## 3 Results
### Phenolic Characterization of Different Ecotypes of Burdock Roots by HPLC-IT-MS  
Table 4 shows that an HPLC-IT-MS analysis of the PLE extracts from the three different ecotypes obtained at 100 \({}^{\circ}\)C allowed the detection of ten different phenolic compounds, of which seven were tentatively identified. Principally, the identified phenolic compounds corresponded to caffeoylquinic acids. In fact, cynarin (number 3) was identified with a molecular ion at \(m/z\) 515 [M-H]\({}^{-}\), exhibiting the characteristic fragmentation of caffeoylquinic acid with intense fragment ions at \(m/z\) 353, 334.9, 190.9, and 178.9, representing the majority phenolic compounds observed in all the ecotypes [35; 36]. Comparing the ecotypes, the SPME extract presented the highest peak area of cynarin compared to OLE and SPLE. In addition, another dicaffeoylquinic acid was observed but with a sodium adduct with a molecular ion at \(m/z\) 537 (number 4). Furthermore, dicaffeoylmaloylquinic acid (number 6) was tentatively identified with a molecular ion at \(m/z\) 631 [M-H]\({}^{-}\). Its fragmentation pattern showed characteristic ions at \(m/z\) 352.8 and \(m/z\) 190.9, corresponding to the sequential loss of one and two caffe acid molecules and malic acid, and a quinic acid molecule [37]. This compound was not detected in the SPME extract; neither were urchin (number 5) and monocaffeoylquinic acid (number 7); however, they were observed in the SPLE and OLE extracts. Particularly, arcitin (number 5) was tentatively identified with a \(m/z\) ion at 533 [M-H]\({}^{-}\), from which an intense fragment at \(m/z\) 371 was observed, with neutral losses of methyl groups, including \(m/z\) 355, as the phenolic compound detected with the lowest peak area in the extracts [38]. In addition to the monocaffoylquinic acid (number 7), another one was identified with a molecular ion at \(m/z\) 374.9 [M-H]\({}^{-}\) with fragment ions at \(m/z\) 190 involving the loss of quinic acid and \(m/z\) 178, which corresponded to the loss of the caffeic acid molecule. This compound was one of the majority phenolic compounds identified in the burdock roots after cynarin in the OLE and SPLE extracts, and after dicaffeoylquinic acid isomer (number 4) in the SPME extract, having its peak area higher in SPME than in the rest of the ecotypes. It is noted that, corresponding to the chromatogram registered at 280 nm (Figure S3), three different phenolic compounds were observed but they cannot be identified by MS (compounds number 8, 9, and 10). In addition, Figure S3 and Table 4 show higher peak areas of identified compounds in the SPME extract compared to the SPLE and OLE extracts. It was observed that higher altitudes in the growing conditions of the burdocks implied a higher synthesis of phenolic compounds identified in the HPLC-IT-MS analysis, except for arcitin, dicaffeoylmaloylquinic acid, and the monocaffeoylquinic acid detected at 53.3 min, whose synthesis was favored when the plant grew at an altitude of 150 m. When the altitude was the same (comparing SPLE and OLE extracts), it was noted that the absence of weeding during plant growth produced an increase in the content of phenolic compounds, as can be seen in Table 4. Finally, cynarin (number 3) presented higher a peak area in the SPLE than in the OLE extract. In addition, Figure S3 displays unknown compound peaks starting at minute 40 that were detected in the OLE ecotype extract but were not observed in the SPLE and SPME extracts. This reflected that weeding treatment favors the synthesis of other secondary metabolites. Evaluation of the Role of Growing Conditions of Burdock Roots in Their Bioactivity, Non-Polar, and Polar Composition  
To study the influence of weeding treatment and the altitude at which the plant was collected on the recovery of bioactive compounds from the PLE extracts obtained at 100 \({}^{\circ}\)C, a multivariate analysis was performed on the TPC values, antioxidant capacity was determined by DPPH\({}^{\bullet}\) and ORAC assays, and anticholinergic capacity was evaluated