## 3 Results
### Characterization of the (Poly)Phenolic Compounds of V. nigrum Extracts  
Table 3 showed the LC-DAD-MS/MS characteristics of the 22 compounds detected. In detail, (poly)phenolic compounds mostly belonged to the phenolic acid subclass of hydroxycinnamic acids, with the flavone luteolin as the only flavonoid identified in the _V. nigrum_ extracts. Luteolin, verbascoside, caffeic acid, and \(p\)-coumaric acid were unambiguously identified by comparison with their corresponding standard compounds. The exact structure of several hydroxycinnamic acid derivatives could not be assigned, but the fragment ions of caffeoyl and feruloyl groups led to their tentative identification as caffeic acid or ferulic acid derivatives. The identification of hydroxycinnamic acid derivatives was helped by DAD, as their maximum absorption peaks were close to 325 nm, a common feature for hydroxycinnamic acids. Besides (poly)phenolic compounds, other unknown compounds were detected. We argue that these species are unlikely to be (poly)phenols because of their UV absorption below 260 nm. The ion abundance of relevant compounds mentioned above is reported in Figure 5. The LC-MS/MS traces of some (poly)phenols are shown in Figure S2 and ion abundances of the unknown compounds in Figure S3. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Compound** & **R.T.** & **Amax (nm)** & **ESI Mode** &
\begin{tabular}{c} **[M-H]\({}^{-}\)/[M]\({}^{*}\)** \\ **(_m/z_)** \\ \end{tabular} & **MS\({}^{2}\) Fragment Ions (_m/z_)** \\ \hline \(p\)-Coumaric acid \({}^{*}\) & 4.83 & 309 & - & 163 & **119** \\ Caffe acid \({}^{*}\) & 4.28 & 323 & - & 179 & **135**, 107 \\ Unknown I & 0.68 & 220 & - & 278 & **113**, 139, 158, 160 \\ Luteolin \({}^{*}\) & 5.90 & 345 & - & 285 & **133**, 151, 175, 199, 217, 213 \\ Caffe acid-hexoside-dramnoside & 3.53 & 328 & - & 487 & **179**, 135, 161 \\ Ferulic acid-hexoside-dramnoside & 4.20 & 303 & - & 501 & **193**, 134, 149 \\ Unknown II & 2.09 & 220 & - & 555 & **129**, 157, 139, 365 \\ Caffe acid derivative I & 3.50 & 330 & - & 619 & **179**, 135 \\ Caffe acid derivative II & 4.97 & 324 & - & 621 & **161**, 179, 135 \\ Caffe acid derivative III & 5.07 & 324 & - & 621 & **161**, 179, 135 \\ Verhascoside \({}^{*}\) & 4.86 & 315 & - & 623 & **161**, 461, 135, 179, 133 \\ Caffe acid derivative IV & 3.15 & 328 & - & 635 & **473**, 291, 309, 141, 179, 135 \\ Caffe acid derivative V & 4.56 & 328 & - & 667 & **179**, 343, 161, 135 \\ Unknown IV & 6.73 & 241, 361 & - & 673 & **249**, 209, 307, 231, 221, 163 \\ Coumaroyd-acetylhammosyl-aucubin & 5.34 & 266 & - & 679 & **187**, 145, 163, 205, 309, 119, 499, 161 \\ Caffeoyl-acetylhammosyl-aucubin & 5.08 & 333 & - & 695 & **161**, 203, 135, 179, 325, 653, 515 \\ Forsythoside B & 4.61 & 329 & - & 755 & **593**, 161, 133, 179, 135, 447, 125 \\ Ferulic acid derivative & 5.21 & 323 & - & 783 & **175**, 193, 149, 125, 160, 134 \\ Unknown V & 2.41 & 220 & - & 929 & **455**, 353, 689, 809, 293, 581, 767, 191, 113 \\ Unknown VI & 2.86 & 340 & - & 987 & **467**, 365, 701, 323, 729, 353, 305, 191, 941 \\ Unknown VII & 3.81 & 252 & + & 557 & **131**, 203, 395 \\ \hline \hline \end{tabular} \({}^{*}\) Unambiguous identification by comparison with the corresponding standard compound. The fragment ions have been reported in decreasing order of ion abundance. The base MS\({}^{2}\) ions used in the MRM analysis are shown in bold.  
\end{table} 
Table 3: LC-DAD-MS/MS characteristics of the main (poly)phenolic/phytochemical compounds identified in _Verhascum nigrum_. Figure 5: _Cont._  
The data reported in Figure 5 show that verbascoside levels were lower in all samples compared to the control sample 29, while luteolin remained at similar levels in all the samples except for sample 65, where a drastic reduction was observed, indicating that a subsequent hydroalcoholic extraction could be beneficial in increasing the extraction yield.