Resveratrol analogues present effective antileishmanial activity against promastigotes and amastigotes from distinct Leishmania species by multitarget action in the parasites

Article abstract of DOI:10.1111/jphp.13177

Objectives: The in vitro antileishmanial effect of analogues of resveratrol (AR) present in the N-aryl imines and N-aryl hydrazones series was investigated. In addition, possible parasite targets were evaluated. Methods: Antipromastigote activity of Leishmania amazonensis, L. braziliensis and L. infantum, as well as the cytotoxicity on macrophages was determined by MTT assay and L. braziliensis-infected macrophages effect by Giemsa stain. After staining, effects on the parasite targets were analysed by flow cytometry or by fluorescence microscopy. Key-findings: Among the tested compounds, the derivative AR26 showed the best effect against promastigotes of all Leishmania species (IC₅₀ < 3.0 μg/ml), being more active than miltefosine, the control drug. AR26 was also effective against amastigotes of L. braziliensis (IC₅₀ = 15.9 μg/ml), with low toxicity to mammalian cells. The evaluation of mechanism of action of AR26 on L. braziliensis promastigotes indicates mitochondrial potential depolarization, plasma membrane permeabilization, interference in the progression of the cell cycle and accumulation of autophagic vacuoles. In addition, any increase of the reactive oxygen species levels was detected in the treated L. braziliensis-macrophages. Conclusions: Data indicate that the antileishmanial activity of AR26 is related to multitarget action, and the resveratrol analogues could be used in future studies as antileishmanial agent.

Full text of DOI:10.1111/jphp.13177

Research Paper
Resveratrol analogues present effective antileishmanial
activity against promastigotes and amastigotes from
distinct Leishmania species by multitarget action in the
parasites
Luciana Maria Ribeiro Antinarellia,b, Raissa Soares Meinelc, Eduardo Antonio Ferraz Coelhoa,d
Adilson David da Silvac and Elaine Soares Coimbrab
,
a
Programa de Pos-Graduaßcao em Ciencias da Saude: Infectologia e Medicina Tropical, Faculdade de Medicina, Belo Horizonte bDepartamento de
ꢀ
~
^
ꢀ
c
^
ꢀ
ꢀ
^
Parasitologia, Microbiologia e Imunologia, Instituto de Ciencias Biologicas, Departamento de Quımica, Instituto de Ciencias Exatas, Juiz de Fora
d
ꢀ
and Departamento de Patologia Clınica, COLTEC, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
Keywords
Abstract
Leishmania; mechanism of action; N-aryl
hydrazones; N-aryl imines; resveratrol
Objectives The in vitro antileishmanial effect of analogues of resveratrol (AR)
present in the N-aryl imines and N-aryl hydrazones series was investigated. In
addition, possible parasite targets were evaluated.
Correspondence
Elaine Soares Coimbra, Departamento de
Parasitologia, Microbiologia e Imunologia,
Methods Antipromastigote activity of Leishmania amazonensis, L. braziliensis
and L. infantum, as well as the cytotoxicity on macrophages was determined by
MTT assay and L. braziliensis-infected macrophages effect by Giemsa stain. After
staining, effects on the parasite targets were analysed by flow cytometry or by flu-
orescence microscopy.
^
ꢀ
Instituto de Ciencias Biologicas.,
Universidade Federal de Juiz de Fora,
ꢀ
Campus Universitario, Juiz de Fora, Minas
Gerais 36036-900, Brazil.
E-mail: elaine.coimbra@ufjf.edu.br
Key-findings Among the tested compounds, the derivative AR26 showed the best
effect against promastigotes of all Leishmania species (IC50 < 3.0 µg/ml), being
more active than miltefosine, the control drug. AR26 was also effective against
amastigotes of L. braziliensis (IC50 = 15.9 µg/ml), with low toxicity to mam-
malian cells. The evaluation of mechanism of action of AR26 on L. braziliensis
promastigotes indicates mitochondrial potential depolarization, plasma mem-
brane permeabilization, interference in the progression of the cell cycle and accu-
mulation of autophagic vacuoles. In addition, any increase of the reactive oxygen
species levels was detected in the treated L. braziliensis-macrophages.
Conclusions Data indicate that the antileishmanial activity of AR26 is related to
multitarget action, and the resveratrol analogues could be used in future studies
as antileishmanial agent.
Received July 16, 2019
Accepted September 14, 2019
doi: 10.1111/jphp.13177
etiologic agent and insect vector involved in the transmis-
sion cycle.[2,3]
Introduction
Leishmaniasis is a disease complex caused by species of
flagellate protozoa of the genus Leishmania. It represents a
serious global health problem, which can affect 350 million
people in more than 98 countries, with an estimated inci-
dence of 20,000–30,000 deaths annually.[1] The infection by
the Leishmania parasite manifests in humans with multiple
clinical symptoms, affecting the skin, mucosal and/or vis-
ceral organs of the patients. The main determining factors
for the diversity of the clinical manifestations are associated
with the immunological status of the hosts, as well as by
The tegumentary leishmaniasis (TL) of the diseases can
manifest as cutaneous leishmaniasis, the main clinical form
characterized by unique lesions with favourable response to
treatment, showing high frequency of spontaneous heal-
ing.[4,5] The mucosal leishmaniasis (ML) is a debilitating
clinical form of TL characterized by the presence of
destructive and metastatic lesions in the oronasal mucosa
and respiratory tract, resulting from the lymphatic or
haematogenous dissemination of the parasites.[6] The cuta-
neous-diffuse leishmaniasis is a severe form of TL, which
© 2019 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2019), pp. **–**
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Antileishmanial activity of resveratrol analogues
Luciana Maria Ribeiro Antinarelli et al.
manifests in patients with deficiency in the immune cellular
response against antigens of the parasite. This infection
begins with a single lesion that is not responsive to treat-
ment and evolves with the formation of multiple non-ul-
cerated lesions, which are characterized by abundance of
parasites.[4,7] Visceral leishmaniasis (VL) is the most serious
clinical syndrome of leishmaniasis, resulting from
haematogenous dissemination of parasites to internal
organs. The disease can manifest as persistent fever, spleno-
megaly, hepatomegaly and severe anaemia with risk of fatal
progression, mainly in cases of secondary infections, mal-
nutrition and organ failure.[8,9]
amastigotes of different Leishmania species, aiming to eval-
uate their action against parasites able to cause TL and VL
in the world. Also, the mechanism of action of the best ana-
logue was evaluated in Leishmania braziliensis, the main eti-
ological agent of localized and mucosal forms of TL in the
Americas.[26]
Materials and Methods
Synthesis of the compounds
Imine resveratrol analogues (AR26 and AR27) and hydra-
zone resveratrol analogues (AR29, AR30 and AR32) were
obtained by condensation of 2-hydroxyaniline, aniline,
phenylhydrazine or 2,4-dinitrophenylhydrazine with the
equimolar amount aromatic aldehydes in ethanol as solids
after being filtered, washed with ethanol and dried in an
oven (yield 65–82%) (Scheme 1). All the compounds were
characterized via one-dimensional nuclear magnetic reso-
nance (1D-NMR) and melting point (Appendix S1).[27,28]
The current treatment against leishmaniasis is considered
unsatisfactory, mainly due to the severe toxicity, high cost
and/or emergence of the parasite resistance.[10] The pentava-
lent antimonials are the first-line drugs, although they pre-
sent limitations, such as the prolonged treatment by
parenteral route and side effects, including cardiotoxicity,
[9,11]
hepatotoxicity and pancreatitis.
Amphotericin B is a
therapeutic option applied in cases of antimonials treatment
failure, presenting higher cure rate against VL and TL. How-
ever, the toxicity of this drug causing renal insufficiency and
hepatotoxicity, along with the long treatment course and the
emergence of treatment failure, represents complications of
this therapeutic process.[12] Pentamidine has been presented
as an option in areas where the treatment failure occurs;
however, its clinical efficacy is unsatisfactory, with its use
currently being restricted to the treatment of infections
caused by L. guyanensis species in the South America.[11]
Miltefosine is the only therapeutic option recommended for
oral use, although its employ is restricted in some countries.
The main inconveniences are the teratogenic potential and
therapeutic failure to treat against TL.[13,14] In this context,
and due to the distinct problems related to the current sce-
nario of the treatment against leishmaniasis, additional
efforts need to be performed aiming to identify new thera-
peutic antileishmanial agents.
Mice
BALB/c mice (female, 8 weeks old, weighing 20–25 g) were
purchased from the Reproductive Biology Center of Federal
University of Juiz de Fora (UFJF), and they were main-
tained under specific pathogen-free conditions. The study
was approved by the Committee of Ethical Handling of
Research Animals of UFJF (protocols number 007/2018
and 008/2018).
Parasites
Leishmania amazonensis (IFLA/BR/1967/PH8) was cultured
in Warren’s medium; L. braziliensis (MHOM/Br/75/
M2903) was cultured in BHI medium supplemented with
20 mM L-glutamine, and L. infantum (MHOM/Br/74/PP75)
was cultured in LIT medium, all supplemented with 10%
Fetal Bovine Serum (FBS; Sigma-Aldrich, St. Louis, MO,
USA), 100 U/ml penicillin and 0.1 mg/ml of streptomycin,
and they were maintained at 25 °C in BOD incubator.[29]
Resveratrol and its derivates represent a chemical diver-
sity of substances with pharmacological relevance, present-
ing activities such as antioxidant, anticancer,
cardioprotective, anti-inflammatory, immunomodulatory,
antibacterial and neuroprotective.[15–17] Moreover, resvera-
trol presents efficacy against chronic chagasic cardiomyopa-
thy in experimental models, promoting significant
reduction of parasite burden and improvement of heart
function, which are associated with antioxidative activity,
reduction in the reactive oxygen species (ROS) levels and
induction of lipid peroxidation.[18]
In vitro antipromastigote activity
Leishmania braziliensis and L. infantum (3 9 106 cells) or
L. amazonensis promastigotes (2 9 106 cells) in logarith-
mic-phase were platted in 96-well plates and exposed to the
resveratrol analogues (concentrations ranging from 0.47 to
30.0 µg/ml), for 72 h at 25 °C. The parasite viability was
determined using the colorimetric reagent 3-(4.5-
dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide
(MTT; Sigma-Aldrich). The optical density values were
read in a microplate spectrophotometer (Multiskan MS
The antileishmanial effect of resveratrol and its analogues
against different Leishmania species have been reported in
the literature.[19–25] However, in this study, we extended
the investigation of effect of a new class of resveratrol ana-
logues against promastigotes and intramacrophage
2
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Luciana Maria Ribeiro Antinarelli et al.
Antileishmanial activity of resveratrol analogues
R1
R1
R2
R3
R5
R2
R3
R5
EtOH
r.t.
O
NH2
N
R4
R4
R6
R6
2-hydroxyaniline
aromatic aldehydes
or
AR26:
AR27:
R1: OH; R2: COOH; R3, R4, R5, R6: H
R1: OH; R2, R3, R4, R5: H; R6: COOH
aniline
R2
R3
R2
R3
R4
R5
R4
R5
O
EtOH
R1
HN N
R1
HN NH2
R6
R6
r.t.
phenylhydrazine or
2,4-dinitrophenylhydrazine
aromatic aldehydes
AR29:
AR30:
AR32:
R1, R2, R3, R4, R5: H; R6: CHO
R1, R3: NO2; R2, R4, R5: H; R6:CHO
R1, R3: NO2; R2, R4: H; R5: OH; R6:OCH3
Scheme 1 Synthesis of resveratrol analogues AR26, AR27, AR29, AR30 and AR32.
Microplate Reader; LabSystems Oy, Helsink, Finland), at
570 nm. AmpB (0.03–1.0 µg/ml, Cristꢀalia, S~ao Paulo, Bra-
zil) and miltefosine (0.63–40.0 µg/ml) were used as drug
controls.[29]
incubated for 4 h at 33 °C in 5% CO2. Free parasites were
removed by extensive washing with PBS (0.1 M, pH 7.4),
and infected macrophages were treated with the com-
pounds (3.1–50.0 µg/ml), miltefosine (0.63–40.0 µg/ml) or
AmpB (0.06–1.0 µg/ml), for 72 h at 33 °C in 5% CO2.
After fixation, cells were stained with Giemsa solution, and
200 macrophages were counted in a light microscope.[29]
The infection index was calculated by: number of amastig-
otes per macrophage 9 percentage of infected macro-
phages/total number of macrophages.
Cytotoxicity
The cytotoxicity assay was assessed in murine macrophages
collected from peritoneal cavity of BALB/c mice, which
were previously stimulated with 2 ml thioglycolate. Then,
macrophages (2 9 105 cells) were prepared in an RPMI-
1640 medium supplemented with 10% FBS and incubated
in 96-well plates at 37 °C in 5% CO2. After 24 h, the non-
adherent cells were removed and the others were incubated
with serial dilutions of the compounds (2.3–150.0 µg/ml),
for 72 h at 37 °C in a humidified atmosphere containing
5% CO2. Miltefosine (3.8–30.0 µg/ml) and AmpB (4.3–
138.6 µg/ml) were used as control. The cell viability was
analysed by the MTT method.[30]
Evaluation of mitochondrial dysfunction
The mitochondrial membrane potential (DΨm) was evalu-
ated in L. braziliensis promastigotes in the log phase (107
cells), which were incubated for 24 h in the absence or pres-
ence of the best resveratrol-analogue (AR26; 1.0 and 2.0 µg/
ml, corresponding to one and two times the IC50 values) at
25 °C. Afterwards, parasites (107 cells) were labelled with
Mitotracker (500 nM; Invitrogen, Eugene, OR, USA) for
40 min at 37 °C. After washing twice with PBS (0.1 M, pH
7.4), samples were added into a black 96-well plate, and fluo-
rescence intensity was measured in a spectrofluorometer
(FLx800; BioTek Instruments, Inc., Winooski, VT, USA), at
both 540 and 600 nm wavelengths. Parasites incubated with
2.0 µM carbonyl cyanide 4-(trifluoromethoxy)phenylhydra-
zone (FCCP; Sigma-Aldrich) for 10 min were used as con-
trol.[30] For measurement of ROS levels, parasites (2 9 107
cells) were marked using the cell-permeable probe
Treatment of L. braziliensis-infected
macrophages
Macrophages (6 9 105 cells) were plated on round glass
coverslips in 24-well plates in RPMI 1640 medium added
with 10% FBS and incubated for 24 h at 37 °C in 5% CO2.
Afterwards, L. braziliensis stationary-phase promastigotes
(6 9 106 cells; at a ratio of 10 parasites per one macro-
phage) were added into the wells, and cultures were
© 2019 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2019), pp. **–**
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Antileishmanial activity of resveratrol analogues
Luciana Maria Ribeiro Antinarelli et al.
H2DCFDA (20,70-dichlorodihydrofluorescein diacetate,
Molecular Probes, Eugene, OR, USA), for 30 min in the dark
at room temperature. ROS levels were measured in a fluori-
metric microplate reader (FLx800, BioTek Instruments),
with excitation filter of 485 nm and emission filter of
528 nm.[30] Parasites incubated with miltefosine (10.4 µg/
ml) for 24 h were used as positive control.
miltefosine (1.3 µg/ml) for 24 h. Then, cells were washed
with PBS (0.1 M, pH 7.4), and incubated with 20 µM
H2DCFDA for 30 min in the dark at 33 °C, 5% CO2. The
fluorescence intensity was measured in a fluorimeter
(FLx800; BioTek Instruments), with 485 nm excitation and
528 nm emission.[30] Infected macrophages were treated
with zymosan derived from Saccharomyces cerevisiae
(250 µg/ml, Sigma-Aldrich) and used as positive control.
Evaluation of the cell membrane integrity
Statistical analysis
Leishmania braziliensis promastigotes in the log phase (107
cells) were either untreated or treated with AR26 (1.0 and
2.0 µg/ml) or miltefosine (10.4 µg/ml), for 24 h at 25 °C.
Afterwards, parasites were washed with PBS (0.1 M, pH
7.4) and stained with 1.0 µg/ml propidium iodide (PI;
Sigma-Aldrich) for 15 min and in the dark. PI-stained par-
asites were analysed in a fluorimetry microplate reader
(FLx800; BioTek Instruments), with excitation wavelength
of 540 nm and emission wavelength of 600 nm.[31]
The compound concentrations necessary to inhibit 50% of
Leishmania viability (IC50 values) and of the macrophages
(CC50 values) were calculated by the Probit program. Data
were analysed by One-way ANOVA followed by Dunnett’s
post-test to compare the groups. GraphPad Prism 5 was
also used (GraphPad Software, San Diego, CA, USA).
Results were expressed as the mean ꢀ standard error of the
mean of three independent experiments, which presented
similar results. Differences were considered significant with
P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).
Analysis of the cell cycle
Leishmania braziliensis promastigotes in the log phase (107
cells) were treated with AR26 (1.0 and 2.0 µg/ml) for 24 h
at 25 °C. After treatment, parasites were permeabilized
with 70% ethanol for 1 h at 4 °C. Then, cells were incu-
bated with 200 lg/ml Ribonuclease A (Sigma-Aldrich) for
1 h at 37 °C and subsequently stained with 7.0 µg/ml PI
(Sigma-Aldrich), for 20 min in the dark at room tempera-
ture. For each sample, 10,000 events were acquired using
the FACsCanto II flow cytometer (Becton Dickinson,
Rutherford, NJ, USA), and obtained data were analysed in
a FlowJo VX software.[32]
Results
Firstly, the antileishmanial activity of a series of resvera-
trol analogues was investigated against promastigotes of
L. amazonensis, L. braziliensis and L. infantum species
and the cytotoxic effect on murine macrophages. The
effect of compounds was assessed using the MTT
method, based on the reduction of dehydrogenase system
of metabolically active/live cells.[34] Although disadvan-
tages, it is easy to use, has a high reproducibility, and
considered as one the most widely assay for evaluation
of cell viability and cytotoxicity for screening the
drugs.[34] In addition to MTT assay, parasites and
macrophages were also observed under the light micro-
scope. Results showed that all molecules exhibited inhibi-
tory effect against L. braziliensis promastigotes, whereas
the analogues AR26, AR30 and AR32 showed better
activity against L. infantum, and only AR26 was effective
against L. amazonensis (Table 1). Comparing all ana-
logues, AR26 was this presenting better action against
the three parasite species, with IC50 values lower than
miltefosine, used as a control. The cytotoxicity on mur-
ine macrophages showed that these analogues were not
toxic for these cells in the highest concentration tested
(CC50 > 30.0 lg/ml) (Table 1). Since the compounds
were more effective (lower IC50 values) against
L. braziliensis species and that AR26 was active against
all parasite species, it was thus tested to treat macro-
phages that were infected with L. braziliensis, and results
showed an IC50 value of 15.9 lg/ml against the intra-
macrophage amastigotes (Table 1). In the evaluation of
Evaluation of the accumulation of
autophagic vacuoles
Leishmania braziliensis promastigotes in the log phase (107
cells) were untreated or treated with AR26 (1.0 and 2.0 µg/
ml) for 24 h. Then, parasites were washed with PBS (0.1 M,
pH 7.4), and parasites were labelled with 100 µM mon-
odansylcadaverin (Sigma-Aldrich) for 1 h in the dark at
25°C. Afterwards, the fluorescence intensity was analysed in
a spectrofluorometer (Varioskanâ Flash, Thermo Scientific,
Waltham, MA, USA), using 335 nm for excitation and
460 nm for emission, respectively. Untreated stationary
promastigotes were used as control.[33]
Measurement of ROS levels in Leishmania-
infected macrophages
Macrophages were infected with L. braziliensis species and
untreated or treated with AR26 (15.88 and 31.76 µg/ml) or
4
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Luciana Maria Ribeiro Antinarelli et al.
Antileishmanial activity of resveratrol analogues
Table 1 Antileishmanial activity and cytotoxicity in murine macrophages of resveratrol analogues
Promastigotes
Promastigotes
Promastigotes
Amastigotes
L. amazonensis
L. braziliensis
L. infantum
Macrophages
L. braziliensis
a
a
a
b
a
Compounds
IC50 µg/ml
IC50 µg/ml
IC50 µg/ml
CC50 µg/ml
IC50 µg/ml
AR26
2.7 (2.3–2.9)
2.6 (2.1–3.0)
>30.0
1.0 (0.8–1.2)
0.7 (0.6–0.9)
3.0 (4.1–4.7)
13.1 (9.7–17.6)
>30.0
>150.0
15.9 (13.9–18.1)
>30.0
AR27
>30.0
AR29
2.7 (2.1–3.3)
7.3 (6.0–8.9)
>30.0
>5.0
AR30
>30.0
15.4 (13.6–17.4)
26.6 (23.4–30.2)
0.12 (0.09–0.14)
10.4 (9.0–12.1)
11.4 (8.2–15.9)
12.7 (8.3–19.6)
0.05 (0.04–0.06)
11.8 (10.0–14.0)
>30.0
AR32
>30.0
>30.0
>30.0
Amphotericin B
Miltefosine
0.11 (0.09–0.12)
9.0 (8.5–9.5)
10.1 (7.8–13.1)
>30.0
0.069 (0.057–0.085)
1.3 (1.16–1.47)
Results correspond to the mean of three independent experiments performed in duplicate. Miltefosine and amphotericin B were used as drug con-
trols. aIC50, Inhibitory concentration of 50% of parasites. bCC50, Cytotoxic concentration of 50% of macrophages.
the chemical composition between the analogues, the
imine analogues (AR26 and AR27) exhibited better activ-
ity than their respective hydrazones counterparts (AR 29,
AR30 and AR32). For imine group, AR26 presenting the
hydroxy group in the ortho position was more active
than AR27 against Leishmania parasites, indicating that
the hydroxy substitution was critical for biological
activity.
miltefosine induced an increase in the ROS levels in the
order of 63.33%.
The plasma membrane integrity of AR26-treated para-
sites was evaluated following the incubation with propid-
ium iodide cell probe (PI). Results showed that the
compound induced permeabilization of plasma membrane
of the promastigote forms, at a dose of 2.0 lg/ml, when
compared with the control group (Figure 4). In addition,
an increase in the order of 107.60% was observed in the
percentage of PI-positive promastigotes that were treated
with miltefosine, indicating also the disrupting of plasma
membrane integrity.
The treatment of L. braziliensis-infected macrophages
using AR26 showed reductions in the order of 96.95%,
63.71%, 31.75%, 25.34% and 19.30%, when 50.0, 25.0,
12.5, 6.25 and 3.12 µg/ml of the compound were used,
respectively (Figure 1). No toxic effect was observed in the
host cell; thus, confirming results obtained previously by
cytotoxicity assay (CC50 > 150 µg/ml), demonstrating a
satisfactory selectivity index against intracellular amastig-
otes of L. braziliensis.
To evaluate the effect of AR26 in the parasite’s cell cycle,
promastigotes were labelled with PI, and the DNA content
was evaluated by flow cytometry. Results showed the dele-
terious effect of treatment with AR26 at 1.0 or 2.0 µg/ml
on the parasite´ cell cycle, with increase of subG0/G1 popu-
lation (2.7- and 2.5-fold greater than untreated parasites,
respectively), as well as a significant reduction of G0/G1
(32.09% and 36.12%, respectively) and G2 (47.17% and
53.91%, respectively) populations (Figure 5).
Due to the promising antileishmanial effect presented by
AR26, more detailed studies were conducted to investigate
the mechanism of action of this compound in Leishmania
parasites. The mitochondrial function was evaluated based
on (i) determination of the mitochondrial transmembrane
electric potential (DΨm) and (ii) by detection of ROS pro-
duction in AR26-treated parasites. The effect of this mole-
cule on the DΨm depolarization was detected using the
cationic probe MitoTrackerâ Red. Results showed that the
treatment with AR26 caused a decrease in DΨm in the
order of 20.66% and 22.68%, respectively, when 1.0 and
2.0 µg/ml of the compound (corresponding to one and
two times the IC50 values, respectively) were used, after
24 h of incubation (Figure 2). The reduction in the DΨm
in FCCP-treated parasites, which was used as a control,
showed reduction in the order of 14.24%.
The study assessed also whether the treatment with
AR26 induces the activation of autophagic cell death. To
quantify the accumulation of autophagic vacuoles, a stain-
ing using the MDC marker was performed. Results showed
that the treatment of promastigotes with AR26 at concen-
trations of 1.0 and 2.0 µg/ml induced increase in the order
of 30.81% and 21.78%, respectively, in the formation of
autophagic vacuoles, when compared with untreated para-
sites (Figure 6).
To investigate whether the effect of AR26 against intra-
macrophage amastigotes was related with the activation of
oxidative mechanisms by host cell, the ROS production
was evaluated in infected macrophages that were thus trea-
ted with this molecule. Results showed that any increase in
the ROS levels was detected in the infected macrophages
after treatment with AR26. As expected, both uninfected
and infected macrophages respond to zymosan stimulation
with increase in the ROS production (Figure 7).
To investigate whether the effect of AR26 on DΨm depo-
larization is associated to the induction of oxidative stress,
ROS levels were evaluated using the sensitive cell permeat-
ing probe, H2DCFDA. Results showed any increase in the
ROS production after 24 h of incubation, when compared
to the untreated parasites (Figure 3). On the other hand,
© 2019 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2019), pp. **–**
5

Antileishmanial activity of resveratrol analogues
Luciana Maria Ribeiro Antinarelli et al.
Figure
3
Reactive oxygen species (ROS) levels in AR26-treated
L. braziliensis promastigotes. Parasites were untreated (control group)
or treated with 1.0 or 2.0 µg/ml of AR26 or miltefosine (10.4 µg/ml)
for 24 h, when they were probed with H2DCFDA. The fluorescence
intensity was measured in a fluorimeter, and results were expressed in
arbitrary units (A.U.) of the mean of three independent experiments.
P < 0.001 (***) showed significant difference compared with the con-
trol group.
Figure 1 Effect of AR26 in the treatment of macrophages infected
by L. braziliensis. Murine macrophages were infected with L. brazilien-
sis promastigotes (in a ratio of 10 parasites per cell) and treated with
AR26 (3.12–50.0 µg/ml) for 72 h at 33 C in 5% CO2. Cells were
stained with Giemsa, and the percentage of reduction of infection
was determined by counting of 200 cells in duplicate in a light micro-
scope. P < 0.001 (***) and P < 0.01 (*) showed significant difference
when compared with the control group.
Figure 4 Evaluation of cell membrane integrity of AR26-treated
L. braziliensis promastigotes. Parasites were untreated (control group)
or treated with 1.0 or 2.0 µg/ml of AR26 or miltefosine (10.4 µg/ml)
for 24 h. Then, cells were loaded with propidium iodide (PI) and the
fluorescence intensity was evaluated using a fluorimeter. P < 0.001
(***) and P < 0.01 (*) indicate statistically significant difference com-
pared with untreated parasites.
Figure 2 Effects of AR26 on the mitochondrial membrane potential
(DΨm) of L. braziliensis promastigotes. Promastigotes were untreated
(control group) or treated with AR26 (1.0 and 2.0 µg/ml) for 24 h,
when they were probed with fluorescent probe Mitotrackerâ Red.
Results were expressed as the percentage of DΨm reduction in com-
parison with untreated parasites. P < 0.001 (***) and P < 0.01 (**)
showed significant difference compared with the control group.
exploited.[17,35] Among the tested compounds, the most
prominent molecule was AR26, which exhibited a selective
action against distinct Leishmania species, presenting IC50
values below of 3.0 µg/ml, and an activity in the treatment
of L. braziliensis-infected macrophages, when IC50 value of
15.9 µg/ml against the intramacrophage amastigotes was
obtained. Interestingly, AR26 was more effective than
resveratrol, which exhibited IC50 values of 75.13 µM
(17.15 µg/ml) and 124.48 µM (28.41 µg/ml) against
L. braziliensis promastigotes and intramacrophage amastig-
otes, respectively, after 72 h of treatment.[24] Trans-
Discussion
In the search for new potential drug targets to be employed
in the treatment against leishmaniasis, in this study, the
antileishmanial action of a series of AR-analogues, a class
of bioactive compounds that has been already showed pre-
senting
distinct
pharmacological
activities,
was
6
© 2019 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2019), pp. **–**

Luciana Maria Ribeiro Antinarelli et al.
Antileishmanial activity of resveratrol analogues
analysed.[25] In addition to the antileishmanial activity, a
recent study showed that AR26 exhibited also promising
in vitro anti-inflammatory, antioxidant and immunomodu-
latory properties, thus demonstrating the pharmacological
potential of this molecule.[35,36]
In our study, we also advanced towards a better under-
standing regarding the distinct pathways of cell death, such
as apoptosis-like, autophagy or even necrosis involved in
the mode of action of this class of resveratrol analogues in
Leishmania, and we evaluated the action of AR26 against
L. braziliensis promastigotes. Investigations into the mech-
anism of antileishmanial effect of resveratrol suggest disor-
ders in the cell cycle of the parasite, reduction of the
mitochondrial membrane potential and distinct morpho-
logical changes revealed by an irregular number of flagella
and nuclei and alteration in cell shape. Moreover, its leish-
manicidal effect is also dependent on the inhibition of
proinflammatory cytokines and ROS production by mur-
ine-infected macrophages.[21] Also, compounds structurally
related to resveratrol, such as stilbene-based compounds
induced similar disorders, altering the cell cycle of the para-
site and inducing depolarization of mitochondrial mem-
brane potential in L. amazonensis promastigote forms and
decreased ROS levels in L. amazonensis-infected macro-
phages.[22]
Figure 5 Analysis of the cell cycle progression of AR26-treated
L. braziliensis promastigotes. Parasites were untreated or treated with
AR26 (1.0 or 2.0 µg/ml) for 24 h, when they were loaded with pro-
pidium iodide (PI), and the DNA content was evaluated by flow
cytometry. The percentage of promastigotes in each phase of cell
cycle was determined. P < 0.01 (*) indicates significant statistical dif-
ference in comparison to the control group.
The single mitochondria of trypanosomatids parasites
presents a peculiar ultrastructure and functional plasticity
that makes this organelle an excellent indicator of cell irre-
versible dysfunction and, consequently, an attractive thera-
peutic target for new therapeutic strategies.[37,38] A critical
event for the monitoring of the proper functioning of the
mitochondrial respiratory chain of parasite is the determi-
nation of the mitochondrial membrane potential
(DΨm).[39,40] Corroborating with this hypothesis, a signifi-
cant reduction in DΨm was detected to parasites exposed
to AR26, suggesting that this molecule directly acted caus-
ing mitochondrial dysfunction.
Figure 6 Detection of autophagic vacuole accumulation in AR26-
treated L. braziliensis promastigotes. The formation of autophagic vac-
uoles in the treated promastigotes was evaluated by fluorimetry, after
24 h of incubation with AR26 (1.0 or 2.0 µg/ml). Stationary pro-
mastigotes were used as positive control. P < 0.001 (***) and
P < 0.01 (*) indicate significant statistical difference in comparison to
the control group.
The drastic alterations in the bioenergetics metabolism
of parasites and the resulting impairment in the function-
ing of respiratory chain as a consequence of drug treatment
could strongly contribute to the excessive generation of
intramitochondrial ROS levels. In turn, such toxic prod-
ucts, when in high levels, present a potent ability to pro-
mote multifactorial cellular responses, causing cell damage
in proteins, DNA and lipids, thus culminating in the activa-
tion of cell death by distinct pathways.[41,42] However, in
this study, no increase in the ROS production was detected
in AR26-treated parasites, suggesting the regulation of ROS
levels by the antioxidant metabolism of the parasites.
To investigate whether the AR26 caused damage to the
integrity of the plasma membrane, an event clearly associ-
ated with cell death by necrosis, a staining with PI was per-
formed. Results showed that the treatment of
resveratrol analogues such as pterostilbene, piceatannol,
polydatin and oxyresveratrol were assayed against L. ama-
zonensis amastigotes, with IC50 values ranging from 6 µg/
ml to 10 µg/ml (29–45 µM), below than that obtained with
AR26.[22] As with these trans-resveratrol analogues, AR26
did not show toxicity against murine macrophages, high-
lighting the selective effect of these resveratrol analogues.
Resveratrol-treated L. major amastigotes revealed antileish-
manial effect, but the authors point out that this biological
activity and toxicity on mammalian cells are closed related
to the host cell, and therefore, the results must be carefully
© 2019 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2019), pp. **–**
7

Antileishmanial activity of resveratrol analogues
Luciana Maria Ribeiro Antinarelli et al.
Figure 7 Production of reactive oxygen species in L. braziliensis-infected macrophages and treated with AR26. Macrophages that were infected
(a) or not (b) with L. braziliensis were treated with AR26 at 15.88 or 31.76 µg/ml, or with amphotericin B (AmB, 0.069 µg/ml) or miltefosine
(1.3 µg/ml), used as control. After 24 h, ROS levels were evaluated by incubation with H2DCFDA (20 lM) for 30 min in the dark. The fluorescence
intensity was measured in arbitrary units (a.u.). Zymosan (250.0 µg/ml) was used as a positive control. P < 0.001 (***), P < 0.01 (**), and
P < 0.05 (*) indicate statistical difference in comparison to the control group.
promastigotes with AR26 resulted in simultaneous impair-
ment of plasma membrane integrity. Such alterations
directly affected the pathway of biosynthesis of sterols in
the parasites, transport of nutrients and ions as well as their
lipid and protein composition; culminating to the deleteri-
ous effects in the growth and viability of these cells.[43,44]
An intense dysregulation in the cell cycle progression was
also evidenced in AR26-treated parasites, since a marked
increase in the level of subG0/G1 phase cells with concomi-
tant decrease of the G0/G1 and G2/M phases´ cell popula-
tion were encountered. The cell cycle arrest in the subG0/G1
peak is suggestive of parasite population with low DNA
content, thus indicating the occurrence of apoptotic-like
death.[45] In accordance with our findings, previous reports
using resveratrol and their analogues have also showed
increase in the promastigotes population in the sub-G0/G1
phase of their cell cycle, culminating with collapse of the
mitochondrial membrane potential.[21,22,46] Moreover,
studies confirmed that resveratrol displays its antiprolifera-
tive action in tumour cell lines by activating cell cycle
arrest, reinforcing that this molecule class has common tar-
gets in different biological targets.[16,47]
exposition to drugs and toxins, among others.[49–51] In try-
panosomatids, autophagy is a biological process required
for the maintenance of parasite life cycle, contributing to
the regulation of cell density in the vectors and mammalian
hosts, thus responding to the cell differentiation and modu-
lation of host immunity.[51] Despite its protective role, the
exacerbation of this autophagic pathway can also lead to
regulated cell death.[37,52] To investigate the activation of
this pathway of cell death caused by AR26, the accumula-
tion of autophagic vacuoles was evaluated by labelling with
MDC. Evidences of autophagic death were observed in the
L. braziliensis promastigotes that were exposed to AR26,
thus indicating that the autophagic responses could be an
attempt to recover the cell homoeostasis, resulting from
extensive damage in the parasite cellular processes. Corrob-
orating with this hypothesis, studies have pointed to the
action of resveratrol in the cell death by autophagy also in
tumour cell lines.[16,53,54]
The action of antileishmanial molecules against parasites
inside the host cell can occur as a direct effect of drug about
the parasites or as a consequence of the activation of micro-
bicidal mechanisms of host cell.[55,56] To explore the mode
of action of AR26 against intracellular amastigotes, the
ROS production in treated and infected macrophages was
analysed and, interestingly, the ROS production was
decreased in macrophages that were infected with
L. braziliensis promastigotes and treated with the molecule,
thus suggesting its antioxidant action. Corroborating our
Autophagy has been also showed as a mechanism of
action of molecule classes presenting antileishmanial
action.[48] Autophagy is a catabolic process of degradation
and recycling of injured organelles and cytosol compo-
nents, where cellular homoeostasis is ruptured in stress
conditions, such as nutrient deprivation, infection,
8
© 2019 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2019), pp. **–**

Luciana Maria Ribeiro Antinarelli et al.
Antileishmanial activity of resveratrol analogues
results, studies also showed that resveratrol and its ana-
logues exert their antileishmanial action by suppressing the
NO and ROS production in murine macrophages.[21,22]
Our findings are also consistent with such works, which
corroborated with the results described here.
antileishmanial agent against Leishmania infection in mam-
malian hosts.
Declarations
Conflicts of interest
Conclusions
The authors confirm that they have no conflicts of interest
in relation to this work.
In summary, this study presents a new class of resveratrol
analogues based on N-aryl imines, which showed selective
antileishmanial action against distinct Leishmania species.
The evaluation of the mechanism of action of the most
effective compound, AR26, suggested that this molecule is
involved in multiple biochemical and cellular alterations in
the parasites, including disruption of mitochondrial func-
tion causing DΨm depolarization, plasma membrane per-
meabilization and interference in the progression of the cell
cycle. Therefore, data showed here encourages the develop-
ment of future studies to evaluate AR26 as an
Acknowledgements
The authors would like to acknowledge the ‘Programa de
Poꢀs-Graduaßc~ao em Ci^encias Bioloꢀgicas (Universidade Fed-
eral de Juiz de Fora)’ for allowing the use of the FACsCanto
II flow cytometer multiuser equipment facility. This work
received financial support from FAPEMIG and CNPq.
LMRA, ADS and EAFC, and ESC receive grants from
CNPq.
and treatment regimens. Infect Dis Clin 18. Vilar-Pereira
G et al. Resveratrol
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Appendix S1. Spectrometric data.
10
© 2019 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2019), pp. **–**