# Deep Eutectic Solvents as Extraction Media for
## 2 Factors Affecting the Extraction of Flavonoids Using DES Separation Techniques
### Toxicity  
Generally, DES are reported as \"safe\" and \"non-toxic\" or of \"low toxicity\", without any other justification than the safety and low toxicity of their components. However, this assumption can be true only in the case of NaDES, which are constituted by naturally occurring compounds, and therefore can be considered as inherently non-toxic. Thus, the literature concerning the toxicity of DES or NaDES is still scarce, and in the majority of the published works involving their use as extraction solvents, no toxicity tests are included. [PERSON] et al. [25] were among the first to study the toxicity and cytotoxicity of DES possessing choline chloride as the HBA and glycerine, ethylene glycol, triethylene glycol, and urea as the HBD. The tested DES and their individual components did not show toxicity against _Bacillus subtilis_, _Staphylococcus aureus_, _Escherichia coli_, or _Pseudomonas aeruginosa_. The interesting finding was that the DES under study showed significantly higher cytotoxicity than their individual components against _Artemia salina_leaches. This striking difference in cytotoxicity was attributed to the hydrogen bonding network present in DES and definitely merits further investigation. The toxicity of a series of NaDES against L929 fibroblast-like cells was studied by [PERSON] et al. [PERSON] et al. (2018). The results indicated that although no clear trend regarding cytotoxicity in relation with structure was observed, the presence of organic acids as HBDs results in increased cytotoxic activity. A series of 28 NaDES containing ChCl as the HBA and a variety of HBDs were tested for their cytotoxicity against the human embryonic kidney cell line (HEK-293) [PERSON] et al. (2018). The results showed that ChCl, as well as the compounds used as HBDs, were less toxic than the corresponding NaDES and that the structure of the HBD and the HBA/HBD ratio play a role in cytotoxicity. [PERSON] et al. [PERSON] et al. (2018, 2019) studied different NaDES regarding their antimicrobial activity against _Salmonella typhimurium._, _Escherichia coli_, _Pseudomonas aeruginosa_, _Proteus mirabilis_, _Staphylococcus aureus_, and _Candida albicans_ as well as their cytotoxicity against human normal and cancer cell lines (HEK29 ST, HeLa, MCF-7). NADES containing an organic acid were found to possess good antimicrobial activity, whereas their individual components were not active. Moreover, the majority of the tested NaDES showed low cytotoxicity, with the exception of ChCl-oxalic acid, which exhibited moderate cytotoxicity selectively against cancer cells. This observation is very important and the authors claim that this can be attributed to the fact that cancer cells have higher energy demands than normal cells. In the work of [PERSON] et al. [PERSON] et al. (2019), a series of DES comprised of ChCl, tetramethylammonium chloride ([N1111]Cl), and tetrabutylammonium chloride ([N4444]Cl) as HBAs, in combination various HBDs, were extensively studied for their cytotoxicity against two human skin cell lines, HaCaT32-35 (chosen as model for cosmetic applications) and MNT-136-38 (selected as a model to understand the potential of the DES under study for the treatment of skin disorders). The ChCl- and [N1111]Cl-containing DES were not cytotoxic, and some of them even increased cell viability. Thus, these DES can be safely characterized as \"benign\", at least for these cell lines, and for skin-related applications. The [N4444]Cl containing DES was cytotoxic against these cell lines, and as no clear trend regarding the relation of cytotoxicity with the HBD used was deduced, this HBA should not be considered safe for further applications. The in vivo safety of DES and NaDES is much less studied. The first published research in which both in vitro and in vivo toxicity of DES was conducted is the work of [PERSON] et al. in 2015 [PERSON] et al. (2015).