## 4 Discussion
### Influence of PLE Temperature on the Recovery of Phenolic Compounds from Burdock Roots
Despite these differences, the bioactivities of the OLE and SPLE extracts were relatively similar, as reflected in the PCA, which grouped both ecotypes in the same cluster (see Figure 3). Differences in the individual quantification of phenolic compounds using HPLC-DAD-IT-MS, TPC values, and bioactivity assays among the ecotypes revealed that burdock's secondary metabolism is influenced by both growing conditions and land treatment. Notably, the higher altitude, atmospheric pressure, cold temperatures, and distinct soil conditions of the SPME ecotype compared to the OLE and SPLE may contribute to its increased phenolic content as a response to these environmental stressors, which is in agreement with that observed by [PERSON] et al. [53] in different species of ephedra collected at different altitudes. Indeed, previous studies have shown that plants growing at high altitudes are more exposed to ultraviolet (UV) radiation, increased atmospheric pressure, and cold temperatures, which trigger the synthesis of phenolic compounds as a protective mechanism against these harsh conditions [14; 15; 16; 17; 18; 19; 20; 21]. Consistent with this, other researchers have found that plants originating from cold climates and high altitudes tend to have higher phenolic contents [50]. These results agree with [PERSON] et al. [33], who observed the same behavior for OLE, SPLE, and SPME extracts obtained by PLE using 2-MTHF. This consistent superiority of the SPME ecotype across different extraction techniques may be attributed to its intrinsically higher concentration and diversity of bioactive phenolic compounds, which remain stable or are more efficiently recovered regardless of the extraction conditions applied. However, it was noted that PLE with 2-MTHF at 100 \({}^{\circ}\)C provided the SPLE, OLE, and SPME burdock root extracts with higher phenolic content (22.9, 48.8, and 64 mg gallic acid/g extract, respectively) and bioactivity (IC\({}_{50}\) of ORAC assay from 3.1 to 9 mg/mL extract) than the extracts obtained by PLE with EtOH:H\({}_{2}\)O (70:30, \(v/v\)) at 50 \({}^{\circ}\)C.  
The majority of the phenolic compounds identified in the extracts were caffeoylquinic acids, characterized by their anti-inflammatory, antiviral, antidiabetic, and antitumor properties [54]. In addition, dicaffeoylquinic acid isomers were also identified, which can be differentiated using the standards. In agreement with a previous study, 1,5-dicaffeoylquinic acid (number 8) was identified as the major phenolic compound in the extracts of burdock leaves [55]. The findings from this study align with data from previous research, including prior studies where the same burdock samples were characterized using Nuclear Magnetic Resonance (NMR) spectroscopy and HPLC-DAD-IT-MS [33; 56]. In these studies, [PERSON] et al. [57] and [PERSON] et al. [33] observed that burdock ecotypes harvested at an altitude of 800 m exhibited the highest concentrations of sugars, amino acids, organic acids, and other bioactive compounds, including dicaffeoylquinic acid. In the current study, it was noted that higher altitudes were associated with the increased synthesis of dicaffeoyldimaloylquinic acid, dicaffeoylmaloylquinic acid, and 1,5-dicaffeoylquinic acid in burdock roots. These compounds were identified as the primary contributors to the antioxidant and anticholinergic capacities of the SPME extract, as shown in Figure 3.