## 3 Results
### Optimizing the PLE Polyphenol Extraction from Burdock Roots
The extracts produced using PLE at 50 \({}^{\circ}\)C and those from conventional SLE exhibited similar total phenolic content (42 \(\pm\) 4 and 38 \(\pm\) 3 mg gallic acid/g extract, respectively), antioxidant capacity (the same for DPPH, 113 \(\pm\) 1 \(\upmu\)mol Trolox/g extract), IC\({}_{50}\) values (2.85 \(\pm\) 0.08 and 1.7 \(\pm\) 0.1 mg/mL for the ORAC assay, respectively), and anticholinergic activity (IC\({}_{50}\) = 442 \(\pm\) 21 and 505 \(\pm\) 136 \(\upmu\)g/mL for AChE; and 791 \(\pm\) 80 and 751 \(\pm\) 33 \(\upmu\)g/mL  
\begin{table}
\begin{tabular}{l c c c c c} \hline
**Extraction** & **Yield (\%)** & **TPC (mg Gallic Acid/g Extract)** & **DPPH (\(\upmu\)mol Trolox/g Extract)** & **ORAC IC\({}_{50}\) (mg/mL)** & **AChE IC\({}_{50}\)** (mg/mL)** & **BChE IC\({}_{50}\) (mg/mL)** \\ \hline OE SLE & 23 \(\pm\) 1 \({}^{\mathrm{b}}\) & 38 \(\pm\) 3 \({}^{\mathrm{a}}\) & 113 \(\pm\) 1 \({}^{\mathrm{b}}\) & 1.7 \(\pm\) 0.1 \({}^{\mathrm{a}}\) & 505 \(\pm\) 136 \({}^{\mathrm{a}}\) & 751 \(\pm\) 33 \({}^{\mathrm{a}}\) \\ \hline OE PLE 50 \({}^{\circ}\)C & 27 \(\pm\) 5 \({}^{\mathrm{b}}\) & 42 \(\pm\) 4 \({}^{\mathrm{a}}\) & 113\(\pm\) 1 \({}^{\mathrm{a}}\) & 2.85 \(\pm\) 0.08 \({}^{\mathrm{b}}\) & 442 \(\pm\) 21 \({}^{\mathrm{a}}\) & 791 \(\pm\) 80 \({}^{\mathrm{a}}\) \\ \hline OE PLE 100 \({}^{\circ}\)C & 26 \(\pm\) 1 \({}^{\mathrm{b}}\) & 33 \(\pm\) 3 \({}^{\mathrm{b}}\) & 104 \(\pm\) 3 \({}^{\mathrm{b}}\) & 2.9 \(\pm\) 0.3 \({}^{\mathrm{b}}\) & 821 \(\pm\) 76 \({}^{\mathrm{b}}\) & 1151 \(\pm\) 76 \({}^{\mathrm{b}}\) \\ \hline OE PLE 150 \({}^{\circ}\)C & 45 \(\pm\) 2 \({}^{\mathrm{a}}\) & 25 \(\pm\) 2 \({}^{\mathrm{c}}\) & 98 \(\pm\) 5 \({}^{\mathrm{b}}\) & 2.3 \(\pm\) 0.1 \({}^{\mathrm{c}}\) & 912 \(\pm\) 45 \({}^{\mathrm{b}}\) & 1292 \(\pm\) 46 \({}^{\mathrm{b}}\) \\ \hline \endfor BChE, respectively). However, it is noteworthy that PLE required only 20 min of extraction time, whereas SLE needed 24 h, thereby consuming significantly more time and energy. Influence of the Extraction Temperatures on the Phenolic Profile of PLE Extracts Obtained from OLE Burdock Roots  
After qualitative analysis, quantitative differences among the extractions were determined by HPLC-DAD-IT-MS using caffeoylquinic acid standards. Figure 1 shows the HPLC-DAD chromatograms (at 280 nm, corresponding to the phenolic compounds) recovered from SLE and PLE at 50, 100, and 150 \({}^{\circ}\)C extracts of the organic burdock ecotypes. Table 2 shows ten different phenolic compounds identified in burdock roots, from which five were exhaustively identified using the following standards: chlorogenic acid (number 1, tR = 7.9 min), 4-\(O\)-caffeolquinic acid (number 2, tR = 8.32 min), cynarine (number 3, tR = 10.25 min), 3,5-dicaffeoylquinic acid (number 7, tR = 21.1 min), and 1,5-dicaffeoylquinic acid (number 8, tR = 21.7 min). Particularly, Figure S1 shows a 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. Mono-caffeoylquinic acids, which include chlorogenic acid (3-O-caffeoylquinic acid) and 4-O-caffeoylquinic acid (numbers 1 and 2; Table 2), were identified by the molecular ion at \(m/z\) 374.9 [M\(--\)H+Na]\({}^{-}\) and 465 [M\(-\)H], respectively. Chlorogenic acid presented fragment ions at \(m/z\) 190 [M\(-\)162]\({}^{-}\), corresponding to the loss of quinic acid, and a fragment ion at \(m/z\) 178 [M\(-\)174]\({}^{-}\), produced by the loss of caffe acid. Chlorogenic acid was the mono-caffeoylquinic acid with the highest content in PLE extracts (1.06 \(\pm\) 0.03 g/100 g of dried PLE extract obtained at 50 \({}^{\circ}\)C). A reduction in the concentration of chlorogenic acid was observed as the extraction temperature increased (0.893 \(\pm\) 0.007 and 0.26 \(\pm\) 0.01 g/100 g of dried PLE extract obtained at 100 \({}^{\circ}\)C and 150 \({}^{\circ}\)C, respectively).