## 4 Discussion  
Influence of Biobased Solvent Composition and Extraction Temperature on the Recovery of Phenolic Compounds from Burdock Roots  
Although burdock roots have been associated with multiple beneficial properties due to their phenolic content, the recovery of these compounds from this plant part using advanced extraction techniques has been scarcely studied. Particularly, the majority of the studies are focused on the recovery of phenolic compounds from burdock roots by ultrasound-assisted extraction (UAE) [39; 40; 41]. To our knowledge, there is only one investigation into the use of PLE to obtain bioactive phenolic compounds from burdock roots, where [PERSON] et al. [42] indicated that PLE recovered a lower phenolic amount than UAE using conventional extraction solvents, like ethanol/water (70:30, \(v/v\)). However, [PERSON] et al. did not optimize the solvent composition and the extraction conditions for the PLE extraction, which are crucial parameters for an efficient recovery of these compounds by PLE. Additionally, several authors have reported that biobased solvents such as 2-methyloxolane, CPME, 2-MTHF, or EtOAc provide higher extraction yields from other matrices than using conventional solvents like hexane or ethanol [16; 18; 43; 44]. Usually, these biobased solvents have been used in conventional solid-liquid extractions, and little attention has been paid to their inclusion in advanced extraction techniques, such as PLE. In this context, [PERSON] et al. [16] observed a high extraction efficiency of tucoxanthin from algae when PLE was combined with ethyl acetate biobased solvent, compared to the use of ethanol. However, there are no studies on combining PLE with biobased solvents for extracting phenolic compounds from burdock roots. Thus, this study investigated the use of biobased solvents combined with PLE. The selection of CPME, 2-MTHF, and ethyl acetate biobased solvents for the study was carried out according to the positive phenolic compound extraction results achieved by different researchers [16; 18; 44; 45]. In this study, a simplex-centroid experimental design allowed us to elucidate the optimal mixture biobased composition to obtain by PLE at 40 \({}^{\circ}\)C the highest phenolic amount determined by the peak areas observed in HPLC-DAD analysis from SPLE burdock roots. This experimental design revealed that the best biobased solvent composition that reported the highest peak areas of compounds determined by HPLC-DAD at 280 nm was 100% 2-MTHF (see Figure 1). In agreement with [PERSON] et al. [45], this could be due to the capacity of 2-MTHF to form hydrogen bonds with phenolic groups compared to CPME and EtOAc, increasing the recovery of phenolic compounds. This hydrogen bond formation implies a more exothermic behavior of 2-MTHF, promoting the extraction of phenolic compounds. Although CPME can also form hydrogen bonds with phenolic groups, it was less effective than 2-MTHF. This fact could be because the 2-MTHF structure is relatively polar due to the oxygenated ring, which facilitates stronger interactions with polar compounds such as phenols. However, CPME has a hydrophobic molecular structure, and this characteristic reduces its affinity for phenolic compounds compared to 2-MTHF. Concerning EtOAc, the HPLC-DAD showed higher peak areas than CPME but lower than 2-MTHF. EtOAc is a moderately polar solvent, with a dielectric constant (\(\sim\)6.02) higher than CPME but lower than 2-MTHF (\(\sim\)7.6). This allows it to dissolve certain compounds, such as phenolic compounds, better than CPME but not with the same efficiency as 2-MTHF. On the other hand, the mixture of biobased solvent could have decreased the proportion of 2-MTHF molecules available to form hydrogen bonds with phenols and the polarity of the mixture could have moved away from the ideal for dissolving the compounds to be extracted. Thus, the use of 100% 2-MTHF provides the highest peak areas of compounds determined at 280 nm in the HPLC-DAD analysis compared to CPME and EtOAc biobased solvents.