## 2 Materials and Methods
### Analysis of Z. officinal Extracts by UPLC-DAD and UPLC-ESI-MS  
A Thermo PDA e\(\lambda\) photodiode array detector (DAD) coupled with a Thermo Ultra-Mate U3000 UPLC system was used for quantification of five gingerols contents, namely 6-gingerol, 8-gingerol, 10-gingerol, 6-gingerdiol and 6-dihydroingerdione, and 6-shogal in the different extracts (\(n\) = 17) under the conditions and the calibration curve described in Section 2.2. On the other hand, to characterize _Z. officinal_ polyphenols, measurements were performed with a method developed in our laboratory, using a Xevo G2-XS QTOF (Waters, Wilmslow, UK) coupled with an AQUITY H Class UPLC system with a quaternary pump. ESI source parameters were set to a capillary voltage of 2 kV, sampling cone of 20 eV, source temperature 150 \({}^{\circ}\)C, source offset 10 \({}^{\circ}\)C, desolvation temperature of 450 \({}^{\circ}\)C, cone gas flow 0 L/h, and desolvation gas flow 900 L/h. The measure was performed in MSe high-resolution negative mode using an acquisition mass range from 100 m/z to 1000 m/z and a scan rate of 0.5 s, where fragmentation was carried out using Independent Data Acquisition for all eluting compounds with collision energy ramp from 20 V to 30 V storing  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Experiment** & **Solvent** & **Volume (mL)** & **Stirring Time (hours)** & **TG [1; 2; 3]** \\ \hline
1 & Ethanol & 25 & 8 & 19.38 \({}^{\mathrm{b}}\)\(\pm\) 1.1 \\
2 & Methanol & 50 & 24 & 27.79 \({}^{\mathrm{ab}}\)\(\pm\) 2.0 \\
3 & Methanol & 50 & 8 & 30.40 \({}^{\mathrm{a}}\)\(\pm\) 2.4 \\
4 & Ethanol & 25 & 24 & 19.50 \({}^{\mathrm{b}}\)\(\pm\) 0.9 \\
5 & Ethanol & 50 & 24 & 26.01 \({}^{\mathrm{ab}}\)\(\pm\) 2.3 \\
6 & Ethanol & 50 & 8 & 20.16 \({}^{\mathrm{b}}\)\(\pm\) 1.5 \\
7 & Methanol & 25 & 24 & 21.20 \({}^{\mathrm{ab}}\)\(\pm\) 1.3 \\
8 & Methanol & 25 & 8 & 21.93 \({}^{\mathrm{ab}}\)\(\pm\) 1.4 \\ \hline \hline \end{tabular} \({}^{\dagger}\)TG: Total Gingerol contents are expressed as mg/g dry material \({}^{2}\) TG values are expressed as mean \(\pm\) standard deviation. \({}^{3}\) Different superscript letters indicate differences are significant at \(p\) < 0.05 using one-way analysis of variance (ANOVA) with a Tukey post hoc as statistical test.  
\end{table}
Table 1: UPLC-DAD quantification of 6-gingerol, 8-gingerol and 10-gingerol in _Z. officinal_ sample NR-1 under various solvents, extraction volumes and stirring time conditions.  
at the high energy function. Instrument calibration was performed in the mass range of the measurement with sodium formate. Lock mass correction was applied directly to the measurement using leucine enkephalin infusion measured each 30 s during the run. The data was analyzed using MassLynx V4.2 software from Waters. One \(\upmu\)L of sample was injected with a flow of 0.5 mL/min using a using a Synergi 4u Polar-RP80A (150 \(\times\) 4.6 mm \(\times\) 4 \(\upmu\)m) (Phenomenex Inc., Torrence, CA, USA) at 30 \({}^{\circ}\)C using a chromatographic gradient starting at 40% B increasing to 100% B at 30 min, the gradient was held for 7 min and then the column was equilibrated for 5 min to initial conditions. Solvents used in the mobile phase were A water with 0.1% formic acid and B acetonitrile with 0.1% formic acid.