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Brazilian Journal of Pharmaceutical Sciences

versão On-line ISSN 2175-9790

Braz. J. Pharm. Sci. vol.55  São Paulo  2019  Epub 24-Out-2019

https://doi.org/10.1590/s2175-97902019000217584 

Original Article

Antileishmanial in vitro activity of essential oil from Myrciaria plinioides, a native species from Southern Brazil

Carla Kauffmann1 

Ana Caroline Giacomin1 

Kelen Arossi1 

Leandra Andressa Pacheco1 

Lucélia Hoehne2 

Elisete Maria de Freitas1 

Gérzia Maria de Carvalho Machado3 

Marilene Marcuzzo do Canto Cavalheiro3 

Simone Cristina Baggio Gnoatto4 

Eduardo Miranda Ethur2  * 
http://orcid.org/0000-0003-1731-3778

1Centro de Ciências Biológicas e da Saúde, University of Vale do Taquari - Univates, Lajeado, RS, Brazil

2Centro de Ciências Exatas e Tecnológicas, University of Vale do Taquari - Univates, Lajeado, RS, Brazil

3Laboratory of Biochemistry of Trypanosomatid, Institute Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ, Brazil

4Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil


ABSTRACT

In South American folk medicine members of the genus Myrciaria are used for the treatment of malaria, diarrhoea, asthma, inflammation and post-partum uterine cleansing. The aim of this work was to evaluate its antileishmanial properties (in vitro) of essential oil derived from leaves of Myrciaria plinioides D. Legrand, a plant species that is native in South of Brazil. The essential oil was obtained by hydro-distillation using fresh leaves of M. plinioides. The chemical composition of this essential oil (MPEO, M. plinioides essential oil) was determined by gas chromatography coupled to mass spectrometry (GC-MS). MPEO was assayed in vitro for antileishmanial properties against promastigotes of Leishmania amazonensis and Leishmania infantum, and for cytotoxicity against murine peritoneal macrophages. The MPEO comprised 66 components and was rich in oxygenated sesquiterpenes (82.66%) containing spathulenol (21.12%) as its major constituent. The MPEO was effective against L. amazonensis with IC50 value of 14.16 ± 7.40 µg/mL, while against L. infantum the IC50 value was higher with 101.50 ± 5.78 µg/mL. The MPEO showed significant activity against L. amazonensis, and presented a selectivity index (SI) of 6.60. The results suggest that the essential oil from leaves of M. plinioides is a promising source for new antileishmanial agents against L. amazonensis.

Keywords: Antileishmanial activity; Myrciaria plinioides; Myrtaceae; Leishmania amazonensis; Leishmania infantum

INTRODUCTION

Leishmaniasis, a parasitic infection caused by protozoa of the genus Leishmania, rates as one of the most pernicious of neglected tropical diseases. Some 350 million people worldwide are at risk of contracting one of the forms of the disease, and around 2 million new cases occur annually, mainly within the poorest populations in developing countries. Various factors have contributed to the increase in the number of cases of the disease, especially the difficulties associated with vector control and the lack of a vaccine (Freitas-Junior et al., 2012). Moreover, drugs such as meglumine antimoniate and pentamidine isethionate that are commonly employed in the treatment of leishmaniasis are of somewhat limited application because of issues relating to routes of administration, adherence to treatment, resistance, toxicity and/or teratogenicity (Buckner, Waters, Avery, 2012).

Alternative therapies, including miltefosine and paromomycin, and new formulations of older medications such as amphotericin B, have been introduced but most are restricted in their use and none provide a satisfactory treatment of the disease (Freitas-Junior et al., 2012). In this context, medicinal plants that have been applied in traditional remedies often represent promising sources of lead compounds for the development of new drugs (Oliveira et al., 2011). Within the last few years, considerable research interest has focused on screening plant extracts as potential sources of drugs for the treatment of leishmaniasis (Vila-Nova et al., 2011; Cota et al., 2012; Ramírez-Macías et al., 2012; Vila-Nova et al., 2012; Santos et al., 2013).

Members of the family Myrtaceae are ubiquitous in Brazil, and the presence of around 1000 species in discrete biomes, principally the Atlantic forest, restinga and cerrado, suggests an ecological importance. Myrciaria is a genus of large shrubs and small trees belonging to the myrtle family, and various species are used in traditional medicine (Souza, Lorenzi, 2012).

The shrub Myrciaria plinioides D. Legrand, popularly known as camboim, cambuim or cambuí, is native to the state of Rio Grande do Sul in southern Brazil. Despite the medicinal potential of this species, very few reports are available concerning its pharmacological activities. For example, tea prepared from the leaves of Myrciaria tenella, popularly known as vassourinha, is employed in the Amazonian region as a post-partum uterine cleansing agent (Coelho-Ferreira, 2009), while the volatile oil obtained from leaves of this species is rich in α-pinene and b-pinene and exhibits antimicrobial activity against Enterobacter spp. and Shigella flexneri (Schneider et al., 2008). Additionally, the leaves and trunk bark of M. cauliflora (popular name jabuticaba) are used to treat diarrhoea, asthma, and throat inflammation (Albuquerque et al., 2007), while ethanolic extracts of the leaves exhibit inhibitory action against Candida and Streptococcus cultures derived from dental plaque (Carvalho et al., 2009; Diniz et al., 2010). Of particular interest is the report (Ruiz et al., 2011) that the edible fruits of M. dubia are employed by Indigenous and Mestizo populations living on the banks of the Nanay river in the Loreto region of Peru as a traditional remedy for the treatment of malaria, which is also a neglected protozoan infection.

In consideration of the above, we have assessed the in vitro activities of essential oil of M. plinioides against Leishmania infantum (syn. L. chagasi), which is the causal agent of visceral leishmaniasis, and against L. amazonensis, a species that has been associated with various clinical forms of the disease including cutaneous, mucosa, diffuse cutaneous and visceral leishmaniasis (Leon et al., 1990). In addition, we have determined the composition of the essential oil derived from this native Brazilian species.

MATERIAL AND METHODS

Plant material

Leaves of Myrciaria plinioides D. Legrand were collected in Lajeado, RS, Brazil during July 2012. The plants were authenticated by the botanist Dr. Elisete Maria de Freitas (Centro Universitário UNIVATES) and a voucher specimen was deposited at the Herbário do Vale do Taquari, Museu de Ciências Naturais UNIVATES under the registration number HVAT1066.

Preparation of essential oil

Fresh leaves (200 g) of M. plinioides were subjected to hydro-distillation for 3.5 h in a Clevenger-type apparatus. The essential oil (MPEO) was dried over anhydrous sodium sulphate, transferred to amber glass bottles and stored at −20 °C, until required for chemical analysis and bioassay.

Chemical analysis of the essential oil

Samples of MPEO were analysed by gas chromatography coupled to mass spectrometry (GC-MS) at the Instrumental Analysis Laboratory, Food Processing Development Centre - FPDC, Univates. Analyses were performed on a Shimadzu GC2010 Plus system, comprising a model AOC-5000 Plus auto injector and a model QP2110 Ultra mass detector, using a Restek Rtx®-5MS fused silica capillary column (30 m x 0.25 mm i.d.; 0.25 µm film thickness). The chromatographic conditions were: carrier gas - helium at a flow rate of 1.00 mL/min; oven temperature - initially at 50 °C and increased at 4 °C/min to 290 °C; injector temperature - 240 °C; injection mode - split with 1:20 ratio and 3 mL/min purge; MS interface temperature - 280 °C; ion source temperature - 260 °C; ionisation energy - 70 eV. Oil samples (15 mg) were dissolved in 1.5 mL of purified ethyl acetate and aliquots in the order of 1 µL were injected for analysis. GC analyses with flame ionisation detection (FID) were carried out using an Agilent J & W HP-5 MS column (30 m x 0.25 mm i.d.; 0.25 µm film thickness) with helium as carrier gas, an FID temperature of 260 °C and the oven temperature program as described for the GC-MS procedure.

Separated components were identified initially from their Kováts retention indices (RI), determined by reference to a series of n-alkanes, and their identities confirmed by comparison of mass spectral data with those obtained using pure standards together with values quoted in the literature (Adams, 2009) and those stored in the Wiley 8 and NIST11 spectral libraries of the analytical system. The relative compositions of the oils were calculated from the peak areas (uncorrected for specific response factors) of the separated components.

Cultivation of Leishmania promastigotes

Promastigotes of L. amazonensis MHOM/BR/77/LTB0016 were grown at 26 ºC in Schneider’s Drosophila medium (Sigma-Aldrich) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) and adjusted to pH 7.2. Promastigotes of L. infantum MCAN/BR/97/P142 were cultivated at the same temperature and pH, but in this case the medium was supplemented with 20% (v/v) FCS, 2% (v/v) human urine, 100 µg/mL streptomycin and 100 U/mL penicillin. Promastigotes were harvested on day 4, when the percentage of infective metacyclic forms was found to be high, and counted in a Neubauer chamber. Parasite suspensions were adjusted to a concentration of 1x107 promastigotes/mL using the supernatant of the respective culture as diluent.

Determination of antileishmanial activity in vitro

Appropriate amounts of MPEO or pentamidine isethionate (as reference drug) (Sideron®) were dissolved in aqueous dimethyl sulphoxide (DMSO; 10 mg/mL) to yield solutions containing analytes in the concentration range of 0.156 to 80 µg/mL. The level of DMSO in each assay solution was below 1.4%, which is the highest concentration that is not hazardous to the parasites.

Suspensions of late log phase promastigotes suspended in Schneider’s Drosophila medium were seeded in Corning™ 96-well flat bottom tissue culture tested plates (1×107 promastigotes/200 µL/well). Aliquots of freshly prepared MPEO and pentamidine were added to the wells and the plates were incubated for 24 h at 26 °C. Promastigote viability was evaluated using a modified version of the dye-reduction assay employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Dutta et al., 2005). Briefly, MTT reagent was added to each well and incubation was continued in the dark for an additional 4 h. After this time, an 80 µL aliquot of DMSO was added to each well and the optical density of the assay solution was determined at 570 nm using a BioTek µQuant™ microplate spectrophotometer. The specific absorbance associated with the formazan so-produced was determined by subtracting the background absorbance from the total absorbance, and the mean percentage viability was calculated from:

MeanspecificabsorbanceoftreatedparasitesMeanspecificabsorbanceofuntreatedparasites×100

Values for IC50, i.e. the concentration that inhibited parasite growth by 50%, were determined.

Assessment of cytotoxicity

Peritoneal macrophages from BALB/c mice were obtained by using the lavage technique, counted in a Neubauer chamber and adjusted to a concentration of 2 x106 cells/mL. Macrophages were transferred to Corning 96-well flat bottom tissue culture tested plates and incubated for 24 h at 37 ºC under a 5% CO2 atmosphere. Freshly prepared solutions, in aqueous DMSO, containing MPOE or pentamidine isethionate at concentrations range 0.156 to 80 µg/mL were then added to the wells. Macrophage viability was evaluated using a modified version of the dye-reduction assay employing MTT. In order to assess macrophage viability, 22 µL of MTT solution (5 mg/mL) was added to each well and the plates were incubated for an additional 2 h. After this time, an 80 µL aliquot of DMSO was added to each well and the optical density of the assay solution was determined at 540 nm using a BioTek µQuant™ microplate spectrophotometer. This study was approved for The Animal Ethics Committee of the Institute Oswaldo Cruz/IOC - FIOCRUZ (license number L-026/2015).

Statistical analysis

Assays were carried out in three independent experiments and each was performed in triplicate. Values of IC50 and CC50 were determined by logarithmic regression analysis using GraphPrism 5 software. Values for in vitro antileishmanial activity and in vitro cytotoxicity were expressed as mean ± standard deviation. The significant differences between samples were evaluated by analysis of variance (ANOVA) and the Tukey test using BioEstat 5.0 software with the alpha level set at 0.05.

RESULTS AND DISCUSSION

Analysis of the essential oil from fresh leaves of M. plinioides

The yield of essential oil obtained from fresh leaves of M. plinioides was 0.05% (w/w). According to the GC analyses, the oil comprised 66 components (Table I) and was particularly rich in oxygenated sesquiterpenes (82.66%) and sesquiterpene hydrocarbons (11.05%). The principal volatile components of the oil were the sesquiterpenes spathulenol (21.12%), caryophyllene oxide (15.20%), α-isolongifolan-7-ol (9.84%), mustakone (5.60%), α-cadinol (5.40%), cis-isolongifolanone (3.38%) and α-copaene (3.27%).

TABLE I Composition of the essential oil from fresh leaves of M. plinioides 

Compound RIEXPa RILITb Relative composition (%)
NIc 1049 - 0.05
α-Terpineol 1194 1189 0.11
α-Ylangene 1373 1375 0.08
α-Copaene 1378 1376 3.27
β-Bourbonene 1386 1388 1.24
β-Elemene 1394 1391 0.26
β-Ylangene 1421 1421 0.09
β-Copaene 1431 1432 0.10
Aromadendrene 1440 1441 0.17
α-Humulene 1455 1455 0.08
allo-Aromadendrene 1462 1460 0.83
γ-Muurolene 1479 1480 0.67
cis-Eudesma-6,11-diene 1487 1490 0.11
trans-Muurola-4(14),5-diene 1496 1494 0.10
α-Muurolene 1503 1500 0.31
δ-Amorphene 1522 1512 0.55
trans-Calamenene 1535 1529 0.15
(E)-γ-Bisabolene 1554 1531 0.06
β-Vetivenene 1559 1533 0.84
Silphiperfol-5-en-3-ol B 1575 1535 1.53
Selina-3,7(11)-diene 1595 1546 0.65
1-nor-Bourbonanone 1601 1563 0.14
Spathulenol 1606 1578 21.12
Caryophyllene oxide 1610 1583 15.20
β-Copaen-4-α-ol 1613 1591 1.25
Khusimone 1617 1604 2.61
Curzerenone 1619 1606 1.22
β-Atlantol 1623 1608 0.43
cis-Isolongifolanone 1626 1613 3.38
Isolongifolan-7-α-ol 1631 1619 9.84
Junenol 1637 1619 0.82
2,(7Z)-Bisaboladien-4-ol 1642 1619 0.99
1,10-Di-epi-cubenol 1646 1619 2.13
trans-Isolongifolanone 1649 1626 0.24
β-Cedren-9-one 1652 1631 0.33
epi- α-Muurolol 1657 1642 2.65
α-Muurolol 1661 1646 1.54
Vulgarone B 1663 1651 0.70
α-Cadinol 1667 1654 5.40
Selin-11-en-4-α-ol 1669 1659 0.63
cis-Calamenen-10-ol 1670 1661 0.26
14-Hydroxy-(Z)-caryophyllene 1673 1667 0.24
trans-Calamenen-10-ol 1677 1669 0.19
14-Hydroxy-9-epi-(E)-caryophyllene 1679 1669 0.22
Cadalene 1682 1676 1.49
Mustakone 1685 1677 5.60
Khusinol 1690 1680 1.60
5-neo-Cedranol 1695 1685 0.36
Germacra-4(15), 5, 10(14)-trien-1- α-ol 1702 1686 0.19
10-nor-Calamenen-10-one 1704 1702 0.45
Mayurone 1707 1704 0.43
E-Apritone 1711 1708 0.24
Longifolol 1716 1714 0.14
E-Nerolidyl acetate 1720 1717 1.70
iso-Longifolol 1728 1729 0.60
Vetiselinenol 1730 1731 0.10
Eremophilone 1735 1736 0.78
E-β-Santalol 1741 1739 0.41
8-α-11-Elemodiol 1748 1747 0.33
NI 1751 - 0.15
α-Bisabolol oxide A 1753 1749 0.45
β-Acoradienol 1767 1763 0.64
β-Costol 1772 1767 0.69
Khusinol acetate 1820 1823 0.18
NI 1839 - 0.43
8S,13-Cedranediol 1904 1897 0.22
Total constituents identified 99.37
Oxygenated monoterpenes 0.11
Sesquiterpene hydrocarbons 11.05
Oxygenated sesquiterpenes 82.66

aExperimental retention Index.

bLiterature retention Index (Adams, 2007).

cNot identified.

Members of the family Myrtaceae are commonly rich in essential oils, many of which possess biological activity (Tietbohl et al., 2012; Borges, Conceição, Silveira, 2014). In the case of M. floribunda, popularly known as camboin amarelo, monoterpenes predominated in the oils derived from leaves and flowers, with 1,8-cineole as the major component accounting for 38.4% of the leaf oil and 22.8% of the flower oil (Tietbohl et al., 2012). In contrast, the stem oil contained mainly sesquiterpenes, of which (2E,6E)-farnesyl acetate represented the major component accounting for 19.9% of the oil.

Oxygenated sesquiterpenes have been identified as major constituents of leaf oils from a number of Myrciaria species (Apel et al., 2006). Thus, the oil of M. cauliflora, similar to that of M. plinioides, contained mainly spathulenol (27.2%) and caryophyllene oxide (21.6%), while β-caryophyllene, caryophyllene oxide and spathulenol predominated in the essential oil of M. edulis. Conversely, the major components of M. trunciflora leaf oil were globulol, bicyclogermacrene and γ-muurolene, while the essential oil of M. cordifolia was rich in α-bisabolol oxide A, α-bisabolol oxide B, α-bisabolol and β-caryophyllene. The oxygenated sesquiterpenes spathulenol and caryophyllene oxide have been identified as major constituents of the essential oils of other members of the Myrtaceae, including Eugenia brasiliensis (Magina et al., 2009), E. calycina (Sousa et al., 2015), Eucalyptus camaldulensis (Verdeguer et al., 2009) and Callistemon citrinus (Petronilho et al., 2013).

In vitro antileishmanial activity and cytotoxicity of the essential oil from fresh leaves of M. plinioides

The essential oil derived from leaves of M. plinioides was effective against L. amazonensis promastigotes (Table II) and presented IC50 value of 14.16 ± 7.40 µg/mL, while the standard drug pentamidine isethionate presented IC50 value of 23.22 ± 9.04 µg/mL. However, activity against L. infantum promastigotes were less pronounced, presented an IC50 value of 101.50 ± 5.78 µg/mL (Table II).

TABLE II IC50 (µg/mL) value of essential oil of M. plinioides against promastigotes of L. amazonensis and L. infantum and CC50 (µg/mL) value of cytoxicity against murine peritoneal macrophages. 

Extract Antiparasitic Activity
IC50 ( µg/mL)
Toxicity
CC50 ( µg/mL)
SI
L. amazonensis L. infantum Macrophages
MPEO 14.16 ± 7.40a 101.50 ± 5.78a 93.50 ± 9.10 6.60
Pentamidine* 23.22 ± 9.04a 34.20 ± 2.50b 61.21 ± 1.40 2.63

*Reference drug. ** Selective Index: ratio CC50/IC50 (L. amazonensis). Data are expressed as mean values ± standard error. Within each column, values followed by dissimilar upper case superscript letters are statistically different (p > 0.05).

American tegumentary leishmaniasis (ATL) affects populations in various regions of the world, including an area extending from southern USA to northern Argentina, with the exception of Chile and Uruguay. The disease can present with diverse clinical forms described as cutaneous, diffuse cutaneous or mucocutaneous. Moreover, ATL can cause injury way beyond its deforming effects, thereby raising issues concerning possible psychological damage and, consequently, social and economic losses (Amato Neto et al., 2008; Garcia et al., 2011).

Antileishmanial activity has been demonstrated for essential oils from a number of plant species including Lippia origanoides (Escobar et al., 2010), L. sidoides (Medeiros et al., 2011; Farias-Junior et al., 2012) and Lantana camara (Machado et al., 2012). In the case of M. plinioides, it is likely that the antileishmanial activity of the leaf oil is associated with the presence of the sesquiterpenes spathulenol and caryophyllene oxide, which represent 36.32% of the total components.

In this context, various studies have demonstrated that terpenes can cause alterations in the mitochondrial membrane potential, modification of the redox index, inhibition of cellular isoprenoid biosynthesis and changes in the plasma membrane (Santos et al., 2008; Rodrigues et al., 2013; Monzote et al., 2014). According to Oliveira et al. (2014), the essential oil of Bocageopsis multiflora is also rich in spathulenol and exhibits in vitro antileishmanial activity against promastigotes of Leishmania amazonensis. Additionally, spathulenol and caryophyllene oxide have been identified as the principle components of the essential oil of Piper angustifolium (Bosquiroli et al., 2015), which also exhibits significant in vitro antileishmanial activity against L. infantum amastigotes.

Monzote et al. (2014) carried out a comparative study of the essential oil of Chenopodium ambrosioides and its major constituents, namely ascaridole, carvacrol and caryophyllene oxide, and found that the natural mixture of the oil was potentially more active than the isolated components. Caryophyllene oxide, for example, exhibited non-specific activity and presented similar IC50 values against L. amazonensis and macrophages. Santin et al. (2009) reported an analogous situation for the essential oil of Cymbopogon citratus in which the principal constituent, citral, exhibited greater toxicity than the natural oil mixture. These results demonstrate the importance of complementary studies to determine whether the leishmanicidal activity against Leishmania promastigotes and cytotoxicity observed for M. plinioides is related to a specific component or mixture thereof.

Pentamidine isethionate and other drugs used in the treatment of leishmaniasis are toxic and their application is limited owing to issues associated with high cost, acquired resistance, routes of administration and difficulties of adherence to treatment (Bucknerm Waters, Avery, 2012). The MPEO showed significant activity against L. amazonensis, and presented selectivity index (SI) of 6.60 (Table II). The activity against L. infantum promastigotas form of sample was less significant, besides that the SI data demonstrated considerable toxicity.

The results obtained in this study reveal that the MPEO is promising as a source for new antileishmanial agents against L. amazonensis. However, more studies are necessary in order to determine the constituents responsible for the antileishmanial activity and the mechanism of action involved. It stands out that the assay against promastigote forms is a preliminary screening to identify possible novel antileishmanial compounds, as it is a low cost and easy to handle method like in amastigotes (Siqueira-Neto et al., 2010). Even so, it is essential to evaluate the activity of MPEO against amastigote forms.

ACKNOWLEDGMENTS

The authors acknowledge to Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; PRONEX-10/0029-0).

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Received: January 12, 2018; Accepted: August 14, 2018

*Correspondence: E. M. Ethur. Centro de Ciências Exatas e Tecnológicas, Universidade do Vale do Taquari - Univates. Avenida Avelino Tallini, 171, Universitário, 95900-000, Lajeado-RS, Brazil. Tel: +55 51 3714-7000 / Fax: +55 51 3714-7001. E-mail: eduardome@univates.br

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