## 3 Results
### Phenolic Characterization of Different Ecotypes of Burdock Roots by HPLC-IT-MS
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**No.** & **Compound** & **Rt (min)** & **[M-H]\({}^{-}\)** & **MS\({}^{2}\) Ions** & **SPLE PLE** & **OLE PLE** & **SPME PLE** \\ \hline
1 & Chlorogenic acid & 8.8 &
\begin{tabular}{c} 374.9 \\ [M+H + Na]\({}^{-}\) \\ \end{tabular} & 353, 2008, 190.9, 178.8, 172.9, & 452 \(\pm\) 79 & 264 \(\pm\) 18 & 2069 \(\pm\) 75 \\ \hline
2 & 4-O-Caffeoylquinic acid & 13.3 & 353.0 & 190.8, 178.8, 172.8, 135, 127 & 167 \(\pm\) 43 & 121 \(\pm\) 10 & 1681 \(\pm\) 4 \\ \hline
3 & \
\begin{tabular}{c} Dcaffeoylquinic acid \\ (Cynarin) \\ \end{tabular} & 20.7 & 515.2 & 379, 353, 334.9, 190.9, 178.9 & 1392 \(\pm\) 81 & 658 \(\pm\) 84 &
\begin{tabular}{c} 14,039 \(\pm\) \\ 56 \\ \end{tabular} \\ \hline
4 & \begin{tabular}{c} Dicaffeoylquinic acid \\ isomer \\ \end{tabular} & 21.8 & \
\begin{tabular}{c} 537 \\ [M+H + Na]\({}^{-}\) \\ \end{tabular} &
\begin{tabular}{c} 515.1, 375, 353, 333, 264.9, 190.9, \\ 178.9, 172.9 \\ \end{tabular} & 60 \(\pm\) 6 & 181 \(\pm\) 21 & 4121 \(\pm\) 214 \\ \hline
5 &
\begin{tabular}{c} Arctin \\ Acid \\ \end{tabular} & 28.9 & 533 & 515, 371, 355, 289, 249, 151, 136, 121 & 61 \(\pm\) 14 & 95 \(\pm\) 6 & - \\ \hline
6 & \
\begin{tabular}{c} Dicaffeoylmaloylquinic acid \\ (Cynarin) \\ \end{tabular} & 29.7 & 631 &
\begin{tabular}{c} 352.8, 325.0, 9, 190.9, 172.9, \\ 160.8, 135 \\ \end{tabular} & 214 \(\pm\) 23 & 395 \(\pm\) 12 & - \\ \hline
7 &
\begin{tabular}{c} Monocaffeoylquinic acid \\ \end{tabular} & 53.3 & 353.1 & 333, 394.9, 232.8, 192.9, 179 & 236 \(\pm\) 21 & - & - \\ \hline
8 & Unknown 1 & 54.1 & 441.3 & 429, 422.2, 399.4, 3834, 340.9, 327.2, & 286 \(\pm\) 7 & 336 \(\pm\) 5 & 479 \(\pm\) 5 \\ \hline
9 & Unknown 2 & 54.5 & 471.4 & 4513, 441.2, 407.2, 367.3, 337, & 298 \(\pm\) 9 & 380 \(\pm\) 26 & 1137 \(\pm\) 16 \\ \hline
10 & Unknown 3 & 58.4 & 383.4 & 365.2, 337.2 & 3115 \(\pm\) 60 & - & 4540 \(\pm\) 37 \\ \hline \hline \end{tabular} (-): Non-detected.  
\end{table} 
Table 4: Mass data spectra and peaks areas of compounds in the three different burdock ecotypes (OLE, SPLE, SPME) obtained by PLE at 100 \({}^{\circ}\)C by HPLC-DAD-IT-MS.  
by the AChE and BChE methods, as well as on the peak areas of the non-polar and polar compounds identified by GC-MS and HPLC-IT-MS, respectively. The burdock root extracts were divided into two groups according to phenolic, antioxidant, and anticholinergic values, and the peak areas of compounds identified by the chromatographic analysis, which were differentiated by colors in a Hierarchical Cluster Analysis (HCA). One cluster grouped the SPME extract (green color) while the other one grouped the OLE and SPLE extracts (blue color) (see Figure 3A). In addition, a Principal Component Analysis (PCA) also separated the extracts according to their bioactive and non-polar and polar composition, selecting the most significant variables (principal components). Two principal components described 100% of the total data variability, grouping the extracts similarly to the HCA (employing the same colors for HCA and PCA). Figure 3: (**A**) HCA dendrogram created using the Ward method and shorter by height from SPLE, OLE, and SPME extracts achieved by PLE under the optimal extraction conditions, (**B**) PCA score plot, and (**C**) PCA loading plot. Extracts from the same group and color presented similar bioactivity and non-polar and polar composition. By overlapping Figure 3B,C, it can be observed that the SPLE and OLE extracts from the burdock roots collected at 150 m (group 2) showed the lowest TPC and antioxidant and anticholinergic values because they were situated in the opposite site to the TPC and DPPH\({}^{\bullet}\) assays by their low phenolic content and antioxidant capacity that are presented in these methods. In addition, they were in the same site as the ORAC, AChE, and BChE methods expressed as IC\({}_{50}\), indicating that they presented higher IC\({}_{50}\) values, that is, lower bioactive capacity.