## 3 Results and Discussion
### Optimization of MAE Extraction Conditions
#### 3.2.1 Optimization of Extraction Conditions for the Content of Total Compounds
Furthermore, the experimental value of the content of total compounds obtained under optimum MAE conditions in this study was 65.9% higher than the value of the sum of individual compounds  
Figure 2: Response surface plots showing combined effects of process variables for total compounds: Temperature (\({}^{\circ}\)C)–% EtOH (a), temperature (\({}^{\circ}\)C)–time (min) (b) and % EtOH–time (min) (c). obtained by [PERSON] et al. [39] at optimum MAE conditions. Besides, the experimental result of this study was 40.5%, 39.9% and 31.3% higher than that obtained in 'Sikitita', 'Picual' and 'Arbequina' olive leaves, respectively, by ultrasonic assisted extraction (UAE) [26]. Also this was 38.7%, 81.3%, 59.8% and 37.6% higher than the means for all 'Arbequina', 'Arbosana', 'Changlot Real', 'Picual', 'Koroneiki' and 'Sikitita' cultivars, respectively, at four sampling times (June, August, October and December) obtained by UAE [25]. Therefore, a high concentration of compounds in olive leaf extract was obtained by using optimum MAE conditions compared with that obtained in previous studies using MAE and UAE. However, this difference may be due to agronomic variability in the sample such as harvesting season, cultivar, etc. [25]. 2.2 Optimization of Extraction Conditions for the Content of Compounds from Olive Leaves with Capacity to Modulate AMPK  
According to [PERSON] et al., there are compounds in different fractions of olive leaf extract that decrease intracellular lipid accumulation through AMPK-dependent mechanisms in hypertrophic adipocyte [22]. However, these bioactive compounds were not quantified in that previous study. For that reason, it has been proposed that these compounds extracted by MAE should be quantified in order to establish the best conditions to obtain their highest recovery. Among a total of 41 compounds determined in olive leaf MAE extracts, 18 of them possess the capacity to activate the AMPK pathway (Table 3) [22]. It is important to note that according to [PERSON] et al., demethyloleouropein, verbascoside, hydroxyoleouropein, rutin, luteolin rutinoside and luteolin glucoside are the compounds that provide the highest activation of AMPK and these were quantified in the previous study. Table 1 shows the total content of these 18 compounds obtained in each extraction. Total AMPK bioactive compounds ranged from \(2.81\pm 0.05\) mg g\({}^{-1}\) d.w. in MAE 2 (50 \({}^{\circ}\)C, 50% EtOH and 40 min) to \(9.39\pm 0.02\) mg g\({}^{-1}\) d.w. in MAE 14 (100 \({}^{\circ}\)C, 50% EtOH and 22.5 min). Therefore, high temperatures, an intermediate percentage of ethanol and extraction time of 22.5 min generated the highest content of total compounds with the capacity to modulate the AMPK activation route. The increasing temperature speeds up the mass transfer of analytes from the sample to the extraction solvent due to the increase in the diffusion speed and solubility, and the decrease in viscosity, surface tension and the strength of the links between compounds and the matrix [41]. Therefore, high temperatures of 100 \({}^{\circ}\)C provided an increase in the recovery of total compounds, whereas an extraction time of 40 min resulted in a decrease \begin{table}
\begin{tabular}{c c} \hline \hline
**Peak** & **AMPK Bioactive Compounds** \\ \hline
9 & Secologanoside isomer b \\
10 & Elenolic acid glucoside isomer c \\
14 & Demethyloleuropein \\
15 & Hydroxyoleuropein/hydroxyoleuroside isomer a \\
16 & Rutin \\
17 & Luteolin rutinoside \\
18 & luteolin glucoside isomer a \\
19 & Verbascoside \\
22 & Oleuropein glucoside isomer a \\
23 & Diosmetin rhamnoside glucoside (diosmin) \\
24 & luteolin glucoside isomer b \\
35 & Oleuropein isomer c \\
36 & Oleuropein isomer d \\
37 & ligstroside \\
38 & Luteolin \\
39 & Quercetin \\
40 & Oleuropein/oleuroside methyl ether \\
41 & Resinoside \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Compounds identified in olive leaf extracts with the potential to modulate AMPK. in the content of these compounds.