molecules

Article
Leishmanicidal Activity and Structure-Activity
Relationships of Essential Oil Constituents
Audrey R. S. T. Silva 1 , Ricardo Scher 2 , Flaviane V. Santos 2 , Sebastio R. Ferreira 3,4 ,
Scrates C. H. Cavalcanti 1 , Cristiane B. Correa 2 , Lilian L. Bueno 3 , Ricardo J. Alves 5 ,
Damio P. Souza 6 , Ricardo T. Fujiwara 3 and Silvio S. Dolabella 1,7, *
1 Department of Pharmacy, Federal University of Sergipe, 49100-000 So Cristvo, Sergipe, Brazil;
audreytavares2@gmail.com (A.R.S.T.S.); socratescavalcanti@yahoo.com.br (S.C.H.C.)
2 Laboratory of Molecular Biology of Pathogenic Bioagents, Department of Morphology,
Federal University of Sergipe, 49100-000 So Cristvo, Sergipe, Brazil; scher@ufs.br (R.S.);
flavianevieirah@gmail.com (F.V.S.); crisbani@gmail.com (C.B.C.)
3 Department of Parasitology, Biological Sciences Institute, Universidade Federal de Minas Gerais,
31270-901 Belo Horizonte, Minas Gerais, Brazil; ferreirasr2008@hotmail.com (S.R.F.);
lilacerdabueno@gmail.com (L.L.B.); fujiwara@icb.ufmg.br (R.T.F.)
4 Health Science Center, Federal University of Roraima, Av. Cap. Ene Garcez, 69310-000 Boa Vista,
Roraima, Brazil
5 Department of Pharmaceutical Products, Federal University of Minas Gerais, 31270-901 Belo Horizonte,
Minas Gerais, Brazil; ricardodylan@farmacia.ufmg.br
6 Department of Pharmaceutical Sciences, Federal University of Paraba, 58051-900 Joo Pessoa, Paraba,
Brazil; damiao_desousa@yahoo.com.br
7 Laboratory of Parasitology and Tropical Entomology, Department of Morphology,
Federal University of Sergipe, 49100-000 So Cristvo, Sergipe, Brazil
* Correspondence: dolabella@ufs.br ; Tel.: +55-79-3194-6619

Academic Editor: Thomas J. Schmidt

Received: 17 March 2017; Accepted: 11 May 2017; Published: 16 May 2017

Abstract: Several constituents of essential oils have been shown to be active against pathogens
such as bacteria, fungi, and protozoa. This study demonstrated the in vitro action of ten
compounds present in essential oils against Leishmania amazonensis promastigotes. With the exception
of p-cymene, all evaluated compounds presented leishmanicidal activity, exhibiting IC50 between
25.4 and 568.1 g mL1 . Compounds with the best leishmanicidal activity presented a phenolic
moiety (IC50 between 25.4 and 82.9 g mL1 ). Alicyclic alcohols (()-menthol and isoborneol)
and ketones (()-carvone) promoted similar activity against the parasite (IC50 between 190.2 and
198.9 g mL1 ). Most of the compounds showed low cytotoxicity in L929 fibroblasts. Analysis of the
structure-activity relationship of these compounds showed the importance of the phenolic structure
for the biological action against the promastigote forms of the parasite.

Keywords: monoterpenes; Leishmania amazonensis; leishmanicidal activity; essential oil

1. Introduction
Leishmaniasis is a disease of worldwide distribution, present in Africa, Latin America,
Asia, and Europe. It is estimated that 1.3 million new cases occur annually in these continents,
with approximately 20,000 to 30,000 deaths due to the disease [1]. Leishmaniasis is transmitted by
sandflies and is usually associated with poverty, malnutrition, poor living conditions, and climate
and environmental changes [1,2]. Leishmaniasis can be caused by different protozoa species of the
genus Leishmania, and the development of the disease is influenced by factors such as parasite species,

Molecules 2017, 22, 815; doi:10.3390/molecules22050815 www.mdpi.com/journal/molecules

Molecules 2017, 22, 815 2 of 10

parasite-host
Molecules 2017, 22, interaction,
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Antimonials and
Antimonials and amphotericin
amphotericin BB are are thethe first-choice
first-choice drugs drugs for for the
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treatment of of leishmaniasis
leishmaniasis in in
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and
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term
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days), high
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andvariable
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strains,
[57]. especiallyin
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and variable
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limiting cure).for Intheaddition, the emergence
use of traditional of resistant [57].
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endemic
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areas,
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for thetheuse
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of traditional
traditional
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been used to chemotherapies
tochemotherapies
chemotherapies
control parasites
parasites [57].
[57].
[57].
for centuries
centuries [8,9].
endemicA areas,
array is also a limiting
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has of traditional
been used chemotherapies
control [57].
for [8,9].
For A
A A vast
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garlic of
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to be active plants
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toto control
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control
human and parasites
parasites
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nonhuman forcenturies
for
for centuries
parasites[8,9].
centuries [8,9].
[8,9].
[10],
A vast array
For example, garlicofoil essential
is known oil-bearing
to be active plantsagainsthas12 been used to
different control
human and parasites
nonhuman for centuries
parasites [8,9].
[10],
For
For example,
example,
For example,
example, garlic
eugenol-containing garlic
garlic
garlic oil oil
oil
oil
basilisis known
known
is known
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and clove to to
to be
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be
be active
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oils possess against
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different
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different human
antiphagocytic human
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activity,and and
and
andand nonhuman
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Mentha crispa parasites
parasites
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crispa [10],
[10],
[10],
essential
For
eugenol-containing basilis and to oilsactive
possess against 12
antiphagocytic activity, and nonhuman
Mentha parasites
essential[10],
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eugenol-containing
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oil is active
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against Trypanosoma
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and clove
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oils
oils possess
possess
possess
[11]. antiphagocytic
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discovery of new
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and
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been crispa
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[11]. The antiphagocytic of activity,
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[11].
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of new
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by the synthesisbrucei [11]. The discovery
of substances of newa natural
that mimic drugs has been based
product, on natural
by modifying an
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an existing for
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existing for
natural years, either
molecule, by byby
or the the the
synthesis
use useof ofthethe natural
substances
natural product
that
product mimic itself
itself [12].
a[12].
natural Among
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anexisting
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existing
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to[12].
[12].
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in addition itselfto [12].
having Amongvast natural products,
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monoterpenes
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monoterpenes
already verified stand
stand
stand
against out
out
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for
for
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their wide
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bacteria, use
use
use in
in in
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the
the industry,
industry,
industry,
and in
inin
parasites, addition
addition
addition
including to
to
to having
having
having
protozoa vast
vast
vast biological
biological
biological
[1321]. activity
activity
Thus,activity
the
monoterpenes
already verifiedstand against out fungi,
for their wide use
bacteria, in the and
viruses, industry, in addition
parasites, including to having
protozoa vast[1321].
biological activity
Thus, the
already
already
already work
present verified
verified
verified
work aimsaims against
against
against fungi,
fungi, bacteria,
bacteria,
fungi, bacteria,
to investigate
investigate bacteria, viruses,
viruses,
viruses, and
the relationship
relationship and
and parasites,
parasites,
andbetween
parasites,
between including
including
including
chemical protozoa
protozoa
protozoaand
structures [1321].
[1321].
[1321].
and Thus,
Thus,
Thus, the
the activity
activity the
the
the
of
already
present verified against
to fungi, the viruses, parasites, including
chemical protozoa
structures [1321].
the Thus, of
present
present
present
different work
work
work aims
aims
aims
monoterpenes to
toto investigate
investigate
investigate
and a the
the
the relationship
relationship
relationship
phenylpropanoide, between
between
between
constituents chemical
chemical
chemical
of essential structures
structures
structures
oils, and
and
and
against thethe
the activity
activity
activity
promastigote ofof
of
present work
different aims to investigate
monoterpenes the relationshipconstituents
and a phenylpropanoide, between chemical of essential structures
oils, againstand the activity of
promastigote
different
different
different
forms monoterpenes
monoterpenes
monoterpenes
of Leishmania
Leishmania and
and a a phenylpropanoide,
phenylpropanoide,
and aa phenylpropanoide,
amazonensis. phenylpropanoide, constituents constituents
constituents
constituents of ofof essential
essential
of essential
essential oils, oils,
oils, against
against
oils, against promastigote
promastigote
against promastigote
promastigote
different
forms of monoterpenes and
amazonensis.
forms
forms
forms of ofof Leishmania
Leishmania
of Leishmania amazonensis.
amazonensis.
Leishmania amazonensis.
amazonensis.
forms
2. Results
2. Results and and Discussion
Discussion
2.2.Results
2.
2.
Resultsand
Results
Results
andDiscussion
and
and
Discussion
Discussion
Discussion
Nine monoterpenes
Nine monoterpenes and one
and one phenylpropanoid
phenylpropanoid were selected selected (Table (Table 1) 1) to
to investigate
investigate the the action
action
of these
these Nine
Nine
Nine monoterpenes
monoterpenes
monoterpenes
compounds against and
and
and L. onephenylpropanoid
one
one phenylpropanoid
phenylpropanoid
amazonensis promastigotes wereand
were
were selected
selected
selected
to (Table1)
(Table
(Table
determine 1)the
1) to
totoinvestigate
investigatethe
investigate
relationship the
the action
action
action
between
of compounds against L. amazonensis promastigotes
Nine monoterpenes and one phenylpropanoid were selected (Table 1) to investigate the action and to determine the relationship between
of
of these
these
of these
these
their compounds
compounds
compounds
chemical structure against
against
against L.
L.L. amazonensis
amazonensis
amazonensis
andbiological
biological promastigotes
promastigotes
promastigotes and
activity. and
and
and to toto determine
determine
to determine
determine the the
the relationship
relationship
the relationship
relationship between between
between
between
their
of chemical
compoundsstructure againstand L. amazonensis activity.
promastigotes
their
their chemical
chemical
their chemical structure
structure
chemical structure
structure and and
and biological
biological
and biological activity.
activity.
biological activity.
activity.
their
Table 1. In vitro
Table leishmanicidal
In vitro activity
leishmanicidal of different
activity essentialessential
of different oils constituents against Leishmania
oils constituents against
Table1.
Table
Table 1.In
1. Inpromastigotes
In
amazonensis
Leishmania vitroleishmanicidal
vitro
vitro leishmanicidal
.
leishmanicidal
amazonensis activityof
activity
activity
promastigotes. ofdifferent
of differentessential
different essentialoils
essential oilsconstituents
oils constituentsagainst
constituents againstLeishmania
against Leishmania
Leishmania
Table 1. In vitro leishmanicidal activity of different essential oils constituents against Leishmania
amazonensispromastigotes
amazonensis
amazonensis promastigotes
promastigotes .. .
amazonensis
Compoundpromastigotes .
Chemical Structure IC50 (g mL11) * IC50 (M) * R22
Compound
Compound
Compound Chemical
Chemical Structure
ChemicalStructure
Structure IC50IC
IC (g mL
(g
(g
5050 mL
mL ))*)**
11 IC
IC50
IC (M)
(M)* **
(M)
5050
RRR2 2
Compound
Compound Chemical Structure
Chemical Structure IC50
IC 50 (g mL1
(g mL )) **
1 IC50
IC 50 (M) *
(M) * R22
R

Carvacrol (1) 25.4 2.4 169.08 15.97 0.70

Carvacrol(1)
Carvacrol
Carvacrol
Carvacrol (1)
(1)
(1) 25.4
25.4
25.4
25.4 2.4
2.4
2.4
2.4 169.08
169.08
169.08
169.08 15.97
15.97
15.97
15.97 0.70
0.70
0.70
0.70
Carvacrol (1) 25.4 2.4 169.08 15.97 0.70

Thymol (2) 26.8 3.7 178.40 24.63 0.81

Thymol(2)
Thymol (2) 26.83.7
26.8 3.7 178.4024.63
178.40 24.63 0.81
0.81
Thymol
Thymol
Thymol (2)
(2)
(2) 26.8 3.7
26.8
26.8 3.7
3.7 178.40
178.40
178.40 24.63
24.63
24.63 0.81
0.81
0.81

3-Carene (3) 72.5 18.5 532.18 135.79 0.81

3-Carene(3)
3-Carene
3-Carene (3)
(3) 72.518.5
72.5
72.5 18.5
18.5 532.18135.79
532.18
532.18 135.79
135.79 0.81
0.81
0.81
3-Carene
3-Carene(3)(3) 72.5 18.5
72.5 18.5 532.18
532.18 135.79
135.79 0.81
0.81

Eugenol (4) 82.9 6.2 504.87 37.75 0.98

Eugenol(4)
Eugenol
Eugenol
Eugenol (4)
(4)
(4) 82.9 6.2
82.9
82.9
82.9 6.2
6.2
6.2 504.87
504.87
504.87
504.87 37.75
37.75
37.75
37.75 0.98
0.98
0.98
0.98
Eugenol (4) 82.9 6.2 504.87 37.75 0.98

Isoborneol
Isoborneol(5)(5) 190.2 9.8
190.2 9.8 1233.06
1233.06 63.53
63.53 0.97
0.97
Isoborneol(5)
Isoborneol
Isoborneol (5)
(5) 190.29.8
190.2
190.2 9.8
9.8 1233.0663.53
1233.06
1233.06 63.53
63.53 0.97
0.97
0.97
Isoborneol (5) 190.2 9.8 1233.06 63.53 0.97

()-Carvone (6) 194.7 16.9 1296.09 112.50 0.94

()-Carvone(6)
()-Carvone
()-Carvone (6)
(6) 194.716.9
194.7
194.7 16.9
16.9 1296.09112.50
1296.09
1296.09 112.50
112.50 0.94
0.94
0.94
()-Carvone (6) 194.7 16.9 1296.09 112.50 0.94
 Eugenol (4) 82.9 6.2 504.87 37.75 0.98
Molecules 2017, 22, 815 3 of 10

Table 1. Cont.
Isoborneol (5) 190.2 9.8 1233.06 63.53 0.97

Compound Chemical Structure IC50 (g mL1 ) * IC50 (M) * R2

()-Carvone(6)(6)
()-Carvone 194.7 16.9
194.7 16.9 1296.09
1296.09 112.50
112.50 0.94
0.94
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()-Menthol
()-Menthol (7)
()-Menthol (7)
(7) 198.9 12.0
198.9
198.9 12.0
12.0 1272.87
1272.87
1272.87 76.79
76.79
76.79 0.96
0.96
0.96
()-Menthol (7)
()-Menthol (7) OH 198.9 12.0
198.9 12.0 1272.87 76.79
1272.87 76.79 0.96
0.96
OH
OH
OH

()-Linalool (8) 276.2 24.0

24.0 1790.59 155.59
155.59 0.91
()-Linalool
()-Linalool (8)
()-Linalool (8)
(8) 276.2 24.0
276.2
276.2 24.0 1790.59
1790.59
1790.59 155.59
155.59 0.91
0.91
0.91
()-Linalool (8) 276.2 24.0 1790.59 155.59 0.91

1,8-Cineole (9)
1,8-Cineole 568.1 56.5 3682.98 366.28 0.89
1,8-Cineole (9)
1,8-Cineole
1,8-Cineole (9)
(9)
(9)
568.1
568.1
568.1
568.1
56.5
56.5
56.5
56.5
3682.98
3682.98
3682.98
3682.98
366.28
366.28
366.28
366.28
0.89
0.89
0.89
0.89

p-Cymene (10)
p-Cymene (10) >1000
>1000 >7450.45
>7450.45 --
p-Cymene (10)
p-Cymene
p-Cymene (10)
(10) >1000
>1000
>1000 >7450.45
>7450.45
>7450.45 ---

c Amphotericin B - 0.05 0.01 0.054 0.01 -

a Amphotericin
c
ICAmphotericin B
B capable ---of inhibiting 50% of 0.05
0.05 0.01
0.01 0.054 0.01
0.054 0.01
b 2 ---
0.054
c = Drug concentration promastigote multiplication;
Amphotericin
c 50
Amphotericin
c B
B 0.05
- of the curve fitting of0.05 0.01
0.01 0.054 0.01R c = Coefficient
0.01
curves; -
of
determinationmeasure for the quality sigmoidal dose-response Amphotericin B
was used as positive control. * Represents the mean of three independent experiments conducted in triplicate and
expressed with means plus or minus standard deviation (SD).
IC50 == Drug
a
IC
a Drug concentration
concentration capable
capable of
of inhibiting
inhibiting 50% 50% ofof promastigote
promastigote multiplication;
multiplication; b R
R2 == Coefficient
b 2 Coefficient of
of
IC50
a
IC
a 50 = Drug concentration capable of inhibiting 50% of promastigote multiplication; b R2 = Coefficient of
50 = Drug concentration capable of inhibiting 50% of promastigote multiplication; b R2 = Coefficient ofc
determinationmeasure
determinationmeasure for the quality of the curve fitting of sigmoidal dose-response curves;
determinationmeasure for for the
the quality
quality ofof the
the curvecurve fitting
fitting of
of sigmoidal
sigmoidal dose-response
dose-response curves;
c
determinationmeasure for the quality curves; cc
Among the compounds
Amphotericin B was used asevaluated
positive in of
this
control. the* curve
study,
Represents fitting
threethe of
mean sigmoidal
presented
of three dose-response
a phenolic
independentmoiety: curves;
carvacrol
experiments
Amphotericin
Amphotericin1B B was
was used
used as
as positive
positive control.
control. ***Represents
Represents the
the mean
mean of of three
three independent
independent experiments
experiments
(IC50 Amphotericin
25.4 g mL
conducted
conducted
B),was used
thymol
in triplicate
in triplicate andas
and
(ICpositive
expressed
expressed
control.
50 26.8 g mL
with
with means
means
1 ), and eugenol
Represents or the
plus or
plus
mean
minus
minus
(IC of three
50 82.9
standard
standard
g mL(SD).
independent
deviation
deviation
1 ). experiments
(SD).The positional
conducted in
conducted in triplicate
triplicate and
and expressed
expressed with
with means
means plus plus or
or minus
minus standard
standard deviation
deviation (SD).
(SD).
isomers carvacrol and thymol were the most active compounds in this series, exhibiting the lowest IC50
Among
valuesAmongamongthe compounds
evaluated compounds.evaluated in this study,
In addition, study,
these three
compoundspresented a phenolic
presented similar moiety: carvacrol
leishmanicidal
Among the
Among the compounds
the compounds
compounds
evaluated
evaluated in
evaluated in this
in this study, three
this 1study, three presented
three presented aaa phenolic
presented phenolic1moiety:
phenolic moiety: carvacrol
moiety: carvacrol
carvacrol
(IC50 25.4
activity,
(IC 25.4 g mL
possibly
g mLsuggesting
1
1),), thymol
thymolthat (IC50
(IC 50 26.8
the g mL
different
26.8 g mL ), and
positions
1), and of eugenol (IC50
the hydroxyl
eugenol (IC 50 82.9
in the
82.9 garomatic
g mL1).). The
mL The positional
ringpositional
does not
(IC50
(IC 50 25.4 g mL1), thymol (IC50 26.8 g mL1), and eugenol (IC50 82.9 g mL1). The positional
50 25.4 g mL1), thymol (IC50 26.8 g mL1), and eugenol (IC50 82.9 g mL1). The positional
isomers
influence
isomers carvacrol
the and
antiparasitic thymol
activity.were the most active compounds in this series, exhibiting the lowest
isomers carvacrol
isomers carvacrol and
carvacrol and thymol
and thymol were
thymol were the
were the most
the most active
most active compounds
active compounds in
compounds in this
in this series,
this series, exhibiting
series, exhibiting the
exhibiting the lowest
the lowest
lowest
IC
IC values
50 The acidity
values among
of phenolic
among evaluated
evaluatedhydroxyl compounds.
groups mayInplay
compounds. In addition,
an important
addition, these
theserole compounds
in explaining
compounds presented similar
higher potencies
presented similar
IC50
IC 50 values among evaluated compounds. In addition, these compounds presented similar
50 values among evaluated compounds. In addition, these compounds presented similar
ofleishmanicidal
phenolic
leishmanicidal activity,
compounds inpossibly
this suggesting
series. Aside fromthat thethe different
hydroxyl and positions
the of
aromatic the hydroxyl
ring, eugenol inalso
the
leishmanicidal activity,
leishmanicidal activity, possibly
activity, possibly suggesting
possibly suggesting that
suggesting that the
that the different
the different positions
different positions of
positions of the
of the hydroxyl
the hydroxyl in
hydroxyl in the
in the
the
aromatic
bears
aromatic a ring
hydrogen does not
bond influence
between the
the antiparasitic
methoxyl in activity.
the ortho position and the phenolic OH, which
aromatic ring
aromatic ring does
ring does not
does not influence
not influence the
influence the antiparasitic
antiparasitic activity.
activity.
reduces Thethe
The acidity
release
acidity of phenolic
of phenolic
of protons by the
hydroxyl
hydroxyl the antiparasitic
groups
OH
groups group maydue
may
activity.
play
play an
toantheimportant
presencerole in explaining
of this intramolecular higherhydrogen
potencies
The
The acidity
acidity of
of phenolic
phenolic hydroxyl
hydroxyl groups
groups may
may play
play an important
an important role
important role in
role in explaining
in explaining higher
explaining higher potencies
higher potencies
potencies
of phenolic
bonding
of [22].compounds
This molecular in thisproperty
series. Asidemightfrom justifytheeugenol
hydroxyl and potency
lower the aromatic ring, eugenol
in relation to both also bears
phenolic
of phenolic
of phenolic compounds
phenolic compounds in
compounds in this
in this series.
this series. Aside
series. Aside from
Aside from the
from the hydroxyl
the hydroxyl and
hydroxyl and the
and the aromatic
the aromatic ring,
aromatic ring, eugenol
ring, eugenol also
eugenol also bears
also bears
bears
a hydrogen
compounds
aa hydrogen bond
deprived betweenof the methoxyl
intramolecular in the
hydrogen ortho position
bonds, and
carvacrol the andphenolic
thymol OH,[23]. which reduces the
hydrogen bond
arelease
hydrogen bond between
bond between the
between the methoxyl
the methoxyl in
methoxyl in the
in the ortho
the ortho position
ortho position and
position and the
and the phenolic
the phenolic OH,
phenolic OH, which
OH, which reduces
which reduces the
reduces the
the
release of protons by
Non-aromatic by the OH group
hydroxylated group due to the
compounds, the presence
such as of this intramolecular
isoborneol and menthol hydrogen
alcohols, bonding
exhibited[22].
release of
release of protons
of protons by
protons by thethe OH
the OH group due
OH group due to
due to the presence
to the presence of
presence of this
of this intramolecular
this intramolecular hydrogen
intramolecular hydrogen
hydrogen
1 and
bonding
bonding [22].
bonding [22].
[22].
1 ,
This
weaker
This molecular
antiparasitic property
action might
than justify
phenolic eugenol
compounds lower
(IC potency
of 190.2 in
g relation
mL to both
198.0 gphenolic
mL
This molecular
This molecular property
molecular property might
property might justify
might justify eugenol
justify eugenol lower
eugenol lower 50potency
lower potency in
potency in relation
in relation to
relation to both
to both phenolic
both phenolic
phenolic
compounds deprived
respectively).
compounds Within the of intramolecular
aliphatic alcohols hydrogen
tested,bonds,linalool carvacrol
was theand leastthymol
active [23].
compound (IC50 of
compoundsdeprived
compounds deprived
deprived
1
of
of intramolecular
of intramolecular hydrogen
intramolecular hydrogen bonds,
hydrogen bonds, carvacrol
bonds, carvacrol and
carvacrol and thymol
and thymol [23].
thymol [23].
[23].
Non-aromatic
276.2 Non-aromatic
g mL ). Carvone, hydroxylated an compounds,
,-unsaturated such
ketone, as isoborneol
exhibited and
similar menthol
results alcohols,
to isoborneol exhibited
and
Non-aromatic hydroxylated
Non-aromatic hydroxylated compounds,
hydroxylated compounds,such
compounds, such
such
1 ). The
as
as isoborneol
as isoborneol and
isoborneol and menthol
and menthol
menthol
alcohols,
alcohols, exhibited
alcohols, exhibited
exhibited
weaker
menthol
weaker antiparasitic
alicyclic
antiparasitic alcoholsaction
action (ICthan
than of phenolic
194.7 g compounds
m (IC
enone 50 of
of 190.2
carvone
50 phenolic compounds (IC50 of 190.2 g mL 1 and 198.0 g mL1 g mL
might 1 and
1 play 198.0
an g
importantmL 1,
1,,
weaker
weaker antiparasitic
antiparasitic action
action than
than fromphenolic
phenolic compounds
compounds (IC 50 of 190.2 g mL1 and 198.0 g mL1
(IC50was of 190.2 g active
mL and 198.0 have
g(IC mL
respectively).
antiparasitic
respectively). Withinas
activity, the aliphatic
expected alcohols tested, linalool
the hydroxyls of alcohols, the
once least
conjugated compound
ketones of,
the
50
respectively).1Within
respectively). Within the
Within the aliphatic
the aliphatic alcohols
aliphatic alcohols tested,
alcohols tested, linalool
tested, linalool was
linalool was the
was the least
the least active
least active compound
active compound (IC
compound (IC50
(IC
of
50 of
50 of
276.2 gtomL
potential mL ). Carvone,
function as a Michael an ,-unsaturated
acceptor by reacting ketone, exhibited
with nucleophilicsimilar results
species of to
theisoborneol
parasite [24]. and
276.2
276.2 g an ,-unsaturated
1).
g mL mL1 1). Carvone,
Carvone, an ,-unsaturated ketone, ketone, exhibited
exhibited similar similar results
results to to isoborneol
isoborneol and and
276.2
menthol g ). Carvone,
alicyclic alcohols an
(IC ,-unsaturated
50 of 194.7 g m1
ketone,
1). The exhibited
enone of similar
carvone results
might to
play isoborneol
an important and
menthol
menthol alicyclic
alicyclic alcohols
alcohols (IC (IC50 of 194.7 g m ). The enone of carvone might play an important
50 of 194.7 g m1). The enone of carvone might play an important
menthol
antiparasiticalicyclic alcohols
activity, (IC
as expected
expected 50 of 194.7 g m1). The enone of carvone might play an important
from the hydroxyls
hydroxyls of alcohols, alcohols, once conjugatedconjugated ketones have have the
antiparasitic activity,
antiparasitic activity,
activity, as as expected from
as expected from the hydroxyls of
the hydroxyls alcohols, once
of alcohols, conjugated ketones
once conjugated ketones have have the the
antiparasitic
potential to function as a Michael from
acceptorthe by reacting ofwith once
nucleophilic species of ketones
the parasite the
[24].
potential
potential to to function
to function as a
function as aa Michael Michael acceptor
Michael acceptor
acceptor by by reacting
by reacting
reacting with with nucleophilic
with nucleophilic
nucleophilic species species
species of of the
of the parasite
the parasite [24].
parasite [24].
[24].
potential importance as
The importance of the
the phenolic
phenolic hydroxyl
hydroxyl group as as forfor leishmanicidal
leishmanicidal activity activity is observed
observed in in
The
The importance of
of the phenolic hydroxyl group
group as for leishmanicidal activity is
is observed in
The importance
carvacrol, thymol, p-cymene, of the phenolic
p-cymene, and menthol hydroxyl
menthol in aa similar group
similar manner, asas for leishmanicidal
as previously observed activity
observed by Ultee is observed
Ultee et al. in
al. [25]
carvacrol,
carvacrol, thymol, p-cymene, and
thymol, p-cymene, menthol in
and menthol similar manner,
in aa similar manner, as as previously
previously observedobserved by Ultee et
by Ultee al. [25]
et al. [25]
carvacrol, thymol,
for monoterpenes with antibacterial and action.inInterestingly, manner,
p-cymene, previously
a precursor of thymol by et
and carvacrol, [25]
Molecules 2017, 22, 815 4 of 10

The importance of the phenolic hydroxyl group as for leishmanicidal activity is observed
in carvacrol, thymol, p-cymene, and menthol in a similar manner, as previously observed by
Ultee et al. [25] for monoterpenes with antibacterial action. Interestingly, p-cymene, a precursor of
thymol and carvacrol, which does not have the hydroxyl group, did not exhibit any activity against
L. amazonensis, unlike the two hydroxylated derivatives that exhibited better results. These results
suggest that the lack of a polar hydroxyl group in the p-menthane ring template, as well as the
absence of aromaticity might render monoterpenes inactive. The importance of aromatic hydroxyls for
antiparasitic activity is again highlighted in the analysis of menthol. Although menthol has a hydroxyl
group, the absence of the aromatic ring contributed to an IC50 well above phenolic compounds.
The importance of the aromatic structure for leishmanicidal activity was also observed in the study of
synthetically modified lactones [26].
Previous studies of carvacrol in the literature found an IC50 of 2.3 g mL1 [27] and
28.0 g mL1 [28] against L. chagasi promastigotes, while another study found an IC50 of 15.3 g mL1
against L. amazonensis promastigotes [29]. The different values of IC50 , when comparing the same
species and compound, are probably related to the experimental design. In a previous study, the use
of longer drug-parasite incubation (72 h) and the assay development performed with the chromogen
p-nitrophenol phosphate to measure viability, determined the IC50 of 15.3 g mL1 for carvacrol [29].
Meanwhile, in the present study (IC50 of 25.4 g mL1 ), the parasites were incubated for 24 h using
the resazurin method.
Several studies have reported the action of thymol on species of Leishmania, such as
L. chagasi (IC50 of 9.8 g mL1 , 12.85 g mL1 , and 65.2 g mL1 ) [28,30,31], L. panamensis
(IC50 of 194.3 g mL1 ) [32] and L. amazonensis (IC50 of 22.6 g mL1 ) [33], presenting similar activity
to the ones observed in our study. Eugenol, on the other hand, was evaluated against promastigote
forms of L. chagasi (IC50 of 56.1 g mL1 ) [30] and L. amazonensis (IC50 of 500 g mL1 and
80.0 g mL1 ) [34,35], and the latter exhibits similar results to our experiment.
The potent behavior of thymol and carvacrol identified in the present study, when compared
with other monoterpenes, has already been observed previously in bacteria. Dorman and Deans [36]
evaluated the action of six essential oils and their major compounds, with 21 monoterpenes and
the phenylpropanoid eugenol, against 25 different bacterial species, and identified that thymol,
carvacrol, and eugenol were much more potent than the other monoterpenes. These authors also
discussed a value of the phenolic hydroxyl present in these compounds for this biological activity.
Nazzaro et al. [37] still emphasized the importance of the delocalization of electrons, besides the
phenolic hydroxyl, as important characteristics for the antibacterial activity of carvacrol and thymol.
However, bacteria and protozoa are microorganisms, thus they are relatively different in structural
and molecular terms.
Isoborneol, a bicyclic alcohol, was previously evaluated against promastigotes of L. infantum,
L. tropica, and L. major. No leishmanicidal activity was found, even at a maximum dose of
400.0 g mL1 [38]. To date, there are no reports of isoborneol activity against L. amazonensis.
In our study, isoborneol exhibited an IC50 of 190.2 g mL1 as leishmanicidal against L. amazonensis.
In this study, linalool, an acyclic tertiary alcohol, exhibited an IC50 of 276.2 g mL1 , considerably
higher than the value of 4.3 ng mL1 found by ROSA et al. [39], which was also obtained on
L. amazonensis promastigotes. Meanwhile, Dutra et al. [35] did not identify changes in the viability
of promastigotes of L. infantum chagasi treated with linalool up to the dose of 750 g mL1 . These
authors believe that the difference between their results and those observed by ROSA et al. [39] may
be related to different concentrations of enantiomers. The literature does not mention the action of
menthol, a cyclic alcohol, in L. amazonensis or other species of Leishmania. In our results, an IC50 of
198.0 g mL1 was obtained for this compound.
Among the monoterpenes, two hydrocarbons 3-carene and p-cymene were tested. No report
was found in the literature of the leishmanicidal activity of the bicyclic hydrocarbon 3-carene on
L. amazonensis. In this study, an IC50 of 72.5 g mL1 was found for 3-carene against L. amazonensis.
Molecules 2017, 22, 815 5 of 10

However, on L. donovani promastigotes, 3-carene exhibited an IC50 of 27.0 g mL1 [40]. p-Cymene,
an aromatic hydrocarbon, was evaluated on L. chagasi and found to have an IC50 of 149.1 g mL1 [28].
On the other hand, in our experiments, no effect on the viability of L. amazonensis promastigotes was
found in concentrations up to 1000.0 g mL1 .
()-Carvone was previously evaluated on L. chagasi promastigotes and exhibited an
IC50 > 300.0 g mL1 [28]. However, no IC50 for carvone was found in the literature against
L. amazonensis. In our trial, an IC50 of 194.7 g mL1 was obtained for ()-carvone against
L. amazonensis. Similar results were also obtained for the alicyclic alcohols isoborneol and menthol.
Machado et al. [38] evaluated the cyclic ether 1,8-cineole on promastigotes of L. infantum, L. tropica,
and L. major in increasing doses up to 400 g mL1 . However, no leishmanicidal activity was
detected. On the other hand, Camargos et al. [41] obtained an IC50 of 4697.0 M against L. amazonensis
promastigotes, which is equivalent to 724.0 g mL1 , similar to that obtained in the present study
(568.1 g mL1 ).
The replacement of the hydroxyl group in menthol by the ether function found in the 1,8-cineole
p-menthane ring resulted in a lower toxic effect (IC50 = 568.1 g mL1 as compared to 198.9 g mL1
of menthol), against the assessed species of Leishmania. This result corroborates the importance of the
aromatic ring and the hydroxyl group to yield more potent compounds.
The interpretation of the results and comparison between studies should also consider similar
species of Leishmania and life stage, such as promastigote or amastigote forms of the parasite,
since different species of the parasite and evolutionary forms have specific biological characteristics
that can produce discordant results [4244]. In addition, the results are dependent on the experimental
model being used [38].
In general, the compounds showed low cytotoxicity on L929 fibroblasts at the concentrations
evaluated, 50.0 and 100.0 g mL1 . The results of the tests are expressed as a percentage of viability
(Table 2).

Table 2. Cytotoxic activity of the compounds on L929 fibroblasts.

Viability (%)
Compounds
50 g mL1 100 g mL1
3-Carene 84.1 6.4 ** 48.7 6.7 *
Carvacrol 51.3 3.0 46.1 2.9 *
()-Carvone 65.1 4.7 58.2 4.2
1,8-Cineole 71.4 0.7 66.9 7.8
Eugenol 78.1 7.0 63.1 1.7
Isoborneol 73.3 9.4 72.9 7.5
()-Linalool 65.7 8.2 66.7 7.8
()-Menthol 83.8 8.0 ** 81.2 2.4 **
Thymol 64.5 4.0 58.5 6.7
p-Cymene 91.2 6.6 ** 87.1 6.7 **
Low cytotoxicity (viability between >50% and <80%); * Moderate cytotoxicity (viability between >30% and <50%);
and ** Non-cytotoxic (viability >80%).

An intensity scale based on the methods of Rodrigues et al. [45] was used to classify
the cytotoxicity of the compounds. At 50.0 g mL1 , 3-carene, menthol and p-cymene were
considered non-cytotoxic. The remaining compounds showed low cytotoxicity. At the concentration of
100.0 g mL1 , only menthol was not considered cytotoxic. Most compounds showed low cytotoxicity,
whereas carvacrol and 3-carene showed moderate cytotoxicity to the cells. Additionally, essential
oil components, such as carvacrol, thymol, 1,8-cineole, isoborneol, menthol, carvone, linalool,
and p-cymene are considered to be safe food additives, an indication of low mammalian toxicity [46].
The structure-activity relationships are important not only to understand how these compounds
act on the parasite, but also to guide future studies on these molecules. Although most of the
Molecules 2017, 22, 815 6 of 10

compounds used in this work had an IC50 higher than 100.0 g mL1 , the tests are still preliminary,
performed only on L. amazonensis promastigotes. Some studies show a greater sensitivity to intracellular
amastigote forms of Leishmania, with an IC50 about 50% lower than that found for promastigotes [47,48].
Thus, a more detailed evaluation of these compounds in the intracellular form of the parasite and in
other mammalian cells is needed to better evaluate the toxicity of these compounds.

3. Materials and Methods

3.1. Compounds
The following compounds were used: 3-carene (96.1% purity) (IUPAC (International Union
of Pure and Applied Chemistry) name: 4,7,7-trimethylbicyclo[4.1.0]hept-3-ene), carvacrol (99.9%)
(IUPAC name: 2-methyl-5-propan-2-ylphenol), ()-carvone (99.4%) (IUPAC name: (5R)-2-methyl-5-
prop-1-en-2-ylcyclohex-2-en-1-one), 1,8-cineole (99.7%) (IUPAC name: 2,2,4-trimethyl-3-oxabicyclo
[2.2.2]octan-6-ol), p-cymene (99.7%) (IUPAC name: 1-methyl-4-propan-2-ylbenzene), isoborneol
(99.0%) (IUPAC name (1R,3R,4R)-4,7,7-trimethylbicyclo[2.2.1]heptan-3-ol), ()-linalool (99.1%)
(IUPAC name: (3R)-3,7-dimethylocta-1,6-dien-3-ol) e ()-menthol (99.4%) (IUPAC name: (1R,2S,5R)-
5-methyl-2-propan-2-ylcyclohexan-1-ol) purchased from Sigma-Aldrich (St. Louis, MO, USA), eugenol
(99.0%) (IUPAC name: 2-methoxy-4-prop-2-enylphenol) purchased from Biodinmica (Ibipor, PR,
Brazil) and thymol (99.0%) (IUPAC name: 5-methyl-2-propan-2-ylphenol) purchased from Synth
(Diadema, SP, Brazil), all with analytical purity.

3.2. Parasites
Culture of L. amazonensis (LTCP 9667 obtained by Giudice et al. [44]) promastigotes were
maintained at 24 C in Schneider0 s Drosophila medium (Sigma-Aldrich, St. Louis, MO, USA; pH 6.7)
supplemented with 10% (v/v) inactivated fetal bovine serum (FBS, Gibco by Thermo Fisher Scientific,
Carlsbad, CA, USA), ampicillin 500 mg m1 , 1% and gentamicin 40 mg mL1 , 0.1% (Sigma-Aldrich,
St. Louis, MO, USA).

3.3. Fibroblasts
Culture of the mouse fibroblast cell line (L929, ATCC CCL-1) were maintained in Dulbecco0 s
Modified Eagle Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10%
heat-inactivated FBS and 1% streptomycin/penicillin (5000 units + 5 mg mL1 , Sigma-Aldrich,
St. Louis, MO, USA), and kept in a humid atmosphere at 37 C and 5% CO2 .

3.4. Evaluation of Leishmanicidal Activity

Promastigotes of L. amazonensis in log-phase growth were distributed in a 96-well plate
(5 105 cells/well) and treated with the evaluated compounds solubilized in dimethyl sulfoxide
(DMSO, Sigma-Aldrich, St. Louis, MO, USA) and diluted in Schneider0 s medium in different
concentrations (between 0.0 and 1000.0 g mL1 ) and incubated for 24 h at 24 C in a biochemical
oxygen demand (BOD) incubator. Promastigotes incubated in the absence of test compounds were
used as a negative control. Promastigotes treated with Amphotericin B (Sigma-Aldrich, St. Louis, MO,
USA) were used as a positive control. The cellular viability of the parasites was evaluated using the
colorimetric method of resazurin, adapted from Kulshrestha et al. [49]. Briefly, after the treatment
time, 50 L of resazurin (2 mM mL1 in phosphate-buffered saline (PBS)) (Sigma-Aldrich, St. Louis,
MO, USA) was added per well and the plates were again incubated for 6 h at 24 C, then read in a
spectrophotometer (Synerg H1, Biotek, Winooski, VT, USA) at 570 and 595 nm. Absorbance was used
to calculate cell viability based on the following equation:

Abs.570 nm ( Abs.595 nm RO) test

Viability (%) = 100 (1)
Abs.570 nm ( Abs.595 nm RO) control
Molecules 2017, 22, 815 7 of 10

wherein:
OD medium with resazurin in 570 nm OD medium without resasurin in 570 nm
RO = (2)
OD medium with resazurin in 595 nm OD medium without resazurin in 595 nm

The IC50 values were obtained by a non-linear regression analysis from the viability values using
the GraphPad Prism 5.0 program. All experiments were performed in triplicate from three independent
experiments and the data were expressed as mean standard deviation (SD).

3.5. Cytotoxicity
Fibroblasts were distributed in 96-well plates (2 104 cells/well) and incubated for 24 h in a
5% CO2 atmosphere at 37 C. After this period, the medium was removed and the adhered cells
were treated with the compounds at concentrations of 50.0 and 100.0 g mL1 for 24 h under the
same incubation conditions. Untreated cells were used as controls and considered with 100% cell
viability. After the treatment period, cell viability was determined by MTT assay as described in ISO
10993-5 [50], with modifications. For this, the cell monolayer was washed twice with PBS (pH 7.4),
and then 200 l MTT (0.5 mg mL1 in PBS, Sigma-Aldrich, St. Louis, MO, USA) was added to each
well. The plates were again incubated under the same conditions as listed above, for a period of
3 h. After the incubation time, the MTT was aspirated and the formazan crystals were solubilized in
200 L of DMSO. After 10 min, the optical density (OD) was measured on a microplate reader at the
wavelength of 570 nm. The results were expressed as percentage of viability according to the following
equation:
Absorbance (treated cell )
Viability = 100 (3)
Absorbance (control cell )
Each experiment was conducted in quadruplicate and repeated at least three times. Data were
expressed as mean standard deviation (SD).

4. Conclusions
In this work, the compounds with phenolic moiety carvacrol, thymol, and eugenol were the most
potent against L. amazonensis promastigotes. In addition, the evaluated compounds exhibited low
cytotoxicity on L929 fibroblasts.
Studies with a clinically more relevant intracellular amastigote form will have to be conducted
in order to further evaluate the potential of these compounds. Even though the overall activity level
of the investigated compounds is low compared to amphotericin B, the results presented in this
work demonstrate there are activity differences among the essential oil constituents distinguishing
between more and less potent compounds that may serve as a basis for the planning of more promising
antileishmanial agents.

Acknowledgments: We gratefully acknowledge financial support received from the Coordenao de

Aperfeioamento de Pessoal de Nvel Superior (CAPES) and Fundao de Apoio Pesquisa e Inovao Tecnolgica
do Estado de Sergipe (FAPITEC).
Author Contributions: S.S.D. and R.S. conceived and designed the experiments; A.R.S.T.S. and F.V.S. performed
the experiments; A.R.S.T.S., R.S., C.B.C., S.S.D. and R.T.F. analyzed the data; S.R.F., S.C.H.C., L.L.B., R.J.A., D.P.S.,
R.T.F. contributed to manuscript writing; A.R.S.T.S. and S.S.D. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
Molecules 2017, 22, 815 8 of 10

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Sample Availability: Not available.

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