## 3 Results and Discussion
### Enzyme-Assisted Extraction (EAE)  
The total phenolic content profile of the EAE obtained extracts showed that, for all enzymes, there was a burst release of phenols at the first hour of the process, but after five hours, their concentration in the extract remained stable. The use of proteolytic (Alcalase 2.4L FG) and pectinolytic (Bioprep 3000L) enzyme resulted in the release of approximately 18% more phenolic compounds compared to cellulolytic and hemicellulose ones. The kinetics curves of total phenolic content (TPC) with extraction times (Figure 1) for the five different enzyme preparations were nonlinearly fitted to Equation (2), using SigmaPlot software. The extraction capacity, C\({}_{\rm e}\), the initial extraction rate, h, the extraction rate constant, \(k\), and the coefficient of determination, \(R^{2}\), for the five different enzymes used are presented in Table 3. The second-order model fitted well the experimental results due to the obtained high coefficient of determination. This confirmed that there are two main stages during phenolics extraction from rosmary: the first stage involves the dissolution of soluble constituents near particle surfaces into the solvent and is characterized by a rapid extraction rate; The second stage involves the mass transfer of soluble constituents from the internal material into the solvent by the diffusion process and is characterized by a slow extraction rate [17; 18]. The kinetic constants for both cellulolytic enzyme preparations (Cellic Ctec2 and Viscozyme L) were found almost similar, while Cellic Htec2 (the hemicellulose enzyme preparation) had a slightly higher initial extraction rate (\(h\)). Furthermore, initial extraction rates (\(h\)) as well as second-order extraction rate constants (\(k\)) of the above enzymes were higher compared to pectinase (Bioprep 3000L) and protease (Alcalase 2.4L FG). On the other hand, pectinase and protease had higher C\({}_{\rm e}\) values (Table 3). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Extraction Method-** & **DPPH Radical Scavenging Ability** & **TPC** \\
**Hydroethanolic Solvent** & **IC\({}_{\rm 50}\) (\(\mu\)g/mL)** & **(mg\({}_{\rm CAE}\)/grosemary)** \\ \hline CEM-ethanol/water 95:5 \(v/v\) & 12.1 \(\pm\) 0.5 & 4.1 \(\pm\) 0.4 \\ CEM-ethanol/water 70:30 \(v/v\) & 16.8 \(\pm\) 1.2 & 8.5 \(\pm\) 0.5 \\ CEM-ethanol/water 50:50 \(v/v\) & 20.6 \(\pm\) 1.1 & 11.1 \(\pm\) 0.4 \\ MAE-ethanol/water 95:5 \(v/v\) & 24.0 \(\pm\) 1.1 & 3.0 \(\pm\) 0.3 \\ MAE-ethanol/water 70:30 \(v/v\) & 19.3 \(\pm\) 0.9 & 7.0 \(\pm\) 0.6 \\ MAE-ethanol/water 50:50 \(v/v\) & 19.2 \(\pm\) 0.7 & 8.9 \(\pm\) 0.6 \\ Gallic acid & 6.1 \(\pm\) 0.3 & - \\ \hline \hline \end{tabular}
\end{table} 
Table 2: The effect of the extraction method and solvent on the antioxidant activity and the total phenolic content recovered from dried rosmary leaves. Pectin constitutes the middle lamella, and its function is to bind the cells together, while it is also part of the primary cell wall matrix and binds water. Degradation of the pectin may lead to a significant increase of the phenolics release by reducing the barrier effect, reported as one of the dominant resistances for mass transfer. This could act against (a) the penetration of the enzymes specifically attacking the polysaccharide-protein-phenol structure linkages and (b) the migration of the phenols released from both the carbohydrate structure of the primary and secondary cell wall and from other parts in the cytoplasm domain-vacuolar and/or nuclear phenols [31]. On the other hand, proteases were added to catalyze the breaking down of the structural proteins stiffening the polysaccharide domain in the primary and secondary cell wall. If weakened, this structure may become more porous and allow a higher penetration of solvent and enzymes, easing the solvent contact and mass transfer of the phenols from the interior of the plant matrix. The antioxidant activity of the extracts was measured at 1 h and 5 h in order to conclude on the optimal EAE conditions that will be used as pretreatment (Table 4).