Subido por Lisbeth Encalada

1-s2.0-S2666246921000033-main

Anuncio
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
Descargar