# Green Ultrasound-Assisted Extraction of Podophyllotoxin from _Juniperus scopulorum_ Needles
## 4 Discussion
It should be noted that this content appears to be consistent with the published content: between 0.2 mg/g DW (_Juniperus communis_) and 22.6 mg/g DW (_Juniperus hermudiana_) [31]. However, compared with the extraction protocol that yielded the highest amount of podophyllotoxin from _Juniperus_ needles [31], there is no statistically significant difference. Thus, this USAE protocol is as effective as the protocol of [PERSON] et al., 2011 [31], which is the reference protocol. Furthermore, this USAE protocol only requires ethanol as the organic extraction solvent, which is an eco-friendly solvent with minor toxicity for humans, unlike the [PERSON] et al. (2011) [31] protocol, which uses methanol, a neurotoxic solvent. Also, the [PERSON] et al. (2011) [31] protocol requires 5 h of maceration at 25 \({}^{\circ}\)C after Ultraturax grinding to extract the maximum podophyllotoxin amount, while this USAE protocol does not require prior grinding and extraction lasts less than an hour. Therefore, USAE is less time-consuming, and thus more economical. Additionally, using Ultraturax, samples must be processed one by one, while the ultrasonic bath allows treating twenty samples simultaneously. Then, the [PERSON] et al. (2011) [31] protocol demonstrates a statistically significant podophyllotoxin loss as early as one hour after extraction, which can pose a problem for the pharmaceutical industry, because once extracted, the podophyllotoxin must immediately be purified. However, the protocol herein developed allows the extract to be stored for 6 days without loss of podophyllotoxin, thus giving more latitude/time for purification of the active ingredient. Furthermore, the impact of the variety and/or year of purchase of the plant on podophyllotoxin content is a result that could be expected. Indeed, [PERSON] and [PERSON] (2019) (2019) and [PERSON] et al. (2020) (2020) (2020) detected differences in the content of ultrasonically extracted phenolic compounds depending on geographical origin, cultivars, or plant species. In addition, regarding podophyllotoxin content specifically, damage to _Podophyllum peltatum_ leaves increases the podophyllotoxin content (2020). A difference has also been shown in the needles of different _Juniperus_ varieties: for instance, the _Juniperus virginiana_ variety Blue Clouds contains 384.5 mg/100 g DW, whereas the variety Kosteri contains 4.3 mg/100 g DW (2020). Additionally, podophyllotoxin is a phytoplankton, i.e., a molecule produced by the plant to protect itself and/or as a response to stress. Therefore, the content of podophyllotoxin will be modified depending on the plant's basic protective ability and/or the stresses suffered by the plant (2020, 2020). In this study, the significant variation in podophyllotoxin levels according to the year of purchase and/or variety shows the impact of the environment and genetics on the production of molecules. To maximize the interest in _J. scopulorum_ as a source of podophyllotoxin, it could be interesting to identify varieties and environmental factors that could stimulate podophyllotoxin production, along the lines of what has been conducted for one of these precursors in flax (2020) or for podophyllotoxin in _Podophyllum peltatum_, considering the way the leaves are harvested and stored (2020, 2020). In conclusion, it is important to remember that podophyllotoxin is a well-known precursor of anti-cancer drugs, and the current plant source, _Sinopodophyllum hexandrum_, is now prohibited for the extraction of podophyllotoxin. A new alternative plant source is therefore being sought. _Juniperus_ species are known to contain podophyllotoxin in their needles. Hence, we developed a protocol for the extraction of podophyllotoxin from _Juniperus scopulorum_ needles. This study focused on a green chemistry approach: ultrasound-assisted extraction to avoid environmental damage, and on commercially available _Juniperus scopulorum_ varieties to ensure the availability of the plant source.