# Green Ultrasound-Assisted Extraction of Podophyllotoxin from _Juniperus scopulorum_ Needles
## 1 Introduction  
Podophyllotoxin is a lignan, a polyphenol, a natural plant metabolite (Figure 1) [1] isolated in the 19 th century [2; 3; 4; 5] and presenting remarkable properties: mitotoxic, neurotoxic, insecticidal, antimicrobial, anti-inflammatory, antispasmodic, hypolipidemic, immunosuppressive, antioxidative, analgesic, and cathartic activities [6]. Furthermore, podophyllotoxin is the unique molecule used as a precursor for the hemisynthesis of some anticancer drugs: etoposide, teniposide, or etopophos with topoisomerase II inhibition ability [7; 8; 9; 10; 11; 12; 13; 14; 15; 16]. These anticancer drugs are involved in the treatment of certain cancers, including lung, breast, testicular, gastric, and blood [17; 18; 19]. The literature also attributes antiviral activity to podophyllotoxin against _Herpes simplex_ type 1 [20], to some derivatives against HIV [21], and etoposide can fight cytokin storms in patients infected with the COVID-19 virus [22]. Despite its clinical importance, the supply of podophyllotoxin is currently problematic. Podophyllotoxin is classically extracted from the rhizomes of _Podophylllum peltatum_ or _Sinopodophylllum hexandrum_ [PERSON] [1; 23], yielding up to 4% DW podophyllotoxin [24]. However, with slow regeneration and without organized cultivation, the _Sinopodophylllum hexandrum_ Royle overexploitation (due to its podophyllotoxin) has led the plant tobeing classified as \"critically endangered\", in Appendix 0.2 of The Convention on International Trading of Endangered Species (CITES). Although complete chemical synthesis of podophyllotoxin is possible, the complex structure makes it economically unviable [15]. Thus, since 2018, there has often been a shortage of etoposide injections, a problem most often solved by switching to topophos injections [25]. However, without podophyllotoxin available, there is no way to produce etoposide or etopophos, and consequently, cancer patients will no longer be able to receive the treatment they vitally need. Therefore, to continue to ensure the future podophyllotoxin needs, one promising idea is to find other biological materials as plant sources containing a significant amount of podophyllotoxin. The _Juniperus_ genus appears to be interesting as an alternative natural source of podophyllotoxin. The occurrence of cytotoxic lignan (especially podophyllotoxin) has been described in the needles of _Juniperus_ species since 1953 [26], then confirmed in _Juniperus virginiana_[27; 28; 29; 30; 31; 32; 33], _Juniperus_\(\times\)_media_[31; 32; 33; 34], _Juniperus rigida_[35; 36; 37], _Juniperus horizontalis_[33; 38], _Juniperus subina_[31; 34; 39], and other species including _Juniperus scopulorum_[31; 32; 33]. The most interesting species is _Juniperus hermudiana_, with 22.6 mg podophyllotoxin/g DW [31]. However, this species is endangered and protected by the International Union for Conservation of Nature (\"[[https://www.iucn.org](https://www.iucn.org)]([https://www.iucn.org](https://www.iucn.org))\" (accessed on 2 November 2023)). Although _Juniperus needles_ don't contain as much podophyllotoxin as _Sinopodophylllum hexandrum_ rhizomes, some _Juniperus_ yet contain around 2% DW podophyllotoxin [31], which is the most credible amount found in an alternative natural source. In addition, some _Juniperus_ species are suitable for cultivation, providing a greater amount of plant material than _Podophyllum_[33]. _Juniperus_ is therefore a potentially creditable source of podophyllotoxin. Among all the _Juniperus_ species, _Juniperus scopulorum_ has not been extensively studied even though it contains podophyllotoxin [31; 32; 33]. Moreover, it has several cultivated varieties, including _Juniperus scopulorum_ Blue Arrow and _Juniperus scopulorum_ Skyrocket. Thus, this work proposes to explore the credibility of cultivated varieties of these two _Juniperus scopulorum_ as a source of podophyllotoxin. However, studying the podophyllotoxin content of these plants requires the development and optimization of an efficient extraction protocol.