Atividade Antioxidante de Eugenia moraviana

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Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02588-3
MEDICINAL
CHEMISTRY
RESEARCH
ORIGINAL RESEARCH
Chemical composition, antileishmanial and antioxidant activity
of Eugenia moraviana (Myrtaceae) fruit extract
Fabiana Borges Padilha Ferreira 1
● Áquila Carolina Fernandes Herculano Ramos-Milaré1 ●
Márcia Regina Pereira Cabral2 ● Danielle Lazarin-Bidóia3 ● Celso Vataru Nakamura3 ● Maria Helena Sarragiotto4 ●
Wanessa de Campos Bortolucci5 ● Carla Maria Mariano Fernandez5 ● Zilda Cristiani Gazim5
●
Izabel Galhardo Demarchi6 ● Thaís Gomes Verzignassi Silveira1,7 ● Maria Valdrinez Campana Lonardoni1,7
Received: 6 April 2020 / Accepted: 5 June 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Leishmaniasis is a severe disease caused by protozoa of the genus Leishmania. The disease affects millions of people.
Treatment is limited to only a few drugs that cause severe side effects. The identification of new compounds to treat
leishmaniasis remains a challenge. This study aimed to determine the chemical composition of Eugenia moraviana fruit
extract (EmCE) and to investigate its effect on the promastigote and amastigote forms of Leishmania amazonensis, the
ultrastructural changes induced by it, its cytotoxicity, and antioxidant effect. Liquid chromatography–high-resolution mass
spectrometry was used to identify EmCE components. Amastigote forms (IC50 0.6 µg mL−1) were more sensitive to EmCE
treatment than promastigote forms (IC50 77.2 µg mL−1). The cytotoxicity concentration 50% was 187.8 µg mL−1 for J774.A1
macrophages and 190.0 µg mL−1 for murine macrophages, with selectivity index of 313 and 316.7, respectively. EmCE
induced 14.7% hemolysis. Promastigotes exposed to EmCE experienced drastic ultrastructural changes, such as intense
cytoplasm vacuolization and mitochondrial swelling. The antioxidant activity IC50 for DPPH was 0.78 µg mL−1, inhibition
of lipid peroxidation was 56.4%, FRAP assays with 1.19 µM ferrous sulfate/mg sample, and total phenolic acid equivalent
was 76.1 µg gallic acid/mg sample.EmCE was not cytotoxic, was highly selective for protozoans, and caused intracellular
death of the parasite. The results indicated the potential value of EmCE in the treatment of leishmaniasis. These results are
the first report of antileishmanial activity and chemical composition for E. moraviana fruits extract.
Keywords Leishmaniasis ● Antileishmanial agents ● Cambuí ● Myrtaceae ● Chemical characterization
Introduction
Leishmaniasis is a neglected disease that affects the most
vulnerable populations and those with poor access to health
services (PAHO 2019). It is estimated that 350 million
people are exposed to the disease worldwide, with
approximately two million new cases of its different clinical
forms being recorded each year. The disease is caused by
* Fabiana Borges Padilha Ferreira
fbpferreira@hotmail.com
1 Postgraduate Program in Health Sciences, Maringá State
University, Maringá, Paraná, Brazil
2 Postgraduate Program in Pharmaceutical Sciences, Maringá State
University, Maringá, Paraná, Brazil
3 Department of Health Sciences, Laboratory of Innovation in Drug
and Cosmetic Development, Maringá State University,
Maringá, Paraná, Brazil
4 Postgraduate Program in Chemistry, Maringá State University,
Maringá, Paraná, Brazil
5 Postgraduate Program in Biotechnology Applied to Agriculture,
University of Paranaense UNIPAR, Umuarama, Paraná State,
Brazil
6 Federal University of Santa Catarina, Florianópolis, Santa Catarina
State, Brazil
7 Department of Clinical Analysis and Biomedicine, Maringá State
University, Maringá, Paraná, Brazil
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02588-3) contains supplementary
material, which is available to authorized users.
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https://doi.org/10.1007/s00044-020-02588-3
https://doi.org/10.1007/s00044-020-02588-3
the parasitic protozoa of the genus Leishmania and is
transmitted to humans through the bite of infected female
sand flies (Alvar et al. 2013). Depending on the species of
the parasite, it is clinically categorized as cutaneous,
mucocutaneous, and visceral forms (Hammi et al. 2019). In
the Americas, leishmaniasis is prevalent in 18 countries, and
the most common clinical form is cutaneous.
As for many other infectious diseases, leishmaniasis
therapy is still a medical challenge (Viegas et al. 2019).
Drugs currently used to treat leishmaniasis are pentavalent
antimonials, amphotericin B, miltefosine, pentamidine, and
paromomycin. These have several limitations, including
pronounced toxicity (Garcia et al. 2018) low efficacy, high
cost, prolonged clinical regimen, and the development of
resistance (Ferreira et al. 2012; Singh et al. 2016). Given
these difficulties, the screening of natural products has been
undertaken as a necessary pathway in the development of
new drugs (Oliveira et al. 2013). Several studies have
validated the effect of natural products as potential sources
of new and selective agents for the treatment of tropical
diseases caused by protozoa and other parasites (Andrade
et al. 2016; Hammi et al. 2019).
The Myrtaceae family consists of approximately 150
genera and 6076 species. It is one of the largest plant
families in South and Central America, and is found
worldwide (Govaerts et al. 2019). The fruits and leaves of
this family are widely used in folk medicine. Many studies
have been conducted to elucidate their medicinal applica-
tions, due to the presence of bioactive compounds in them
that can be used in the prevention and treatment of various
diseases (Asmari et al. 2014; Lemos et al. 2011). Edible
fruits of Eugenia species have anticonvulsant, anti-
microbial, and insecticidal actions (Silva et al. 2017).
The genus Eugenia is one of the largest in the Myrtaceae
family and is notable for the important economic and
pharmacological potential of its species. This potential is
evidenced by many documents and scientific publications
and by the commercial exploitation of its edible fruits,
wood, and essential oils (Queiroz et al. 2015). The species
of this genus are distributed mainly in climatic and sub-
tropical regions of America and climatic regions of Asia
(Reynertson et al. 2008).
Eugenia moraviana Otto Berg has been very promising
in the sphere of plants with medicinal potential. It is a native
plant from Brazil, where it is popularly known as Cambuí.
E. moraviana has been studied for its potential in the
treatment of human immunodeficiency virus, some types of
tumors, malaria and inflammatory processes (Lunardi et al.
2001), diarrhea, and intestinal disorders (Hajdu and Hoh-
mann 2012). Triterpenic derivatives have been identified in
leaves and stems of E. moraviana (Lunardi et al. 2001) and
in the β-caryophyllene, β-elemene, and α-copaene essential
oils (Apel and Sobral 2002). However, to our knowledge,
there have been no reports in the literature about the che-
mical composition of E. moraviana fruits and their antil-
eishmanial activity.
In the search for new agents that have reduced toxicity
and increased efficacy in the treatment of leishmaniasis, this
study aimed to determine the chemical composition of E.
moraviana fruit extract and to investigate its effect on the
promastigote and amastigote forms of L. amazonensis, in
addition to the ultrastructural changes induced by the
extract, its cytotoxicity, and antioxidant effect.
Materialand methods
Isolation of red cells from human blood
Human blood samples were obtained by venipuncture from
six healthy women donors, 21–30 years old, nonsmoking,
who were not on any medication. Each donor answered a
questionnaire and signed informed consent forms. The
study was approved by the “Standing Committee on Ethics
in Research with Human Beings,” State University of
Maringá (Code no. 31009320.0.0000.0104/COPEP/UEM),
and complied with Resolution No. 446/2012 of the National
Council of Health, Ministry of Health of Brazil.
Blood was collected in heparinized tubes, centrifuged at
1500 rpm for 10 min, the red cells pellet was washed three
times in sterile 1% glucose saline solution, and resuspended
in sterile 1% glucose saline solution to give a 6% (v/v) cell
suspension.
Animals
Female 4–6-week-old BALB/c mice were used. The
experiments were approved by the “Experimental Animal
Use Ethics Committee” of the Maringá State University,
Paraná, Brazil (Code No. 7977050517/CEUA/UEM).
Collection of plant material
E. moraviana Otto Berg fruits were collected in May 2016
in Diamante do Norte, Paraná, Brazil (S 22° 35’, W 52°
53’). The species has been identified and is deposited in the
Educational Herbarium of Maringá State University
(HUEM), Maringá, Paraná, Brazil under No. 21428. This
species is registered in the National Genetic Heritage
Management System and Associated Traditional Knowl-
edge (SisGen) under No. A031F14.
Fruit extract of Eugenia moraviana (EmCE)
The fruits of E. moraviana were ground in a knife mill
(Tecnal®). The extract was obtained by dynamic
Medicinal Chemistry Research
maceration using 96° GL alcohol as a solvent at 10%
(w/v). The extract was concentrated in a rotary evaporator
(Tecnal®, Piracicaba, Brazil) at 40 °C to yield crude
extract (CE).
Ultra-high performance liquid chromatography
analysis high-resolution mass spectrometry (UHPLC-
HRMS/MS)
EmCE was analyzed by UHPLC (Nexera X2 device,
Shimadzu, Japan) coupled with HRMS (QTOF Impact II,
Bruker Daltonics Corporation, USA) equipped with an
electrospray ionization source. The capillary voltage
operated in negative ionization mode set at 4500 V and
with an endplate offset potential of −500 V. The dry gas
parameters were set to 8 L min−1 at 200 °C with a neb-
ulization gas pressure of 4 bar. Data were collected from
50 to 1300 m/z with an acquisition rate of 5 spectra
per second. The ions of interest were selected by auto MS/
MS scan fragmentation. Chromatographic separation was
performed using a C18 column (75 × 2.0 mm i.d.; 1.6 μm
Shim-Pack XR-ODS III). The gradient mixture of solvents
A (H2O) and B (acetonitrile) was as follows: 5% B;
0–1 min, 30% B; 1–2 min, 95% B; 2–8 min, maintained at
95% B; 8–12 min at 40 °C. The identification of these
compounds was proposed in a review of the genus Euge-
nia, in addition to the mass error value. Only molecular
formula with ≤5 ppm of error was considered in this study
(Brenton and Godfrey 2010).
Antileishmanial activity against promastigote forms
of Leishmania amazonensis
The evaluation of the antileishmanial activity of EmCE
fruit extract was performed against promastigote forms of
L. amazonensis (MHOM/BR/1977/LTB0016). Aliquots
of a containing 4 × 107 mL−1 in the log phase of growth
were solubilized using 0.2% Tween 80 and dispensed in
the wells of a 96-well microplate in the presence of
decreasing EmCE concentrations (1000–0.5 µg mL−1) and
amphotericin B (125–0.24 µg mL−1) (AmB, Laboratory
Cristália, São Paulo, Brazil). The plate was incubated at
25 °C for 24 h. To assess cell viability, 100 µL of 2,3-bis-
(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-car-
boxanilide (XTT; Sigma-Aldrich, St. Louis, EUA) was
added to each well and the plate was incubated for 3 h at
37 °C. After the incubation period, the results were
determined using an ASYS Expert Plus microplate reader
(ASYS Hitech GmbH, Eugendorf, Austria) at 450/
620 nm. The 50% inhibitory concentration of parasites
(IC50) was calculated from the linear regression of the
percentage of death. The tests were performed in triplicate
and repeated at least three times.
Antileishmanial activity against amastigotes forms
of Leishmania amazonensis
Peritoneal macrophages from BALB/c mice were incubated
in a 24-well plate at a concentration of 1 × 106 mL−1 on
13mm diameter sterile glass coverslips for 2 h at 37 °C in an
atmosphere containing 5% CO2. After this period, macro-
phages were infected with promastigote forms of L. amazo-
nensis (5 × 105 parasites/well) and incubated for 4 h at 37 °C
in an atmosphere of 5% CO2. After incubation, EmCE was
added at concentrations of 50, 25, and 12.5 µgmL−1. After
24 h, the coverslips were washed in PBS, fixed in 95%
ethanol solution, and stained with hematoxylin and eosin. A
total of 200 cells were counted using an optical microscope.
The infection index was calculated from the percentage of
infected macrophages multiplied by the average number of
parasites per macrophage. The tests were performed in tri-
plicate and repeated at least three times.
Cytotoxicity in macrophages J774.A1
The cytotoxicity assay was performed using J774.A1 strain
macrophages. A macrophage suspension containing 1 × 106
cells mL−1 was prepared. One hundred microliter aliquots
were added to each well in a 96-well plate and incubated for
48 h at 37 °C and 5% CO2. EmCE was added to the wells at
concentrations from 1000 to 0.5 µgmL−1. After 24 h, 100 μL
of XTT was added to each well. After incubating the
microplate for 3 h at 37 °C, cytotoxicity was determined using
the aforementioned ASYS Expert Plus microplate reader at
450 and 620 nm. The tests were performed in triplicate.
Cytotoxic concentration 50% (CC50) was defined as the dose
of compound that reduced macrophage survival by 50%
compared with untreated macrophages (viability control).
Murine macrophage cytotoxicity
Peritoneal macrophages (1 × 106 cells mL−1) were dis-
tributed in a 96-well plate and incubated for 2 h at 37 °C in
5% CO2. Subsequently, they were treated with different
concentrations of EmCE (1000 to 0.5 µg mL−1) and AmB
(125 to 0.24 µg mL−1) for 24 h at 37 °C in 5% CO2. Cell
viability was assessed by the XTT colorimetric reduction
method and determined on the ASYS Expert Plus micro-
plate reader at 450 and 620 nm. Cytotoxic concentration
(CC50) was defined as the dose of compound that reduced
macrophage survival by 50% compared with untreated
macrophages (viability control).
Hemolytic activity
A suspension of 6% human red blood cells in saline solu-
tion was prepared as modified from Valdez et al. (2009).
Medicinal Chemistry Research
One hundred microliter aliquots of the suspension were
added to the wells of a 96-well plate containing diluted
EmCE (1000 to 0.5 µg mL−1). After incubation of the
plate for 2 h at 37 °C, the absorbance of the supernatant
was determined at a wavelength of 550 nm to estimate
hemolysis. Triton X-100 (Sigma-Aldrich, USA) was used
at 4% as a positive control for hemolysis and a red cell
suspension was used as a negative control. Results were
expressed as a percentage of hemolysis based on the
equation:
%ð Þ ¼ Asð Þ= Apð Þ½ � � 100;
where As and Ap are the absorbance of the test sample and
positive control, respectively. Hemolysis was performed in
two triplicate experiments.
Transmission electron microscopy
Promastigotes forms of L. amazonensis were treated with
IC50 EmCE (77.2 µg mL−1) for 24 h at 25 °C. After this
period, the parasites were fixed with 2.5% glutaraldehyde
(Sigma-Aldrich, USA) in 0.1 M cacodylate. They were
washed three times with 0.1 M cacodylate buffer and
postfixed with 1% osmium tetroxide, 0.8% potassium fer-
ricyanide, and 5 mM calcium chloride in 0.1 M cacodylate
buffer for 30 min. The postfixed parasites were dehydrated
in acetone and embedded in EMBed-812. Untreated para-
sites were used as controls. Ultrathin sections were stained
with uranyl acetate and lead citrate, and examined using a
JEM 1400 electron transmission microscope (JEOL, Tokyo,
Japan).
Determinationof mitochondrial membrane
potential (ΔΨm)
To analyze the effect of EmCE on L. amazonensis mito-
chondria, Rhodamine 123 (Rh123; Sigma-Aldrich, USA)
was used as a marker. L. amazonensis promastigotes (2 ×
107 parasites/mL) were treated with IC50 for 1, 4, and 24 h
at 25 °C. Subsequently, the parasites were incubated with
5 μg mL−1 Rh123. Results were obtained and analyzed
using a flow cytometer (Becton Dickinson, Rutherford, NJ,
USA) equipped with CellQuest software (Joseph Trotter,
Scripps Research Institute, La Jolla, USA). Ten thousand
events per sample were analyzed. Changes in ΔΨm polar-
ization were measured by the variation index (IV) obtained
by the equation (MT-MC)/MC, where MC is the median of
fluorescence control intensity and MT the median fluores-
cence of treated cells. Negative IR values correspond to
mitochondrial membrane depolarization. Hydrogen per-
oxide (Sigma-Aldrich, USA) was used as a positive control
(Dagnino et al. 2018).
Antioxidant activities
Evaluation by DPPH radical scavenging
To determine the free radical scavenging capacity of 2,2-
diphenyl-1-picryl-hydrazil (DPPH) by EmCE, we used the
methodology described by Rufino et al. (2007). Aliquots of
0.1 mL of the different EmCE concentrations (1.0, 0.75,
0.50, and 0.25 mg mL−1) were added to 3.9 mL of DPPH
methanolic solution (60 µM). For negative control, 0.1 mL
of methanol in DPPH solution (60 µM) was used. Absor-
bance reduction was measured at 515 nm on a UV/VIS
spectrophotometer. The total antioxidant capacity of
extracts and fractions was calculated using a standard
quercetin solution (60 µM) as a 100% reference. From the
correlation between absorbance and antioxidant sample
concentration, the concentration required to reduce 50% of
free radicals (IC50) was determined.
Ferric reducing antioxidant power (FRAP)
The assay was performed as described by Rufino et al.
(2006b). The FRAP reagent comprised 25mL of acetate
buffer (0.3M), 2.5 mL of 2,4,6-Tris (2-pyridyl) triazine
(TPTZ, 10mM) aqueous solution, 2.5 mL of aqueous ferric
chloride (20 mM) and 3mL of distilled water. Ten microliters
of EmCE at different concentrations (1.0, 0.75, 0.50, and
0.25mgmL−1) and 290 μL of FRAP reagent were added to
wells of a 96-well plate. The mixture was placed in the
SpectraMax Plus 384 Microplate Reader (Molecular devices,
Sunnyvale, CA, USA) and maintained at 37 °C for 30min
following which the absorbance was read at 595 nm. Using a
standard ferrous sulfate curve (0–2000 μM), the percentage
antioxidant activity was calculated. The antioxidant activity
was expressed as μM ferrous sulfate/mg of the sample.
Co-oxidation of β-carotene/linoleic acid method
The antioxidant capacity of EmCE samples was evaluated
as described by Rufino et al. (2006a). The reaction was
monitored by spectrophotometry and the loss of β-carotene
staining at 470 nm. An emulsion was prepared and absor-
bance was adjusted to 0.7 at 470 nm. Antioxidant activity
was determined by mixing 280 µL emulsion with 20 µL
samples at different concentrations (1.0, 0.75, 0.50, and
0.25 mg mL−1) of EmCE in 96-well microplates. Absor-
bance was determined at 470 nm. Trolox (0.2 mg mL−1)
was used as a reference standard. The results were expres-
sed as a percentage of oxidation inhibition as calculated by
Eq. (1). The reduction of antioxidant system absorbance
was considered 100% oxidation. From the absorbance cal-
culated in Eq. (2), the oxidation percentage correlated with
Medicinal Chemistry Research
the absorbance of the sample decreasing with the system
absorbance; the oxidation percentage of each sample was
subtracted from 100 (Eq. (3)) to determine the oxidation
inhibition percentage (%).
Absorbance reduction ¼ Ainitial � Afinal ð1Þ
% oxidation ¼ ReductionAð Þsample�100
h i
= Reduction Að Þsistem
ð2Þ
% of protection ¼ 100� % oxidationð Þ ð3Þ
Total phenols
The analysis of the phenolic compounds of EmCE was per-
formed using the Folin–Ciocalteau reagent method (Viuda-
Martos et al., 2010). Samples were diluted in methanol to a
concentration of 1.0 mgmL−1. Sample methanol solution
(0.3 mL) was added and mixed with 2.5 mL 10% aqueous
Folin–Ciocalteau reagent and 2.0 mL 7.5% (w/v) sodium
carbonate. The mixture was kept for 15min in a water bath at
50 °C. Absorbance was read on a model 1601PC UV/VIS
spectrophotometer (Shimadzu) at 760 nm. Methanol was
used as control. Phenol concentration was calculated from
the standard gallic acid curve (0–2000 µM). Results were
expressed as µg gallic acid equivalent/mg sample.
Results and discussion
The compounds identified in the EmCE by UHPLC-HRMS/
MS in the negative mode are shown in Table 1. Twelve
phenolic compounds were identified by comparing the high-
resolution mass data obtained with the literature data. It was
possible to verify the presence of phenolic acids that
included gallic acid (Fig. 1b) and ellagic acid (Fig. 1c),
ellagic acid derivative (ellagic acid 4-O-xylopyranoside)
(Fig. 1g), eschweilenol C (Fig. 1k) and syringic acid
(Fig. 1h), flavonoids like isoquercetin (Fig. 1i) and
kaempferol pentoside (Fig. 1l), glycosylated flavonoids
including rutin (Fig. 1f), myricetin-3-O-pentoside (Fig. 1d),
and quercetin-O-pentoside (Fig. 1e), and anthocyanins like
pelargonidin-3-glucoside (Fig. 1j). These compounds are
the first to be identified in this species of Eugenia.
Lunardi et al. (2001) isolated the extract of the leaves and
stem of E. moraviana and identified the triterpene 6α-
hydroxybetulinic acid, along with three other substances
(platanic acid, betulinic acid, and β-sitosterol). Studies with
other Eugenia species have identified myricetin and gallic
acid in the leaves and bark of E. malaccensis stem (Oliveira
et al. 2006). Simirgiotis et al. (2008) isolated quercetin,
quercitrin, myricitrin, gallic acid, and ellagic acid from the
fruits of E. javanica. Compounds identified in the leaves of
E. jambolana were gallic acid, kaempferol, ellagic acid, and
myricetin (Mahmoud et al. 2001). Myricetin-3-O-pentoside
has also been identified in the fruits of E. uniflora (Celli
et al. 2011).
In this study, we observed that EmCE inhibited the
growth of L. amazonensis promastigotes after 24 h of
treatment, with an IC50 of 77.2 µg mL−1. The IC50 for AmB
was 0.5 µg mL−1 (Table 2). For L. amazonensis amasti-
gotes, the IC50 was 0.6 µg mL−1 (Table 2). Amastigote
forms were more sensitive to EmCE treatment than pro-
mastigote forms. This was an important result, as amasti-
gotes forms are responsible for clinical manifestations in the
Table 1 Constituents of the
Eugenia moraviana crude
extract
Molecular
formula
Exact mass
(m/z) (M-H)−
Precursor ion
(m/z) (M-H)−
Mass
error (ppm)
tR/min Identification
C7H12O6 191.0548 191.0554 −3.14 2.98 Quinic acid
C7H6O5 169.0130 169.0137 −4.14 3.03 Gallic acid
C14H6O8 300.9977 300.9968 2.99 4.75 Ellagic acid
C20H18O12 449.0713 449.0693 4.45 4.11 Myricetin-3-O-
pentoside
C20H18O11 433.0763 433.0753 2.31 4.45 Quercetin-O-pentoside
C27H30O16 609.1448 609.1413 5.75 4.55 Rutin
C19H14O12 433.0400 433.0383 3.92 4.71 Ellagic acid 4-O-
xylopyranoside
C9H10O5 197.0443 197.0448 −2.54 4.40 Syringic acid
C21H20O12 463.0871 463.0856 3.24 4.23 Isoquercetin
C21H20O10 431.0972 431.0966 1.39 4.83 Pelargonidin-3-
glucoside
C20H16O12 447.0556 447.0543 2.91 4.59 Eschweilenol
C20H18O10 417.0814 417.0803 2.64 4.66 Kaempferol pentoside
Data of the compounds identified by UHPLC-HRMS/MS in negative mode
Medicinal Chemistry Research
vertebrate host and are the main target of chemotherapy for
leishmaniasis (Cunningham 2002; McConville and Hand-
man 2007). Several reports in the literature have described
the antileishmanial activity of other Eugenia species, such
as the extract of the bark of E. monteverdensis (Monzote
et al. 2014), methanolic extract of E. umbelliflora fruits
(Filho et al. 2013), and methanolic extract of E. uniflora
leaves (Braga et al. 2007). As far as we know, there are no
other reports on the antileishmanial activity of E. moraviana
fruit extract.
The anti-Leishmaniastructure-activity relationship is
described in the literature, and it has been shown that the
flavonoid class consists of potential antiprotozoal agents
(Cota et al. 2012; Iqbal et al. 2017; Rocha et al. 2019;
Tasdemir et al. 2006). A study by Vila-Nova et al. (2012)
reported that the antileishmanial activity of the quercetin
and rutin flavonoids was similar to those of pentamidine and
amphotericin B, against promastigote and amastigote forms
of L. chagasi. Mittra et al. (2000) demonstrated the inhi-
bitory effect quercetin on the growth of promastigote and
amastigote forms of L. donovani, in both in vitro and
in vivo studies. According to Mehwish et al. (2019) rutin
and quercetin displayed an IC50 of 91.2 and 182.3 µg mL−1,
respectively, against the promastigote form of L. tropica
Fig. 1 Chemical structures of the
constituents of the raw fruit
extract of Eugenia moraviana.
(a) Quinic acid, (b) Gallic acid,
(c) Ellagic acid, (d) Myricetin-3-
O-pentoside, (e) Quercetin-O-
pentoside, (f) Rutin, (g) Ellagic
acid 4- O-xylopyranoside, (h)
Sirinic acid, (i) Isoquercetin,
(j) Pelargonidin-3-glucoside,
(k) Eschweilenol C, (l)
Kaempferol pentoside
Medicinal Chemistry Research
and an IC50 of 101.3 and 137.4 µg mL−1, respectively,
against the amastigote form. These results were superior to
those exhibited by EmCE, indicating the promising antil-
eishmanial activity of this extract.
EmCE displayed low cytotoxicity in J774.A1 macro-
phages (187.7 µgmL−1) and murine macrophages (190.0 µg
mL−1) compared with the reference medicine AmB (2.3 µg
mL−1) (Table 2). Another very favorable result for EmCE
was its high selectivity index, which suggested that EmCE is
more selective for the parasite than for mammalian cells.
EmCE showed hemolytic activity of 14.7% at the highest
concentration tested (1000 µgmL−1) (Table 2). Research
evaluating the in vitro cytotoxicity of E. moraviana is scarce.
However, Santos et al. (2017) observed low toxicity of an
ethanolic extract of E. uniflora against J774 macrophages,
corroborating our results. Roesler et al. (2010) analyzed the
cytotoxicity of E. dysenterica fruits with 3T3 cells and found
no cytotoxic effect up to 300 µgmL−1. Castro et al. (2019)
examined the effects of E. uniflora leaf extract using Vero
cells and observed no cytotoxicity at 250 μgmL−1.
To investigate and identify the organelles that may be
potential targets of EmCE in promastigote forms, trans-
mission electron microscopy was performed. Untreated
promastigote forms displayed a nuclear morphology and
preserved cytoplasmic organelles (Fig. 2a). However, after
treatment with EmCE (IC50 77.2 µg mL−1), drastic changes
were observed. These comprised cell disorganization, rup-
tured organelles (Fig. 2b, e), induction of abnormal lipid
secretion, intense cytoplasm vacuolization, membrane pro-
files in the inside of the organelle, and mitochondrial
swelling (Fig. 2b–f).
The presence of multivesicular bodies and various
vacuoles with membrane profiles and cellular debris are
related to the presence of lysosomes and secondary orga-
nelles, which are probably involved in the degradation of
damaged structures (Rodrigues and Souza 2008). EmCE
caused severe cellular damage (Fig. 2b, c), with visible
damage to the plasma membrane, culminating in extravasa-
tion of the cytoplasmic content and loss of cellular integrity.
Similar observations were also reported by Almeida-Souza
et al. (2016) with the fruits of Morinda citrifolia Linn.
Kinetoplasts could not be identified due to the drastic altera-
tions after the treatment, as well as the disorganization of the
nucleus with an intense process of cell destruction (Fig. 2c, f).
The mitochondrial membrane potential of the L. ama-
zonensis promastigote forms treated with EmCE indicated
considerable membrane hyperpolarization after 1 h of
treatment and increased fluorescence intensity for Rh123
(Fig. 3a, d, e). After 4 and 24 h, mitochondrial depolariza-
tion of the parasites incubated with EmCE was evident
compared with untreated parasites, inducing loss of fluor-
escence Rh123 (Fig. 3b, c, e). In many cases, mitochondrial
depolarization is preceded by transient hyperpolarization
that is often considered the last attempt of cells to prevent
death (Jiménez-Ruiz et al. 2010). Changes in mitochondrial
membrane potential may lead to a decrease in adenosine
triphosphate and reduced transcription and translation of
mitochondrial genes, which may result in cell death due to
both apoptosis and necrosis (Mayer and Oberbauer 2003).
The results of evaluation of the antioxidant activity of
EmCE by DPPH, β-carotene/linoleic acid, and FRAP
methods are presented in Table 3. EmCE displayed content
of phenolic compounds content of 76.1 µg gallic acid/mg
sample. The content of phenolic compounds for other
Eugenia species found in a study by Magina et al. (2010)
was 162.6 mg for E. brasiliensis, 138.0 mg for E. beau-
repaireana, and 128 mg gallic acid/g sample for E.
umbelliflora. These values are higher than the phenolic
compounds content found for EmCE. DPPH assay revealed
that EmCE had an IC50 of 0.78 mg mL−1. In their study
involving the analysis of antiradical activity by DPPH,
Tomborelli et al. (2018), reported the IC50 values of ethyl
acetate and butanolic fraction of E. dysenterica as 0.152 and
1.053 mg mL−1, respectively.
In the β-carotene/linoleic acid peroxidation assay, EmCE
showed 56.4% inhibition of the free radicals generated
during acid peroxidation. Mello et al. (2017) obtained
similar results with E. catharinensis extract showing 61.3%
inhibition of lipid peroxidation. In the FRAP assay, the
EmCE value of 1.19 µM ferrous sulfate/mg sample differed
from the results of Bagetti et al. (2011) with the red and
orange E. uniflora extracts displaying 1.4 and 1.1 mmol
Trolox 100 g−1 of ferric reduction, respectively. These dif-
ferences may be caused by the action of environmental and
genetic influences and chemical and quantitative modula-
tion of the compounds present in the extracts and are factors
Table 2 Anti-Leishmania activity and in vitro cytotoxicity of Eugenia
moraviana fruits extract
µg mL−1
Eugenia moraviana Amphotericin B
CC50
J774.A1 Macrophages 187.8 ± 6.0 –
Murine macrophages 190.0 ± 0.3 2.3 ± 0.06
Hemolysis (%) 14.7% ± 0.01 –
IC50
Promastigotes 77.2 ± 14.9 0.5 ± 0.01
Amastigotes 0.6 ± 0.06 –
ISM1 313.0 4.6
ISM2 316.7 –
The results are expressed as mean ± standard error
IC50 inhibitory concentration of 50%, CC50 cytotoxic concentration of
50%, SI selectivity index, SIM1 SI from J774.A1 macrophages, SIM2 SI
from murine macrophages, SI CC50/IC50, – not calculated
Medicinal Chemistry Research
Fig. 2 Transmission electron microscopy of Leishmania amazonensis
promastigotes forms treated with crude extract of Eugenia moraviana.
a Promastigote forms not treated with preserved organelles. b–f Pro-
mastigote forms treated with the IC50 (77.2 µg mL−1) of Eugenia
moraviana crude extract. Asterisks indicate cytoplasm vacuolation
(b–e), in some cases appearing as autophagosome-like structures (#)
(b and f). Black arrows indicate loss of membrane integrity (b and c).
N nucleus, M mitochondria and K kinetoplast
Fig. 3 Change in mitochondrial membrane potential after incubation
with crude Eugenia moraviana extract. Parasites (2 × 107) were incu-
bated with IC50 (77.2 µg mL−1) of Eugenia moraviana crude extract
for 1 h (a), 4 h (b) and 24 h (c) at 25 °C. Untreated promastigote forms
(negative control) and promastigotes treated with 2 mM H2O2 (positive
control) were used. Rh123-stained Leishmania amazonensis samples
were analyzed by flow cytometry, and a total of 10.000 events were
acquired. Representative histograms (a–c) and graphical representation
of percent fluorescence intensity relative to treatment group negative
and positive controls (d). Variation index obtained from the equation
(MT-MC)/MC, where MC is the mean fluorescence intensity of the
control and MT is the mean treated cell fluorescence (e). Results were
expressed as mean ± standard deviationMedicinal Chemistry Research
that will directly influence the antioxidant activity (Angél-
ico et al. 2012; Gobbo-Neto and Lopes 2007).
The phenomenon of oxidation occurs naturally in cellular
processes as part of the biochemical reaction mechanism,
cellular energy production, intercellular signaling, and pha-
gocytosis (Barreiros et al. 2006). However, excessive oxi-
dation can cause cell damage, resulting in the evolution or
aggravation of disease (Alam et al. 2013). In this study,
EmCE reduced DPPH levels, inhibited oxidation by the β-
carotene/linoleic acid method, and iron ion reduction by
FRAP, proving to be a potential chemopreventive molecule.
Conclusion
EmCE was effective against promastigote and amastigote
forms of L. amazonensis, with low toxicity to macrophages
and erythrocytes. Furthermore, it induced remarkable
effects on the morphology and ultrastructure of this parasite
which led to its death. The presence of important secondary
metabolites, such as phenolic compounds, may contribute to
the strengthening of the body against oxidative stress
caused by leishmaniasis. These results suggest that EmCE is
promising for the treatment of cutaneous leishmaniasis.
Acknowledgements This study was financed in part by Fundação
Araucária the Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior - Brazil (CAPES) Finance Code 001 and Complexo de
Centrais de Apoio a Pesquisa (COMCAP).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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Medicinal Chemistry Research
	Chemical composition, antileishmanial and antioxidant activity of�Eugenia moraviana (Myrtaceae) fruit extract
	Abstract
	Introduction
	Material and methods
	Isolation of red cells from human blood
	Animals
	Collection of plant material
	Fruit extract of Eugenia moraviana (EmCE)
	Ultra-high performance liquid chromatography analysis high-resolution mass spectrometry (UHPLC-HRMS/MS)
	Antileishmanial activity against promastigote forms of Leishmania amazonensis
	Antileishmanial activity against amastigotes forms of Leishmania amazonensis
	Cytotoxicity in macrophages J774.A1
	Murine macrophage cytotoxicity
	Hemolytic activity
	Transmission electron microscopy
	Determination of mitochondrial membrane potential (ΔΨm)
	Antioxidant activities
	Evaluation by DPPH radical scavenging
	Ferric reducing antioxidant power (FRAP)
	Co-oxidation of β-carotene/linoleic acid method
	Total phenols
	Results and discussion
	Conclusion
	Compliance with ethical standards
	ACKNOWLEDGMENTS
	References