molecules

Article
Natural Deep Eutectic Solvents (NADES) as a Tool for
Bioavailability Improvement: Pharmacokinetics of
Rutin Dissolved in Proline/Glycine after Oral
Administration in Rats: Possible Application
in Nutraceuticals
Marta Faggian 1 , Stefania Sut 1 , Beatrice Perissutti 2 , Valeria Baldan 1 , Iztok Grabnar 3 and
Stefano Dall’Acqua 1, *
1 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5,
35131 Padova, Italy; marta.faggian@studenti.unipd.it (M.F.); stefania_sut@hotmail.it (S.S.);
valeria.baldan.4@studenti.unipd.it (V.B.)
2 Department of Chemical and Pharmaceutical Sciences, University of Trieste, P.le Europa 1,
34127 Trieste, Italy; bperissutti@units.it
3 Faculty of Pharmacy, University of Ljubljana, Askerceva cesta 7, SI-1000 Ljubljana, Slovenia;
Iztok.Grabnar@ffa.uni-lj.si
* Correspondence: stefano.dallacqua@unipd.it; Tel.: +39-049-827-5344; Fax: +39-049-827-5366

Academic Editor: Josef Jampilek

Received: 27 September 2016; Accepted: 4 November 2016; Published: 14 November 2016

Abstract: There is a need for innovation in plant-derived pharmaceuticals, food supplements

and nutraceutical products regarding the use of more eco-sustainable solvents for their extraction.
Furthermore, the poor oral bioavailability of several phytochemicals with health promoting effects
stimulates the research in the field of pharmaceutical formulations. Natural Deep Eutectic Solvents
(NADES) are formed by natural compounds, and can be considered as future solvents being
especially useful for the preparation of nutraceuticals and food-grade extracts. In this paper
various NADES were prepared using sugars, aminoacids and organic acids. Rutin (quercetin-3-O-
α-L-rhamnopyranosyl-(1→6))-β-D-glucopyranose) was used as a model compound to study
NADES. Moreover, the effect of various eutectic mixtures on rutin’s water solubility was studied.
Proline/glutamic acid (2:1) and proline/choline chloride (1:1) mixtures have a solubility comparable
to ethanol. The proline/glutamic acid (2:1) eutectic containing rutin was used in a pharmacokinetic
study in Balb/c mice while bioavailability was compared to oral dosing of water suspension.
Plasmatic levels of rutin were measured by HPLC-MS/MS showing increased levels and longer
period of rutin permanence in plasma of NADES treated animals. This paper reports the possible use
of non-toxic NADES for pharmaceutical and nutraceutical preparations.

Keywords: NADES; HPLC-MS; rutin; pharmacokinetics

1. Introduction
Health promoting products such as herbal medicines, food supplements or nutraceuticals obtained
by solvent extraction from plants or foods are widespread. However, the conventional extraction
methods used have several drawbacks, namely low selectivity and residual solvent in the final
products. Due to their safety level, the most commonly used solvents for nutraceutical production
are water, ethanol or aqueous ethanol mixtures. Unfortunately, these mixtures may be scarcely
efficient in extraction due to the variable nature and polarity of extractable bioactive compounds [1–4].
Additionally, many claimed active ingredients of nutraceuticals have poor bioavailability. Thus the

Molecules 2016, 21, 1531; doi:10.3390/molecules21111531 www.mdpi.com/journal/molecules

Molecules 2016, 21, 1531 2 of 11

need for further research in the field of nutraceutical and pharmaceutical formulation to enhance
the oral absorption of such compounds. A new approach in this area may be represented by the
so called deep eutectic solvents (DES) or Low Transition Melting Mixtures (LTMM). These solvents
are mixtures of organic compounds having significantly lower melting points than their individual
components. They have been developed as an alternative to other solvents, namely, the ionic liquids
(IL), i.e., salts possessing particular physicochemical properties (viscosity, density, hydrophilicity,
solubility), which may be tuned by combining different cations and anions [5]. IL are not allowed in
food or food supplement production due to their potential toxicity and are generally avoided due
to their “non-natural” origin. In recent years, some special DES that were produced using natural
products have been studied and generally called Natural Deep Eutectic Solvents. These new solvents
have gained much attention from the scientific community, especially in the green chemistry area,
for they have a potential for replacing common organic solvents presenting inherent toxicity and
high volatility, thereby releasing volatile organic compounds in the atmosphere [6–8]. In this context,
Natural Deep Eutectic Solvents (NADES) comprising natural compounds, such as organic acids, amino
acids and sugars, have been put forward. NADES are obtained by the complexation of a hydrogen
acceptor and a hydrogen-bond donor. Such solvents are almost non-volatile at ambient condition,
are chemically and thermally stable, non-flammable, and have good solvent properties for several
organic compounds [5,7,9].
While the poor solubility of several bioactive compounds in water and ethanol mixtures is a severe
limitation in the extraction of food supplements and nutraceutical bioactive ingredients [3,10–13],
any alternative ideal solvent should present high level of safety and eco-sustainability as well as
improved extraction performances. The DES capacity for the extraction of bioactive natural products
is correlated with their physical–chemical properties, including H-bonding interactions, polarity,
viscosity, and pH. The high extractability of phenolic acids with DES may be attributed to H-bonding
interactions between DES molecules and phenolic compounds. The polarity of DESs is an important
factor affecting their extraction efficiency. Nevertheless, NADES can be considered also as “ingredients”
in a nutraceutical or functional food, and offer the possibility of combining various molecules, leading
to the preparation of tailor-made solvents for solutes. The combination of various molecules in different
NADES leads to the preparation of tailor-made solvents designed to extract solutes with different
properties (polarity, charge etc.) [4,6–8,14,15]. Radošević at al. studied various phenolic grape skin
extracts obtained by using NADESs and tested their biological activity in two human tumor cell lines
(HeLa and MCF-7). Results show that NADES components could be chosen not only to fine-tune
solvent physicochemical characteristics but also to enhance biological activity of extracts prepared in
NADESs [16]. Due to the food grade property of these ingredients, it is assumed that extracts obtained
by NADESs may be directly used in products for human consumption without the need for expensive
downstream purification steps [17,18]. This was demonstrated by in vitro cytotoxicity assays using
two human cell lines (MCF-7 and HeLa) of a few tailor made NADES. The tested NADESs possessed
low cytotoxicity, which makes them good candidates for the green extraction, leading to the novel
application of NADES in food and pharmaceutical industry [16].
DES have also been taken into consideration for pharmaceutical applications. Morrison et al.
considered them as solvents for low water soluble drugs, including griseofulvin, itraconazole and
danazol [19,20]. Recently deep eutectic solvent derivatives (DESD) were used for the solubilization of
poorly water-soluble drugs such as itraconazole, piroxicam, lidocaine, and posaconazole while the
enhanced drug solubility and the DESD properties were considered attractive for topical formulations
of such drugs [21].
The use of NADES as extraction solvents appears a promising approach in the field of
nutraceuticals, especially for natural products with poor bioavailability. To our knowledge,
the literature in this field is very scarce. In the present paper, NADES were investigated as solubilizers
using rutin as a model compound. Rutin (30 ,40 ,5,7-tetrahydroxy-flavone-3-rutinoside) is a common
dietary glycosylated flavonoid present in fruits, vegetables and in many plant-derived beverages
Molecules 2016, 21, 1531 3 of 11

such as tea and wine [22]. It has been extensively studied due to its anti-inflammatory [23],
antibacterial [24], cancer chemopreventive [25] and antidepressive [26] activities. Furthermore,
it is used in pharmaceutical and nutraceutical products as a phlebotonic drug, although literature
supporting these effects is still limited [27]. Rutin has been widely used for the treatment of chronic
venous insufficiency; further uses that have been proposed are glaucoma, hay fever, haemorrhoids,
varicose veins, poor circulation, oral herpes, cirrhosis, stress, low serum calcium, and cataract [28].
Pharmacokinetics and oral absorption of rutin and aglycone quercetin were previously studied
in healthy volunteers at various dose levels [19]. Quercetin glucuronides and/or sulfates were
measured in plasma, and no rutin was detectable, indicating the intense metabolism of these
compounds. The authors claimed that prior to absorption rutin is hydrolized. Additionally,
there are two pharmacokinetic studies with oral administration of rutin in rats. Recently, rutin
and quercetin as patented polyherbal formulations were co-administered in rats by gavage, and
bioavailability was compared to that of the co-administration of the two pure compounds at equivalent
doses [29]. The observed differences indicated that from the polyherbal formulation bioavailability
of rutin had increased, while bioavailability of quercetin has decreased when compared to the
co-administration [29]. In a methodological paper describing an HPLC-MS/MS method for rutin
quantification in rat plasma, preliminary pharmacokinetic data were published showing the maximum
concentration (1659 ng/mL) 5 min after 2.5 mg/kg sublingual vein administration of rutin [28].
He et al. reported that the three flavonoid glycosides (rutin, astragalin and isoquercitin) were rapidly
absorbed and eliminated from rat plasma after oral administration of total flavonoids from mulberry
leaves [30].
The aim of this study was to evaluate the use of food-components NADES as vehicles for rutin
administration and to estimate their influence on rutin bioavailability. Thus a series of NADES
were prepared using various compounds. Rutin solubility was evaluated in various eutectic systems
comparing the results with water and ethanol. In order to study the possibility of using NADES as
administration vehicles, the proline/glutamic acid (2:1) eutectic was tested in a pharmacokinetic study
in Balb/c mice using 10 mg/kg oral dose of rutin, and compared to the same dose of rutin suspended
in water.

2. Results

2.1. Rutin Solubility in the Different Prepared NADES

Various NADES composed of urea, amino acids, sugars and choline were used to solubilize rutin.
Thirty NADES were selected and used for evaluation of rutin solubility. Water and ethanol were used as
reference solvents due to their favorable use as solvents in the production of extracts for nutraceuticals
and functional foods. Water was selected as a reference, because of the poor rutin solubility (120 µg/mL
at 22 ◦ C). For comparison purposes, the solubility ratio (solvent/water) was used. The solubility and
solubility ratios of rutin in each solvent are reported in Table 1 and summarized in Figure 1.

Table 1. Solubility of rutin (mean ± SD of 3 determinations) in each solvent. The solubility in all
solvents were significantly different from solubility in water (p-values < 0.05).

Rutin Solubility at 22 ◦ C
Solvent Class Mixture Number Solvent
(µg/mL)
Water 120.0 ± 4.9
Reference solvent Ethanol 2369.7 ± 93.2
Methanol 2053.7 ± 89.7
1 Urea–Glucose–Citric Acid 1:1:1 378.7 ± 8.5
2 Urea–Glucose–Fructose 1:1:1 961.3 ± 30.6
Urea based
3 Urea–Tartaric Acid 1:1 679.8 ± 19.0
4 Urea–Choline chloride 1:1 1883.3 ± 48.1
Molecules 2016, 21, 1531 4 of 11

Table 1. Cont.

Rutin Solubility at 22 ◦ C
Solvent Class Mixture Number Solvent
(µg/mL)
5 Glucose–Fructose–Water 1:1:1 81.9 ± 2.5
Sugar based
Molecules 2016, 21, 1531 6 Glucose–Fructose–Sorbitol 1:1:1 90.8 ± 1.9 4 of 11
7 Citric Acid–Fructose 1:1 205.1 ± 5.1
Sugar and organic acid 108 Proline–Glutamic Acid 1:1 2255.9
Tartaric Acid–Fructose 1:1 504 ± ±16.4
63.4
based
119 Proline–Glutamic Acid 2:11:1:1
Glucose–Citric Acid–Water 2938.4 ±117.9
175.2 ± 3.7
12
10 Proline–Oxalic Acid
Proline–Glutamic 1:11:1
Acid 749.3 ±±22.5
2255.9 63.4
13
11 Proline–Tartaric
Proline–GlutamicAcid 1.12:1
Acid 546.9±
2938.4 ± 19,9
117.9
12
14 Proline–OxalicAcid
Ornitine–Tartaric Acid 1:1 209.7±±22.5
749.3 5.7
13
15 Proline–TartaricAcid
Arginine–Tartaric Acid 1:1
1.1 362.7±
546.9 19,9
± 11.2
14 Ornitine–Tartaric Acid 1:1 209.7 ± 5.7
16 Citrulline–Tartaric Acid 1.1 370.4 ± 13.1
15 Arginine–Tartaric Acid 1:1 362.7 ± 11.2
17
16 Arginine–Oxalic Acid
Citrulline–Tartaric 1:11.1
Acid 414.3 ±
370.4 ± 13.6
13.1
Organic acid and amino 18
17 Proline–Malic Acid 1:1
Arginine–Oxalic Acid 1:1 900.3
414.3 ±± 31.1
13.6
Organicacids
acid based
and amino 19
18 Arginin–Malic
Proline–MalicAcid
Acid1:1
1:1 457.4±
900.3 ± 17.8
31.1
acids based 19
20 Arginin–MalicAcid
Ornitine–Malic Acid1:1
1:1 408.0±
457.4 17.8
± 14.8
20
21 Ornitine–MalicAcid
Citrulline–Malic Acid1:1
1:1 454.5±
408.0 14.8
± 18.5
21 Citrulline–Malic Acid 1:1 454.5 ± 18.5
22 Proline–Citric Acid 1:1 672.5 ± 26.2
22 Proline–Citric Acid 1:1 672.5 ± 26.2
23
23 Arginine–Citric
Arginine–CitricAcid
Acid1:1
1:1 414.3±
414.3 ± 13.3
13.3
24
24 Ornitine–Citric Acid1:1
Ornitine–Citric Acid1:1 424.7 ±
424.7 ± 17.2
17.2
25
25 Citrulline–Citric
Citrulline–Citric Acid1:1
Acid 1:1 459.7±
459.7 ± 14.4
26
26 Proline–Glucose1:1
Proline–Glucose1:1 878.7±
878.7 31.3
± 31.3
27
27 Proline–Fructose1:1
Proline–Fructose 1:1 1563.9±
1563.9 43.8
± 43.8
28
28 Proline–Choline
Proline–Choline Chloride1:2
Chloride 1:2 2642.8±
2642.8 101.3
± 101.3
Choline
Cholinechloride
chloridebased
based 29
29 Proline–Choline
Proline–Choline Chloride1:3
Chloride 1:3 2799.2±
2799.2 103.3
± 103.3
30 Choline Chloride–Malic Acid 1:1 702.0 ± 22.3
30 Choline Chloride–Malic Acid 1:1 702.0 ± 22.3

Figure 1. Solubility ratio of rutin (solvent/water) in each prepared NADES. The solubility in all
Figure 1. Solubility ratio of rutin (solvent/water) in each prepared NADES. The solubility in all
solvents were significantly different from solubility in water (p-values < 0.05).
solvents were significantly different from solubility in water (p-values < 0.05).
These eutectics, prepared using different starting materials, can be divided into five groups, the
These
first is basedeutectics,
on urea prepared using different
(1–4), the second starting
on polyalcohols materials,
and canthe
sugars (5–6), be third
divided into five
on organic groups,
acid and
thesugars
first is(7–9),
based onfourth
the urea (1–4), the second
on organic on polyalcohols
acid and and sugars
amino acid (10–27), (5–6),
and the thegroup
fifth third was
on organic
preparedacid
and sugars
using (7–9),
choline the fourth
chloride, on and
sugars organic
aminoacid and
acid aminoOur
(28–30). acidresults
(10–27), andthe
show theability
fifth group wasNADES
of various prepared
to dissolve
using cholinerutin. To our
chloride, knowledge,
sugars and amino no published data
acid (28–30). areresults
Our available
showrelated to the of
the ability in various
vivo effects
NADESof
toNADES
dissolveasrutin.
administration tools for bioactive
To our knowledge, constituents.
no published data are available related to the in vivo effects of
NADES as administration tools for bioactive constituents.
2.2. HPLC-MS/MS Method Validation

2.2.1. Specificity, linearity, LOQ and LOD

Exemplary multiple reaction monitoring (MRM) chromatograms for rutin-spiked plasma (rutin
20 ng/mL, ISTD 100 ng/mL) are reported in Figure 2. Five calibration mixtures prepared mixing
Molecules 2016, 21, 1531 5 of 11

2.2. HPLC-MS/MS Method Validation

2.2.1. Specificity, linearity, LOQ and LOD

Exemplary multiple reaction monitoring (MRM) chromatograms for rutin-spiked plasma
(rutin 20 ng/mL, ISTD 100 ng/mL) are reported in Figure 2. Five calibration mixtures prepared
mixing different
Molecules ratios of rutin/ISTD (namely, 0.053, 0.0106, 0.0181, 0.0363, 0.0727, 0.1453) were
2016, 21, 1531 5 ofused
11
to prepare calibration curve (y = area of rutin/area of ISTD; x = quantity of rutin/quantity of ISTD)
calibration
that was linear curve
and(yreliable
= area of rutin/area
over of ISTD; xcalibration
the considered = quantity range.
of rutin/quantity of ISTD) that was
Limit of quantification linear
(LOQ) was
and reliable over the considered calibration
3 ng/mL and limit of detection was 1 ng/mL for rutin. range. Limit of quantification (LOQ) was 3 ng/mL and
limit of detection was 1 ng/mL for rutin.

Figure
Figure2. 2.HPLC-MS/MS
HPLC-MS/MSchromatograms
chromatograms corresponding transitions 609
corresponding to transitions 609>>301
301for
forrutin
rutinand
and481
481> > 124
124
forfor ISTD
ISTD (silimarin)ofofblank
(silimarin) blankplasma
plasma(traces
(traces red
red and green for
for rutin
rutinand
andISTD
ISTDrespectively)
respectively)and plasma
and plasma
spiked
spiked with
with rutinand
rutin andISTD
ISTD(traces
(tracesyellow
yellowand
and blue
blue for
for rutin and ISTD
ISTD respectively).
respectively).

2.2.2.
2.2.2. Accuracyand
Accuracy andPrecision
Precision
Spikedsamples
Spiked sampleswere
wereassayed
assayedfor
forintra-day
intra-day and
and inter-day
inter-day precision
precisionand
andaccuracy
accuracyatatconcentrations
concentrations
of 10, 20, 80 ng/mL. Data are summarized in Table
of 10, 20, 80 ng/mL. Data are summarized in Table 2. 2.

Table 2. Intra-day and inter-day precision and accuracy at different concentrations.

Table 2. Intra-day and inter-day precision and accuracy at different concentrations.
Nominal Measured
RSD (%) * Accuracy (%)
Nominal Concentration
Concentration (ng/mL) Measured
(ng/mL)
(ng/mL) RSD (%) * Accuracy (%)
(ng/mL)
10 10.76 ± 0.30 3.2 107.5
10 20 19.64±± 0.30
10.76 0.79 3.2
4.9 107.5
98.2
Intra day (n = 5)
Intra day (n = 5)
20 40 38.87±± 0.79
19.64 1.10 4.9
2.8 98.2
97.1
40 38.87 ± 1.10 2.8 97.1
80 80.46 ± 1.88 2.4 100.5
80 80.46 ± 1.88 2.4 100.5
10 10.22 ± 0.40 3.9 102.2
10 20 10.22 ± 0.40
19.44 ± 0.61 3.9
3.2 102.2
97.2
Inter day (n = 5) 20 19.44 ± 0.61 3.2 97.2
Inter day (n = 5) 40 39.11 ± 1.86 4.7 97.8
40 39.11 ± 1.86 4.7 97.8
80 80 78.79 ± 1.80
78.79 ± 1.80 2.9
2.9 98.4
98.4
**Relative StandardDeviation.
Relative Standard Deviation.

2.3. Pharmacokinetics in Mice of Proline-Glutamic Acid Rutin Eutectic and Rutin Water Suspensions
Considering the solubility properties nine NADES (2, 3, 4, 10, 11, 17, 28, 29, 30) were able to
solubilize half of the rutin compared to the best solvent (ethanol). On the other hand, eutectic 11 and
28 (proline/glutamic acid 2:1 and proline/choline chloride 1:2 respectively) resulted to be the most
promising NADES presenting rutin solubilization higher than ethanol and methanol. These results
showed the ability of different NADES to dissolve rutin. Mixture 11 (Proline–Glutamic Acid 2:1) was
Molecules 2016, 21, 1531 6 of 11

2.3. Pharmacokinetics in Mice of Proline-Glutamic Acid Rutin Eutectic and Rutin Water Suspensions
Considering the solubility properties nine NADES (2, 3, 4, 10, 11, 17, 28, 29, 30) were able to
solubilize half of the rutin compared to the best solvent (ethanol). On the other hand, eutectic 11 and
28 (proline/glutamic acid 2:1 and proline/choline chloride 1:2 respectively) resulted to be the most
promising NADES presenting rutin solubilization higher than ethanol and methanol. These results
showed the ability of different NADES to dissolve rutin. Mixture 11 (Proline–Glutamic Acid 2:1) was
selected to this purpose, because it contains only two amino acids and is therefore suitable for oral
administration.
Molecules 2016, Selected
21, 1531 rutin NADES 11 formulation (Proline–glutamic acid: 2:1) and rutin as 6 ofa11water
suspension were administered orally to mice, and plasma levels were determined up to 4 h after
administration.
administration. Selected rutin
As reported NADES
in Figure 11 formulation
3 and (Proline–glutamic
Table 3, different plasma levels acid: 2:1)observed.
were and rutin as a
water suspension were administered orally to mice, and plasma levels
Rutin (10 mg dose dissolved in 0.5 mL of solvent) in NADES 11 and as water suspension were determined up to 4 h was
after administration. As reported in Figure 3 and Table 3, different plasma levels were observed.
administered orally to mice and the plasma levels were determined up to 4 h after administration.
Rutin (10 mg dose dissolved in 0.5 mL of solvent) in NADES 11 and as water suspension was
As demonstrated in Figure 3 and Table 3, incorporation of rutin in NADES markedly affected rutin
administered orally to mice and the plasma levels were determined up to 4 h after administration.
plasmaAs levels.
demonstrated in Figure 3 and Table 3, incorporation of rutin in NADES markedly affected rutin
With
plasma both formulations, absorption of rutin was fast with tmax at 15 min following administration
levels.
of suspension and formulations,
With both 60 min following the administration
absorption of NADES
of rutin was fast with tmax at 15(Figure 3). Theadministration
min following elimination from
plasma was also rapid
of suspension and 60with
min afollowing
terminalthe half-life of 106 min
administration and 86(Figure
of NADES min for 3). suspension
The eliminationandfrom
NADES,
plasma was also rapid with a terminal half-life of 106 min and 86 min for suspension
respectively. Despite the difference in tmax , MRT of rutin with the two formulations was similar and NADES,
respectively.
(158 min Despite
vs. 131 min) the difference
indicating that in tmax,is
there MRT of rutin with difference
no remarkable the two formulations was similar
in the absorption (158
rate. min was,
There
vs. 131 min) indicating that there is no remarkable difference in the absorption
however, a large difference in Cmax and AUC. Relative bioavailability of NADES vs. suspension rate. There was,
however, a large difference in Cmax and AUC. Relative bioavailability of NADES vs. suspension was 2,
was 2, indicating approximately 100% improvement in the extent of rutin absorption with NADES,
indicating approximately 100% improvement in the extent of rutin absorption with NADES, presumably
presumably due to improved solubility. Thus, the use of NADES 11 can be considered as an interesting
due to improved solubility. Thus, the use of NADES 11 can be considered as an interesting tool for
tool for solubilization
solubilization and administration
and administration of rutinofdue
rutin due to valuable
to valuable pharmacokinetic
pharmacokinetic properties. properties.

70

Suspension
60 NADES

50
Concentration (ng/ml)

40

30

20

10

0
0 50 100 150 200 250 300

Time (min)

Figure 3. Time course (mean ± SD) of plasma concentration time course of rutin in Balb/c mice
Figure 3. Time course (mean ± SD) of plasma concentration time course of rutin in Balb/c mice
following oral administration of 10 mg as proline/glutamic NADES or suspension in water.
following oral administration of 10 mg as proline/glutamic NADES or suspension in water.
Table 3. Non-compartmental pharmacokinetic parameters of rutin following oral administration in
3. Non-compartmental
TableBalb/c mice (dose 10 mg). pharmacokinetic parameters of rutin following oral administration in
Balb/c mice (dose 10 mg).
Pharmakokinetic Parameter Suspension NADES
tmax (min) 15 60
Pharmakokinetic Parameter Suspension NADES
Cmax (ng/mL) 28.6 58.3
t1/2 λtzmax (min)
(min) 15 106 60 86
AUClastC(ng (ng/mL)
max min/mL) 28.6 2225 58.3 4862
t1/2 λz (min) 106 86
AUC (ng min/mL) 2888 5806
AUClast (ng min/mL) 2225 4862
AUC (%Extrapolated) 23 16
AUC (ng min/mL) 2888 5806
MRT
AUC (min)
(%Extrapolated) 23 158 16 131
MRT (min) 158 131
3. Discussion
Recently Dai et al. investigated the possibility to modulate NADES properties as solvents for
poorly soluble natural compounds, also by adding small amount of water decreasing viscosity and
improving conductibility [8]. For example, a previously published paper considered the mixtures
sucrose-choline chloride, lactic acid–glucose and proline–malic acid; these NADES were reported to
be efficient for polyphenol extraction from Cartamus tinctorius L. [18]. The solubility and physical
Molecules 2016, 21, 1531 7 of 11

3. Discussion
Recently Dai et al. investigated the possibility to modulate NADES properties as solvents for
poorly soluble natural compounds, also by adding small amount of water decreasing viscosity and
improving conductibility [8]. For example, a previously published paper considered the mixtures
sucrose-choline chloride, lactic acid–glucose and proline–malic acid; these NADES were reported to
be efficient for polyphenol extraction from Cartamus tinctorius L. [18]. The solubility and physical
properties of NADES can be modulated by adding water to the composition. The supermolecular
complex structures of proline choline NADES are preserved when the content of water is below
50% while further dilution produces a solution of the free forms of the individual components in
water, showing that gradual changes in the structure of NADES during dilution may affect their
physicochemical properties and also their applications [8]. Recently, Aroso et al. proposed therapeutic
deep eutectic systems prepared using choline chloride or menthol conjugated with three different active
principles, namely, acetylsalicylic acid, benzoic acid and phenylacetic acid. Their results indicated the
potential of these eutectics as dissolution enhancers in the development of novel and more effective
drug delivery systems. However, their experiments were limited to in vitro trials only [31].

4. Experimental Section

4.1. Chemicals
NADES have been prepared using mixtures of sugars (glucose, fructose), amino acids (glutamic
acid, proline, arginine, citrulline, ornithine), organic acids (citric acid, malic acid, oxalic acid, tartaric
acid), and other compounds containing nitrogen (urea and choline chloride). As regards sugars and
polyols, glucose were purchased from Carlo Erba (Milan, Italy), fructose, from Sigma-Aldrich (Milan,
Italy). As regards organic acids, citric and oxalic acids were purchased from Riedel-De-Haen AG
(Seelze, Germany), tartaric acid from Codex (Turin, Italy), malic acid from Carlo Erba. As regards
amino acids and derivative, alanine was purchased from Merck (Vimodrone (MI), Italy), histidine from
Sigma-Aldrich, proline, arginine, citrulline and ornithine from Fagron (Bologna, Italy). Choline chloride
was purchased from Sigma-Aldrich and urea from Alfa Aesar (Karlsruhe, Germany). Rutin and internal
standard silimarin were purchased from Sigma-Aldrich. Solvents as acetonitrile and methanol of
HPLC grade were purchased from Scharlab (Riozzo di Cerro al Lambo (MI), Italy), formic acid from
Carlo Erba reagents.

4.2. NADES Preparation

For NADES preparation we used the previously reported approaches described in the review of
Dai et al. 2013 [6].

4.3. Solubility Trials and Quantification of Solubilized Rutin in the NADES by HPLC-DAD
Exactly weighed quantity of rutin was suspended in water, ethanol, methanol and in the various
prepared NADES with a concentration of 2.5 mg/mL. Samples were stirred on a magnetic stirrer (Stuart,
Bibby Scientific Ltd., Stone, Staffordshire, UK) for one hour at room temperature and centrifuged for
21 min at 13,000 rpm with Eppendorf 5415 R centrifuge The time of one-hour stirring was chosen
because it was sufficient for all the prepared mixtures to reach maximum concentration of rutin.
For quantitative measurement of solubilized rutin, a portion (100 µL) of the clear supernatant obtained
after centrifugation was diluted 1:5 mL in DMSO. Dilution is necessary to assess the amount of rutin
because of the high concentration of solutions and because of the high viscosity of NADES.
For quantification standard solution of rutin (100 µg/mL) was prepared dissolving rutin in
methanol with an ultrasound bath. Calibration curve was obtained injecting standard solution
at different concentrations namely (50, 25, 10, 5 and 1 µg/mL) Calibration curve was as follows
y = 17.832 x + 9.7822 (R2 was 0.9998). Limit of Quantification was 1 µg/mL.
Molecules 2016, 21, 1531 8 of 11

For HPLC-DAD a series 1260 HPLC instrument (Agilent, Cernusco Sul Naviglio (MI), Italy)
equipped with a quaternary pump, a diode-array detector, an auto sampler and a column oven
compartment was used. Analyses were performed on Eclipse XDB C8 column (5 µm, 4.6 mm × 150 mm,
Agilent). The mobile phase was (A) water-formic acid (99.9:0.1, v/v) and (B) acetonitrile. A gradient
program was used as follows: [0 → 10th min: A:B (90:10) → A:B (60:40) 10 → 11th min: A:B (60:40) →
A:B (0:100) 11 → 12th min: A:B (0:100) → A:B (0:100) 12 → 13th min: A:B (0:100) → A:B (90:10) 13 →
14th min: A:B (90:10) → A:B (90:10)]. The mobile phase flow rate was 1.2 mL/min and the injection
volume was 10 µL. The chromatogram was recorded at 350 nm and spectral data for all peaks were
obtained in the range of 190–400 nm. The retention time of rutin in the analysis conditions was 3.8 min.

4.4. Animals Blood Collection and Extraction

All experimental protocols involving animals were reviewed and approved by the Ethical
Committee for animal Experiments of the University of Padua (CEASA; 49571). Female, Balb/c
mice (8–10 weeks old) were housed (three per cage) in polycarbonate cages and kept on a 12 h
light/dark cycle. Food and water were given ad libitum. Mice, randomly divided into two groups of
15 animals each, received 10 mg of rutin (20 mg/mL) by oral gavage as water suspension or proline
glutamic acid 2:1 NADES. A single blood sample was collected by cardiac puncture from each animal
at 15, 30, 60, 120, or 240 min after dosing. Whole blood samples were heparinized. Three samples were
obtained per each time point and each treatment group.

4.5. HPLC-MS Plasma Analysis

Standard stock solutions for determination of rutin in mice blood were prepared by dissolving
rutin and internal standard silimarin in methanol (100 µg/mL and 150 µg/mL respectively).
Rutin stock solution was diluted 1:100 (100 µg/mL → 1 µg/mL) and silimarin (ISTD) stock
solution was diluted 1:10 (150 µg/mL → 15 µg/mL). The calibration curve was obtained mixing
100 µL of 15 µg/mL IS with different volume (400, 200, 100, 50, 25, 10, 5 and 2.5 µL) of 1 µg/mL rutin
standard solution in order to obtain different rutin/silimarin quantity ratios. Mixture of IS and rutin
were added to blank plasma samples and used for sample and calibration curve preparation. To 400 µL
of whole blood 100 µL of IS solution were added (161 ng) and acidified methanol was added (300 µL)
in order to precipitate proteins. Sample was vortexed and subjected to five minute ultrasound bath
at room temperature. The sample was then centrifuged and the clear supernatant was transferred to
an Eppendorf tube and dried under nitrogen flow. 200 µL of methanol were then used to redissolve
the solid and used for HPLC-MS/MS measurements.
For measurement an Agilent series 1260 HPLC chromatograph equipped with a Prostar 410
autosampler (Varian, Cernusco Sul Naviglio (MI), Italy) and coupled with Varian 320 TQD MS
spectrometer was used. The mass spectrometer was equipped with electrospray ionization (ESI) source
as the interface and analysis was conducted in negative ion mode for both the analytes. Analyses
were performed on a Pursuit XRs 3 C18 column (50 mm × 2.0 mm, Varian). The mobile phase
was (A) water-formic acid (99:1 v/v) and (B) acetonitrile. A gradient program was used as follows:
[0 → 1th min: A:B (85:15) → A:B (85:15) 1 → 7th min: A:B (85:15) → A:B (0:100) 7 → 8th min: A:B
(0:100) → A:B (85:15) 8→11th min: A:B (85:15) → A:B (85:15)]. The mobile phase flow rate was
220 µL/min. The injection volume was 10 µL.
The ESI source was set in negative ionization mode. Quantification was performed using multiple
reaction monitoring (MRM) with m/z 609 → 301 transition for rutin and m/z 481 → 124 transition for
IS. The MS parameters were capillary voltage −115 V, needle voltage −4400 V, shield voltage 600 V,
collision energy 26 V, Q1 voltage 0.5 V and Q3 voltage 2.6 V, nebulising gas pressure 50 psi and drying
gas pressure 25 psi. Calibration curve using the ratio Peak area Rutin/Peak area IS versus quantity
rutin/quantity IS was y = 2.8515 x + 0.0002. The Limit of Detection was 0.3 ng/mL and the Limit of
Quantification was 1.2 ng/mL.
Molecules 2016, 21, 1531 9 of 11

4.6. Method Validation

Assay specificity was evaluated comparing the chromatograms of standard-spiked plasma with
blank plasma from three different sources. Calibration curves were fitted by least square regression
analysis to plot peak area ratio of rutin/ISTD relatively to the ratio of the amount of rutin/ISTD.
Limit of Quantification (LOQ) was calculated as the lowest amount with a relative standard deviation
< 20%. Intra and inter day stability, extraction recovery, matrix effects were measured. Precision and
accuracy were evaluated using QC samples (n = 5) at concentrations of 10, 20 40 and 80 ng/mL on
three different days. Different plasma samples were used for intra- and inter-day stability, extraction
recovery, and matrix effects with five replicates.

4.7. Pharmacokinetic Analysis

Non-compartmental pharmacokinetic analysis was performed using WinNonlin Version 2.1
(Pharsight Corporation, Mountain View, CA, USA) software. The area under the mean plasma
concentration-time curve extrapolated to infinity (AUC) was calculated using the linear trapezoidal
method. Maximum concentration and the time when it was observed (Cmax and tmax , respectively)
were reported as observed. Terminal half-life (t1/2 ) was calculated as t1/2 = ln2/λz , where λz is the
slope of the terminal phase of the plasma concentration-time curve in the semi-log plot calculated by
linear regression. Mean residence time (MRT) was calculated as AUC/AUMC, where AUMC is the
area under the first moment curve calculated by linear trapezoidal method. Relative bioavailability of
NADES versus suspension was estimated as a ratio of AUC following the administration of NADES
and AUC following the administration of the suspension.

5. Conclusions
In this study we showed the possibility of producing amino acid-based NADES with good
capability of dissolving the polyphenol rutin. The most promising mixture was proline/glutamic acid
(2:1) being able to dissolve a comparable amount of rutin as ethanol and twenty times higher than
water. The compounds selected for NADES preparation in this paper are available in a normal diet and
can be administered orally at moderate doses without major health hazards. Administration of rutin
equidoses in mice, as water suspension or as solution in NADES, resulted in different pharmacokinetic
profiles. Rutin absorption was fast in both cases, yet four times slower than in water suspension.
The elimination from plasma was also rapid, but with a longer terminal half-life of NADES. For this
reason, proline/glutamic acid (2:1) NADES may improve bioavailability due to the increase of rutin
solubility. The obtained in vivo data indicate that the oral administration of rutin with proline/glutamic
acid (2:1) NADES improve bioavailability of this polyphenol compared to the water suspension.
This effect may be related to the fact that the NADES formulation allow the administration of rutin as
a solution being more available for the absorption by the gastrointestinal tract. This preliminary study
showed the potential of NADES as solubilizing and formulating agents for polyphenols administration.
Further studies are needed to deeply understand the role of different NADES in the bioavailability of
poorly soluble natural products.

Acknowledgments: Authors thank MIUR for financial support.

Author Contributions: M.F., S.S., V.B. and S.D.: experiment design and supervision of all experiments and results
S.S., S.D., B.P. and I.G.: analysis of the results and manuscript preparation. All authors read and approved the
final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

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