## 5 Conclusions  
This study provides the first evidence that both the extraction temperature applied during PLE with aqueous-organic solvents and the altitude of the cultivation (150 or 800 m) site play a significant role in determining the phenolic composition and biological activity of burdock root extracts. PLE at 50 \({}^{\circ}\)C yielded extracts from the OLE ecotype with a higher total phenolic content (55 mg gallic acid/g extract) with antioxidant (113 umol Trolox/g extract for DPPH and with an IC\({}_{50}\) value of 2.85 mg/mL for the ORAC assay) and anticholinergic activities (with IC\({}_{50}\) values of 442 and 791 ug/mL for the AChE and BChE assays, respectively) than that of the employed PLE temperatures of 100 and 150 \({}^{\circ}\)C. Furthermore, burdock roots cultivated at higher altitudes (800 m) showed significantly greater phenolic content (90 mg gallic acid/g extract) and antioxidant activity (IC\({}_{50}\) ORAC assay = 0.55 mg/mL; DPPH = 526 umol Trolox/g extract) (SPME extract) compared to those grown at lower elevations (150 m) (OLE and SPLE extracts). These findings support the hypothesis that plants exposed to higher-altitude environments experience increased abiotic stress, such as lower temperatures, greater UV radiation, and reduced oxygen availability, which likely stimulates the biosynthesis of phenolic compounds as part of their adaptive response. Analysis by HPLC-DAD-IT-MS allowed the identification and quantification of ten phenolic compounds in the extracts, with 1,5-dicaffeoylquinic acid being the most abundant across all ecotypes (from 2.1 to 4.62 g/100 g of dried extract). In conclusion, this work contributes to the still-limited research examining the impact of altitude on the extraction and bioactivity of phenolic compounds from burdock roots, while also presenting valuable findings on their anticholinergic properties. This study provides preliminary knowledge that may serve as a basis for future investigations on the bioavailability and blood-brain barrier permeability of these compounds to exert their neuroprotective effects. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app15136978/s1](https://www.mdpi.com/article/10.3390/app15136978/s1)]([https://www.mdpi.com/article/10.3390/app15136978/s1](https://www.mdpi.com/article/10.3390/app15136978/s1)), Figure S1: Heatmap visualization of the abundance of compounds identified in SLE and PLE extracts obtained at 50 \({}^{\circ}\)C, 100 \({}^{\circ}\)C, and 150 \({}^{\circ}\)C by HPLC-DAD-IT-MS. Red > Orange > Yellow > Green. Figure S2: Average temperature (A), humidity (B), and days of rain (C) recorded in Collepardo in the time range of January 2020-October 2021 and January 2021-October 2022, and in Isola del Liri in the time range of January 2020-October 2021. 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., G.D.-R. and L.M. ; 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--original draft 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, funded by MCIN/AEI/10.13039/501100011033 and The European Union Next Generation EU/PRTR, as well as INCSLO0019 (Bioprotection of local agricultural resources, a way to achieve the Objectives of Sustainable development). In addition, this work was supported by the FITO-BIO project from the Sapienza University of Rome with SARANDREA Marco & C S.r.l., Fibreno Officianli, and HERBA SAPIENS companies.