Full length article
Anti-Leishmania
activity of essential oil of
Myracrodruon urundeuva
(Engl.) Fr. All.: Composition, cytotoxity and possible mechanisms of
action
C.E.S. Carvalho
a,*, E.P.C. Sobrinho-Junior
a, L.M. Brito
a, L.A.D. Nicolau
a, T.P. Carvalho
a,
A.K.S. Moura
b, K.A.F. Rodrigues
a, S.M.P. Carneiro
a, D.D.R. Arcanjo
a, A.M.G.L. Cit
o
b,
F.A.A. Carvalho
aaMedicinal Plants Research Center, Federal University of Piauí, 64049-550, Teresina, PI, Brazil bDepartment of Chemistry, Federal University of Piauí, 64049-550, Teresina, PI, Brazil
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
MuEO inhibits the proliferation of L. amazonensis promastigotes and axenic amastigotes.
MuEO exhibited selectivity indexes (SI) greater than reference drugs. MuEO reduced the infection index of
macrophages byL. amazonensis. MuEO is more effective against
intracellular amastigotes than promastigotes.
MuEO increase phagocytic activity in macrophages.
a r t i c l e
i n f o
Article history:
Received 18 April 2016 Received in revised form 23 January 2017 Accepted 7 February 2017 Available online 9 February 2017
Keywords:
Myracrodruon urundeuva
Anti-Leishmaniaactivity
Leishmania amazonensis
Cytotoxity Essential oil
a b s t r a c t
Myracrodruon urundeuva(Engl.) Fr. All., commonly known as“aroeira-do-sert~ao”, is a medicinal plant from Anacardiaceae family. In this study, the chemical composition ofM. urundeuvaessential oil (MuEO) was evaluated by gas chromatography-mass spectrometry (GC-MS), as well as its anti-Leishmania po-tential, cytotoxicity, and macrophage activation capability as possible antiprotozoal mechanism of action were assessed. Fourteen compounds were identified, which constituted 94.87% of total oil composition. The most abundant components were monoterpenes (80.35%), with b-myrcene (42.46%),a-myrcene (37.23%), and caryophyllene (4.28%) as the major constituents. The MuEO inhibited the growth of pro-mastigotes (IC50205±13.4mg mL 1), axenic amastigotes (IC50104.5±11.82mg mL 1) and decreased percentage of macrophage infection and number of amastigotes per macrophage (IC50 of
44.5 ±4.37mg,mL 1), suggesting significant anti-Leishmaniaactivity. The cytotoxicity of MuEO was
assessed by MTT test in Balb/c murine macrophages and by human erythrocytes lysis assay and low cytotoxicity for these cells was observed. The CC50value against macrophages were 550±29.21mg mL 1,
while cytotoxicity for erythrocytes was around 20% at the highest concentration assessed, with HC50>800mg mL 1. While MuEO-induced anti-Leishmaniaactivity is not mediated by increases in both lysosomal activity and nitric oxide production in macrophages, the results suggest the antiamastigote
Abbreviations:IC50, Half-maximal inhibitory concentration; CC50, Half-maximal cytotoxicity concentration; HC50, Half-maximal hemolytic concentration; MIC, Minimum
inhibitory concentration; SI, Selective index; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; DMSO, Dimethyl sulfoxide; FBS, Fetal bovine serum; BOD, Biological oxygen demand; PBS, Phosphate-buffered saline; MuEO, Essential oil ofMycracrodruon urundeuva; GC-MS, Gas Chromatography-Mass Spectrometry; NO, Nitric Oxide; ANOVA, Analysis of variance.
*Corresponding author. Medicinal Plants Research Center, Federal University of Piauí, s/neIninga. 64049-550, Teresina, PI, Brazil.
E-mail address:camilaernanda@hotmail.com(C.E.S. Carvalho).
Contents lists available atScienceDirect
Experimental Parasitology
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / y e x p r
http://dx.doi.org/10.1016/j.exppara.2017.02.012
0014-4894/©2017 Elsevier Inc. All rights reserved.
activity is associated with an immunomodulatory activity of macrophages due to an increase of phagocytic capability induced by MuEO. Thus, MuEO presented significant activity againstLeishmania amazonensis, probably modulating the activation of macrophages, with low cytotoxicity to murine macrophages and human erythrocytes.
©2017 Elsevier Inc. All rights reserved.
1. Introduction
Leishmaniasis is a complex of infectious parasitic diseases caused by protozoa from Trypanosomatidae family andLeishmania genus. It is considered a public health problem which affects more than 12 million people throughout the world, with 2e3 million new cases in each year. Furthermore, leishmaniasis is included in the group of neglected tropical diseases (NTD) (Feasey et al., 2010; Who, 2014).
The parasites from Leishmaniagenus have a heteroxenic life cycle, which is characterized by two distinct developmental forms: the mobile promastigote form, with outwardlyflagellum and pre-sent in the gastrointestinal tract of the insect vector, and the amastigote form, with innerflagellum and acting as an obligate intracellular form in mononuclear phagocyte cells. The amastigote forms are responsible for the clinical manifestations of leishmani-asis (De Almeida et al., 2003; Chappuis et al., 2007). Clinical man-ifestations are diverse, and can vary from the mucocutaneous form, which is characterized by the presence of ulcerative and nodular lesions in the skin, to the visceral form, the most severe and potentially fatal form (David and Craft, 2009).Leishmania
(Leish-mania)amazonensisis one of the species distributed throughout the
New World, specially in Latin America, and is associated with different clinical forms of leishmaniasis. It is the main agent of diffuse cutaneous leishmaniasis, which is commonly refractory to the currently available treatments (Guimar~aes-Costa et al., 2009).
Currently, the conventional therapy recommended for leish-maniasis has some drawbacks, such as long-term treatment with high doses, severe toxic adverse effects, and increased chemo-resistance of the parasite. The pentavalent antimonials have been thefirst choice treatment since 1945. The second-line drugs, such as amphotericin B, pentamidine and paromomycin, are used in cases of resistance to antimonials. However, they have high cost, and may be even more toxic than the antimonials (Croft and Coombs, 2003; Chappuis et al., 2007). Therefore, there is a glob-ally necessity for the discovery of new antileishmanial drugs.
In the ongoing search for leishmanicidal compounds, many studies have shown that plant-derived products may be promising sources for the development of new drugs (Machado et al., 2012). In this context, essential oils are composed by a wide diversity of small hydrophobic molecules such as monoterpenes, sesquiterpenes, and phenylpropanoids, and have demonstratedin vitroandin vivo
anti-Leishmaniaactivity against promastigote and amastigote forms of
Leishmaniaspp. The effective action of essential oil fromEugenia
unifloraL. (Rodrigues et al., 2013b),Pistacia veraL. (Mahmoudvand et al., 2016), Cymbopogoncitratus(D.C.) Staff (Machado et al., 2012),
andCopaifera cearensisHuber ex Ducke (Santos et al., 2008) against
Leishmaniaspp. shows that essential oils can be a promising source
of new drugs with anti-Leishmaniaactivity.
Myracrodruon urundeuva(Engl.) Fr. All., commonly known as
“aroeira-do-sert~ao”, is a medicinal plant species from Anacardida-ceae family. It is native from South America, and commonly found in regions with tropical and subtropical climate, including the northeastern region of Brazil (Sa et al., 2009). Phytochemical in-vestigations have shown the presence offlavonoids, polyphenols,
chalcones, tannins, and terpenoids (Viana et al., 2003; Calou et al., 2014; Figueredo et al., 2014). In Brazil,M. urundeuva is used in traditional medical practices for the treatment of mycoses, candi-diasis, bacterial infections, and allergy, as well as an anti-inflammatory agent (Silvino et al., 2014). Interestingly, pharmaco-logical assays have demonstrated its antifungal, anti-inflammatory, antiulcerogenic, antihistamine, antibradicinina and analgesic ac-tivities (Barbara Caroline et al., 2015).
Considering the potential pharmacological benefits of M. urundeuva, allied to the interest about discovering essential oils with anti-Leishmaniaactivity, the aim of this study was to deter-mine the chemical composition of essential oil from the leaves of
M. urundeuva, and investigate its anti-Leishmaniaactivity, as well as
its cytotoxicity and underlying mechanisms of action.
2. Materials and methods
2.1. Chemicals
Dimethyl sulfoxide (DMSO: 99%), anhydrous sodium sulfate, glacial acetic acid, ethanol, formaldehyde, sodium chloride, calcium acetate, zymosan, and neutral red were purchased from Merck Chemical Company (Germany). The n-alkane (C8eC24) homolo-gous series, Schneider's medium, RPMI 1640 medium, heat-inactivated fetal bovine serum (FBS), Azul de Alamar®
(AbD Sero-tec, Oxford, UK, MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide), Griess reagent (1% sulfanilamide in H3PO410% (v/v) in Milli-Q water), and the antibiotics penicillin
and streptomycin were purchased from Sigma Chemical (St. Louis, MO, USA). The antibiotic amphotericin B (90%) was purchased from Cristalia (S~ao Paulo, SP, Brazil).
2.2. Extraction of the essential oil of Myracrodruon urundeuva (MuEO)
M. urundeuvaleaves were collected from a mature tree in the
flowering stage in Teresina city, Piauí state, located in the Northeast Region of Brazil (5502000S e 42480700W). A voucher specimen (no. 20,026) was deposited at the Graziela Barroso Herbarium from the Federal University of Piauí. The plant material was air-dried for 7 (seven) days, subjected to hydrodistillation using a Clevenger-type apparatus (300 g, 6 h). Afterwards, the essential oil was dried over anhydrous sodium sulfate,filtered, and weighed, and then the oil yield was determined as 0.2% (w/w). MuEO was then stored in a darkflask and refrigerated at 4C until use. For thein vitrotests, MuEO was solubilized in DMSO, diluted in culture medium, and added to the cultures.
2.3. Gas chromatography-mass spectrometry analysis of the essential oil
MuEO was analyzed using a SHIMADZU GC-17/MS-QP5050A instrument (Shimadzu, Duisburg, Germany) under the following conditions: DB-5 HT (i.d.; 0.25 mm, 1.0
m
m film thickness) fused silica capillary column, with the following temperature program:50C, subsequently increased by 3C/min up to 240C; injector temperature: 260C; carrier gas: helium (high purity), adjusted to a linear velocity of 32 cm/s (measured at 100C). The oil sample was diluted to 1:100 in hexane solution (v/v), and the volume injected was 1.0
m
L. The splitflow was adjusted to 1:50 ratio, and the septum sweep was constant at 10 mL/min. All mass spectra were acquired in electron impact (EI) mode with an ionization voltage of 70 eV over a mass scan range of 35e450 amu. The temperature of the ion source and connection parts was 200C.2.4. Identification and quantification of constituents
The quantitative data regarding the volatile constituents were obtained by peak-area normalization using a FOCUS GC/ FID oper-ated under conditions similar to those used in the Gas Chromatography-Mass Spectrometry (GC-MS) assay, except that the carrier gas was nitrogen. The retention index was calculated for all the volatile constituents by usingn-alkane (C8eC24) homologous
series. The peak assignment was performed by comparison of both mass spectrum and GC retention data by using authentic com-pounds previously analyzed and stored in our private library, as well as with the aid of commercial libraries containing retention indices and mass spectra of volatile compounds commonly found in essential oils (NIST 05, 2005; Adams, 2007). Relative percentage of the identified compounds was computed from the GC peak area.
2.5. Parasites and murine macrophage cultures
Leishmania (Leishmania) amazonensis (IFLA/BR/67/PH8) was
used for the determination of the anti-Leishmania activity. The parasites were grown in supplemented Schneider's medium (10% FBS, 100 U,mL 1penicillin, and 100
m
g mL 1streptomycin at 26C). Extracellular axenic amastigote forms were obtained by the methodology described byUeda-Nakamura et al. (2006). Promas-tigotes forms ofL. amazonensisat the stationary growth phase were differentiated into axenic amastigotes by the combination of a temperature increase (32C) and pH decrease (5.5). Murine mac-rophages were collected from the peritoneal cavities of 4e5 week old male and female BALB/c mice from the Medicinal Plants Research Center (NPPM/CCS/UFPI), located at Teresina, PI, Brazil. The mice were maintained at a temperature of 24±1C and a 12 h light/dark cycle. All protocols were approved by the Animal Research Ethics Committee (CEEAPI no. 008/2012).2.6. Anti-Leishmania activity assay in promastigotes
Promastigotes in the logarithmic growth phase were seeded in 96-well cell culture plates (Biosystem, PR, BRA) at 1 106
L. amazonensis per well in 100
m
L of supplemented Schneider'smedium. Then, MuEO was added to the wells in serial dilutions of 6.25, 12.5, 25, 50, 100, 200, 400 and 800
m
g mL 1. The plate was kept at 26C in a biological oxygen demand (BOD) incubator, and pro-mastigotes were observed and counted at 400magnification us-ing a Neubauer hemocytometer after 24, 48, and 72 h in order to observe parasite growth and viability. 0.5% DMSO in supplemented Schneider's medium was used as control (100% viability) (Carneiro et al., 2012).2.7. Anti-Leishmania activity assay against axenic amastigote
The axenic amastigotes were added in an amount of 1106per well to 96-well plates at 32C and with the eight concentrations of the MuEO (6.25, 12.5, 25, 50, 100, 200, 400 and 800
m
g mL 1), then incubated for 48 h. After the incubation period, 20m
L of alamar Blue®solution was added and the plate was incubated again for
another 6 h. At the end of thefirst incubation, a reading was taken at 550 nm using a BioTek plate reader (Model Elx800, Winooski, VT, USA). Assays were performed in triplicate and expressed as growth inhibition (%). The negative control was carried out using 0.5% DMSO in Schneider's medium adding 1106of axenic amastigotes (100% of viability). The positive control had just Schneider's me-dium (0% of viability).
2.8. Anti-Leishmania activity assay against intracellular amastigote
Murine peritoneal macrophages were plated in 24-well culture plates at a concentration of 2105cells/500
m
L in RPMI 1640 per well, containing sterile 13 mm round coverslips. They were then incubated at 37C in 5% of CO2for 2 h to allow cell adhesion. The
medium was then aspirated, and a new medium containing pro-mastigotes at a ratio of 10 propro-mastigotes to 1 macrophage. After 4 h of incubation in 5% CO2at 37 C, the medium was aspirated to
remove free promastigotes, and the MuEO were added at nontoxic concentrations to the macrophages (100 and 200
m
g mL 1). 0.5% DMSO in supplemented RPMI medium was used as control (100% viability). This preparation was then incubated for 48 h, after which the coverslips were removed,fixed in methanol, and stained with Giemsa. For each coverslip, 100 cells were evaluated and both the number of infected macrophages and the amount of parasites per macrophage were counted. The survival index was determined by multiplying the number of infected macrophages to the number of amastigotes per infected macrophage. These values were used to determine the half-maximal inhibitory concentration (IC50) valueson intramacrophagic amastigotes (Medeiros et al., 2011).
2.9. Cytotoxicity determination
Cytotoxicity of MuEO was assessed using the MTT test. In a 96-well plate, 100
m
L of supplemented RPMI 1640 medium and about 1105murine peritoneal macrophages were added per well. They were then incubated at 37C in 5% of CO2for 2 h to allow cell
adhesion. After this time, 2 washes with supplemented RPMI 1640 medium were performed to remove cells that did not adhere. Subsequently, MuEO was added, in triplicate, after being previously diluted in supplemented RPMI 1640 medium to afinal volume of 100
m
L for each well at the tested concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800m
g mL 1). Cells were then incubated for 48 h. At the end of the incubation, 10m
L of MTT diluted in phosphate-buffered saline (PBS) was added at a final concentration of 5m
g mL 1(10% of volume, i.e., 10m
L for each 100m
L well) and was incubated for an additional 4 h at 37C in 5% CO2. The supernatant
was then discarded, and 100
m
L of DMSO was added to all wells. The plate was stirred for 30 min at room temperature to complete formazan dissolution. Finally, spectrophotometric reading was conducted at 550 nm in an ELISA plate reader. 0.5% DMSO in sup-plemented RPMI medium was used as control (100% viability) (Medeiros et al., 2011).2.10. Hemolytic activity
The hemolytic activity was investigated by incubating 20
m
L of serially diluted MuEO in phosphate-buffered saline (PBS; 6.25, 12.5, 25, 50, 100, 200, 400 and 800m
g mL 1) with 80m
g mL 1of a sus-pension of 5% red blood cells (human Oþ) for 1 h at 37C in assay tubes. The reaction was slowed by adding 200
m
L of PBS, and then the suspension was centrifuged at 1000 g for 10 min. Cell lysis was then measured with an ELISA plate reader (540 nm). The absence of hemolysis (blank control) or total hemolysis (positive control) was determined by replacing the essential oil solution with an equal volume of PBS or Milli-Q sterile water, respectively. The resultswere determined by the percentage of hemolysis compared to the positive control, and the experiments were performed in triplicate (Lofgren et al., 2008).
2.11. Lysosomal activity
Measurement of the lysosomal activity was carried out ac-cording to the method ofGrando et al. (2009). Murine peritoneal macrophages were plated at a concentration of 2105cells in 96-well culture plates and incubated with MuEO in serial dilutions of 6.25, 12.5, 25, 50, 100, 200, 400 and 800
m
g mL 1. After 4 h of in-cubation at 37C in 5% CO2, 10
m
L of neutral red solution was addedand incubated for 30 min. Once this time had elapsed, the super-natant was discarded, the wells were washed with 0.9% saline at 37+C, and 100
m
L of extraction solution was added (glacial acetic acid 1% v/v and ethanol 50% v/v dissolved in bidistilled water) to solubilize the neutral red inside the lysosomal secretion vesicles. After 30 min on a Kline shaker (model AK 0506), the plate was read at 550 nm by using an ELISA plate reader. 0.5% DMSO in supple-mented RPMI medium was used as control.2.12. Phagocytosis test
Murine peritoneal macrophages were plated at a concentration of 2105cells in 96-well culture plates and incubated with MuEO in serial dilutions of 6.25, 12.5, 25, 50, 100, 200, 400 and 800
m
g mL 1. After 48 h of incubation at 37C in 5% CO2, 10
m
L ofzymosan color solution was added at afinal concentration of 0.02% and incubated for 30 min at 37C. Afterwards, 100
m
L of Baker's fixative (formaldehyde 4% v/v, sodium chloride 2% w/v, and calcium acetate 1% w/v in distilled water) was added to stop the phagocy-tosis process. Thirty minutes later, the plate was washed with 0.9% saline in order to remove zymosan that was not phagocytized by macrophages. The supernatant was removed and added to 100m
L of extraction solution. After solubilization in a Kline shaker, the absorbances were measured at 550 nm by using an ELISA plate reader. 0.5% DMSO in supplemented RPMI medium was used as control (Grando et al., 2009).2.13. Nitric oxide (NO) production
In 96-well plates, 2105murine peritoneal macrophages were added per well and incubated at 37C in 5% CO
2for 4 h to allow cell
adhesion. A new medium containing promastigotes (in the sta-tionary phase) at a ratio of 10 promastigotes per macrophage was added to half of the wells. The MuEO was added after being pre-viously diluted in a culture medium containing different concen-trations (6.25, 12.5, 25, 50, 100, 200, 400 and 800
m
g mL 1) in the absence or presence of L. amazonensis, and was then incubated again at 37C in 5% CO2for 24 h. After this period, the cell culture
supernatant was collected and transferred to another plate for measurement of nitrite. A standard curve was prepared with so-dium nitrite at varying concentrations of 1, 5, 10, 25, 50, 75, 100, and 150
m
M diluted in a culture medium. At the time of dosing, 100m
L of either the samples or the solutions prepared for obtaining the standard curve was mixed with equal volume of Griess reagent. The analysis was performed using an ELISA plate reader at 550 nm. 0.5% DMSO in supplemented RPMI medium was used as control (Grando et al., 2009).2.14. Statistical analysis
All assays were performed in triplicate and in 3 independent experiments. The half-maximal inhibitory concentration (IC50),
half-maximal cytotoxicity concentration (CC50), and half-maximal
hemolytic concentration (HC50), were calculated using a probit
regression model at confidence interval of 95%. Analysis of variance (ANOVA) followed by a Bonferroni's post-hoc test were performed, consideringp<0.05 as the minimum level required for statistical significance.
3. Results and discussion
3.1. Identification of the constituents of essential oil of MuEO
The Total Ion Chromatogram (TIC) of the MuEO (Fig. 1) exhibited the presence of two isomers from the myrcene (1 and 2). The un-resolved mixture is probably related to
a
- andb
-isomers, which corresponds to nearly 80% of the total area of the chromatogram. The low relative abundance of constituents 4 and from 6 to 12 did not allow their detections by TIC. The monitoring of the ion m/z 204, relative to the molecular mass of some terpenes, demonstrates the presence of these compounds. Fourteen constituents were identified, representing 94.87% of the total mixture. The chemical constituents are listed inTable 1. The prevalence of monoterpenes and sesquiterpenes hydrocarbons as the most abundant chemical classes was observed in MuEO. Theb
-myrcene was the major constituent (42.46%), followed bya
-myrcene (37.23%) and car-yophyllene (4.28%). The results found in this study correspond to the results of previous report; the essential oil of leaves fromM. urundeuva collected in Brasília, center region of Brazil, was
determined to contain 67.5% of monoterpene hydrocarbons, with
b
-myrcene (66.4%) being the major constituent (Costa et al., 2014). Otherwise, the essential oil ofM. urundeuvafrom Penarforte, Ceara, northeast region of Brazil, was observed to be mainly composed of monoterpene hydrocarbond
-Carene (80.41%) (Figueredo et al., 2014).Montanari et al. (2012)identified the monoterpene hydro-carbond
-3-carene (78.8%) to be the major chemical component of the essential oil ofM. urundeuvaobtained from Minas Gerais State, Southeast of Brazil. The similarities and differences between the chemical profiles of M. urundeuva essential oils are probably because the plants were collected from different regions. Genetic variability, geographical and environmental conditions, and phys-iopathological traits influence composition of secondary metabo-lites in plants (Teixeira et al., 2012; Dias et al., 2015).3.2. Anti-Leishmania activity
The assessment of MuEO-induced inhibitory effect against
L. amazonensispromastigotes showed a significant
concentration-dependent decrease (p < 0.001) in parasite viability, with 100% inhibition of promastigote growth at concentrations of 800 and 400
m
g,mL 1(Fig. 2). The IC50was 205±13.4m
g mL 1at 48 h ofexposure (Table 2). In promastigote cultures treated with MuEO, morphological alterations, such as cells with rounded or completely spherical shapes, as well as the presence of cellular debris, typical of cell lysis, were observed by optical microscopy, suggesting the leishmanicide activity for the MuEO (data not shown).
b
-Myrcene, the main monoterpene identified in MuEO, has shown anti-Leishmaniaactivity as previously reported (Machado et al., 2012). In this study,b
-myrcene was identified as the third major constituent of essential oil from leaves ofC. citratus, and showed inhibitory effects againstLeishmania(Leishmania)infantum promastigote forms, with IC50value of 164m
g,mL 1, while the essential oil showed IC50value of 25m
g,mL 1. Many isolated major constituents from essential oils possess lower anti-Leishmania ac-tivity then the whole mixture. The differences between the phar-macological activities of essential oils and their isolated compounds result from additive or synergistic effects between thecompounds, which increase their biological activities (Bassole and Juliani, 2012). Interestingly, this observation represents an addi-tional advantage for the MuEO rather than its major constituent
b
-myrcene, which encourages futher studies related to the anti-leishmanial potential of MuEO.Experimental models based on the activity against amastigotes forms ofLeishmania spp.are important because the life stage of the
parasite responsible for the different clinical manifestations of leishmaniasis is considered. Therefore, the anti-Leishmaniaactivity of MuEO was performed against axenic amastigote forms of L. amazonensis. The inhibitory action of MuEO was statistically significant at concentrations from 25 to 800
m
g mL 1(Fig. 3). TheIC50for 48 h of incubation was 104.6±11.82
m
g mL 1(Table 2).Based on the anti-Leishmaniaeffects observed against axenic amastigotes, the effect of MuEO against intracellular amastigotes was evaluated in an experimental model of Leishmania-infected murine macrophages. This experimental model represent the most effective way to relate the in vitro and in vivo antileishmanial Fig. 1.Total Ion Chromatogram (TIC) and Monitoring Ion Chromatogramm/z204, obtained from the essential oil analysis ofMyracrodruon urundeuva.
Table 1
Chemical composition and retention indices of the constituents ofMyracrodruon urundeuvaessential oil (MuEO).
Number aCompounds bRRIs cRRIs dArea (%)
1 b-Myrcene 990 988 42.46
2 a-Myrcene 992 988 37.23
3 Limonene 1030 1031 0.66
4 Copaene 1373 1376 0.15
5 Caryophyllene 1418 1418 4.28
6 Aromadendrene 1438 1437 0.32
7 a-humulene 1453 1452 0.49
8 cis-muurola-4 (14),5-diene 1485 1465 0.19 9 Allo-aromadendrene 1454 1458 0.68 10 Allo-aromadendrene (isomer)^ 1455 1458 0.63
11 d-amorfene 1532 – – 0.57
12 No Identify 1596 – – 4.22
13 Hexadecanoic acid 1974 1975 3.13 14 9-Hexadecenoic acid – – – – 1.34 15 Octadecanoic acid – – – – 0.53
Monoterpene hydrocarbons 80.35
Sesquiterpene hydrocarbons 7.31
Fatty acids 5
TOTAL IDENTIFIED 94.87
aCompounds listed in order of elution on the DB-5ms column.
b Relative retention indices (RRIs) experimentally determined againstn-alkanes
by using the DB-5ms column.
c RRIs reported in the literature (NIST 05, 2005; Adams, 2007).
d Content expressed as percentages obtained by integration of the GC peak area.
Fig. 2.Effects ofMyracrodruon urundeuva(MuEO) on growth ofLeishmania ama-zonensispromastigote forms. Cells in log-phase (1106) were incubated in different
concentrations of MuEO. Values are expressed as the mean±standard error of the average of three independent experiments performed in triplicate, considering the control (0.5% DMSO in supplemented RPMI medium) as 100% viability and ***p<0.001
vs.control.
activity of a test-drug, since the microbicidal effect of macrophages allied to the direct effects of the test-drug against the parasite is considered (Russell and Talamas-Rohana, 1989). In this study, a significant reduction of infection of macrophages at 48 h was observed at the concentrations of 100 and 200
m
g mL 1. The reduction in infection rate was 42% and 100% at concentrations of 100 and 200m
g mL 1, respectively (Fig. 4). MuEO also decreased the number of amastigotes per infected macrophage in 86.24% and 100% at concentrations of 100 and 200m
g mL 1, respectively (Fig. 4). The IC50 of exposure to MuEO at 48 h was44.5±4.37
m
g mL 1(Table 2). Additionally,Fig. 5shows a quali-tative representation of the effect of MuEO against infected mac-rophages, where the integrity of macrophages was preserved after 48 h of treatment.Interestingly, MuEO provided a lower IC50value (44.5
m
g mL 1)against intracellular amastigotes than other essential oils previ-ously reported as promising anti-Leishmaniaagents, such as:Piper
demeraranum (Miq.) C. DC., with IC50 of 78
m
g mL 1 againstL. amazonensis(Moura do Carmo et al., 2012);Piper obrutunTrel.&
Yunck with IC50of 89.02
m
g mL 1againstL. infantum(Leal et al.,2013);Croton cajucara Benth with IC50 of 66.7
m
g mL 1 againstLeishmania(Leishmania)chagasi(Rodrigues et al., 2013a); and
Oci-mum gratissiOci-mumL. with minimum inhibitory concentration (MIC)
of 150
m
g mL 1 against L. amazonensis (Ueda-Nakamura et al., 2006).3.3. Cytotoxicity against mammalian cells
In order to assess the potential application of a test-drug in the treatment of leishmaniasis, the absence of toxicological effect against the host is required. Therefore, the possible cytotoxic effects of MuEO against murine peritoneal macrophages and human red
blood cells were evaluated. MuEO decreased the viability of mac-rophages by 32.9%, 21.8%, and 16.6% at concentrations of 800, 400, and 200
m
g mL 1, while the other concentrations demonstrated absence of cytotoxicity (Fig. 6A), with CC50of 550±29.21m
g mL 1 (Table 2). MuEO presented selective index (SI) of 12.3 to macro-phages. The SI represents how much the activity of a test-drug is selective to the parasite, which represents a safety profile of MuEO to the host cells. Outstandingly, the selectivity of MuEO for the parasites was much more pronounced when compared with reference drugs; MuEO was 5.0- and 83.3-fold safer than meglu-mine antimoniate and amphotericin B, respectively.Cytoxicity tests against erythrocytes are based on the release of hemoglobin caused by either the total rupture or the formation of pores in the cell membranes (Orsine et al., 2012). This is a well-accepted model used for the preliminary evaluation of the protec-tive and toxic effects of substances on humans (Freshney, 2005). In this study, the MuEO induced hemolysis by 20% against human blood type Oþ
erythrocytes only at the highest concentration tested (800
m
g mL 1), with HC50 higher than 800m
g mL 1 (Fig. 6B,Table 2). The selection index of MuEO for erythrocytes is higher than 17.97 (Table 2), indicating low cytoxicity against erythrocytes. Similarly to macrophages, MuEO demonstrated to be safer against erythrocytes than reference drugs; MuEO was 21.5- and 1.36-fold safer than meglumine antimoniate and amphotericin B, respec-tively. Hence, the high selectivity of MuEO to the parasite leads towards furtherin vivostudies as well as the elucidation of mech-anisms of action possibly involved in the MuEO-induced
anti-Leishmaniaactivity.
Table 2
Anti-Leishmaniaand cytotoxic effects ofMyracrodruon urundeuvaessential oil (MuEO).
Promastigote Amastigote axenic Amastigotes in Macrophage Macrophage Red Blood Cell SIama SIrb
IC50(mg$mL 1) 48 h IC50(mg$mL1) 48 h IC50(mg$mL1) 48 h CC50(mg$mL1) 48 h HC50(mg$mL 1) 1 h
MuEO 205±13.4 104.6±11.82 44.5±4.37 550±29.21 >800 12.3 >17.97 Meglumine antimoniate 1200±97.6 624.5±35.27 167.4±9.06 412.9±30.25 139.7±14.7 2.46 0.83 Amphotericin B 0.18±0.04 0.22±0.09 1.19±0.06 0.18±0.01 15.59±1.94 0.15 13.1
SIama(Selectivity Index)¼CC50macrophages/IC50amastigotes in macrophage. SIrd(Selectivity Index)¼HC50red blood cell/IC50amastigotes in macrophage. Data represent the
mean±standard error of 3 experiments carried out in triplicate.
Fig. 3.Effects ofMyracrodruon urundeuva(MuEO) on growth of axenic amastigotes of
Leishmania amazonensis. Cells in log-phase (1106) were incubated in different
concentrations of MuEO. Values are expressed the mean± standard error of the average of three independent experiments performed in triplicate, considering the control (0.5% DMSO in supplemented Scheneider's medium) as 100% viability and **p<0.01vs.control and ***p<0.001vs.control.
Fig. 4.Effects ofMyracrodruon urundeuva(MuEO) on murine peritoneal macrophage infection withLeishmania amazonensis. Values are expressed as the mean±standard error of mean of three independent experiments performed in triplicate, considering the control (0.5% DMSO in supplemented RPMI medium) as 100% viability. ***p<0.001
vs.control.
3.4. Macrophages activation
The effects of MuEO in promastigotes and amastigotes may involve several possible mechanisms of action. Lipophilic com-pounds from essential oils are able to readily cross the cytoplasmic membrane, thus affecting the structure of the different layers of polysaccharides, fatty acids and phospholipids, and then altering their permeability (Buchbauer and Jirovetz, 2006; Medeiros et al., 2011). In addition, essential oil affects the cytoplasm by disrup-tion of metabolic pathways of lipids and proteins, interfering with the potential of the mitochondrial membranes, which can lead to increase of oxidative stress and cell dealth (Tariku et al., 2011).
A comparative analysis of MuEO-induced anti-Leishmania ac-tivity against three different forms ofL. amazonensisshows that the MuEO was more effective against intracellular amastigotes (IC50 ¼ 44.5
m
g mL 1) than promastigotes (IC50 ¼ 205 ± 13.4m
g mL 1) or axenic amastigotes (IC50 ¼ 104.6 ± 11.82m
g mL 1). This observation could be an indicative of macrophage activation, leading to a microbicidal effect allied to a direct action of MuEO (Kolodziej and Kiderlen, 2005). In order to exert their microbicide activities, macrophages develop both structural (spreading, phagocytosis, vacuolization, and increased lysosomal volume) and molecular mechanisms (changes in the NO profile, altered cytokine levels, and matrix metal-loproteinase secretion) (Bonatto et al., 2004; Bhattacharya et al., 2013). Phagocytosis and lysosomal system play a critical role in the microbicide capability of the macrophages, due to their in-volvements in internalization, degradation, and eventually pre-sentation of antigens. The internalization of pathogens results in the formation of a membrane-bound vacuole called‘phagosome’, which is innocuous to the cell. Thus, in order to destroy the path-ogens, the phagosome is converted into a‘phagolysosome’capable of kill the pathogens (Flannagan et al., 2012). The phagolysosome is a compartimentfilled of acid hydrolases and reactive oxygen spe-cies where the most degradation of the encompassed contentoccurs. Internalized pathogens are killed within phagolysosome. In addition, antigen presentation occurs through phagocytosis and targeting towards endosomal/lysosomal systems (Lee et al., 2003; Niedergang and Chavrier, 2004).
In this sense, two structural mechanisms of anti-Leishmania activity (lysosomal and phagocytic activities), as well as the determination of NO content related to the macrophages activation were evaluated in order to assess the possible role of immuno-modulatory mechanisms. The lysosomal activity of macrophages treated with MuEO showed no significant increase at tested con-centrations (Fig. 7A). On the other hand, the phagocytic capability of macrophages incubated with MuEO (Fig. 7B) was increased only at concentrations of from 400 to 800
m
g mL 1, which are higher than the concentration able to induce the maximal leishmanicidal effect against macrophage-internalized amastigotes (200m
g mL 1). Besides, NO production is stimulated by protective cytokines like IFN-g
, and it is extremely reactive, causing damage to proteins and DNA of parasite (Carvalho et al., 2012). However, NO production was determined indirectly by measuring the nitrite produced by macrophages treated with MuEO, and stimulated or not-stimulatedbyL. amazonensispromastigotes. The results revealed that the NO
production was not promoted after incubation with MuEO (Fig. 8). Therefore, the promising MuEO-induced activity against
L. amazonensis probably does not occur by immunomodulatory
mechanisms directly involving these evaluated parameters.
4. Conclusions
This study demonstrates that MuOE promotes significant ac-tivity against L. amazonensis, preferably against intracellular amastigote forms, which is more related with the clinical man-isfestation of leishmaniasis. Furthermore, an acceptable selectivity index for the parasite was observed, indicating lower cytotoxicity against the host cells. Moreover, the activity of MuOE against
L. amazonensis is not probably mediated by increase of NO
Fig. 5.Optical microscopy observation of murine peritoneal macrophages infected withLeishmania amazonensisamastigotes in the absence and presence of theMyracrodruon urundeuva(MuEO). The slides were stained with Giemsa and observed at 1000magnification. (A) Control, (B) cells incubated with 100mg mL1MuEO, (C) cells incubated with
200mg mL1MuEO.
Fig. 6.Cytotoxicity effects ofMyracrodruon urundeuva(MuEO) on macrophage (A) and on human erythrocytes (B). Murine macrophages were incubated for 48 h in the presence of different concentrations. Macrophage viability was measured by tetrazolium salt (MTT) assay. Hemolytic activity was assessed in a 5% suspension of human erythrocytes after 1 h of incubation. Data are expressed as means±standard error of three experiments performed in triplicate, considering **p<0.01vs.control and ***p<0.001vscontrol.
production, but an increase of phagocytic capability was observed. Further investigation is necessary in order to elucidate which additional mechanisms are involved in these effects. These results suggest MuEO as a promising agent for the treatment of leishmaniasis.
Acknowledgments
The authors are grateful to the CAPES, CNPq and UFPI for financial grants by the concession of scholarships andfinancial support.
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