## 3 Results and Discussion
### EAE as a Pretreatment Method of CEM  
The next step was to investigate the pretreatment of the rosemary leaves with pectinase for 1 h or 5 h followed by conventional extraction using ethanol/water 50:50 for 24 h. \begin{table}
\begin{tabular}{c c c} \hline \hline  & \multicolumn{3}{c}{**Extraction Time**} \\ \cline{2-3}
**Enzyme** & \(t=1\)**h** & \(t=5\)**h** \\ \cline{2-3}  & \multicolumn{3}{c}{**DPPH Radical Scavenging Ability**} \\  & \multicolumn{3}{c}{**IC\({}_{50}\) (\(\mu\)g/mL)**} \\ \hline Alcalase 2.4L FG & 32.1 \(\pm\) 3.4 & 18.9 \(\pm\) 0.9 \\ Cellic CTec2 & 37.9 \(\pm\) 2.5 & 28.3 \(\pm\) 1.1 \\ Viscozyme L & 46.0 \(\pm\) 1.4 & 27.2 \(\pm\) 1.3 \\ Cellic HTec2 & 31.1 \(\pm\) 1.4 & 27.6 \(\pm\) 1.8 \\ Bioprep 3000L & 33.6 \(\pm\) 2.9 & 13.2 \(\pm\) 1.4 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Effect of the enzyme type on the antioxidant activity during enzymatic extraction. Figure 2: TPC (mgGAE/g rosemary) values for a 24 h enzyme-assisted extraction in water using (\(\bullet\)) 2.5% or (\(\odot\)) 5.0% (\(w/w\)) of Bioprep 3000L. The TPC and DPPH radical scavenging ability of the extracts obtained by this combined methodology is presented in Table 5.  
The data of Table 5 indicated that the highest recovery was achieved for 1-h enzymatic pretreatment followed by 24 h conventional extraction. It was also observed that, despite the fact that the 5 h pretreatment step solely resulted in higher antioxidant activity (Table 4), the combination of the two aforementioned methods was more efficient for 1-h enzyme pretreatment. Comparing the results between the different types of extraction, the EAE presented the highest values for both antioxidant activity and TPC. This can be assigned to the degradation and disruption of the cell wall matrix, as pectinase depolymerizes cell-wall polysaccharides and hydrolyzes the glycosidic linkages between phenolic compounds and cell wall polymers. Consequently, not only the structure of the cell wall can be weakened and broken down, whereby intracellular materials are more exposed to extraction, but also the solubility of the target substances can be improved [33]. In order to determine the optimum time for the combined extraction method, 1 h pretreatment with pectinase, followed by 1 to 24 h CEM was conducted. Figure 3 shows the differences in TPC values of the different extraction times. Figure 4 depicts the curves of total phenolics with extraction times for CEM and combined EAE and CEM. The curves, as above, were nonlinearly fitted to Equation (2), using SigmaPlot software. The extraction capacity, Ce, the initial extraction rate, h, the extraction rate constant, k, and the coefficient of determination, R\({}^{2}\), for the two different methods used are presented in Table 6. As above, the second-order model fitted well the experimental results owing to the obtained high coefficient of determination. Figure 3: The effect of extraction time on the TPC (mg\({}_{\mathrm{GAE}}\)/g\({}_{\mathrm{Fos}}\)mary) (\(\bullet\)). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Extraction Method** & **DPPH Radical Scavenging Ability** & **TPC** \\  & **IC\({}_{\mathrm{50}}\) (\(\mathrm{\SIUnitSymbolMicro mL}\))** & **(mg\({}_{\mathrm{GAE}}\)/g\({}_{\mathrm{Ros}}\)mary)** \\ \hline
1 h enzymatic pretreatment + 24 h & 14.3 \(\pm\) 0.8 & 15.2 \(\pm\) 0.3 \\ CEM & 17.2 \(\pm\) 1.2 & 13.0 \(\pm\) 0.5 \\ CEM & & \\ \hline \hline \end{tabular}
\end{table} 
Table 5: TPC (mgGAE/g) and DPPH radical scavenging ability [IC\({}_{\mathrm{50}}\) (\(\mathrm{\SIUnitSymbolMicro g}\)/mL)] of the extracts obtained by enzyme pretreatment followed by conventional extraction. The results showed that the \(h\), \(k\), and \(C_{\text{e}}\) were affected by the type of extraction method applied (Table 5). Combined EAE and CEM exhibited approximately 3.0 times higher Ce value compared to conventional ones. Therefore, a combination of EAE and CEM can improve the extraction of antioxidants from rosmenary leaves.