## 3 Results and Discussion
### Optimization of Subcritical Water Extraction  
The effects of variables and their interactions on the RES yield were elucidated by 3D response surface observations and 2D contour plots (Figure 3) that described the regression of Equation (2) by means of a graphic approach, which provided a method to visualize the relationships between RES yield and any two independent variables, while the third was fixed at its zero level (center value of the testing ranges). The maximum predicted response was located on the peak of the 3D response surface,  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Run** & \multicolumn{4}{c}{**Parameters and Levels**} & **RES Yields (\#g/g Material)** \\ \cline{2-4}  & **A: Pressure (MPa)** & **B: Time (min)** & **C: Temperature (\({}^{\circ}\)C)** & \\ \hline
1 & 0.5 & 25 & 130 & 4.59 \(\pm\) 0.02 \\
2 & 0.5 & 30 & 150 & 5.39 \(\pm\) 0.0  
the corresponding point for which was defined by the smallest ellipse in the contour diagram of the 2D contour plot [29].  
Presented in Figure 3a,b are the effects of extraction pressure, time, and their interactions on RES yield. The RES yield increased with increasing extraction pressure, up to 1.02 MPa, and increasing extraction time, up to 24.89 min. Beyond an extraction pressure of 1.02 MPa or extraction time of 24.89 min, there was decrease in the RES yield. In general, the increase in pressure increased the rupture speed of the cell wall, enhancing extraction efficiency. However, the structure of RES was unstable and it could be damaged if the pressure was too high. The increase of extraction time increased exposure of the reactive site to the effective extraction process, which in turn enhanced RES yield. The reduction in RES yield was due to partial degradation of RES over extended time and high temperature. Figure 3c,d displayed the effects of extraction pressure and temperature on RES yield and their interactions. RES yield increased with increased pressure and temperature. The effects of extraction time, temperature, and their interactions on RES yield are depicted in Figure 3e,f. From these observations, we found that RES yield increased with temperature and time. This phenomenon was due to the fact that water at a higher temperature had a lower dielectric constant, reducing the energy required for division in solute-matrix interactions, and also reduced polarity due to weakened hydrogen bonds [30]. Furthermore, water in its subcritical state exhibited hydrolytic properties as its ionization constant increased, producing an increase in the RES extraction efficiency as temperature increased [31]. Beyond an extraction time of 24.89 min or extraction temperature of 152.32 \({}^{\circ}\)C, there was no further increase in the RES yield.  
Figure 3 shows the optimal extraction conditions for the entire SWE process to be an extraction pressure at 1.02 MPa, extraction time of 24.89 min and extraction temperature at 152.32 \({}^{\circ}\)C. Furthermore, RSM predicted a maximum RES yield of 7.14 \(\upmu\)g/g. The validation experiment was performed the above optimized extraction conditions, where RES yield reached 6.90 \(\upmu\)g/g. This was closed with the predicted yield value.