Potential Antileishmanial Activity of Essential Oils of Native Species from Southern Brazil

Leishmaniasis are a neglected tropical diseases that affecting 98 countries on three continents. Every year, 1.3 million of people are infected with the disease and 50.000 persons die because of this. The aim of this work was to evaluate antileishmanial activities in vitro from native species of South of Brazil belonging to the Myrtaceae family. The essential oils from leaves of Calyptranthes grandifolia, Calyptranthes tricona, Eugenia anomala, Eugenia arenosa, Eugenia pyriformis, Myrrhinium atropurpureum and Psidium salutare were analyzed in vitro for antileishmanial activity against promastigotes of Leishmania amazonensis, employed MTT assay. The essential oils from leaves of C. grandifolia, C. tricona, E. arenosa and E. pyriformis presented IC50 values of 31.27 ± 6.40 μg/mL, 26.13 ± 8.60 μg/mL, 13.72 ± 8.65 μg/mL and 19.73 ± 5.40 μg/mL, respectively, and not are statistically different from pentamidine (IC50 = 23.22 ± 9.04 μg/mL), the reference drug. The results show the potential of essential oils from leaves of C. grandifolia, C. tricona, E. arenosa and E. pyriformis as antileishmanial, as well as the importance of continuing studies to in order to advance in the search and development of new therapeutic options from of brazilian flora sources.


Introduction
Leishmaniasis are considered a neglected tropical diseases (NTDs), affecting 98 countries on three continents, with Brazil among the countries where visceral leishmaniasis (VL) cases, cutaneous leishmaniasis (CL) and mucocutaneuos leishmaniasis (MCL) are more frequent, ie, it is considered an endemic area.NTDs are generally infections diseases, prevalent in tropical or subtropical regions, which typically affect the poorest populations.Annually 300.000 new cases de VL and one million of new cases of CL are registrated, totalizing 1.3 million people stricken by illness, besides up to 50.000 deaths per year.The areas of disease transmission has been enlarged and, consequently, the reported cases increase exponentially.Among the causes related to the propagation of this NTD can be cited unfavorable socioeconomic conditions, malnutrition, climate and environmental changes, increased of migration populational, conflicts, coinfection with HIV that generates immunosuppression, accelerated urbanization (World Health Organization [WHO], 2015;WHO, 2016).
Medications for the treatment of NTDs are generally not research targets of the pharmaceutical industries, and the people affected by these diseases suffer from the deficiency of effective and safe pharmacotherapy.In addition, there are no vaccines available for leishmaniasis and vector disease control is also deficient.Thus, the solution for NTDs is complex, is necessary to improve people's living conditions, increase investment in research and development, as well as provide access to preventive or curative therapy for all populations, especially in developing countries (Brasil, 2010;Garcia, De Magalhães, Aurea, Dos Santos, & De Almeida, 2011;Boechat & Magalhães, 2012;L. C. Dias, Dessoy, Guido, Oliva, & Andricopulo, 2013).
The World Health Organization (WHO) recommends the use of plants as a therapeutic resource, in addition, Brazil is renowned for its biodiversity (Brasil, 2009), and thus the search for new therapeutic agents from plants becomes an attractive alternative.Among the botanical families present in our flora, the Myrtaceae family stands out, for its wide distribution and number of species, adding to the fact that many of these, despite the popular usage reporting, not present studies on its effectiveness or safety.About 1.000 species from Myrtaceae family are found in Brazil, distributed among different biomes, especially the Atlantic Rain Forest, the Restinga and the Cerrado.Species from family are used in spice industry (Syzygium aromaticum), in the wood industry and paper production (Eucalyptus spp.), pharmaceutical and cosmetic industries (Pimenta racemosa, Melaleuca spp., Callistemon e Leptospermum).The family also is a source of edible fruit, like guava (Psidium guajava), jabuticaba (Myrciaria cauliflora), cherry (Eugenia uniflora), guabiroba (Campomanesia spp.), araçá (Psidium cattleyanum), jambo (Syzygium spp.) and jambolão (Syzygium cumini) (Govaerts et al., 2008;Souza & Lorenzi, 2012).Species from Myrtacea family are used popularly in treatment of diarrhea and inflammations, as Myrciaria cauliflora and Eugenia uniflora, while Myrciaria dubia is employed as antimalarial (Simões, Mentz, Schenkel, Irgang, & Stehmann, 1989;Boscolo & Valle, 2008;Ruiz et al., 2011).In addition, studies have shown the potential biological of species from family (Schneider et al., 2008;Diniz, Macêdo-Costa, Pereira, Pereira, & Higino, 2010;Magina et al., 2012;Ferreira et al., 2014;Sousa et al., 2015).
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).The screening for new antileishmanial compounds in endemic areas is suggested by employing assays against promastigotes forms of Leishmania spp., because these tests are easier and cheaper compared to analyses against amastigotes forms.Also, for small molecule, as terpenes, results in assays against promastigotes forms have demonstrate similar to those in amastigote forms (Siqueira-Neto et al., 2010).Thus, considering the problem of leishmaniasis as NTDs and the need to develop new therapeutic alternatives for this disease, this study aimed to evaluate the antileishmanial activity in vitro of native species of the State of Rio Grande do Sul, southern Brazil, belonging to Myrtaceae family, against promastigote forms of Leishmania amazonensis.

Plant Materials
Leaves samples from native species of Myrtaceae family were collected in various municipalities of the State of Rio Grande do Sul, Brazil, during June and July 2012, and identified by the botanist Dr. Elisete Maria de Freitas.The GPS data are listed in Table 1.
Table 1.Sources of Myrtaceae species employed in the study.

Preparation of Essential Oil
Fresh leaves from each species of Myrtaceae family were subjected to hydrodistillation for 3.5 h in a Clevengertype apparatus.The essential oil was dried over anhydrous sodium sulfate, transferred to amber glass bottles and stored at −20 °C, until required for chemical analysis and bioassay.

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 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 1x10 7 promastigotes/mL using the supernatant of the respective culture as diluent.

Determination of Antileishmanial Activity in vitro
Appropriate amounts of samples or pentamidine isethionate (as reference drug) were dissolved in aqueous dimethyl sulfoxide (DMSO; 10 mg/mL) to yield solutions containing analytes in the concentration range 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 (1x10 7 promastigotes/200 μL/well).Aliquots of freshly prepared analyte solutions 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-2yl)-2,5-diphenyl tetrazolium bromide (MTT) (Dutta, Bandyopadhyay, Mandal, & Chatterjee, 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 (1): x 100 (1) Values for IC 50 , i.e. the concentration that inhibited parasite growth by 50%, were determined.

Statistical Analysis
Assays were carried out in three independent experiments and each was performed in triplicate.Values of IC 50 were determined by logarithmic regression analysis using GraphPrism 5 software.Values for in vitro antileishmanial activity were expressed as mean ± standard deviation.Results were analyzed by Student's T using BioEstat 5.0 software, and differences were considered significant when p < 0.05.
In Brazil, Myrtaceae constitutes one of the main families of national flora, with 22 genera and about 1000 species, specifically in the southern, species are found in areas of Atlantic Forest as in Pampa biome.Members of the genus Eugenia are of particular interest because produce edible fruits and many are used in popular medicine, especially in the treatment of gastrointestinal disorders and infectious diseases.Species from Psidium genus have attractives fruits with high concentration of vitamin C. While, leaves from Myrrhinium atropurpureum are employed popularly in the treatment of hypercholesterolaemia and diabetes.Still, the omnipresence of Myrtaceae species in threatened biomes suggests an ecological importance for family (Govaerts et al., 2008;Franzon, Campos, Proença, & Silva, 2009;Souza & Lorenzi, 2012;Sobral et al., 2013).
Pentamidine 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 (Buckner, Waters, & Avery, 2012).Pentamidine isethionate was used as drug reference in the assay and showed IC 50 values of 23.22 ± 9.04 µg/mL ( Antileishmanial activity is demonstrated for essential oils from various species.The terpenes cause alterations in the mitochondrial membrane potential, modification of the redox index, inhibition of cellular isoprenoid biosynthesis and changes in the plasma membrane, that can explain the antileishmanial activity (Santos et al., 2008;Rodrigues et al., 2013;Monzote et al., 2014).Essential oil from leaves of Annona foetida showed activity against promastigotes forms of L. guyanensis (Costa et al., 2009), while essential oil of Annona coriacea showed activity against promastigotes forms of L. chagasi (Siqueira et al., 2011).The essential oil of Lippia origanoides demonstred activity against promastigotes of L. chagasi, with IC 50 value of 4.4 μg/mL, and showing no toxicity to mammalian cells (Escobar, Leal, Herrera, Martinez, & Stashenko, 2010).Antileishmanial activity in vitro against promastigotes forms of L. chagasi was checked for the essential oil extracted from leaves of Lippia sidoides (Farias-Junior et al., 2012).For the essential oil from leaves of Lantana camara L., Verbenaceae, was demonstrated significant antileishmanial activity in vitro against promastigotes of L. amazonensis, with IC 50 value of 0.25 μg/mL (Machado et al., 2012).The essential oil of Piper auritum proved to be active in vitro against promastigotes forms of L. major, L. mexicana, L. braziliensis and L. donovani (Monzote, García, Montalvo, Scull, & Miranda, 2010).Essential oils obtained from species of the Myrtaceae family have also presented antileishamanial activity.The essential oil from leaves of Eugenia uniflora showed antileishmanial activity in vitro against promastigotes (IC 50 =3.04g/mL) and amastigotes (IC 50 =1.92g/mL) forms of L. amazonensis, and preferential toxicity to amastigotes compared to macrophages.The activity was related to activation of macrophages, evidenced by the increase in phagocytic capacity and lysosomes activity (Rodrigues et al., 2013).
Others species from Myrtaceae family were evaluated about potential antileishmanial activity.Bioguided study with fractions isolated from the crude extract of leaves from Blepharocalyx salicifolius, a Brazilian native species, showed the potential in vitro of eight fractions against amastigotes forms of L. amazonensis at concentrations ranging 19 a 29 μg/mL (Siqueira et al., 2010).Antileishmanial activity in vitro was observed for the hexanic extract of fruits of Eugenia umbelliflora, that showed IC 50 values of 14.3 g/mL and 5,7 g/mL against promastigotes forms of L. amazonensis and L. brasiliensis, respectively (Cechinel Filho et al., 2013).
The antileishmanial activity in vitro of aqueous and hidroethanolic extracts from leaves of Campomanesia eugenioides, native species from brazilian flora, was evaluated against promastigotes forms of L. amazonensis and showed IC 50 values of 388 ± 53 µg/mL and 555 ± 64 µg/mL, respectively (Moura-Costa et al., 2012).The extracts from bark and leaves of Myrcia linearifolia, native species of Brazilian Cerrado, not presented considerable activity in vitro against promastigotes of L. amazonensis, with IC 50 values higher than 100 µg/mL (Costa et al., 2014).The hidroethanolic extracts from leaves of Psidium guajava, in concentration of 100 μg/mL, inhibited the growth of amastigotes and promastigotes of L. amazonensis in 65.4% ± 5.4 and 52.0% ± 2.1, respectively (Luize et al., 2005).
In Brazil, L. amazonensis cause skin ulcerations and in mucous membranes, and has been associated with various clinical forms of the disease including cutaneous, mucosa, diffuse cutaneous and visceral leishmaniasis (Leon, Machado, Paes, Grimaldi Jr., 1990;WHO, 2010).The results obtained for the essential oils of species Calyptranthes grandifolia, Calyptranthes tricona, Eugenia arenosa and Eugenia pyriformis are promising compared to activity demonstrated for essential oil from E. jambolana and comparable to those observed with extracts of Blepharocalyx salicifolius and Eugenia umbelliflora.Still, the results are hopeful, because these four essential oils showed activity comparable to pentamidine against this parasite.
The assay in vitro against promastigotes forms of L. amazonensis, which is easy to perform and inexpensive, allowed to define native species of Myrtaceae family with the greatest potential antileishmanial.Thus, the results showed the potential of essential oils from leaves of C. grandifolia, C. tricona, E. arenosa and E. pyriformis as antileishmanial, as well as the importance of continuing studies to in order to advance in the search and development of new therapeutic options from of brazilian flora sources.In this sense, studies to evaluate the chemical constitution of essential oils and their cytotoxicity are important.In addition, further study of bioguided form is important to optimize the process and rationalize cost.

Table 2 .
IC 50 (µg/mL) value of essential oil from fresh leaves of Myrtaceae species employed in the study against promastigotes of Leishmania amazonensis.
*Values are statistically different from reference drug (p ≤ 0.05).