# Green Ultrasound-Assisted Extraction of Podophyllotoxin from _Juniperus scopulorum_ Needles
## 3 Results
### Development of a Multifactorial Approach  
Knowing that interaction between the independent variables is possible, an experimental factorial design was performed and completed by statistical analysis and 3D surface response plots [55]. This method allows the ultrasound-assisted extraction of podophyllotoxin from _Juniperus scopulorum_ needles to be optimized quickly and accurately. Preliminary experiments have shown that the four variables affecting podophyllotoxin extraction are: extraction duration, ultrasonic frequency, aqueous ethanol concentration, and extraction temperature. Nevertheless, the frequency cannot be exactly adjusted in this ultrasonic bath. The frequency can only be set to 0, 37, or 80 kHz. Therefore, the 37 kHz frequency was chosen as it provides the highest podophyllotoxin extraction yield. Hence, the experimental factorial design will be realized considering only three variables: aqueous ethanol concentration (\(X_{1}\), ranging from 60 to 90% (\(v/v\))), extraction duration (\(X_{2}\), ranging from 30 to 80 min), and extraction temperature (\(X_{3}\), ranging from 40 to 80 \({}^{\circ}\)C). The codes used for each independent variable are shown in Table 1. Moreover, based on our preliminary experiment, a concentration of 10 mg DW/L of plant material was chosen, without previous grinding. Herein, a Box-Behnken design was performed. For the experiments, eighteen different conditions were randomized using independent process variables. Each condition consists of 5 independent replicates, and the podophyllotoxin content was determined by HPLC (Table 2). The results show that the podophyllotoxin extracted from freeze-dried _Juniperus scopulorum_ needles ranges from 5.81 mg/g DW (Obs10) to 7.14 mg/g DW (Obs15). Using multiple regression analysis, the following second-order polynomial equation was obtained: Y = 6.946 + 0.012 \(X_{1}\) - 0.034 \(X_{2}\) - 0.108 \(X_{3}\) - 0.318 \(X_{1}\)\({}^{2}\) - 0.371 \(X_{1}\)\(X_{2}\) - 3.33.10\({}^{-3}\)\(\times\)\(X_{1}\)\(X_{3}\) - 0.4 \(X_{2}\)\({}^{2}\) + 0.251 \(X_{2}\)\(X_{3}\) - 0.473 \(X_{3}\)\({}^{2}\) to represent the podophyllotoxin content (Y) as a function of the different variables (\(X_{1}\), \(X_{2}\), and \(X_{3}\)). The results of the statistical analysis (Table 3) show that the quadratic coefficients \(X_{1}\)\({}^{2}\), \(X_{2}\)\({}^{2}\) and \(X_{3}\)\({}^{2}\), as well as the interaction coefficients \(X_{1}\)\(X_{2}\) and \(X_{2}\)\(X_{3}\), seem to significantly influence the extraction of podophyllotoxin from freeze-dried _Juniperus scopulorum_ needles. Concomitantly, each linear coefficient (\(X_{1}\), \(X_{2}\) and \(X_{3}\)) and the interaction coefficient \(X_{1}\)\(X_{3}\) were not significant (\(p\) > 0.05). Thus, each variable (incubation duration, temperature, and percentage of ethanol) did not have a direct impact on the podophyllotoxin extraction. However, the interaction between the variables, percentage of ethanol incubation time, and incubation time-temperature induced a significant indirect impact on the extraction yield of podophyllotoxin from the freeze-dried _J. scopulorum_. Table 4 presents the results of the variance analysis (ANOVA) and the fit of the model obtained. Considering the model presenting the predicted podophyllotoxin extraction yield versus the experimentally obtained podophyllotoxin extraction yield, the high \(F\)-value (10.82) and the low \(p\)-value (1.3 \(\times\) 10\({}^{-3}\)) show that the model is highly significant. Then, the coefficient of determination, R\({}^{2}\) (0.924), and the adjusted R\({}^{2}\) (0.839) values confirm that the model for podophyllotoxin extraction yield from freeze-dried _Juniperus scopulorum_ needles closely matches the predicted model. Also, the low value of the coefficient of variation (CV = 1.79%) demonstrates that the model fits the experimental values. According to the analysis, the 3D plots (Figure 3) clearly highlight that the extraction of podophyllotoxin from freeze-dried _Juniperus scopulorum_ needles reached a maximum.