## 3 Materials and Methods
### Phenolic Acids and Polyphenols  
To prepare the extracts, 20 g of dried _Ilex_ leaves were transferred to a glass vial and 100 mL of pure methanol (Sigma-Aldrich, Steinheim, Germany) was added. Samples were shaken on a laboratory bench shaker for 24 h, then the primary extract was transferred to a round bottom flask, and extraction was repeated by adding another 100 mL of pure methanol. The samples were then centrifuged in a laboratory benchtop centrifuge (13,000 rpm, 10 min), and both extracts were filtered, combined, and concentrated in a laboratory evaporator. The extracts were diluted 1:200 and subjected to LC-MS analysis. The fraction prepared this way was then used for biological assays by getting the extract to the appropriate concentration in dedicated solvents (see Sections 3.6 and 3.7 for information).  
LC-MS/MS analysis was performed on a Shimadzu HPLC Prominence-i LC-2030C coupled to a triple quadrupole mass spectrometer (Shimadzu LCMS-8045). Separation of compounds in the extract was carried out on a reversed-phase column (Luna 3 \(\upmu\)m C18 100A, 150 \(\times\) 2.1 mm, Phenomenex, Torrance, Germany) at 40 \({}^{\circ}\)C. Two mobile phases were used for the analysis: water with 0.1% formic acid (eluent A) and acetonitrile (eluent B). The solvents used had a purity of >99.95%. The phase flow rate through the column was 0.35 mL/min, and the gradient was set as follows: start with 10% of solvent B, increase it to 20% over 5 min, then to 60% over 10 min, and then reduce it to 10% over 13 min and maintain it at 10% for 17 min. A 10 \(\upmu\)L volume of the sample was used for the injection. The analysis was performed in triplicate. The analysis was carried out in negative electrospray ionisation (ESI) and the sputtering voltage was set at 4000 V. Bioactive compounds were analysed in the multiple reaction monitoring (MRM) mode; the MRM analysis parameters are given in Table 7. Other MS parameters were set as follows: nebulisation gas flow 3 L min\({}^{-1}\), heating gas flow 10 L min\({}^{-1}\), interface temperature 300 \({}^{\circ}\)C, desolvation temperature 526 \({}^{\circ}\)C, heat block temperature 400 \({}^{\circ}\)C, and drying gas flow 10 L min\({}^{-1}\). Quantitative analysis was performed using pure standard substances and calibration curves. Interpretation of the results obtained was performed using the LabSolution Postrun Analysis version 5.12 software.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & **Precursor _m/z_** & **MRM Transitions _m/z_** & **Q\({}_{1}\) Pre Bias** & **Collision** & **Q\({}_{3}\) Pre Bias** \\  & **(M \(-\) H)\({}^{-}\)** & **(Q\({}_{1}\)\(\rightarrow\) Q\({}_{3}\))** & **(V)** & **Energy** & **(V)** \\ \hline \multirow{4}{*}{Ctric acid} & \multirow{4}{*}{191.400} & 191.400 \(\rightarrow\) 110.950 & 21.0 & 12.0 & 20.0 \\ \cline{3-5}  & & 191.400 \(\rightarrow\) 86.950 & 13.0 & 17.0 & 17.0 \\ \cline{3-5}  & & 191.400 \(\rightarrow\) 85.000 & 13.0 & 17.0 & 16.0 \\ \cline{3-5}  & & 133.400 \(\rightarrow\) 114.900 & 14.0 & 16.0 & 23.0 \\ \cline{3-5}  & & 133.400 \(\rightarrow\) 71.000 & 14.0 & 15.0 & 12.0 \\ \hline \multirow{4}{*}{Quinic acid} & \multirow{4}{*}{191.200} & 191.200 \(\rightarrow\) 85.2500 & 14.0 & 21.0 & 30.0 \\ \cline{3-5}  & & 191.200 \(\rightarrow\) 147.150 & 14.0 & 11.0 & 24.0 \\ \cline{3-5}  & & 191.200 \(\rightarrow\) 93.100 & 15.0 & 23.0 & 29.0 \\ \hline \multirow{4}{*}{Caffeic acid} & \multirow{4}{*}{301.200} & 301.200 \(\rightarrow\) 151.000 & 22.0 & 21.0 & 15.0 \\ \cline{3-5}  & & 301.200 \(\rightarrow\) 179.000 & 21.0 & 18.0 & 11.0 \\ \cline{1-1} \cline{3-5}  & & 301.200 \(\rightarrow\) 121.000 & 22.0 & 27.0 & 24.0 \\ \hline \hline \end{tabular}
\end{table}
Table 7: Multiple reaction monitoring (MRM) transitions and conditions of the mass spectrometer.