*3.3. Optimization of the Extraction Conditions by a Box-Behnken Design*

In order to determine the best extraction conditions for the extraction of phenolic compounds from walnut leaves, a Box-Behnken experimental design was applied. The independent variables selected were temperature, time, and ethanol concentration. The extraction conditions together with the experimental results obtained for the dependent variables: Extraction yield (*Y*1), FRAP (*Y*2), DPPH (*Y*3), and ABTS (*Y*4) antioxidant activities are shown in Table 2, that also shows the good agreement between experimental and predicted values. The fitting model (Equation (1)) was found appropriate to represent all the responses analyzed (*p* < 0.05). Table 4 shows the significant regression coefficients together with the statistical parameters used to evaluate the fitting results. For all the responses, the quadratic effect of %EtOH (in negative mode) was the most significant, followed by the linear effect of temperature (in positive mode), and the effects on which extraction time was involved were the least significant or not significant at all. Equations (2)–(5) show the dependence of the responses *Y*<sup>1</sup> to *Y*<sup>4</sup> on the significant independent variables, double interactions, and quadratic effects:

$$Y\_1\left(\%\right) = 26.28 + 3.024\,\mathrm{x}\_1^\* + 0.905\,\mathrm{x}\_2^\* - 1.444\,\mathrm{x}\_3^\* - 0.865\,\mathrm{x}\_1^\*\mathrm{x}\_3^\* - 3.973\,\mathrm{x}\_3^{\*2}\tag{2}$$

$$\text{Y } \text{ $Y\_2$  (mmol AAE/mg extract d.b.) } = 1395.25 \text{ + } 13.25 \text{ x}\_1^\*-354.50 \text{ x}\_3^{\*2} \tag{3}$$

$$\text{Y}\_3 \text{ (mmol TRE/g extract d.b.)} = 1.223 \times 0.173 \,\text{x}\_1^\* - 0.093 \,\text{x}\_1^\* \text{x}\_3^\* - 0.291 \,\text{x}\_3^{\*2} \tag{4}$$

$$\text{Y}\_4 \text{ (mmol TRE/g extract d.b.) } = 1.315 \text{ + } 0.143 \text{ x}\_1^\* - 0.083 \text{ x}\_1^{\*2} - 0.125 \text{ x}\_2^{\*2} - 0.315 \text{ x}\_3^{\*2} \tag{5}$$

Figure 2a–c show the response surfaces for the extraction yield (*Y*1) in the function of temperature and ethanol concentration for extraction times of 30 (a), 75 (b), and 120 min (c). Extraction yield varied between 17.80% and 30.55% depending on the extraction conditions. Regarding the influence of the linear effects, temperature and time were significant in the positive mode, whereas %EtOH in the negative mode. Only the quadratic effect of %EtOH and the double interaction temperature *x* %EtOH had significant influences on the response, both in the negative mode. The highest extraction yield in the ranges essayed of 30.55% was attained at the highest temperature and extraction time essayed, 75 ◦C and 120 min, and at an intermediate ethanol concentration, 38%.

Figures 3–5 show, respectively, the response surfaces for FRAP (*Y*2), DPPH (*Y*3), and ABTS (*Y*4) antioxidant activities in function of temperature and ethanol concentration. All show a similar trend with respect to the influence of temperature and %EtOH, showing the presence of a maximum located in the vicinity of the design space for FRAP and DPPH and in the case of ABTS within it. Additionally, the responses for FRAP and DPPH were independent of time. FRAP ranged between 908 and 1529 nmol AAE/mg extract d.b. (Figure 3) and depended only on the negative quadratic effect of % EtOH and the linear effect of temperature in positive mode, reaching the highest value in the range assayed, at 75 ◦C and 50% ethanol. With respect to DPPH, it depended additionally on the interaction temperature *x* %EtOH in the negative mode, and a value of 1.40 mmol TRE/g extract d.b. was reached at 75 ◦C and 44% ethanol (Figure 4). ABTS depended on the quadratic effects of %EtOH, time and temperature, in this order of importance, all in the negative mode, and on the positive linear effect of temperature. For ABTS, a maximum located in the region studied, 1.38 mmol TRE/g extract d.b., was reached at 71.3 ◦C, 75 min, and 50% ethanol (Figure 5).

In summary, solvent concentration, temperature, and extraction time, in this order, are important factors influencing the extraction of phenolic compounds from walnut leaves. Both extraction yield and extract antioxidant activity depended significantly on the % ethanol used and aqueous solutions in the range of 38% to 50% ethanol were found to be superior to the more concentrated ones in any of the solvents, behavior that might be explained by the "likes dissolves like" principle [19]. High ethanol concentrations dissolve more lipophilic compounds, whereas higher proportions of hydrophilic compounds are extracted at low ethanol concentrations [20]. Otherwise, extraction yield increases when decreasing %EtOH up to 38%, whereas extract antioxidant capacity diminishes, which can be explained due to the solubilization of other types of compounds such as proteins and polysaccharides that impair selectivity [21]. According to these results, the ethanol concentration selected as the optimum was 50%. Concerning extraction temperature, a temperature rise of up to 75 ◦C increased the extraction yield as the extraction process was favored by temperature as the solubility of phenolic compounds and the mass transfer rate were enhanced [22]. Moreover, FRAP, DPPH, and ABTS antioxidant activity of the extracts also increased with increasing extraction temperature to 75 ◦C. However, greater extraction temperatures were not recommended as oxidation, epimerization, and degradation of phenolic compounds was promoted [17] with the consequent decrease of the extract antioxidant activity. Therefore, 75 ◦C was selected as the optimum temperature. Finally, 120 min was the selected time to favor the extraction yield, as its influence on antioxidant properties was

almost negligible. Longer extraction times were not proposed since the effects were the same as those previously indicated for high temperatures [17].

In brief, the optimal conditions for the extraction of phenolic compounds from *Juglans major 209 x Juglans regia* leaves selected by analyzing together the response surfaces for all the dependent variables (Figures 2–5) were those corresponding to Experiment 12 (Table 2): 75 ◦C, 120 min, and 50% ethanol. Under these conditions, the predicted responses were: *Y*1, pred = 30.21%, *Y*2, pred = 1529 nmol AAE/mg extract d.b., *Y*3, pred = 1.396 mmol TRE/g extract d.b. and *Y*4, pred = 1.25 mmol TRE/g extract d.b. These values were equal or very close to those corresponding to the model optimum for each response that were *Y*1,pred = 30.55% at 75 ◦C, 120 min, and 38% EtOH, *Y*2,pred = 1529 nmol AAE/mg extract d.b. at 75 ◦C and 50% EtOH, *Y*3,pred = 1.40 mmol TRE/g extract d.b. at 75 ◦C and 44% EtOH and *Y*4,pred = 1.38 mmol TRE/g extract d.b. at 71.3 ◦C, 75 min and 50% EtOH. Moreover, these extraction conditions were close to those found by Vieira et al. [7] as the global optimum conditions for the extraction of some of the main phenolic compounds found in this work, namely, 3-*O*-caffeoylquinic acid and quercetin 3-*O*-glucoside, from walnut (*Juglans regia*) leaves by maceration with aqueous ethanol (61.3 ◦C, 112.5 min, and 50.4% ethanol). 50% ethanol also led to higher antioxidant activities for walnut (*Juglans regia*) green husk extracts than pure water and ethanol, although the highest extraction yield was obtained with water [23].


**Table 4.** Coefficients of the models (Equation (1)) and statistical parameters.

NS: Non-significant for a 95% confidence level; SE: Standard error; *p*: Probability; *R*2: Regression coefficient; *R*<sup>2</sup> c: Corrected regression coefficient; *a*0: Scaling constant; *a*i: Linear coefficients; *a*ij: Interaction coefficients; *a*ii: Quadratic coefficients; *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.).

**Figure 2.** Response surfaces for extraction yield (*Y*1, %) in function of temperature and ethanol concentration for extraction times of (**a**) 30, (**b**) 75, and (**c**) 120 min.

**Figure 3.** Response surfaces for FRAP antioxidant activity (*Y*2, nmol AAE/mg extract d.b.) in the function of temperature and ethanol concentration.

**Figure 4.** Response surfaces for DPPH antioxidant activity (*Y*3, mmol TRE/g extract d.b.) in the function of temperature and ethanol concentration.

**Figure 5.** Response surfaces for ABTS antioxidant activity (*Y*4, mmol TRE/g extract d.b.) in the function of temperature and ethanol concentration for extraction times of (**a**) 30, (**b**) 75, and (**c**) 120 min.
