## 4 Discussion
### Bioactive, Trepenoid, and Phenolic Profile Comparison Among Different Ecotypes of Burdock Roots  
Several authors have recognized that growing conditions impact the synthesis of phenolic compounds in plants, and with them, the bioactivity of their extracts [48,49]. However, the influence of the growing conditions of burdock roots on their bioactivity and phenolic profile has scarcely been studied. Considering that the climatic conditions in 2021 and 2022 in the collection areas of the burdock roots (Collepardo and Isola del Liri) were the same, based on the meteorological information reported by The National Meteorological Service of Italy (ItaliaMeteo), the differential factors among the ecotypes were the weeding treatment and the altitude where the plant was seeded. An important influence of the weeding treatment was observed between the ecotypes collected at the same altitude (150 m). When the plant was subjected to weeding treatment (OLE extracts), its extracts exhibited higher TPC and antioxidant capacity values than the plants subjected to non-weeding treatment (SPLE extracts). This reflected that when plants are subjected to weeding (removal of weeds or nearby competitors), they may experience less competition for resources such as light, water, and nutrients. This less competitive environment allows plants to focus more of their energy and resources on their secondary metabolism, which includes the production of phenolic compounds. These compounds, in addition to acting as antioxidants, may serve as defense mechanisms against other environmental stressors [50]. However, the SPLE extracts presented a higher anticholinergic capacity than the OLE extracts. This could be because, in an environment with competition (like for SPLE burdock roots without weeding), plants are under greater biotic and abiotic stress. Under these conditions, they may prioritize the synthesis of specific secondary metabolites that are related to chemical defense, such as compounds with anticholinergic activity. These compounds are usually produced as a defense strategy against herbivores or pathogens, since they interfere with the nervous systems of the organisms that attack them [50,51]. In fact, most insecticides used in the world are based on inhibitors of AChE. As a result, there is a loss of AChE activity, leading to overstimulation of the insect's effector organ. Once a critical proportion of the enzyme tissue mass is deactivated, the symptoms and signs of cholinergic poisoning emerge, ultimately resulting in the insect's death [52]. In this sense, when a plant has not been weeded, as in SPLE, it is more attractive to herbivorous animals. As a defense mechanism, the plant can synthesize anticholinergic compounds to cause a buildup of acetylcholine, resulting in overstimulation of the muscles, paralysis, and, in severe cases, the death of the herbivore. This makes plants containing these compounds less attractive or even toxic to herbivores [51]. This could explain why the OLE extract presented higher antioxidant capacity and SPLE had higher anticholinergic capacity. However, the PCA grouped both extracts together, because they presented bioactivities and GC-MS and HPLC-MS profiles that were not very different from each other but were different to SPME (see Figure 3). This fact suggests that the content of bioactive compounds in burdock roots depends primarily on the altitude at which the plant grows, not on the weeding treatment. Higher TPC values and antioxidant and anticholinergic capacities were observed in the SPME extracts from the burdock roots collected at 800 m compared to the OLE and SPLE extracts from the burdock roots collected at 150 m. Various studies have shown that at higher altitudes, plants are exposed to increased UV radiation, reduced atmospheric pressure, and colder temperatures [53; 54; 55]. To protect themselves from the damage caused by these conditions, they produce bioactive phenolic compounds as a defensive response, including antioxidant and anticholinergic compounds.