## 5 Conclusions  
This study introduced a novel extraction strategy using PLE with biobased solvents to simultaneously obtain non-polar and polar compounds with antioxidant and anticholinergic capacities from burdock roots. It also evaluated the impact of weeding and altitude on bioactive composition across three burdock ecotypes: two collected at 150 m (one weeded during growth: OLE; the other not weeded: SPLE) and a third at 800 m without weeding (SPME). Using a simplex-centroid mixture design, 100% 2-MTHF was identified as the best solvent for extracting compounds with high bioactivity from SPLE, thanks to its polarity and strong hydrogen bonding with phenolic compounds. The optimal extraction temperature was 100 \({}^{\circ}\)C, balancing bioactive compound recovery without yield loss or undesirable reactions observed at 40 \({}^{\circ}\)C and 160 \({}^{\circ}\)C. Extracts from all the ecotypes were compared to assess the influence of altitude and weeding. Weeding during plant growth reduced anticholinergic capacity but enhanced antioxidant compound production (e.g., dicaffeoylquinic acid, 2-methylphenol). However, altitude had the most significant impact: the SPME extracts (800 m) showed the highest bioactivity and greater amounts of non-polar and polar compounds identified via GC-MS and HPLC-IT-MS. The key compounds contributing to bioactivity included caffeoylquinic acids, cyanophyllene oxide, spathulenol, and bisnorallocholanic acid. In conclusion, the results confirmed the hypothesis raised, since PLE extraction with biobased solvents was more efficient in the recovery of bioactive compounds from burdock roots. In addition, to obtain extracts with the highest level of antioxidant and neuroprotective properties from burdock roots, plants should be harvested at high altitudes and without weeding during growth. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app15052491/s1](https://www.mdpi.com/article/10.3390/app15052491/s1)]([https://www.mdpi.com/article/10.3390/app15052491/s1](https://www.mdpi.com/article/10.3390/app15052491/s1)), Table S1: Coefficients of the quadratic model obtained from the simplex-centroid mixture design for the PLE with CPME, EtOAc, and 2-MTHF biobased solvents that best fitted the response (total peak area of chromatogram achieved at 280 nm by HPLC-DAD) with the extraction parameters (% biobased solvents). Figure S1. HPLC-DAD chromatograms at 280 nm of phenolic compounds recovered from SPLE by PLE at 40, 100, and 160 \({}^{\circ}\)C, and by maceration. Figure S2. GC-MS chromatograms of non-polar compounds recovered from SPLE, OLE, and SPME ecotypes by PLE at 100 \({}^{\circ}\)C from burdock roots. * Numbers correspond to the identified compounds by MS (see Table 3). Figure S3. HPLC-DAD chromatograms at 280 nm of phenolic compounds recovered from SPLE, OLE, and SPME ecotypes by PLE at 100 \({}^{\circ}\)C from burdock roots. * Numbers correspond to roots. * Numbers correspond to the identified compounds by MS (see Table 4). Conceptualization, E.R., G.D.-R., E.I. and A.C.; methodology, E.R. and G.D.-R.; validation, G.D.-R., A.C. and E.I. ; formal analysis, E.R. and G.D.-R.; investigation, E.R., G.D.-R., L.M., A.C. and E.I. ; resources, L.M., A.C. and E.I. ; data curation, G.D.-R. and E.I. ; writing--originaldraft preparation, E.R. and G.D.-R.; writing--review and editing, E.R., G.D.-R., L.M., A.C. and E.I. ; visualization, G.D.-R., L.M. and E.I. ; supervision, G.D.-R., A.C. and E.I. ; project administration, L.M., A.C. and E.I. ; funding acquisition, L.M., A.C. and E.I. All authors have read and agreed to the published version of the manuscript. This research was supported by projects PID2020-113050 RB-I00 and PDC2021-120814-I00 and funded by MCIN/AEI/10.13039/501100011033 and The European Union's NextGenerationEU/PTRR, as well as INCGLO0019 (Bioprospection of local agricultural resources, a way to achieve the Objectives of Sustainable Development).