## 4 Discussion
### Influence of PLE Temperature on the Recovery of Phenolic Compounds from Burdock Roots
Moreover, the OLE PLE extracts obtained at 50 \(\mathrm{\SIUnitSymbolCelsius}\) exhibited comparable TPC values and bioactivity to the OLE SLE extracts. However, SLE requires a longer exposure time, leading to increased phenolic degradation due to prolonged oxidative reactions and higher energy consumption. Nevertheless, [PERSON] et al. [33] observed that PLE extracts obtained from burdock roots at 100 \(\mathrm{\SIUnitSymbolCelsius}\) using 2-MTHF resulted in higher TPC values (84 mg gallic acid/g extract) than maceration (9.1 mg gallic acid/g extract). This could be due to 2-MTHF providing a protective effect against the degradation of phenolic compounds during the extraction that was not observed using the EtOH:H\({}_{2}\)O solvent in this study. In fact, the sensitivity of the principal phenolic compounds in burdock roots to extraction temperature and extended exposure during SLE was further examined through quantitative analysis using HPLC-DAD-IT-MS. The results revealed that both higher PLE temperatures and prolonged exposure during SLE resulted in a reduction in all identified phenolic compounds when EtOH:H\({}_{2}\)O was used as the extraction solvent (see Table 2). The shorter extraction times employed in PLE minimize the exposure to oxygen and thereby reduce oxidative reactions, yielding higher-quality extracts. Consequently, PLE offers a more environmentally friendly alternative, overcoming many of the drawbacks associated with SLE. Influence of Growing Conditions of Burdock Roots on the Phenolic Composition and Bioactivity of Its PLE Extracts  
It is well established that climatic conditions play a significant role in influencing the phenolic synthesis in plants [9; 10; 33; 50]. To evaluate the impact of environmental factors on the bioactivity and phenolic composition of burdock roots, the specific climatic conditions of the areas where the roots were harvested were taken into account. Notably, as shown in Figure S2, the climatic conditions in Collepardo and Isola del Liri (Italy) were consistent across the years of harvest (2021 and 2022), suggesting that the observed differences among the ecotypes were primarily attributable to altitude and the unique growing conditions of the plants [33]. Regarding the OLE and SPLE ecotypes, which were harvested at the same altitude (150 m), significant differences were observed between the two, indicating that land treatment played a crucial role in modulating the bioactivity of the burdock roots [14; 15; 16; 17; 18; 19; 20; 21]. Specifically, the exclusion of other plant species during OLE cultivation resulted in extracts with higher TPC, enhanced antioxidant capacity, as determined by the ORAC assay, and greater AChE inhibition [33]. In contrast, the untreated SPLE extracts exhibited higher antioxidant capacity, as measured by the DPPH assay, and a stronger inhibition of BChE [33]. Although the OLE extracts contained a higher concentration of phenolic compounds than the other ecotypes, these compounds may be more general in nature and less specialized in responding to biotic stress. This could be attributed to the reduced competition for resources such as light, nutrients, and water in the OLE cultivation, which leads to lower synthesis of phenolic compounds that are typically produced in response to biotic stress [19; 21; 51; 52]. In contrast, the SPLE ecotype, which was exposed to a more natural growing environment, could synthesize phenolic compounds that are more specialized for combating abiotic stress. As a result, and in agreement with [PERSON] et al. [33], the SPLE extracts demonstrated higher levels of chlorogenic acid, cynarine, and dicaffeoylanoylquinic acid compared to the OLE extracts, compounds that have demonstrated interesting bioactive properties [33]. It is probable that they are responsible for the enhanced antioxidant activity (evaluated by DPPH) and stronger BChE inhibition observed in the SPLE extracts of this study.