Current Research in Chemical Biology 1 (2021) 100003 Contents lists available at ScienceDirect Current Research in Chemical Biology journal homepage: www.journals.elsevier.com/current-research-in-chemical-biology Untangling the bioactive properties of therapeutic deep eutectic solvents based on natural terpenes Eduardo Silva a, b, Filipe Oliveira c, Joana M. Silva a, b, **, Rui L. Reis a, b, Ana Rita C. Duarte c, * a 3B's Research Group – Biomaterials, Biodegradable and Biomimetic, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark, Barco, Guimar~ aes, 4805-017, Portugal b ICVS/3B's PT Government Associated Laboratory, Braga/Guimar~ aes, Portugal c LAQV-REQUIMTE, Chemistry Department, NOVA School of Science and Technology, Caparica, 2829-516, Portugal A R T I C L E I N F O A B S T R A C T Keywords: Deep eutectic systems Terpenes Thymol Menthol Antimicrobial Anticancer Treatment of microbial-related infections remains a clinical challenge that has been slowly aggravating over recent years, due to the dissemination of resistance against currently applied treatment protocols. In this current scenario, the design of novel treatment approaches is of great importance, being a prominent focus of the scientific community. In recent years deep eutectic systems (DES) have gained the attention of the scientific community due to their remarkable physicochemical and biological properties, versatility, and compliance with the green chemistry metrics. In this work, DES containing a monoterpenoid (thymol (THY) and menthol (ME)) in combination with ibuprofen (IBU) were formulated and characterized via thermal analyses and NMR spectroscopy. The biological activity of the most promising formulations was then explored, with focus on its antimicrobial and anticancer activity. Both ME and THY-based formulations presented relevant antibacterial activity against the panel of microorganisms tested. Among the THY-based formulation tested, THY:IBU 3:1 M ratio, showed the highest antibacterial activity, affecting all tested microorganisms, while ME:IBU 3:1 M ratio was only effective against Gram-positive bacteria and Candida albicans. Furthermore, both ME and THY-based formulations revealed cytotoxic effect towards the cancer cell model used (HT29), where ME:IBU 3:1 stood out as the most selective towards cancer cells without compromising normal cells viability. Overall, the results obtained highlight the potential use of terpene-based THEDES formulations that, due to their enhanced thermal properties, may represent a versatile alternative in several biomedical applications where an effective antimicrobial or anticancer therapeutic action remains a challenge. 1. Introduction From a historical perspective, plant extracts and essential oils isolated from plants have been used as medicinal alternative treatments in several health problems (Lummiss et al., 2012). Essential oils are rich in aromatic plants secondary metabolites and are characterized by their hydrophobicity, volatility and strong odor due to the presence of various compounds such as terpenes (Lummiss et al., 2012; Burt, 2004). The biological properties of essential oils are vast, including antimicrobial, anti-inflammatory, antioxidative, anti-mutagenic, spasmolytic, analgesic and sedative properties (Burt, 2004; Galeotti et al., 2002; Kamatou et al., 2013; Salehi et al., 2018; Abbaszadeh et al., 2014). The plant species from which it is possible to obtain essential oils are widely distributed among the Plantae kingdom, being Thymus vulgaris (Thyme), Mentha canadensis L. (corn mint) and Mentha x piperita L (peppermint) well-known representative examples (Galeotti et al., 2002; Kamatou et al., 2013; Salehi et al., 2018; Abbaszadeh et al., 2014). Thyme contains high concentrations of terpenes, including thymol (THY), carvacrol, but also p-cymene among others. THY is a phenol monoterpenoid derivative of p-cymene, that was extracted for the first time from Thyme in 1719 (Salehi et al., 2018; Abbaszadeh et al., 2014). Over the years, several enticing biological properties have been associated with THY such as antibacterial, antifungal, sedative and antioxidant, among others. In fact, THY's pharmacological properties may lead to therapeutic effect against several types of conditions ranging from malignant diseases to infections (Nagoor Meeran et al., 2017; Marchese * Corresponding author. ** Corresponding author. 3B's Research Group – Biomaterials, Biodegradable and Biomimetic, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark, Barco, Guimar~aes, 4805-017, Portugal. E-mail addresses: joana.marques@i3bs.uminho.pt (J.M. Silva), ard08968@fct.unl.pt (A.R.C. Duarte). https://doi.org/10.1016/j.crchbi.2021.100003 2666-2469/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). E. Silva et al. Current Research in Chemical Biology 1 (2021) 100003 adenocarcinoma cell line (HT29) and carcinoma-derived human oral keratinocyte cells (H413). In another example, Badisa et al. (Badisa et al., 2009) (Badisa et al., 2009) evaluated two different DES, piperidinyl:diethylstilbestrol and pyrrolidinyl:diethylstilbestrol, on MCF-7 cancer cell line in comparison with tamoxifen, a drug used in the treatment of breast hyperplasia. The results reveled that these DES had a higher selectivity towards cancer cells in comparison with the standard used drug. In a more recent report, Pereira et al. (Pereira et al., 2019) reported the anticancer activity of Limonene-based THEDES towards the HT29 cancer cell line, where the eutectic system was able to inhibit selectively the cancer cells proliferation without compromising normal cells viability. The range of reports on the eutectic systems activity as anticancer agents have revealed not only the opportunity to produce systems able to cope with the cancer cell proliferation challenge, while in line with the green chemistry metrics, but also the versatility comprised in these systems which ultimately can be designed in case-by-case manner. Herein, we investigate THEDES based on natural terpenes and IBU in terms of their physicochemical, antimicrobial, and anticancer properties, to boost their use as a versatile therapeutic agent for both therapeutic and preventive applications. et al., 2016). In Mentha genus plants, menthol (ME) is the major constituent of its essential oils. ME is a monocyclic monoterpenoid, that was successfully extracted by steam distillation in 1771. Similarly to THY, ME also possesses remarkable biological properties with therapeutic value (e.g., antibacterial, antifungal, antioxidant, and wound healing) (Galeotti et al., 2002; Abbaszadeh et al., 2014; Corvis et al., 2015). While the chemical structure of THY and ME are similar, they possess a key difference, namely the presence of an aromatic ring, in the case of THY, whereas in ME, a cyclohexane ring. In this sense, THY and ME were selected to form deep eutectic systems (DES), and the consequences on its biological activity resulting from having a different chemical structure were evaluated. These molecules were combined with ibuprofen (IBU), a chiral nonsteroidal antiinflammatory drug (NSAID) of the 2 arylpropionic acid (2-APA) class, that was discovered in 1960s and has been widely used from ever since (Davies, 1998). Beyond its classical use as an anti-inflammatory drug, recent reports have also shown IBU's potential as an anti-cancer drug as it is able to inhibit cell proliferation and angiogenesis as well as enhance the anticancer effect of other compounds such as cis-platin (Akrami et al., 2015; Endo et al., 2014). In fact, combination of IBU and terpenes to form DES with desirable therapeutic properties has been previously explored and meet with resounding success (Aroso et al., 2015; Pereira et al., 2019; Silva et al., 2020; Stott et al., 1998). The term DES was first coined by Abbot and coworkers, being defined as a mixture of two or more solid components, which at a certain molar ratio present a lower melting point than that either of its components (Smith et al., 2014; . del Monte et al., 2014; Zhang et al., 2012; Mbous et al., 2017; Abbott et al., 2004). The depression on the melting point is usually ascribed to hydrogen bond interactions and occasionally, electrostatic interactions and van der Waals interactions (Mbous et al., 2017; Pena-Pereira & Namiesnik, 2014; Abo-Hamad et al., 2015; Liu et al., 2015). The unique and attractive properties of DES, such as biodegradability, biocompatibility, together with the virtual unlimited number of possible combination, allow the creation of tailor-made systems for any desired application, which have attracted attention from different fields, ranging from electrochemistry to biomedical applications (Smith et al., 2014; . del Monte et al., 2014; Zhang et al., 2012). When an active pharmaceutical ingredient (API) is one of the counterparts used to form the DES, the formulation is designated as a therapeutic deep eutectic system (THEDES). In the biomedical field, various studies have been pursued to explore the potential of THEDES since when in eutectic form some APIs show a significant enhancement of solubility and permeability (Aroso et al., 2015; Duarte et al., 2017; Morrison et al., 2009; Stott et al., 1998; Nguyen et al., 2021). Previous studies already demonstrated that the pharmacokinetics parameters of IBU can be greatly improved in THEDES when compared with its powder form (Aroso et al., 2015; Stott et al., 1998; Duarte et al., 2017). However, from the best of our knowledge the range of bioactive properties, such as antimicrobial and anticancer, of THY and ME combined with IBU has not yet been addressed. The preparation of DES with antimicrobial properties have been reported in recent publications, showing great potential as novel alternatives to classical treatments (Radosevic et al., 2018; Wikene et al., 2015). For instance, Juneidi et al. (Juneidi et al., 2016) investigated the antifungal potential of choline chloride-based DES against 4 different fungi strains showing relevant inhibitory effects as well as a lower acute toxicity, in DES form, against Cyprinus carpio fish. Additionally, Silva et al. (Silva et al., 2019) recently published studies exploring the antimicrobial properties of fatty acid-based eutectic blends as therapeutic agents themselves or incorporated into novel medical devices via supercritical particle generation. As versatile tailor-made systems, DES bioactive potential have also been associated with anticancer properties. Hayyan et al. (Hayyan et al., 2015) described promising selective cytotoxic profile of different ammonium-based eutectic systems on several cancer cell lines, such as human breast cancer cell line (MCF-7), human prostate cancer cell line (PC3), human malignant melanoma cell line (A375), human liver hepatocellular cell line (HepG2), human colon 2. Materials and methods 2.1. Preparation of terpene-based THEDES Racemic ME (ref. M2772, Sigma Aldrich), THY (ref. T0501, Sigma Aldrich) and IBU (ref. I4883, Sigma Aldrich), were used as received. Briefly, THY was gently mixed with IBU at different molar ratios and constantly stirred at 70 C, until the formation of a clear liquid. Additionally, ME:IBU was prepared according as previously reported works. Briefly, compounds were weighed and mixed, followed by heating at 40 C until a clear solution was obtained (Aroso et al., 2015; Duarte et al., 2017). All THEDES were prepared immediately before conducting an experiment, being kept at 4 C, for a maximum of 48 h, in conveniently sealed containers. 2.2. Thermal properties - differential scanning calorimetry (DSC) DSC experiments were carried out in a TA instrument DSC Q100 model (Thermal analysis & analysers), using aluminium pans. The mass added to the pans of pure compounds and formulations ranged from 4.5 to 10 mg. ME and its correspondent THEDES formulation were equilibrated at 40 C for 2 min, followed by cooling to 40 C at 5 C/min, an isothermal period for 5 min and finally heating to 90 C at 5 C/min. THY and its correspondent THEDES were equilibrated at 20 C for 2 min, followed by heating to 110 C at 10 C/min, isothermal for 2 min and finally cooling to 20 C at 10 C/min. 2.3. Nuclear magnetic resonance (NMR) measurements NMR experiments were recorded at a 400 MHz Bruker Advance II and Mestrenova 12.0 software (Mestrelab Research) was used for spectral processing (peak assignment and integration). THEDES and pure compounds were dissolved in dimethyl sulfoxide-d6 (LOT. STBH4385, Sigma Aldrich). The experiments were recorded when the eutectic systems were in equilibrium, as elsewhere reported (Silva et al., 2020). 2.4. Solubility assessment The solubility of the pure API and the previously prepared THEDES was determined as described in a previous work (Silva et al., 2020). Briefly, an excess amount of IBU and THEDES were added to a phosphate-buffered saline solution (PBS, Sigma–Aldrich, USA) in separate vials, and the samples were then incubated at 37 for 72 h in a shaking water bath. Prior to the solubility measurement, the samples were filtered using a hydrophilic PTFE syringe filter with a 0.22 μm pore 2 E. Silva et al. Current Research in Chemical Biology 1 (2021) 100003 HT29 cells were seeded at a density of 1 105 cells/well in 96-well culture plates. After 24 h, cells were incubated with either culture media (control), pure compounds or THEDES, diluted in culture medium. Cell proliferation was measured after 24 h using the MTS viability reagent, as previously described. Three independent experiments were performed in triplicate. Furthermore, THEDES selectivity indexes were calculated as a ratio between the half maximal effective concentrations (EC50) from the cytotoxic and antiproliferative profiles previously obtained. size (Filter Lab, Barcelona, Spain). The determination of IBU solubility was performed by HPLC, using a Knauer Smartline (Knauer, Berlin, Germany). The column used was a Thermo keystone kromasil C18 5 μm particle size, pore size 100 Å, L I.D. 250 mm 4.6 mm (Thermo Scientific, USA) and the column temperature was 25 C. The chromatographic separation was performed using a mobile phase consisting of 50% (v/v) acetonitrile:50 mM KH2PO4 at pH 4.2. The flow rate was 1 mL/min, following the procedure described by Jahan et al. (Jahan et al., 2014) with the absorbance of the solutions measured at 222 nm. A calibration curve using the respective THEDES and the API as standards was prepared for quantification. The experiment was performed in triplicate. 3. Results 3.1. Physicochemical characterization of terpene-based THEDES 2.5. Antimicrobial activity assessment In this work, THEDES based on the mixture of terpenes ME and THY in combination with IBU, were developed at either balanced or imbalanced molar ratios (Table 1). Different formulations were produced, namely THY:IBU at molar ratios of 1:1; 1.5:1; 1:2; 2:1 and 3:1. Additionally, a ME:IBU was only produced with a 3:1 M ratio d as described elsewhere (Aroso et al., 2015; Redasani & Bari, 2012). DSC, for all the eutectic formulations as well as pure compounds was performed to observe the thermal events, namely changes in the melting point of the mixtures when compared with the pure compounds (see Fig. 1). The obtained thermograms are presented in Fig. 2. The thermograms obtained for pure THY and IBU show a sharp and well-defined endothermic peak at 51.25 C and 77.73 C, respectively, being in agreement with previous reports (Cevallos et al., 2010; Kararli et al., 1989). In particular, the thermogram for racemic ME presents two endothermic peaks at 28.10 C and 33.80 C, which are attributed to ME's isomeric forms (Corvis et al., 2015). For all formulations, an acute depression of the endothermic peak temperature, when compared with the parent compounds, can be observed (35.10–38 C) culminating in a complete suppression of endothermic events at 3:1 M ratio. This suggests the formation of DES-like interactions between the parent compounds. However, for THY:IBU 1:1 and 1:2, specifically, a second endothermic event is observed at 60.96 C and 59.00 C, respectively. This second peak results from excess of one component, most likely IBU, as it will not be able to establish intermolecular interactions since it is in excess over its counterpart THY which results in the appearance of a second endothermic peak (Silva et al., 2018). 1 H NMR spectra were obtained which are presented in Fig. 3 (pure compounds) and Fig. 4 (THEDES formulations) with the respective peak assignments. By analyzing the results it is possible to verify the purity of THY, ME and IBU, as the spectra presented in Fig. 3 are in accordance with previous reports in the literature (Johnson et al., 2007; Salager et al., 2009). Furthermore, the existence of intermolecular interactions via hydrogen bonding, a hallmark of eutectic interactions, can be verified in the spectrum of ME:IBU 3:1 formulation. ME's hydrogen –OH that corresponds to a well-defined doublet with δ ¼ 3.9 ppm (Fig. 3B) becomes a larger singlet with δ ¼ 4.28 when ME is mixed with IBU at a 3:1 M ratio (Fig. 4B). Furthermore, in both systems a suppression of the peak correspondent to IBU's-OH group's hydrogen atom (δ ¼ 12.21), beyond what the molar ratio should reflect, is observed is also an indicative of the possible establishment of hydrogen bond interaction between the components of the THEDES. The antimicrobial activity of THEDES was determined against a panel of clinically relevant microorganisms, namely, Staphylococcus aureus ATCC 700698 (Methicillin-resistant strain, MRSA), S. epidermis ATCC 35984 (Methicillin-resistant strain, MRSE), Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and Candida albicans ATCC 90029. The assessment was carried out as described in a previous work via a two-step methodology. Briefly, the formulations are first subjected to a disk diffusion assay, using loaded blank discs (CT0998B, Oxford), as a preliminary screening, followed by MIC/MBC/MFC determination using aqueous solutions of THEDES formulations that passed the preliminary test. For comparison purposes, the pure compounds were included in both steps, as internal controls. Additionally, suitable antibiotics were used as positive controls for antibacterial activity. The concentration range tested for MIC/MBC/MFC determination was between 2500 and 156.25 μg/ml. Experiments were carried out in triplicate, using independent microbial cultures to account for biological variance. 2.6. Cytotoxic and antiproliferative activity assessment The THEDES anticancer potential was evaluated in terms of their cytotoxicity and antiproliferative effects, and resulting selectivity index, as described in a previous work (Silva et al., 2020). The cytotoxic effect was assessed using a continuous cell line culture of heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2) (ACC 169, DSMZ, Braunschweig, Germany) (Sambuy et al., 2005). Briefly, the cells were subcultured in RPMI medium (Corning, Corning, NY, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Corning, USA) and a 1% penicillin–streptomycin solution (PS, Corning, NY, USA). The cell culture was maintained in a humidified atmosphere at 37 C with 5% CO2. The cytotoxicity assay was performed in accordance with ISO/EN 10993 guidelines. Caco-2 cells were seeded into 96-well plates at a density of 2 104 cells/well and allowed to grow for 7 days, with medium renewal every 48 h. At day 7, cells were incubated with either culture media (control), pure compounds or THEDES, diluted in culture medium. After 24 h, cells were washed twice with PBS and the cell viability was assessed using a CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, Wi, USA), containing an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) viability reagent. Briefly, 100 μL of the viability reagent was added at each well in a 1:10 dilution and incubated for 3 h. The absorbance was measured at 490 nm using a microplate reader (VICTOR NivoTM, PerkinElmer, Waltham, MA, USA) and cell viability was expressed in terms of percentage of living cells relative to the control. Three independent experiments were performed in triplicate. The antiproliferative effect towards cancer cells was assessed using a continuous cell culture of human Caucasian colon adenocarcinoma (HT29) (ACC 299, DSMZ, Germany). These cells form a well-differentiated colorectal adenocarcinoma (CRC), and thus have been accepted as a CRC cell model in 2D and 3D in vitro cultures. The cells were subcultured as described above. For the antiproliferative assay Table 1 Summary of the different THEDES prepared in this study. RT – Room temperature (25 C); Tm – Melting peak temperature. THEDES Molar Ratio Visual aspect at RT Tm ( C) THY:IBU 1:1 1.5:1 1:2 2:1 3:1 3:1 Solid Solid Solid Solid Liquid Liquid 38.00; 60.96 36.78 36.95; 59.00 35.10 – – ME:IBU 3 E. Silva et al. Current Research in Chemical Biology 1 (2021) 100003 Fig. 1. Schematic illustration of the developed monoterpene-based DES for therapeutic applications. THEDES thermograms also suggest that the molar ratio strongly influences the intensity and shift of the peaks, which was elsewhere reported for other THEDES (Nowosielski et al., 2020). Overall, the results obtained by DSC suggest the establishment of intermolecular interactions between the mixture's components forming a DES supramolecular structure, namely hydrogen bond interaction and Van der Waals forces, fact that had already been previously observed for ME:IBU 3:1 (Aroso et al., 2015; Duarte et al., 2017; Al-Saidan, 2004). To further characterize the obtained formulations and explore possible intermolecular interactions typically found in THEDES, NMR spectroscopy was carried out for the formulations that showed complete peak suppression in the DSC thermogram and for the pure compounds. In this sense, both the change of multiplet to singlet, as well as the deprotection of ME's –OH hydrogen, reflected in the upward chemical shift, suggest the establishment of hydrogen bond interactions between ME and IBU's –OH group. 3.2. Solubility of developed formulations In the present work, the potential use of terpene-based eutectic mixture for IBU solubility enhancement was evaluated. Particularly, the solubility assessment was performed in the previously obtained THEDES which were liquid at RT, THY:IBU and ME:IBU both at 3:1 M ratio, in comparison with IBU alone and both systems individual components in a physical mixture (THY þ IBU and ME þ IBU). For this matter, the physical mixtures were considered as the two individual components dissolved in PBS using the same concentration as in the THEDES. The results are summarized in Fig. 5. 3.3. Antimicrobial potential of developed formulations To evaluate the antimicrobial capabilities of the THEDES formulations, firstly a disk diffusion assay was carried out against a selected panel of clinically relevant microorganisms. The antimicrobial activity was only evaluated for the systems that were in a liquid state at RT, namely THY:IBU and ME:IBU, both at a 3:1 M ratio. The results obtained are presented in Table 2. Additionally, representative images of the obtained plates can be found in the supplementary information (Table A1–A5). To obtain a more robust and accurate measure of the antibacterial potential presented by these THEDES, aqueous solutions of the formulations were subjected to MIC/MBC/MFC determination, considering the preliminary results obtained. The determined MIC and MBC/MFC values are presented in Tables 3 and 4, respectively. As the systems are majorly comprised of THY/ME in absolute mass (Fig. A1), never actually reaching the prementioned values and, as such, inevitably reflecting the antibacterial capabilities of the most representative components. 3.4. Anticancer potential of developed formulation In this work the anticancer activity of aqueous solutions of THEDES previously obtained was explored towards an in vitro CRC cell model. For that, the eutectic systems ME:IBU and THY:IBU, both at 3:1 M ratio, were considered since they are both liquid at 37 C. The obtained EC50 values and corresponding selectivity indexes are presented in Table 5. 4. Discussion THEDES formulations are characterized by their eutectic point, which corresponds, in a phase diagram indicating chemical composition and temperature, to a coordinate that represents the lowest melting point of a mixture of two or more components (Smith et al., 2014; Gutierrez et al., 2009). Thereby, analysis by DSC is an efficient method to probe possible eutectic interactions between a mixture of two or more compounds. Regarding the thermograms of the eutectic formulations, a depression of the melting point when compared with the counterparts can be observed with complete peak suppression at a molar ratio of 3:1 for both formulations, which corresponds to a liquid state at RT. Additionally, these Fig. 2. DSC thermograms obtained for (A) pure compounds and (B) THEDES formulations with different molar ratios. Peaks rising above the baseline represent endothermic peaks. 4 E. Silva et al. Current Research in Chemical Biology 1 (2021) 100003 Additionally, the accuracy of the THEDES molar ratios can also be verified since peak integral values of IBU are reduced to a third in eutectic form when compared with the spectrum of pure IBU. In which concerns the bioavailability of an API, such as IBU, it is most often limited by its poor solubility (Kawabata et al., 2011; Savjani et al., 2012). Although IBU is a well-known and widely used NSAID for a range of situations related to acute or chronic pain, inflammation and antipyresis, this API presents very low solubility in water (21 mg/L at 25 C) hindering its therapeutic effectiveness (Savjani et al., 2012; C. United States Pharmacopeial, 1995; Filippa & Gasull, 2013). Considering the broad use of this therapeutic agent it is of our utmost interest the overcome of such a limitation. It has been reported that eutecticity is able to increase the solubility of biomolecules and APIs (Duarte et al., 2017; Morais et al., 2018; Goud et al., 2012). NSAIDs, such as IBU, when in combination with ME as a eutectic mixture experiences a solubility enhancement of 12.76-fold in a physiological like media (Duarte et al., 2017). In another example, the combination of IBU with limonene in a molar ratio of 1:4 it resulted in a 4.3-fold solubility enhancement of the Fig. 4. 1H NMR spectra of (A) IBU (B) THY and (C) THY:IBU 3:1. Peak assignment and integration were fully performed. API. By increasing the ratio of limonene to 8, the solubility enhancement also increases by 5.63-fold, when comparing to the pure form of IBU (Pereira et al., 2019). From the results obtained in this work (Fig. 5), it is possible to verify that just by manipulating IBU's physical state, changing from solid to liquid as a eutectic system, it is possible to obtain an increase in the API solubility, in comparison with the API in powder form. This was achieved for the ME:IBU as expected from previous reports (Aroso et al., 2015). Interestingly, the corresponding physical mixture (ME þ IBU) did not present the same output of increased solubility, confirming ME:IBU eutectic mixture as a different entity rather than an aqueous solution. In contrast, the results obtained for the THY:IBU mixture did not follow this enhancement trend, since the combination of THY and IBU as a eutectic mixture does not result in an increase in the API's solubility. Nevertheless, it is of outmost interest to highlight the potential of these systems to provide a tailor-made solution, since by only varying one component of the system one can obtain an increase in the solubility of a particular therapeutic agent. The eutectic capacity to solubilize poorly water-soluble compounds is designated by hydrotrope Fig. 3. 1H NMR spectra of the pure compounds (A) IBU (B) ME and (C) ME:IBU 3:1. Peak assignment and integration were fully performed. 5 E. Silva et al. Current Research in Chemical Biology 1 (2021) 100003 Table 2 Inhibition halo measurements (diameter (mm)SD) for the various THEDES formulations, individual counterparts, and controls. Results are presented by formulation/ compound for each microbial strain tested. NI – no inhibition, NT – Not tested. Compound/THEDES E. coli P. aeruginosa MRSA MRSE C. albicans THY ME IBU THY:IBU 3:1 ME:IBU 3:1 Sterile water 43.33 2.05 NI NI 38.67 0.47 NI 0.00 0.00 11.67 1.70 NI NI 10.33 1.25 NI 0.00 0.00 45.67 1.70 14.33 0.47 NI 40.67 2.49 14.67 1.70 0.00 0.00 43.67 1.25 13.33 0.47 NI 38.00 1.00 15.33 1.70 0.00 0.00 60.00 1.41 18.33 0.47 NI 68.67 2.49 31.33 2.49 0.00 0.00 bacterial strains and C. albicans, where the lack of an outer membrane, which serves as an additional protective and selective layer against aggression, results in a greater exposure of hydrophobic compounds, such as THY and ME, to the inner cytoplasmic cell membrane, allowing them to exert their effects within the cell (Marino et al., 1999; Karapinar & Aktuǧ, 1987; Ouattara et al., 1997). On the other hand, Gram-negative bacteria such as P. aeruginosa or E. coli, present a more complex membrane structure, with several reports in the literature stating that the presence of lipopolysaccharides on the outer cell wall prevent hydrophobic compounds from reaching the cell and exerting their cytotoxic effects (Vaara & Nurminen, 1999; Wiener & Horanyi, 2011). Regarding ME and its correspondent THEDES formulation, the antimicrobial effects verified are not as pronounced as THY's. In fact, not only are the observed effects on the Gram-positive bacteria and yeast strain used less pronounced, but also no inhibitory effect was verified for the Gram-negative strains. However, these results are not outside the expected since P. aeruginosa possesses documented resistance to ME's antibacterial effects and, as previously stated, Gram-negative bacteria, tend in general to be more resistant to the action of hydrophobic compounds such as essential oils and their components (Aguilar et al., 2006; Cox & Markham, 2007; Vaara & Nurminen, 1999; Wiener & Horanyi, 2011). The difference observed between ME and THY, may reside in key differences in the chemical structure of the compound's cyclic rings. This fact possesses added significance particularly when looking at P. aeruginosa, since previous reports in the literature show that several bacteria in the Pseudomonas genus possess the necessary molecular machinery to carry out the metabolism of terpenes (Aguilar et al., 2006; Esmaeili & Hashemi, 2011; Cox & Markham, 2007; Marmulla & Harder, 2014). As such, it would not be surprising that THY, that possesses a benzene ring, would be more resistant to possible degradation than ME, that possesses a cyclohexane ring, which results in different antibacterial effects (Aguilar et al., 2006; Marmulla & Harder, 2014). The obtained concentration values confirm the previously obtained results, highlighting once again the superior effectiveness of THY over ME, both as pure compound and in THEDES form across all test subjects. Furthermore, the determined MIC/MBC/MFC values are concurrent with the inhibition halo measurements obtained previously, especially regarding THY's, both as pure compound and in THEDES form, activity against P. aeruginosa since the determined concentration values are much higher for this microorganism. Regarding E.coli, a decrease in MIC/MBC values of THY:ME 3:1 Table 3 MIC values of individual counterparts and THEDES. Results are presented by formulation for each microbial strain tested. ND- Not dissolved. Compound/THEDES THY ME IBU THY:IBU 3:1 ME:IBU 3:1 MIC (μg/ml) E. coli P. aeruginosa MRSA MRSE C. albicans 625 NT ND 312.5 NT 1250 NT ND 1250 NT 312.5 1250 ND 312.5 1250 312.5 1250 ND 312.5 1250 312.5 625 ND 312.5 625 Table 4 MBC values of individual counterparts and THEDES. Results are presented by formulation for each microbial strain tested. ND- Not dissolved. Compound/THEDES THY ME IBU THY:IBU 3:1 ME:IBU 3:1 MBC/MFC (μg/ml) E. coli P. aeruginosa MRSA MRSE C. albicans 1250 NT ND 625 NT 2500 NT ND 2500 NT 625 2500 ND 625 2500 625 2500 ND 625 2500 625 1250 ND 625 1250 (Soares et al., 2017). Hydrotropes are able to enhance the solubility of hydrophobic molecules in water in a different phenomenon than micellar solubilization (Cl audio et al., 2015; Sintra et al., 2018). Thus, from these results it is possible to emphasize the potential use of eutectics in overcoming the solubility challenge of poor water-soluble APIs. The antimicrobial properties assessed by the inhibition halo measurements, revealed that both pure THY and THY:IBU eutectic system display significant antibacterial activity against all tested microbes, including P. aeruginosa which is normally resilient to the effects of plant derived terpenes, with some strains being in fact able to metabolize these compounds (Aguilar et al., 2006; Esmaeili & Hashemi, 2011; Cox & Markham, 2007). The obtained results are in accordance with previous accounts in the literature, that report THY's effectiveness as an antibacterial and antifungal agent against several organisms, including E. coli, C. albicans, MRSA and MRSE clinical isolates and P. aeruginosa, among many others (Marino et al., 1999; Olasupo et al., 2003). Also as expected, THY's effects are, overall, more pronounced in the Gram-positive Table 5 EC50 values for the cytotoxicity and antiproliferative assays, and corresponding selectivity indexes, for the selected THEDES, individual compounds. Results were obtained from at least three independent experiments performed in triplicate. System/Compound EC50 (mM) Selectivity index Cytotoxicity assay THY ME IBU ME:IBU 3:1 THY:IBU 3:1 a 6.73 5.09 2.89 8.92 1.07 Antiproliferative assay 1.69 0.73 0.06a 1.39 0.37 5.22 4.31 2.35 4.30 0.30 Reported by Pereira et al. (Pereira et al., 2019). 6 1,16 0,63 0.09a 0,71 0,04 1.29 1.18 1.23 2.07 3.50 E. Silva et al. Current Research in Chemical Biology 1 (2021) 100003 both THEDES are terpene-based, and THY and ME differ in an aromatic or cyclohexane ring, from the obtained results it is possible to verify that their presence in the eutectic formulation results in a different contribution for their cytotoxic profiles, were THY appears as the most cytotoxic. Interestingly, the cytotoxic capability of THY appears to be highly increased as in a eutectic system in both cytotoxic and antiproliferative assays, when in comparison with THY as an individual component. Therefore, and taking into account that the cytotoxicity assay is herein working as a preliminary safety indicator since Caco-2 represent normal colonic cells, although THY:IBU presents the most cytotoxic action towards the cancer cell line, it is also highly cytotoxic towards the normal cell line, excluding its possible application as a selective anticancer agent. In contrast, the result obtained for the ME:IBU reveals a system with less cytotoxicity towards normal colonic cells and higher cytotoxicity towards cancer cells, both in comparison with ME alone. Hence, ME:IBU in a 3:1 M ratio appears to be a promising candidate for further establishment as a selective therapeutic agent for the treatment of colorectal cancer. Fig. 5. Solubility of IBU in powder form, in comparison with IBU as part of a THEDES (ME:IBU 3:1 ratio and THY:IBU 3:1 ratio), and IBU in a physical mixture (ME þ IBU 3:1 ratio and THY þ IBU 3:1 ratio). The results were obtained from three independent experiments performed in a PBS solution at physiological-like conditions. 5. Conclusion Over the years, modern medicine has been facing highly demanding challenges. Either to overcome the defiance of microorganisms, to classical drug options or to keep up with the complexity of the cancer challenge, the scientific community has gradually concentrated increasing efforts into the discovery of novel alternative therapeutics from a range of different sources. In the present study, THEDES formulations were produced combining terpenes with IBU. The THY-based formulation revealed to be the most effective towards microbial cells, and the MEbased formulation the most promising to be explored as an anticancer agent. An improved solubility of IBU was observed for the system ME:IBU 3:1, in contrast to a decrease observed for the system composed by THY:IBU 3:1. The antimicrobial effect verified, highlights the potential of using these systems as alternative antimicrobial agents, coupled with the advantage of having an anti-inflammatory drug. Moreover, both ME and THY-based formulations displayed promising potential to reduce cancer cell proliferation as a result of their cytotoxic effect towards the cancer cell model used (HT29). Nevertheless, the system ME:IBU 3:1 stood out as the most selective towards cancer cells without compromising normal colonic cells viability. Overall, this study shows the potential of THEDES as future effective therapeutic agents alternative for both microbial infection and anticancer applications. when compared with pure THY was obtained. This suggests that the eutectic interactions formed may lead to a molecular organization that facilitates permeation/destabilization of Gram-negative membrane archetype. This is not observed for P. aeruginosa, also a Gram-negative bacteria, most likely due to its higher resistance to this class of compounds (Cox & Markham, 2007; García-Salinas et al., 2018). Another point of note is that, in the conditions established, it was not possible to dissolve IBU. While this compound is primarily known for its anti-inflammatory capabilities some accounts in the literature report antibacterial activity against certain microorganism such as S. aureus, E. coli and Bacillus subtilis (MIC ¼ 1025–2500 μg/ml, when IBU is dissolved in a methanol stock solution) (Ahmed et al., 2016; AL-Janabi, 2010). Overall, the obtained results show that the adoption of a DES supramolecular does not hamper the antimicrobial capabilities of THY and ME and may contribute to a greater permeation/destabilization of Gram-negative type bacteria membranes. The systems designed also find an application in the anticancer therapies. The cancer burden remains as a most prominent health care challenge nowadays, occupying a remarkable place on the worldwide mortality scale. With common cancer such as the colorectal cancer being among the most lethal, mostly due to an alarming ineffectiveness of conventional anticancer therapies in the case of advanced or metastatic phases, the urge for efficient and selective therapeutics has proven to be essential in the cancer battle (Pucci et al., 2019). As previously mentioned, natural occurring molecules often provide promising bioactivities which result in a wide range of therapeutic outputs. The cytotoxic action of terpenes, such as ME and THY, towards cancer cell lines have revealed the potential use of such molecules as anticancer agents (Kamatou et al., 2013; Islam et al., 2019). However, their high volatility and consequent high toxicity and poor water solubility, have been representing great challenges to their effective application. Besides the solubility enhancement by hydrotrope, the supramolecular arrangement experienced by the molecules within a eutectic system helps to reduce their natural volatility, therefore representing a promising system to fulfil terpenes therapeutic potential. Since angiogenesis and inflammation have an important role in tumor progression, NSAID such as IBU can potentially play an important role in its containment (Coussens & Werb, 2002). In light of such a potential, the eutectic systems ME:IBU and THY:IBU, both at 3:1 M ratio, were considered since they are both liquid at 37 C, the normal temperature of the human body. Although Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work received funding from Horizon 2020 through the ERC2016-CoG-725034 Des.Solve (ERC Consolidator Grant) and from Foundation for Science and Technology (FCT), through project PTDC/BBB490 EBB/1676/2014 – Des.Zyme, UID/Multi/50026/2019. E.S. and JMS would also like to acknowledge the financial support by the FCT through the doctoral grant with reference number SFHR/BD/143902/2019 and post-doctoral contract with reference number CCEEIND/01026/2018, respectively. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.crchbi.2021.100003. 7 E. Silva et al. Current Research in Chemical Biology 1 (2021) 100003 References migration kinetics of ibuprofen (()-(R, S)-2-(4-isobutylphenyl) propanoic acid), its metabolites, and analogues. Anal Chem. 79, 8720–8727. Juneidi, I., Hayyan, M., Ali, O.M., 2016. Toxicity profile of choline chloride-based deep eutectic solvents for fungi and Cyprinus carpio fish. Environ Sci Pollut Control Ser. 23, 7648–7659. Kamatou, G.P., Vermaak, I., Viljoen, A.M., Lawrence, B.M., 2013. Menthol: a simple monoterpene with remarkable biological properties. Phytochemistry. 96, 15–25. Karapinar, M., Aktuǧ, Ş.E., 1987. Inhibition of foodborne pathogens by thymol, eugenol, menthol and anethole. Int J Food Microbiol. 4, 161–166. Kararli, T.T., Needham, T.E., Seul, C.J., Finnegan, P.M., 1989. Solid-state interaction of magnesium oxide and ibuprofen to form a salt. Pharmaceut Res. 6, 804–808. Kawabata, Y., Wada, K., Na katani, M., Yamada, S., Onoue, S., 2011. Formulation design for poorly water-soluble drugs based on biopharmaceutics classification system: basic approaches and practical applications. Int J Pharm. 420, 1–10. Liu, P., Hao, J.-W., Mo, L.-P., Zhang, Z.-H., 2015. Recent advances in the application of deep eutectic solvents as sustainable media as well as catalysts in organic reactions. RSC Adv. 5, 48675–48704. Lummiss, J.A., Oliveira, K.C., Pranckevicius, A.M., Santos, A.G., dos Santos, E.N., Fogg, D.E., 2012. Chemical plants: high-value molecules from essential oils. J Am Chem Soc. 134, 18889–18891. Marchese, A., Orhan, I.E., Daglia, M., Barbieri, R., Di Lorenzo, A., Nabavi, S.F., Gortzi, O., Izadi, M., Nabavi, S.M., 2016. Antibacterial and antifungal activities of thymol: a brief review of the literature. Food Chem. 210, 402–414. Marino, M., Bersani, C., Comi, G., 1999. Antimicrobial activity of the essential oils of Thymus vulgaris L. measured using a bioimpedometric method. J Food Protect. 62, 1017–1023. Marmulla, R., Harder, J., 2014. Microbial monoterpene transformations—a review. Front Microbiol. 5, 346. Mbous, Y.P., Hayyan, M., Hayyan, A., Wong, W.F., Hashim, M.A., Looi, C.Y., 2017. Applications of deep eutectic solvents in biotechnology and bioengineering—promises and challenges. Biotechnol Adv. 35, 105–134. Morais, E.S., Mendonça, P.V., Coelho, J.F., Freire, M.G., Freire, C.S., Coutinho, J.A., Silvestre, A.J., 2018. Deep eutectic solvent aqueous solutions as efficient media for the solubilization of hardwood xylans. Chem Sus Chem. 11, 753–762. Morrison, H.G., Sun, C.C., Neervannan, S., 2009. Characterization of thermal behavior of deep eutectic solvents and their potential as drug solubilization vehicles. Int J Pharm. 378, 136–139. Nagoor Meeran, M.F., Javed, H., Al Taee, H., Azimullah, S., Ojha, S.K., 2017. Pharmacological properties and molecular mechanisms of thymol: prospects for its therapeutic potential and pharmaceutical development. Front Pharmacol. 8, 380. Nguyen, C.-H., Augis, L., Fourmentin, S., Barratt, G., Legrand, F.-X., 2021. Deep eutectic solvents for innovative pharmaceutical formulations. In: Fourmentin, S., Costa Gomes, M., Lichtfouse, E. (Eds.), Deep eutectic solvents for medicine, gas solubilization and extraction of natural substances. Springer International Publishing, Cham, pp. 41–102. Nowosielski, B., Jamr ogiewicz, M., Łuczak, J., Smiechowski, M., Warmi nska, D., 2020. Experimental and predicted physicochemical properties of monopropanolaminebased deep eutectic solvents. J Mol Liq. 309, 113110. Olasupo, N., Fitzgerald, D., Gasson, M., Narbad, A., 2003. Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium. Lett Appl Microbiol. 37, 448–451. Ouattara, B., Simard, R.E., Holley, R.A., Piette, G.J.-P., Begin, A., 1997. Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms. Int J Food Microbiol. 37, 155–162. Pena-Pereira, F., Namiesnik, J., 2014. Ionic liquids and deep eutectic mixtures: sustainable solvents for extraction processes. Chem Sus Chem. 7, 1784–1800. Pereira, C.V., Silva, J.M., Rodrigues, L., Reis, R.L., Paiva, A., Duarte, A.R.C., Matias, A., 2019. Unveil the anticancer potential of limomene based therapeutic deep eutectic solvents. Sci Rep. 9, 1–11. Pucci, C., Martinelli, C., Ciofani, G., 2019. Innovative approaches for cancer treatment: current perspectives and new challenges. Ecancermedicalscience 13. Radosevic, K., Canak, I., Panic, M., Markov, K., Bubalo, M.C., Frece, J., Srcek, V.G., Redovnikovic, I.R., 2018. Antimicrobial, cytotoxic and antioxidative evaluation of natural deep eutectic solvents. Environ Sci Pollut Control Ser. 25, 14188–14196. Redasani, V.K., Bari, S.B., 2012. Synthesis and evaluation of mutual prodrugs of ibuprofen with menthol, thymol and eugenol. Eur J Med Chem. 56, 134–138. Salager, E., Stein, R.S., Pickard, C.J., Elena, B., Emsley, L., 2009. Powder NMR crystallography of thymol. Phys Chem Chem Phys. 11, 2610–2621. Salehi, B., Mishra, A.P., Shukla, I., Sharifi-Rad, M., Contreras, M.d.M., SeguraCarretero, A., Fathi, H., Nasrabadi, N.N., Kobarfard, F., Sharifi-Rad, J., 2018. Thymol, thyme, and other plant sources: health and potential uses. Phytother Res. 32, 1688–1706. Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M., Stammati, A., Zucco, F., 2005. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culturerelated factors on Caco-2 cell functional characteristics. Cell Biol Toxicol. 21, 1–26. Savjani, K., Gajjar J, A., Savjani, K., 2012. Drug solubility: importance and enhancement techniques. ISRN Pharm. 2012, 1–10. Silva, J.M., Reis, R.L., Paiva, A., Duarte, A.R.C., 2018. Design of functional therapeutic deep eutectic solvents based on choline chloride and ascorbic acid. ACS Sustainable Chem Eng. 6, 10355–10363. Silva, J.M., Akkache, S., Araújo, A.C., Masmoudi, Y., Reis, R.L., Badens, E., Duarte, A.R.C., 2019. Development of innovative medical devices by dispersing fatty acid eutectic blend on gauzes using supercritical particle generation processes. Mater Sci Eng C. 99, 599–610. Abbaszadeh, S., Sharifzadeh, A., Shokri, H., Khosravi, A., Abbaszadeh, A., 2014. Antifungal efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth of food-relevant fungi. J Mycol Med. 24, e51–e56. Abbott, A.P., Boothby, D., Capper, G., Davies, D.L., Rasheed, R.K., 2004. Deep eutectic solvents formed between choline chloride and carboxylic acids: versatile alternatives to ionic liquids. J Am Chem Soc. 126, 9142–9147. Abo-Hamad, A., Hayyan, M., AlSaadi, M.A., Hashim, M.A., 2015. Potential applications of deep eutectic solvents in nanotechnology. Chem Eng J. 273, 551–567. Aguilar, J., Zavala, A., Diaz-Perez, C., Cervantes, C., Diaz-Perez, A., Campos-Garcia, J., 2006. The atu and liu clusters are involved in the catabolic pathways for acyclic monoterpenes and leucine in Pseudomonas aeruginosa. Appl Environ Microbiol. 72, 2070–2079. Ahmed, E.F., El-Baky, R.M.A., Ahmed, A.B.F., Fawzy, N.G., Aziz, N.A., Gad, G.F.M., 2016. Evaluation of antibacterial activity of some non-steroidal anti-inflammatory drugs against Escherichia coli causing urinary tract infection. Afr J Microbiol Res. 10, 1408–1416. Akrami, H., Aminzadeh, S., Fallahi, H., 2015. Inhibitory effect of ibuprofen on tumor survival and angiogenesis in gastric cancer cell. Tumor Biol. 36, 3237–3243. AL-Janabi, A.A.H.S., 2010. In vitro antibacterial activity of ibuprofen and acetaminophen. J Global Infect Dis. 2, 105. Al-Saidan, S., 2004. Transdermal self-permeation enhancement of ibuprofen. J Contr Release. 100, 199–209. ^ Dionísio, M., Barreiros, S., Reis, R.L., Paiva, A., Aroso, I.M., Craveiro, R., Rocha, A., Duarte, A.R.C., 2015. Design of controlled release systems for THEDES—therapeutic deep eutectic solvents, using supercritical fluid technology. Int J Pharm. 492, 73–79. Badisa, R.B., Darling-Reed, S.F., Joseph, P., Cooperwood, J.S., Latinwo, L.M., Goodman, C.B., 2009. Selective cytotoxic activities of two novel synthetic drugs on human breast carcinoma MCF-7 cells. Anticancer Res. 29, 2993–2996. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods—a review. Int J Food Microbiol. 94, 223–253. C. United States Pharmacopeial, 1995. In: R. Committee of, the United States pharmacopeia, 23rd. United States Pharmacopeia. Cevallos, P.A.P., Buera, M.P., Elizalde, B.E., 2010. Encapsulation of cinnamon and thyme essential oils components (cinnamaldehyde and thymol) in β-cyclodextrin: effect of interactions with water on complex stability. J Food Eng. 99, 70–75. Cl audio, A.F.M., Neves, M.C., Shimizu, K., Lopes, J.N.C., Freire, M.G., Coutinho, J.A., 2015. The magic of aqueous solutions of ionic liquids: ionic liquids as a powerful class of catanionic hydrotropes. Green Chem. 17, 3948–3963. Corvis, Y., Wurm, A., Schick, C., Espeau, P., 2015. New menthol polymorphs identified by flash scanning calorimetry. Cryst Eng Comm. 17, 5357–5359. Coussens, L.M., Werb, Z., 2002. Inflammation and cancer. Nature. 420, 860–867. Cox, S.D., Markham, J., 2007. Susceptibility and intrinsic tolerance of Pseudomonas aeruginosa to selected plant volatile compounds. J Appl Microbiol. 103, 930–936. Davies, N.M., 1998. Clinical pharmacokinetics of ibuprofen. Clin Pharmacokinet. 34, 101–154. del Monte, Carriazo, D., Serrano, M.C., Gutierrez, M.C., Ferrer, M.L., 2014. Deep eutectic solvents in polymerizations: a greener alternative to conventional syntheses. Chem Sus Chem. 7, 999–1009. Duarte, A.R.C., Ferreira, A.S.D., Barreiros, S., Cabrita, E., Reis, R.L., Paiva, A., 2017. A comparison between pure active pharmaceutical ingredients and therapeutic deep eutectic solvents: solubility and permeability studies. Eur J Pharm Biopharm. 114, 296–304. Endo, H., Yano, M., Okumura, Y., Kido, H., 2014. Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70. Cell Death Dis. 5, e1027 e1027. Esmaeili, A., Hashemi, E., 2011. Biotransformation of myrcene by Pseudomonas aeruginosa. Chem Cent J. 5, 26. Filippa, M.A., Gasull, E.I., 2013. Ibuprofen solubility in pure organic solvents and aqueous mixtures of cosolvents: interactions and thermodynamic parameters relating to the solvation process. Fluid Phase Equil. 354, 185–190. Galeotti, N., Mannelli, L.D.C., Mazzanti, G., Bartolini, A., Ghelardini, C., 2002. Menthol: a natural analgesic compound. Neurosci Lett. 322, 145–148. García-Salinas, S., Elizondo-Castillo, H., Arruebo, M., Mendoza, G., Irusta, S., 2018. Evaluation of the antimicrobial activity and cytotoxicity of different components of natural origin present in essential oils. Molecules. 23, 1399. Goud, N.R., Suresh, K., Sanphui, P., Nangia, A., 2012. Fast dissolving eutectic compositions of curcumin. Int J Pharm. 439, 63–72. Gutierrez, M.a.C., Ferrer, M.a.L., Mateo, C.R., del Monte, F., 2009. Freeze-drying of aqueous solutions of deep eutectic solvents: a suitable approach to deep eutectic suspensions of self-assembled structures. Langmuir. 25, 5509–5515. Hayyan, M., Looi, C.Y., Hayyan, A., Wong, W.F., Hashim, M.A., 2015. In vitro and in vivo toxicity profiling of ammonium-based deep eutectic solvents. PLoS One. 10, e0117934. Islam, M.T., Khalipha, A.B., Bagchi, R., Mondal, M., Smrity, S.Z., Uddin, S.J., Shilpi, J.A., Rouf, R., 2019. Anticancer activity of thymol: a literature-based review and docking study with Emphasis on its anticancer mechanisms. IUBMB Life 71, 9–19. Jahan, M.S., Islam, M.J., Begum, R., Kayesh, R., Rahman, A., 2014. A study of method development, validation, and forced degradation for simultaneous quantification of Paracetamol and Ibuprofen in pharmaceutical dosage form by RP-HPLC method. Anal Chem Insights. 9, 75. Johnson, C.H., Wilson, I.D., Harding, J.R., Stachulski, A.V., Iddon, L., Nicholson, J.K., Lindon, J.C., 2007. NMR spectroscopic studies on the in vitro acyl glucuronide 8 E. Silva et al. Current Research in Chemical Biology 1 (2021) 100003 Stott, P.W., Williams, A.C., Barry, B.W., 1998. Transdermal delivery from eutectic systems: enhanced permeation of a model drug, ibuprofen. J Contr Release. 50, 297–308. Vaara, M., Nurminen, M., 1999. Outer membrane permeability barrier in Escherichia coli mutants that are defective in the late acyltransferases of lipid A biosynthesis. Antimicrob Agents Chemother. 43, 1459–1462. Wiener, M.C., Horanyi, P.S., 2011. How hydrophobic molecules traverse the outer membranes of Gram-negative bacteria. Proc Natl Acad Sci Unit States Am. 108, 10929–10930. Wikene, K.O., Bruzell, E., Tønnesen, H.H., 2015. Characterization and antimicrobial phototoxicity of curcumin dissolved in natural deep eutectic solvents. Eur J Pharmaceut Sci. 80, 26–32. Zhang, Q., Vigier, K.D.O., Royer, S., Jerome, F., 2012. Deep eutectic solvents: syntheses, properties and applications. Chem Soc Rev. 41, 7108–7146. Silva, E., Oliveira, F., Silva, J.M., Matias, A., Reis, R.L., Duarte, A.R.C., 2020. Optimal design of THEDES based on perillyl alcohol and ibuprofen. Pharmaceutics. 12, 1121. Sintra, T.E., Shimizu, K., Ventura, S.P., Shimizu, S., Lopes, J.C., Coutinho, J.A., 2018. Enhanced dissolution of ibuprofen using ionic liquids as catanionic hydrotropes. Phys Chem Chem Phys. 20, 2094–2103. Smith, E.L., Abbott, A.P., Ryder, K.S., 2014. Deep eutectic solvents (DESs) and their applications. Chem Rev. 114, 11060–11082. Soares, B., Tavares, D.J., Amaral, J.L., Silvestre, A.J., Freire, C.S., Coutinho, J.o.A., 2017. Enhanced solubility of lignin monomeric model compounds and technical lignins in aqueous solutions of deep eutectic solvents. ACS Sustainable Chem Eng. 5, 4056–4065. Stott, P.W., Williams, A.C., Barry, B.W., 1998. Transdermal delivery from eutectic systems: enhanced permeation of a model drug, ibuprofen. J Contr Release. 50, 297–308. 9