## 3 Results  
### Optimizing the PLE Polyphenol Extraction from Burdock Roots  
To assess the influence of PLE temperature on the bioactivity and phenolic composition of _Arctium lappa_ L. roots, an optimization study was initially conducted using samples from OLE, cultivated at an altitude of 150 m under agronomic practices involving the exclusion of other plant species. Once the optimal extraction conditions were established for the OLE samples, these conditions were subsequently applied to two additional burdock rootecotypes, as follows: SPLE, consisting of wild-grown roots at 150 m, and SPME, comprising wild-grown roots at 800 m, both without being subjected to weeding. The phenolic profiles and bioactivities of the three ecotypes (OLE, SPLE, and SPME) were then compared to elucidate the impact of environmental conditions and agronomic practices on the phenolic composition and associated bioactivities of burdock roots. Considering that PLE parameters have been widely optimized for phenolic recovery from natural matrices, only the extraction temperature was selected for optimization in this study, with the specific objective of maximizing phenolic compound recovery while minimizing potential thermal degradation [34; 39; 40; 41]. Three extraction temperatures (50 \({}^{\circ}\)C, 100 \({}^{\circ}\)C, and 150 \({}^{\circ}\)C) were evaluated using an EtOH/H\({}_{2}\)O (70:30, \(v/v\)) solvent system under 1500 psi for 20 min, following the protocol described by [PERSON] et al. [34]. The resulting extracts were subsequently compared based on their TPC, antioxidant capacity (DPPH and ORAC assays), and anticholinergic activity (AChE and BChE inhibitory assays). In addition, the PLE extracts were compared to a conventional solid-liquid extract (SLE) obtained at room temperature over 24 h using the same solvent system. As shown in Table 1, the PLE at 150 \({}^{\circ}\)C yielded the highest extraction efficiency (45 \(\pm\) 2%) in terms of extract mass. However, this extract exhibited the lowest TPC values (25 \(\pm\) 2 mg gallic acid/g extract) compared to those obtained by PLE at 50 \({}^{\circ}\)C (42 \(\pm\) 4 mg gallic acid/g extract) and 100 \({}^{\circ}\)C (33 \(\pm\) 2 mg gallic acid/g extract), with SLE (38 \(\pm\) 3 mg gallic acid/g extract) producing the highest phenolic content among all conditions tested. Table 1 shows that higher TPC values implied higher bioactivity. In this sense, the PLE extract obtained at 50 \({}^{\circ}\)C exhibited the highest antioxidant capacity (113 \(\pm\) 1 \(\upmu\)mol Trolox/g extract for DPPH and an IC\({}_{50}\) value of 2.85 \(\pm\) 0.08 mg/mL for the ORAC assay) compared to the rest of the PLE extracts, but without statistically significant differences compared to SLE concerning the DPPH assay (113 \(\pm\) 1 \(\upmu\)mol Trolox/g extract). Nevertheless, the SLE extract presented a higher antioxidant capacity, determined by the ORAC assay (IC\({}_{50}\) 1.7 \(\pm\) 0.1 mg/mL), than the PLE extract obtained at 50 \({}^{\circ}\)C (IC\({}_{50}\) 2.85 \(\pm\) 0.08 mg/mL). In terms of anticholinergic activity, the PLE extract obtained at 50 \({}^{\circ}\)C exhibited the highest AChE and BChE inhibitory effect, yielding the lowest IC\({}_{50}\) values (442 \(\pm\) 21 and 791 \(\pm\) 80 \(\upmu\)g/mL, respectively). The bioactivity of these extracts was in line with those obtained from the conventional SLE method and was notably higher than those obtained at increased extraction temperatures.