# Green Ultrasound-Assisted Extraction of Podophyllotoxin from _Juniperus scopulorum_ Needles
## 3 Results  
### Preliminary Single Factor Experiments  
Single-factor experiments were chosen to evaluate the relative impact of different conditions on the extraction yield of podophyllotoxin from _Juniperus scopulorum_ needles. Previously, we developed an HPLC separation gradient enabling precise identification and quantification of podophyllotoxin (Figure 1). The impact of different parameters identified as important in the literature was tested: extraction duration, ultrasound frequency, ethanol concentration, solvent/material ratio, prior grinding, and extraction temperature. These preliminary single-factor experiments determined the main limiting factors. In the second stage, in order to measure the direct impact and the interaction effect between these parameters, they will be applied in the design of the experiments. On the other hand, as the objective is to develop a green extraction protocol, an extraction solvent suitable for green chemistry must be chosen. Conventionally, different organic solvents can be used to extract podophyllotoxin from _Juniperus_ needles: methanol, ethanol, and chloroform [31]. Ethanol appears to be the best choice because it is non-toxic, has minimal environmental impact, and can be mixed with water to improve its ability to solubilize podophyllotoxin. As there is no protocol for podophyllotoxin USAE, we determine the parameter ranges to test based on the secoisolariciresinol USAE study [49]. For secoisolariciresinol, the optimal content is obtained with an extraction time of 60 min and a sample/solvent concentration of 5 mg/mL. Therefore, for the podophyllotoxin USAE, we tested an extraction time of up to 80 min and a sample/solvent concentration from 5 to 20 mg/mL. As the optimal solvent concentration was not exactly determined for secoisolariciresinol USAE, we tested from 40% to 100% ethanol, considering podophyllotoxin solubility. For the other parameters, we tested the largest condition allowed by the apparatus (temperature between 20 and 80 \({}^{\circ}\)C, frequencies equal to 0, 37, or 80 KHz). \begin{table}
\begin{tabular}{c c c c c c} \hline
**Run ID** & **Run Order** & \(\mathbf{X_{1}}\) & \(\mathbf{X_{2}}\) & \(\mathbf{X_{3}}\) & **PT (mg/g DW)** \\ \hline Obs1 & \(11\) & \(-1\) & \(-1\) & 0 & 5.9913053 \\ Obs2 & \(6\) & \(1\) & \(-1\) & 0 & 6.51152504 \\ Obs3 & \(9\) & \(-1\) & \(1\) & 0 & 6.69284701 \\ Obs4 & \(15\) & \(1\) & \(1\) & 0 & 5.72977268 \\ Obs5 & \(14\) & \(-1\) & 0 & \(-1\) & 6.20516809 \\ Obs6 & \(4\) & \(1\) & 0 & \(-1\) & 6.48178819 \\ Obs7 & \(12\) & \(-1\) & 0 & 1 & 5.83502077 \\ Obs8 & \(10\) & \(1\) & 0 & 1 & 6.09834046 \\ Obs9 & \(17\) & \(0\) & \(-1\) & \(-1\) & 6.40098953 \\ Obs10 & \(8\) & \(0\) & \(1\) & \(-1\) & 5.80521013 \\ Obs11 & \(5\) & \(0\) & \(-1\) & 1 & 5.84521745 \\ Obs12 & \(18\) & \(0\) & \(1\) & 1 & 6.25163654 \\ Obs13 & \(3\) & \(0\) & 0 & 0 & 7.07106035 \\ Obs14 & \(1\) & \(0\) & 0 & 0 & 6.88392753 \\ Obs15 & \(16\) & \(0\) & 0 & 0 & 7.13525193 \\ Obs16 & \(2\) & \(0\) & 0 & 0 & 6.77948881 \\ Obs17 & \(13\) & \(0\) & 0 & 0 & 7.01239831 \\ Obs18 & \(7\) & \(0\) & 0 & 0 & 6.79463394 \\ \hline \end{tabular}
\end{table} 
Table 2: Results of experimental design. A preliminary experiment was conducted with several extraction durations (10, 20, 30, 40, 60, and 80 min) using fixed ultrasound frequencies (37 kHz), the aqueous ethanol concentration (80%), the plant material concentration (10 g DW/L), and temperature (40 \({}^{\circ}\)C), but without prior grinding. The results presented in Figure 2a highlight that increasing the extraction duration improves the podophyllotoxin extraction yield by up to 40 min and remains at this maximum for at least 80 min. The sonication frequency can also influence extraction. Three ultrasound frequencies (0, 37, and 80 kHz) were tested with a fixed extraction duration (40 min), aqueous ethanol concentration (80%), plant material concentration (10 g DW/L), and temperature (40 \({}^{\circ}\)C), but without prior grinding.