# Green Ultrasound-Assisted Extraction of Podophyllotoxin from _Juniperus scopulorum_ Needles
## 4 Discussion  
The preliminary experiment highlights the involvement of ultrasound frequency in the podophyllotoxin extraction process (Figure 2b). The literature explains that ultrasound frequency can improve the solubility of podophyllotoxin in the extraction solvent, due to the cavitation effect and the modification of the diffusion coefficient of the targeted  
Figure 5: Comparison of podophyllotoxin content in _Juniperus scopulorum_ needles obtained using the optimized extraction protocol vs. [PERSON] et al., 2011 protocol [31]. Values are means \(\pm\) SD of 5 independent replicates. Different letters represent significant differences between the various extraction conditions (\(p<0.05\)). Figure 6: Stability study of podophyllotoxin content in _Juniperus scopulorum_ needles obtained using the optimized extraction protocol. Values are means \(\pm\) SD of 3 independent replicates. Different letters represent significant differences between the various extraction conditions (\(p<0.05\)). molecule. Up to 60 kHz, the frequency does not affect the stability of phenolic compounds after their extraction [50]. However, at 80 KHz, a significant impact is observed. [PERSON] et al. (2015) [56] also measured a decrease in total phenolic compound concentration in mulberry above and below 33.82 KHz. It has already been established that the high frequency of ultrasound can lead to the degradation of phenolic compounds and, therefore, also of podophyllotoxin. [51; 52]. Additionally, a similar study conducted on secoisolaritiesinol, a podophyllotoxin precursor, showed better extraction yield at 30 kHz than at 45 kHz [49], illustrating the negative impact of high frequencies on lignans. Thus, using an ultrasound frequency of 37 kHz allows a better extraction yield of podophyllotoxin compared to 0 and 80 kHz. The multifactorial approach demonstrates that the interaction between the percentage of ethanol and incubation duration is involved in the extraction of podophyllotoxin from _Juniperus scopulorum_ needles. Indeed, incubation duration has a significant impact: the longer the treatment lasts, the more cavitation bubbles emerge and collapse, and the more the proportion of molecules released from cells increases [45; 47]. Concomitantly, in order to release the molecules from the cell, it is essential to solubilize the molecule. De facto, the percentage of ethanol will unduly affect the extraction yield of the podophyllotoxin. Moreover, the percentage of ethanol modifies the viscosity of the solvent, whereas the cavitation threshold depends on the viscosity of the solvent [47; 57]. A modification of the cavitation threshold will lead to a modification of the incubation duration, resulting in cell perforation. Then, the collapse of the cavitation bubble is more intense at low vapor pressure, which depends on the percentage of ethanol [58]. Thus, this interaction and its impact on podophyllotoxin extraction yield seem obvious. The interaction of temperature and incubation duration has a significant favorable impact on the extraction yield of podophyllotoxin from _Juniperus scopulorum_ needles. Temperature decreases solvent viscosity and surface tension, leading to attenuating the effects of sonication [58]. Then, a positive effect of temperature is associated with an increase in cavitation bubbles [59]. As the temperature can increase the number of cavitation bubbles, the extraction duration will define how long these numerous bubbles will act (perforate the cell to extract the podophyllotoxin), explaining the benefit of the interaction between temperature and incubation duration. In this study, the optimized protocol was established to extract up to 10.34 mg podophyllotoxin/g DW from freeze-dried _Juniperus scopulorum_ needles. Protocols with podophyllotoxin extraction yields of 8.5 mg/g DW [33] and 4.9 mg/g DW [32] have been reported in the literature, but this amount of podophyllotoxin has never been extracted from _Juniperus scopulorum_ needles.