## 4 Discussion
### Influence of PLE Temperature on the Recovery of Phenolic Compounds from Burdock Roots
In contrast, 2-MTHF, a less polar and more thermally stable solvent, provides a protective environment that minimizes degradation, even at 100 \({}^{\circ}\)C, which is likely due to reduced water content, lower radical formation, and the selective extraction of phenolic compounds without co-extracting pro-oxidant components [33]. The observed differences in antioxidant capacity among the extracts can be attributed not only to their phenolic composition, but also to the specific assay employed for evaluation, as each method relies on distinct mechanisms of action. Phenolic compounds, due to their structural features, particularly the presence of multiple hydroxyl groups and conjugated systems, can act as antioxidants through various pathways. However, their measured antioxidant activity may vary significantly depending on whether the assay favors electron transfer (ET) or hydrogen atom transfer (HAT) reactions. [38]. In this study, the PLE extract obtained at 50 \({}^{\circ}\)C exhibited the highest antioxidant activity when assessed using the DPPH assay, which is based on an ET mechanism. This method measures the ability of antioxidant compounds to transfer an electron to the DPPH radical, thereby stabilizing it [48]. In contrast, the SLE extract demonstrated superior antioxidant performance in the ORAC assay, which operates via a HAT mechanism. The ORAC method evaluates the ability of antioxidants to quench peroxyl radicals (ROO\(\bullet\)) through hydrogen atom donation, a mechanism that closely mimics the oxidative processes occurring in biological systems, as peroxyl radicals are among the most prevalent reactive oxygen species in the human body [48]. These findings highlight that the apparent antioxidant capacity of a given extract is highly dependent on the assay used, due to the differing radical species involved and the dominant reaction mechanism. While DPPH is useful for assessing electron-donating capacity, ORAC provides a more physiologically relevant estimate of antioxidant potential, as it reflects the extract's ability to neutralize biologically relevant radicals. Therefore, relying on a single method may offer an incomplete or misleading assessment of antioxidant efficacy. The combined use of both ET- and HAT-based assays allows for a more comprehensive understanding of the antioxidant behavior of phenolic compounds under conditions that approximate those found in vivo [33; 49]. Additionally, comparing extraction solvents, ethanolic PLE extracts exhibited higher antioxidant capacity determined by an ORAC assay (IC\({}_{50}\) from 0.55 to 2.85 mg/mL extract) than 2-MTHF PLE extracts (IC\({}_{50}\) from 3.1 to 9 mg/mL extract) obtained by [PERSON] et al. [33] from burdock roots. However, the 2-MTHF extracts presented higher anticholinergic capacity determined by AChE (IC\({}_{50}\) from 29 to 94 \(\mathrm{\SIUnitSymbolMicro}\)/mL) and BChE assays (IC\({}_{50}\) from 43 to 83 \(\mathrm{\SIUnitSymbolMicro}\)/mL) than the ethanolic extracts of this study (AChE IC\({}_{50}\) from 313 to 442 \(\mathrm{\SIUnitSymbolMicro}\)/mL, BChE IC\({}_{50}\) from 298 to 791 \(\mathrm{\SIUnitSymbolMicro}\)/mL). This difference may be explained by the chemical selectivity of the solvents. EtOH/H\({}_{2}\)O mixtures tend to extract polar phenolic compounds, many of which are known for their strong antioxidant activity, particularly through hydrogen atom donation mechanisms measured by the ORAC assay [41]. In contrast, 2-MTHF is a less polar, more lipophilic solvent that favors the extraction of less polar phenolics and other bioactive compounds, such as certain flavonoid aglycones or terpenoids, which may exhibit stronger inhibitory effects on cholinesterase enzymes (AChE and BChE) [33]. These nonpolar or moderately polar compounds might not contribute significantly to antioxidant capacity in ORAC assays, but they can be more effective in targeting enzymatic active sites, thereby explaining the higher anticholinergic activity observed in the 2-MTHF extracts [33].