## 3 Results
### Characterization of Non-Polar Compounds in Different Ecotypes of Burdock Roots by GC-MS  
A characterization of non-polar compounds from the PLE extracts obtained with 100% 2-MTHF at 100 \({}^{\circ}\)C from the SPLE, OLE, and SPME ecotypes was achieved by GC-MS. A total of 14 different non-polar compounds were tentatively identified in all ecotypes (see Table 3 and Figure S2), including 6 fatty acids, 4 terpenoids, 1 phenolic compound, 2 sterols, and 1 steroid derivative. Among the ecotypes, the highest diversity of non-polar compounds was found in SPLE, where oleic acid amide (number 7) fatty acid and the hotrienol (number 8) monoterpene were detected, but these were not found in the OLE and SPME ecotypes. Oleic acid amide was tentatively identified with a molecular ion at \(m/z\) 281 and fragment ions at 114 [M-C\({}_{8}\)H\({}_{14}\)], 100 [M-C\({}_{10}\)H\({}_{20}\)], 86 [M-C\({}_{6}\)H\({}_{12}\)], 72, 69, 67, 43, and 41 from smaller alkyl fragments resulting from further chain cleavage [32]. This fatty acid was one of the most abundant non-polar compounds detected in the SPLE extract, \begin{table}
\begin{tabular}{l c c c} \hline \hline
**Method** & **SPLE** & **OLE** & **SPME** \\ \hline TPC (mg gallic acid/g extract) & 22.9 \(\pm\) 0.9 \({}^{\circ}\) & 48.8 \(\pm\) 0.2 \({}^{\text{b}}\) & 64 \(\pm\) 5 \({}^{\text{a}}\) \\ DPPH\({}^{\bullet}\)(uMrolox/g extract) & 0.21 \(\pm\) 0.01 \({}^{\circ}\) & 0.43 \(\pm\) 0.03 \({}^{\text{b}}\) & 0.739 \(\pm\) 0.005 \({}^{\text{a}}\) \\ ORAC (IC\({}_{50}\), mg/mL extract) & 9 \(\pm\) 1 \({}^{\text{b}}\) & 4.8 \(\pm\) 0.3 \({}^{\text{a}}\) & 3.1 \(\pm\) 0.3 \({}^{\text{a}}\) \\ AChE (IC\({}_{50}\), mg/mL extract) & 59 \(\pm\) 8 \({}^{\text{b}}\) & 94 \(\pm\) 2 \({}^{\text{c}}\) & 29 \(\pm\) 2 \({}^{\text{a}}\) \\ BChE (IC\({}_{50}\), mg/mL extract) & 75 \(\pm\) 2 \({}^{\text{b}}\) & 83 \(\pm\) 2 \({}^{\text{b}}\) & 43 \(\pm\) 3 \({}^{\text{a}}\) \\ \hline \hline \end{tabular} \({}^{\text{a,b,}^{\text{c}}}\) Letters show the significant differences between extracts (\(p\leq\) 0.05).  
\end{table} 
Table 2: Total phenolic content (Folin–Ciocalteu assay), antioxidant capacity (DPPH\({}^{\bullet}\) and ORAC assays), and anticholinergic capacity (AChE and BChE assays) from SPME, OLE, and SPME extracts obtained by PLE under the optimized extraction conditions (100% 2-MTHF, 100 \({}^{\circ}\)C). according to their peak areas. The other compound detected in the SPLE extract but not in the OLE and SPME extracts was hortienol, which presented a molecular ion at \(m/z\) 152 and characteristic fragment ions at 119 [M-CH\({}_{3}\)CO], 105 [M-C\({}_{4}\)H\({}_{4}\)], 91 [M-C\({}_{7}\)H\({}_{11}\)], 82, 71, 67, and 55 alkyl fragments [33]. As can be observed in Table 3, the non-polar profile was the same among the ecotypes, excepting the two compounds identified in the SPLE extracts. Only the differences already discussed were observed in the peak areas of the other identified compounds. However, the SPLE extract presented the highest total peak area of non-polar compounds identified by GC-MS, compared to the OLE and SPME extracts. Particularly, the most abundant non-polar compound detected in all the ecotypes was y-sitosterol (number 11) but without statistically significant differences with methyl linoleicidate (number 5) in the SPME extract. y-sitosterol was detected with a molecular ion at \(m/z\) 414 and characteristic fragments at 396 [M-CH4]; 213, which corresponds to the fragmentation of a part of the steroid backbone; 159, 147, and 145, involving the loss of a small fragment or part of the steroid structure; 69, which is common in sterols; and 107, 97, 81, 67, involving different losses of alkyl fragments (see Table 3) [34]. One more sterol was identified in these burdock roots, which corresponds to \(\beta\)-stigmasterrol but at a lower level than the \(\gamma\)-sitosterol. According to the peak areas, \(\beta\)-stigmasterrol (number 10) was one of the major compounds detected in the OLE ecotype, obtaining higher peak areas than the SPLE and SPME ecotypes.