## 3 Results  
### Optimization of PLE Extraction by a Simplex-Centroid Experimental Design  
A simplex-centroid experimental design was conducted to optimize the optimal mixture proportions of biobased solvents (CPME, 2-MTHF, and EtOAc) for the recovery ofa high amount of phenolic compounds from burdock roots by PLE (at 40 \({}^{\circ}\)C for 20 min), particularly from the SPLE ecotype. SPLE was selected as the most representative sample of burdock roots, because it was collected at 150 m like the OLE and without weeding treatment like the SPME. For the optimization, the peak areas determined by HPLC-DAD at 280 nm from the 7 experiments established by the simplex-centroid mixture design were used as response variables, and the results are shown in Table 1.  
In addition, the ANOVA results and the adequacy of the model with their corresponding coefficients, shown in Table S1, exhibited that the regression model could explain 99% of the results' variability by HPLC-DAD analysis. In addition, ANOVA revealed a non-significant effect (\(p\)-value > 0.05) of different variables on the response variable. For instance, the effect of the biobased solvent mixture proportions achieved by the simplex-centroid experimental design can be observed in the contour plots in Figure 1. This figure shows that the combination of CPME, 2-MTHF, and EtOAc did not allow significant effects (\(p\)-value > 0.05) on the peak area of phenolic compounds determined by HPLC-DAD. However, the contour plots exhibited that the highest peak areas of phenolic compounds determined by HPLC-DAD were achieved at the highest 2-MTHF percentage. In fact, this experimental design revealed that the optimal extraction conditions to obtain high phenolic content from SPLE roots were 100% 2-MTHF. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{3}{c}{**Independent Variables**} & \multicolumn{2}{c}{**Dependent Variable**} \\ \hline  & \multicolumn{3}{c}{Mixture (\% solvent, \(\upsilon/\upsilon\))} \\ Run & CPME & 2-MTHF & EtOAc & Total Peak Area at 280 nm \\
1 & 50 & 0 & 50 & 4181 \(\pm\) 389 [FOOTNOTE:]Footnote : a:\(\upsilon\)\(\lambda\)\(\lambda\)\(\lambda\)\(\epsilon\) Letters indicate statistically significant differences among extractions.[ENDFOOTNOTE] \\
2 & 33 & 33 & 33 & 6843 \(\pm\) 93 [FOOTNOTE:]Footnote : a:\(\upsilon\)\(\lambda\)\(\lambda\)\(\epsilon\) Letters indicate statistically significant differences among extractions.[ENDFOOTNOTE] \\
3 & 100 & 0 & 0 & 5998 \(\pm\) 15 [FOOTNOTE:]Footnote : a:\(\upsilon\)\(\lambda\)\(\lambda\)\(\epsilon\) Letters indicate statistically significant differences among extractions.[ENDFOOTNOTE] \\
4 & 50 & 50 & 0 & 8308 \(\pm\) 258 [FOOTNOTE:]Footnote : a:\(\upsilon\)\(\lambda\)\(\lambda\)\(\epsilon\) Letters indicate statistically significant differences among extractions.[ENDFOOTNOTE] \\
5 & 0 & 0 & 100 & 9484 \(\pm\) 17 [FOOTNOTE:]Footnote : a:\(\upsilon\)\(\lambda\)\(\lambda\)\(\epsilon\) Letters indicate statistically significant differences among extractions.[ENDFOOTNOTE] \\
6 & 0 & 100 & 0 & 9363 \(\pm\) 135 [FOOTNOTE:]Footnote : a:\(\upsilon\)\(\lambda\)\(\epsilon\) Letters indicate statistically significant differences among extractions.[ENDFOOTNOTE] \\
7 & 0 & 50 & 50 & 8062 \(\pm\) 306 [FOOTNOTE:]Footnote : a:\(\upsilon\)\(\lambda\)\(\epsilon\) Letters indicate statistically significant differences among extractions.[ENDFOOTNOTE] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Simplex-centroid mixture design and response variable that corresponds to the total peak areas of HPLC-DAD chromatograms at 280 nm of extracts obtained by PLE with CPME, 2-MTHF, and EtOAc bicubosed solvents from SPLE burdock roots under the established preliminary conditions. Figure 1: Contour plots show the effect of biobased solvent mixture among CPME, 2-MTHF, and EtOAc on the peak areas of phenolic compounds determined by HPLC-DAD at 280 nm from the SPLE extracts obtained by PLE.