## 3 Results
### Optimizing the PLE Polyphenol Extraction from Burdock Roots
Figure 1: HPLC-DAD chromatograms at 280 nm of phenolic compounds recovered from SLE and PLE at 50, 100, and 150 \({}^{\circ}\)C of organic burdock ecotypes. Numbers correspond to identified compounds in Table 2. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**No.** & **Compound** & **Rt (min)** & **[M\(-\)H]\({}^{-}\)** & **MSE\({}^{2}\) Ions** & **SLE** & **PLE 50 \({}^{\circ}\)C** & **PLE 100 \({}^{\circ}\)C** & **PLE 150 \({}^{\circ}\)C** \\  & & & & **(g/100 g of** & **(g/100 g of** & **(g/100 g of** & **(g/100 g of** \\  & & & & **Dried Extract)** & **Dried Extract)** & **Dried Extract)** \\ \hline
1 & Chlorogenic acid & 7.9 & 374.9 & 353.2, 190.7, 178.7 & 0.67 \(\pm\) 0.02 c  & 1.06 \(\pm\) 0.03 a  & 0.893 \(\pm\) 0.007 b  & 0.26 \(\pm\) 0.01 d  \\
2 & 4-O-Caffeoylquinic acid & 8.32 & 353.0 & 190.8, 178.8, 135.0 & 0.015 \(\pm\) 0.002 a  & 0.0095 \(\pm\) 0.0002 b  & 0.0039 \(\pm\) 0.0004 c  & ND \\
3 & Cymarine & 10.25 & 515.1 & 353.4, 190.8, 178.8, 135.0 & 0.55 \(\pm\) 0.02 c  & 0.99 \(\pm\) 0.05 a  & 0.862 \(\pm\) 0.005 b  & 0.30 \(\pm\) 0.02 d  \\
4 & Déaffeey/Inlayoliquitmic acid & 17.9 & 631 & 573.3, 514.9, 468.6, 467.37, 39.79 & 0.07 \(\pm\) 0.004 c  & 0.27\(\pm\)03 a  & 0.18\(\pm\)0.03 b  & 0.102 \(\pm\) 0.004 c  \\
5 & Déaffeey/Inlayoliquitmic acid & 19.3 & [M\(-\)H\({}_{\rm{x}}\)N\({}_{\rm{a}}\)] & 353.4, 1910.7, 173.1, 135 & 0.074 \(\pm\) 0.008 c  & 0.18 \(\pm\) 0.02 a  & 0.11 \(\pm\) 0.01 b  & 0.062 \(\pm\) 0.003 c  \\
6 & Déaffeey/Inlayoliquitmic acid \({}^{\star}\) & 20.2 & 536.8 & - & - & - & - & - & - \\
7 & 3.5-Déaffeey/Inlayoliquitmic acid & 21.1 & 515 & 353.1, 190.9, 178.9 & 0.06 \(\pm\) 0.004 b  & 0.145 \(\pm\) 0.007 a  & 0.0982 \(\pm\) 0.003 b  & 0.044 \(\pm\) 0.001 d  \\
8 & 1.5-Déaffeey/Inlayoliquitmic acid & 21.7 & 515 & 353.1, 190.9, 178.9 & 1.06 \(\pm\) 0.04 c  & 2.1 \(\pm\) 0.1 a  & 1.485 \(\pm\) 0.001 b  & 0.84 \(\pm\) 0.04 d  \\
9 & Déaffeey/Inlayoliquitmic acid isomer \({}^{\star}\) & 22.6 & 631 & 515, 490.9, 469.4, 449.53, & 0.041 \(\pm\) 0.003 c  & 0.13 \(\pm\) 0.01 a  & 0.09 \(\pm\) 0.02 b  & 0.051 \(\pm\) 0.004 c  \\
10 & Déaffeey/Inlayoliquitmic acid \({}^{\star}\) & 24.7 & 747 & 631, 515.6, 469.4, 451, 433.3, 355.1 & 0.118 \(\pm\) 0.008 c  & 0.30 \(\pm\) 0.02 a  & 0.21 \(\pm\) 0.03 b  & 0.117 \(\pm\) 0.003 c  \\ \hline \hline  & & & & & & & & & \\ \end{tabular}
\end{table} 
Table 2: Mass data spectra and quantification (g/100 g of dried extract) of phenolic compounds in OLE burdock not extracts obtained by SLE and PLE at different temperatures (50, 100, and 150 \({}^{\circ}\)C) by HPLC-DAD-IT-MS.  
On the other hand, four dicaffeoylquinic acids were identified with a molecular ion at \(m/z\) 515 [M\(-\)H]\({}^{-}\), which sometimes can be found forming a sodium adduct with a molecular ion at \(m/z\) 537 [M\(-\)H\(+\)Na]\({}^{-}\). In fact, dicaffeoylquinic acids (numbers 3, 6, 7, 8; Table 2) were observed in this study with a molecular ion at \(m/z\) 537, with their corresponding fragment ions at \(m/z\) 353 [M\(-\)162]\({}^{-}\), 190 [M\(-\)324]\({}^{-}\), and 178 [M\(-\)336]\({}^{-}\) originating from the loss of one and two caffe acid molecules and a quinic acid molecule, respectively. In addition, dicaffeoylquinic acids such as cynarine (number 3), 3,5-dicaffeoylquinic acid (number 7), 1,5-dicaffeoylquinic acid (number 8), and other dicaffeoylquinic acid isomers were observed. Moreover, another dicaffeoylquinic acid was observed at tR = 20.2 min (number 6), which cannot be exhaustively identified and differentiated from its isomers by the standards; therefore, its content was expressed as g equivalents of the 1,5-dicaffeoylquinic acid/100 g extract. As can be seen in Table 2, these four phenolic compounds were obtained with the highest content using PLE at 50 \({}^{\circ}\)C, decreasing their content when higher PLE temperatures were applied.