*2.3. Extraction*

Walnut leaves were extracted with aqueous ethanol in an orbital shaker (UNITRONIC-OR, Selecta (Barcelona, Spain) with temperature control at a shaking speed of 90 rpm. The solid/liquid ratio, temperature, time, and EtOH concentration were fixed for each experiment according to the established experimental planning (Tables 1 and 2). The extract was separated by vacuum filtration, concentrated in a Büchi R-210 rotavapor and finally dried under vacuum to obtain a dry powder. Extraction yield was determined as the weight loss percentage of the initial walnut leaves.

The choice of the independent variables to be analyzed and their respective variation intervals was based on previous investigations on the extraction of phenolic compounds from various lignocellulosic materials using aqueous alcohols [10–12]. In the first stage, the influence of the solid/liquid ratio (1/5, 1/7.5 and 1/10 g/mL) was analyzed for fixed values of the other variables: Temperature, 50 ◦C, time, 60 min, and ethanol concentration, 50% (Table 1). Extraction yield and extract ferric reducing antioxidant power (FRAP) antioxidant activity were determined. All the assays were replicated, and the results expressed as mean value and standard deviation. The existence of significant differences among the results depending on the solid/liquid ratio used was analyzed by applying one-way ANOVA together with the Tukey's test at a confidence level of 95% using the IBM SPSS Statistics 24 software (New York, NY, USA).

Once S/L ratio was fixed at 1/10, a Box-Behnken experimental design was applied to analyze the influence of temperature (*x*1; 25, 50, and 75 ◦C), time (*x*2; 30, 75, and 120 min) and aqueous ethanol concentration (*x*3; 10%, 50%, and 90%) on extraction yield (*Y*1, g extract/100 g leaves on dry basis (d.b.)) and extract antioxidant activity determined according to the FRAP (*Y*2, nmol AAE/mg extract d.b.), DPPH (*Y*3, mmol TRE/g extract d.b.), and ABTS (*Y*4, mmol TRE/g extract d.b.) assays. The phenolic profile of the extract selected as the optimum was analyzed by ultra-performance liquid chromatography coupled with electrospray ionization and time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS).

**Table 1.** Influence of the solid/liquid ratio on extraction yield and ferric reducing antioxidant power (FRAP) antioxidant activity of walnut leaf extracts (50 ◦C, 60 min, and 50% aqueous ethanol).


Values are presented as mean ± standard deviation. a–c In each column, values with different letters are significantly different (*p* < 0.05).


**Table 2.** Box-Benkhen experimental design with the experimental and predicted values of the responses.

Experimental values are presented as mean ± standard deviation. *Y*1, extraction yield (g extract/100 g leaves d.b.); antioxidant activity: *Y*2, FRAP (nmol AAE/mg extract d.b.); *Y*3, DPPH (mmol TRE/g extract d.b.); *Y*4, ABTS (mmol TRE/g extract d.b.); *x*∗ <sup>1</sup>, codified temperature; *x*<sup>∗</sup> <sup>2</sup>, codified time; *x*<sup>∗</sup> <sup>3</sup>, codified ethanol concentration.
