walnut leaf extract selected as the optimum (*T* = 75 ◦C, *t* = 120 min, EtOH concentration = 50%, S/L ratio = 1/10 (g/mL).
