## 3 Results and Discussion  
### Determination of Phenolic and Other Compounds in Olive Leaf Samples by HPLC-ESI-TOF-MS  
A total of 41 compounds were identified in MAE olive leaf extracts, which were identified by considering their mass spectra as determined via TOF-MS and considering the data reported in the literature [22; 26; 27; 28]. Figure 1 shows the extracted ion chromatogram obtained by HPLC-ESI-TOF-MS for each compound in the olive leaf extract from MAE 4. The parameters that enabled the identification of these phenolic compounds were retention time, experimental and calculated m/z, error, mSigma and the molecular formula (Supplementary Materials, Table S1). \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**MAE** & \(\mathbf{X_{1}}\) & \(\mathbf{X_{2}}\) & \(\mathbf{X_{3}}\) & \
\begin{tabular}{c} **Total Compounds** \\ **(mg g\({}^{-1}\) d.w.)** \\ \end{tabular} &
\begin{tabular}{c} **Total AMPK Bioactive Compounds** \\ **(mg g\({}^{-1}\) d.w.)** \\ \end{tabular} \\ \hline
1 & 50 & 100 & 22.5 & \(35.72\pm 0.17\)\({}^{\text{b}}\) & \(3.70\pm 0.10\)\({}^{\text{b}\text{c}}\) \\
2 & 50 & 50 & 40 & \(21.85\pm 0.16\)\({}^{\text{a}}\) & \(2.81\pm 0.05\)\({}^{\text{a}}\) \\
3 & 100 & 50 & 22.5 & \(57.37\pm 0.07\)\({}^{\text{f}}\) & \(8.64\pm 0.08\)\({}^{\text{b}}\) \\
4 & 150 & 100 & 22.5 & \(74.24\pm 0.50\)\({}^{\text{i}}\) & \(7.93\pm 0.21\)\({}^{\text{g}}\) \\
5 & 100 & 100 & 5 & \(67.18\pm 0.60\)\({}^{\text{h}}\) & \(6.99\pm 0.16\)\({}^{\text{f}}\) \\
6 & 150 & 0 & 22.5 & \(51.36\pm 0.37\)\({}^{\text{d}}\) & \(6.56\pm 0.21\)\({}^{\text{f}}\) \\
7 & 100 & 100 & 40 & \(63.55\pm 0.90\)\({}^{\text{g}}\) & \(7.05\pm 0.24\)\({}^{\text{f}}\) \\
8 & 50 & 0 & 22.5 & \(21.84\pm 0.37\)\({}^{\text{a}}\) & \(4.06\pm 0.35\)\({}^{\text{c}\text{d}}\) \\
9 & 50 & 50 & 5 & \(21.02\pm 0.02\)\({}^{\text{a}}\) & \(2.87\pm 0.10\)\({}^{\text{a,b}}\) \\
10 & 100 & 0 & 40 & \(39.19\pm 0.09\)\({}^{\text{c}}\) & \(6.97\pm 0.13\)\({}^{\text{f}}\) \\
11 & 100 & 50 & 22.5 & \(58.25\pm 0.37\)\({}^{\text{f}}\) & \(9.04\pm 0.53\)\({}^{\text{h}}\) \\
12 & 100 & 0 & 5 & \(35.73\pm 0.07\)\({}^{\text{b}}\) & \(6.29\pm 0.06\)\({}^{\text{f}}\) \\
13 & 150 & 50 & 40 & \(38.81\pm 0.57\)\({}^{\text{c}}\) & \(5.25\pm 0.21\)\({}^{\text{e}}\) \\
14 & 100 & 50 & 22.5 & \(62.49\pm 0.45\)\({}^{\text{g}}\) & \(9.39\pm 0.02\)\({}^{\text{h}}\) \\
15 & 150 & 50 & 5 & \(53.47\pm 0.31\)\({}^{\text{e}}\) & \(4.80\pm 0.002\)\({}^{\text{d,e}}\) \\ \hline \hline \end{tabular}  
\(\text{X}_{1}\): Temperature (\({}^{\text{c}}\)), \(\text{X}_{2}\): % EtOH/H\({}_{2}\)O (%(v(v))) and \(\text{X}_{3}\): Time (min). All results are expressed as mg g\({}^{-1}\) of dry matter of olive leaves. Different letters in the same column indicate significant differences (\(p<0.05\)).  
\end{table} 
Table 1: Concentration of total compounds and total AMP-activated protein kinase (AMPK) bioactive compounds in olive leaf extracts from microwave-assisted extraction (MAE) obtained by Box Behnken design. Figure 1: Extracted ion chromatogram (EIC) obtained from HPLC-ESI-TOF-MS analysis of olive leaf extract obtained by MAE. Peaks have been numbered according to the elution order. Consequently, individual compounds in olive leaf extracts were quantified by calibration curves of the standards: hydroxytrosol, oleupreein and apigenin. All calibration curves revealed good linearity among different concentrations, and the determination coefficients were higher than 0.9958 in all cases (Supplementary Materials, Table S2). Quantification of individual compounds were classified by families of simple phenols, secoiridoids, flavonoids, other phenolic compounds and phenolic acids (Supplementary Materials, Table S3). With regard to the simple phenols and derivatives, a total of three compounds were quantified. According to these results, the extraction of simple phenols improved with the use of water as their maximum content was obtained in MAE 12 and MAE 6 with 0% EtOH.