## 3 Results
### Extraction of Bioactive Phenolic Compounds from Different Ecotypes of Burdock Roots  
The optimized PLE conditions, specifically the extraction temperature of 50 \({}^{\circ}\)C, established using OLE burdock roots, were applied to extract bioactive molecules from the SPME and SPLE ecotypes. This approach allowed for the comparison of three burdock root ecotypes, each harvested from different altitudes (150 m and 800 m). Differences in TPC and bioactivity were observed among the ecotypes (see Table 3). As shown, the ecotype harvested at the higher altitude of 800 m (SPME) exhibited the highest TPC values (90 \(\pm\) 7 mg gallic acid/g extract), along with superior anticholinergic (IC\({}_{50}\) = 313 \(\pm\) 4 and 298 \(\pm\) 21 \(\mu\)g/mL for the AChE and BChE assays, respectively) and antioxidant activities (526 \(\pm\) 138 \(\mu\)mol Trolox/g extract for the DPPH assay and an IC\({}_{50}\) value of 0.55 \(\pm\) 0.08 mg/mL for the ORAC assay), when compared to both the OLE and SPLE ecotypes, which were collected at 150 m.  
As shown in Table 3, the OLE extract exhibited higher TPC (55 \(\pm\) 1 mg gallic acid) and greater AChE inhibition (IC\({}_{50}\) = 442 \(\pm\) 5 \(\mu\)g/mL) than the SPLE extract (32 \(\pm\) 3 mg gallic acid and IC\({}_{50}\) = 791 \(\pm\) 80 \(\mu\)g/mL). Conversely, the SPLE extract demonstrated superior antioxidant and anticholinergic activities, as assessed by the ORAC (IC\({}_{50}\) = 1.09 \(\pm\) 0.06 mg/mL) and BChE (IC\({}_{50}\) = 527 \(\pm\) 33 \(\mu\)g/mL) assays, respectively. Furthermore, a comparative phenolic profile analysis and quantification were conducted among the ecotypes using HPLC-DAD. As depicted in Figure 2 and summarized in Table 4, it was observed that chlorogenic acid, cynarine, 1,5-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid were present at concentrations more than twice as high in the SPME extract compared to both the OLE and SPLE extracts. Notably, 4-O-caffeoylquinic acid was the only phenolic compound found at a higher concentration in the SPLE extract (0.020 \(\pm\) 0.002 g/100 g of dried extract). In addition, differences were observed between the OLE and SPLE extracts. Specifically, the SPLE extracts contained higher amounts of 4-O-caffeoylquinic acid and dicaffeoyl-maloylquinic acid (0.020 \(\pm\) 0.002 and 0.30 \(\pm\) 0.01 g/100 g of dried extract, respectively), whereas the OLE extracts had greater concentrations of dicaffeoylmaloylquinic acid isomer 1, 3,5-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid, and dicaffeoylmaloylquinic acid (0.13 \(\pm\) 0.02, 0.145 \(\pm\) 0.007, 2.1 \(\pm\) 0.1, and 0.30 \(\pm\) 0.02 g/100 g of dried extract, respectively) (see Table 4). \begin{table}
\begin{tabular}{c c c c c c} \hline
**Ecotype** & **Yield** & **TPC (mg Gallic** & **ORAC IC\({}_{\mathbf{50}}\)** & **DPPH (\(\mathbf{\mu }\)mol** & **AChE IC\({}_{\mathbf{50}}\)** & **BChE IC\({}_{\mathbf{50}}\)** \\  & **(\%)** & **Acid/g Extract** & **(mg/mL)** & **Trolox/g Extract** & **(\(\mathbf{\mu }\)g/mL)** & **(\(\mathbf{\mu }\)g/mL)** \\ \hline OLE & 27 \(\pm\) 5 \({}^{\mathrm{b}}\) & 55 \(\pm\) 1 \({}^{\mathrm{a}}\) & 2.85 \(\pm\) 0.08 \({}^{\mathrm{c}}\) & 113 \(\pm\) 2 \({}^{\mathrm{c}}\) & 442 \(\pm\) 5 \({}^{\mathrm{a}}\) & 791 \(\pm\) 80 \({}^{\mathrm{c}}\) \\ \hline SPLE & 32 \(\pm\) 3 \({}^{\mathrm{b}}\) & 42 \(\pm\) 2 \({}^{\mathrm{b}}\) & 1.09 \(\pm\) 0.06 \({}^{\mathrm{b}}\) & 268 \(\pm\) 24 \({}^{\mathrm{b}}\) & 975 \(\pm\) 11 \({}^{\mathrm{b}}\) & 527 \(\pm\) 33 \({}^{\mathrm{b}}\) \\ \hline SPME & 38 \(\pm\) 3 \({}^{\mathrm{a}}\) & 90 \(\pm\) 7 \({}^{\mathrm{a}}\) & 0.55 \(\pm\) 0.08 \({}^{\mathrm{a}}\) & 526 \(\pm\) 138 \({}^{\mathrm{a}}\) & 313 \(\pm\) 4 \({}^{\mathrm{a}}\) & 298 \(\pm\) 21 \({}^{\mathrm{a}}\) \\ \hline \end{tabular}
\end{table}