Chemical composition, anti-Trypanosoma cruzi and cytotoxic activities of the essential oil from green fruits of Protium ovatum (BURSERACEAE)

1MSc, Instituto Federal Goiano, Rio verde-GO, Brazil. E-mail: elisangelaborges49@hotmail.com 2PhD, Instituto Federal Goiano, Rio verde-GO, Brazil. E-mail: cassiacefetrv@gmail.com 3PhD, Universidade de Franca, Franca-SP, Brazil. E-mail: viviane.sampaio@unifran.edu.br 4PhD, Instituto Federal do Sudeste de Minas Gerais. Barbacena-MG, Brazil. E-mail: criscazal@yahoo.com.br 5MSc, Instituto Federal do Mato Grosso do Sul, Coxim-MS, Brazil. E-mail: allex_fonseca@yahoo.com.br 6 PhD, Instituto Federal do Sul de Minas Gerais, Pouso Alegre-MG, Brazil. E-mail: mayker.miranda@ifsuldeminas.edu.br Abstract Chagas disease is a major neglected tropical disease caused by Trypanosoma cruzi. It has been treated with the antiparasitic drugs nifurtimox and benzonidazole, which cause several side effects. The market for natural products has considerably grown lately and the use of plants has become an alternative to the development of novel drugs to cure the disease. Therefore, this study aimed at describing the chemical analysis of the essential oil from green fruits of Protium ovatum and evaluating their trypanocidal and cytotoxic potential. The essential oil was obtained by Clevenger hydrodistillation whereas its chemical composition was determined by gas chromatography coupled to mass spectrometry (GC-MS). The major compounds found in the essential oil from green fruits of P. ovatum were β-myrcene (62.0 %), α-pinene (11.3 %) and limonene (7.3 %). To the best of our knowledge, this was the first time that the chemical composition of the essential oil from green fruits of P. ovatum was described. Results showed that the essential oil had strong trypanocidal activity against trypomastigote forms of theY strain of Trypanosoma cruzi (IC50 = 1.2 μg/mL). In addition, the essential oil from green fruits of P. ovatum did not display cytotoxicity against LLCMK2 adherent epithelial cell at the concentration range under analysis (CC50 = 550.3 μg/mL). As a result, it is an excellent option for the development of novel antiparasitic drugs.


Introduction
American trypanosomiasis, also known as Chagas disease (CD), is a neglected tropical disease which is endemic in Latin America.The World Health Organization estimates that approximately 6-7 million Latin Americans have been infected with Trypanosoma cruzi since CD has been mainly found in endemic areas of Latin American countries (PAULA et al. 2015).
The disease has been treated with drugs which display either high toxicity against the host's body or low efficiency against the pathogen (IZUMI et al. 2012).So far, no vaccine has been developed for CD and the current treatment has been conducted with nifurtimox (Nf) or benzonidazole (Bz).However, their well-known toxicity, as well as their limited effect on different parasite isolates and on the chronic phase of the disease, has called for the development of new drugs to treat it (SOEIRO; CASTRO, 2011).
The market for natural products, such as extracts, isolated compounds and essential oils from plants, and the use of plants as an alternative to the development of novel drugs for the treatment of various diseases, including CD, have annually increased in view of the great potential of these compounds (AFFONSO et al. 2012;LEITE et al. 2010).The Cerrado (Brazilian savannah), a natural heritage site owing to its diversity and endemism of biological species, is an important source of novel natural substances with different biological properties (SILVA et al. 2015).
Essential oils from different plant sources carry out several biological activities, such as antibacterial, anticancer, anti-inflammatory, antimutagenic, antifungal, antioxidant and antiprotozoal ones.As a result, the vast arsenal of bioactive compounds found in essential oils has increasingly attracted researchers' intense attention in the last years (CARNEIRO et al. 2017).
The Burseraceae family, for example, comprises 21 genera with 600 species and the genus Protium is its main family member with 135 species.In the literature, species of the Burseraceae family have been described as the ones which are commonly used for treating wounds and ulcers.Besides, they act as anti-inflamatory and repellent agents.Triterpenes, mono-and sesquiterpenes from species of the genus Protium are well-known for their significant biological properties and their anti-inflammatory and acaricidal activities (MORAES et al. 2013).
Protium ovatum Engl. is a herbaceous plant found in the Brazilian Cerrado.In the literature, several studies have described the anti-inflammatory, antinociceptive, immunostimulant and anticancer properties of resins (SIANI et al. 2011).However, reports have described neither the chemical composition nor the trypanocidal potential and cytotoxicity of the essential oil from specimens of Protium ovatum.
This study aims at describing the chemical composition of the essential oil from green fruits of P. ovatum, its in vitro activities against trypomastigote forms of Trypanosoma cruzi and its cytotoxic activity against LLCMK 2 adherent epithelial cells.

Material and Methods
Green fruits of Protium ovatum were collected in Rio Verde, GO, Brazil, in September 2015.The plant was identified by the botanist Erika Amaral (Instituto Federal de Educação, Ciência e Tecnologia Goiano, Campus Rio Verde, GO, Brazil).A voucher specimen (no.HJ 7420) was deposited at the Herbarium Jataiense Professor Germano Guarin Neto, which belongs to the Instituto Federal Goiano, Brazil.
The essential oil was extracted from fresh green fruits of Protium ovatum (100 g) by a modified clevengertype apparatus and hydrodistillation for 2 h.The oil was separated and dried over anhydrous sodium sulfate, stored in hermetically sealed glass containers and kept under refrigeration at 5 °C until analysis and trypanocidal and cytotoxicity assays.Total oil yield was expressed as a percentage value (g/100 g of fresh plant material).All experiments were carried out in triplicate.
Gas chromatography -mass spectrometry (GC-MS) analysis was carried out by a Shimadzu QP2010 with an AOC-20i auto-injector and a DB-5MS column (30 m x 0.25 mm, 0.25 mm in thickness).The carrier gas was He with pressure of 57.4 kPa and flow rate of 1.00 mL/ min.The split ratio was 1/30, the injector temperature was 250 °C and the injected volume was 1 µL.Temperature programming was the following : 60 -240 °C, increasing 3 °C/min.MS were recorded on the electron ionization (EI) mode, with ionization energy of 70 eV (scan time: 2 scans/s).Identification of the constituents was based on the retention indices (the calculation used from C 9 to C 22 alkanes) and on the comparison of the mass spectra with libraries (Wiley 7 and Nist 62) and references to previously published data (ADAMS, 2007).
1D-1 H-and 13 C-NMR spectroscopic data were recorded at room temperature in CDCl 3 (Cambridge Isotope Laboratories, Andover, MA, USA) by a Bruker DPX-300 spectrometer (Karlhue, Germany) operating at 300 MHz ( 1 H)/75 MHz ( 13 C).Standard pulse sequences were used for homo-and heteronuclear correlation experiments.Chemical shifts are reported in ppm, using TMS as an internal standard (δ = 0 ppm) whereas coupling constants (J) are expressed in Hertz.
LLCMK 2 adherent epithelial cells were grown in RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 5 % inactivated fetal calf serum.They were kept at 37 °C in 5 % CO 2 .A cell suspension was seeded at a concentration of 1 x 10 6 cells/mL in a 96-well microplate with RPMI 1640 medium.Thereafter, cells were treated with essential oil at different concentrations (6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL).Plates were incubated at 37 °C for 24 h and the biological activity was evaluated by the MTT colorimetric method [MTT; 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] in a microplate reader at 540 nm.RPMI 1640 medium was the positive control whereas DMSO and RPMI 1640 media were the negative ones.All experiments were performed in triplicate.The percentage of cell viability was determined by the following formula: % cell viability = 1 -[(Y-N)/ (N-P)] x 100, where Y = absorbance of wells containing cells and essential oil at different concentrations; N = negative control; and P = positive control (ESPERANDIM et al. 2013).
β-myrcene, α-pinene and limonene (Figure 1), which are the three major components of the essential oil from green fruits of P. ovatum, were found to be among the constituents of the oils from other species of the same genus (ZOGHBI et al. 2005).In previous studies of the efficiency of the essential oil from leaves of P. ovatum, the result was 0.10 % and a complex mixture of terpene constituents was determined by 1 H NMR, 13 C NMR and IR (CASTELO et al. 2010).Other species of Protium were analyzed and a complex mixture of monoterpenes and sesquiterpenes were determined in essential oils from resins, foliar rachises, branches and leaves (CARVALHO et al. 2013;PINTO et al. 2010;CARVALHO et al. 2010).The identification of β-myrcene (62.0 %) in the mixture, the main substance found in the essential oil from fruits of Protium ovatum, was carried out by the analysis of 1 H (Figure 2) and 13 C (Figure 3) NMR spectra and by comparison with literature data (PESSINI et al. 2005).The spectroscopic data on β-myrcene are as follows: β-myrcene: NMR 1 H (300 MHz, CDCl 3 ): δ 6.35 (dd, J = 10.8 e 17.7 Hz, H-4); 5.43-4.60(m, H-7, H-9 and H-10); 2.24-2.02(m, H-5 and H-6); 1.69 (s, H-1); 1.64 (s, H-2).The essential oil from green fruits of P. ovatum has high trypanocidal activity against trypomastigotes of Trypanosoma cruzi.Increased infeasibility of trypomastigote cells was observed with increasing concentration of essential oil.High activity was obtained at IC 50 = 1.2 µg/mL (Table 2) and it was lower than the one of the positive control with benzonidazole, which was IC 50 = 9.8 µg/mL.
The literature has reported that samples with trypanocidal activity of IC 50 < 10 µg/mL, IC 50 > 50 < 100 µg/mL and IC 50 > 100 µg/mL are considered highly active, active/moderately active and inactive, respectively (ALVES et al. 2012).The trypanocidal properties of the major components of the essential oil from green fruits P. ovatum, β-myrcene (62.0 %), α-pinene (11.3 %) and limonene (7.3 %) were previously reported (SANTOS et al. 2014;SARTORELLI et al. 2012;ZENG et al. 2010).It was proposed that the activity of essential oils against trypanosomatids is mainly due to its terpene composition.Terpenes are responsible for the hydrophobic character of essential oils, thus allowing their diffusion through the parasite cell membrane and affecting intracellular metabolic pathways and organelles (BORGES et al. 2012).This is the first report of the trypanocidal activity of the essential oil from green fruits of Protium ovatum.Despite great advances made by modern medicine in recent decades, plants are still considered very important in regard to health care (CALIXTO, 2000).Several studies of essential oils have shown that some plants have trypanocidal activity against T. cruzi (BALDISSERA et al. 2013;ESCOBAR et al. 2010).
Cultures of LLCMK 2 adherent epithelial cells were treated with essential oil at concentrations of 6.25, 12.5, 25.0, 50.0, 100, 200 and 400 μg/mL for 24 h.Results showed that the essential from green fruits did not have toxicity at the concentration evaluated with CC 50 550.3μg/ mL (Table 3), by comparison with benzonidazole positive control with CC 50 147.3μg/mL.
It is important to point out that the essential oil from green fruits of P. ovatum did not display cytotoxicity against adherent epithelial cells at the concentration range under analysis.There is evidence that, owing to their lipid solubility, essential oils have low density and rapid diffusion across cell membranes.As a result, they could damage the parasite cell membrane structure and lead to cellular lysis (ANTHONY et al. 2005).In addition, there could be synergistic and/or additive effects from constituents of the essential oil (CARNEIRO et al. 2017).

Conclusion
In summary, the results of this study showed that the essential oil from green fruits Protium ovatum found in the Brazilian Cerrado, which is located in the central-west region of the country, has promising antiparasitic potential with no cytotoxicity towards LLCMK 2 adherent epithelial cells.The high concentration of β-myrcene (62.0 %) in the essential oil from green fruits investigated by this study is a prospect of a new source of this secondary metabolite as a raw material in the synthesis of new medication.Further studies with in vivo and field experiments must be carried out to ascertain its efficiency.However, to the best of our knowledge, this was the first time that the chemical composition of the essential oil from green fruits of P. ovatum was described.It is very important to the knowledge of this botanical species.

RI a :
Linear retention indices found in the literature.RI b : Retention indices calculated from retention times in relation to those of the series of n-alkanes on a 30 m DB-5 capillary column.

Table 1 -
Chemical composition of the essential oil from green fruits of Protium ovatum.

Table 2 -
Trypanocidal activity of the essential oil from green fruits of Protium ovatum against trypomastigote forms of Trypanosoma cruzi .