Chemical composition, antioxidant and antiprotozoal activity of Eugenia gracillima Kiaersk. leaves essential oil

Abstract This work evaluated the volatile composition, antioxidant and antiprotozoal activities of the essential oil obtained from leaves of Eugenia gracillima Kiaersk. (EGEO) grown in Brazilian Northeast area (Araripe, Brazil). The volatile compounds of EGEO were analyzed by GC and GC–MS and its chemical composition is mainly composed of sesquiterpene hydrocarbons (91.22%), oxygenated sesquiterpenes (7.45%) and monoterpene (1.01%). The most abundant volatile constituents of the EGEO were germacrene D (16.10%), γ-muurolene (15.60%), bicyclogermacrene (8.53%), germacrene B (7.43%), and Δ-elemene (6.06%). The oil showed weak to moderate antioxidant activity. EGEO was highly selective to Leishmania braziliensis and Leishmania infantum promastigotes with selective indexes of 73.66 and 71.41, respectively. EGEO did not inhibit Trypanosoma cruzi. These data suggest that the E. gracillima essential oil is a relevant source of lead compounds for development of anti-Leishmania drugs.


Plant material
The leaves of E. gracillima were collected in the area of native vegetation, located in Serra dos Paus Dóias in Chapada do Araripe (Exu, Pernambuco; latitude 7° 21"S, longitude 39° 53" W and altitude 884m), between July and August 2017, under authorization of the responsible authority Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) using the license SISBIO 59070. The plant material was identified by Dr. Maria Arlene Pessoa da Silva from the Laboratory of Microbiology and Molecular Biology of the Regional University of Cariri (URCA). The voucher (registration number: 13.055) was deposited in the URCA Herbarium (Herbário Caririense Dárdano de Andrade Lima; HCDAL).

Sample preparation and chemical characterization of E. gracillima essential oil
The essential oil from the leaves of E. gracillima (EGEO) was obtained by hydrodistillation method. The EO was filtered, weighed and stored in a dark at 4° C until use. The EO was analysed by gas chromatography coupled to Shimadzu GCMS-QP2010 mass spectrometry (CG-MS) using Rtx®-5MS capillary column (30 m x 0.25 mm x 0.25 μm). The electron ionization mode was used at 70 eV. and helium (99.999%) was employed as the carrier gas (1 mL/min). The following temperature program was used: 100 °C (3 min) at 310 °C (3.5 °C/min). The identification of the individual volatile components was carried out by comparison with values of retention indices, obtained by co-injection of oil samples and a set of C 9 -C 30 linear hydrocarbons, and the MS data acquired for each volatile component were matched with the data available in the mass spectral library -MassFinder 4 (Dr. Hochmuth scientific consulting, Hamburg, Germany); NIST08 Mass Spectral Library (ChemSW Inc. Fairfield, CA, USA); Wiley Registry™ of Mass Spectral Data 9th Edition (Wiley, Hoboken, NJ, USA) and with other published mass spectral data (Adams, 2007). For the quantitative analysis, a Shimadzu GC2010 Plus gas chromatograph equipped with a FID detector, with the same column and operating conditions above described were used. The percentage volatile composition was performed by the normalization method from the GC peak areas, in relation to the combined area of all peaks. All analyses were carried out in triplicate.

Cell cultures
Tests with T. cruzi were performed using clone CL-B5 (Buckner et al., 1996) carrying Escherichia coli β-galactosidase (lacZ) gene, kindly supplied by Dr. The cytotoxicity assays were performed using NCTC-929 murine fibroblasts obtained from the National Collection of Culture Types. The cells were cultured in Minimum Essential Medium supplemented with SBF (10%), penicillin G (100 U/ml) and streptomycin (100 mg/ml). The cultures were maintained at 37 °C in a humidified atmosphere with 5% CO 2 .

Anti-T. cruzi assay
Aliquot of epimastigote forms of T. cruzi suspension (200 μL at 1 × 10 5 per milliliter) were treated with different concentrations of EGEO (15.75 μg/mL to 1000 μg/mL) at 28 ° C. After 72h, 200 μM of red-β-D-galactopyranoside chlorophenol (CPRG; dissolved in a solution of 0.9% Triton X-100) were used in order to evaluate parasite viability. The plates were incubated at 37 °C for 6 h and then the absorbance was determined at 595 nm. Benznidazole was the reference drug.

Cytotoxicity evaluation
NCTC-929 were seeded (100 μL; 3 × 10 4 cells/well) in 96-well plates with of RPMI 1640 culture medium per well. Cells were grown overnight at 37 °C and 5% CO 2 . The medium was removed, and the new medium were added containing EGEO 15.75 μg/mL to 1000 μg/mL. Cell viability was evaluated using resazurin as described above. The toxicity to the fibroblast cell and the activity against the protozoan were compared by using the selectivity index (SI) ratio (IC 50 for NCTC-929 cells/IC 50 for protozoan).

Antioxidant potential
The antioxidant potential of EGEO were evaluated by the traditional methods of DPPH and ABTS radicals inhibition and phosphomolybdenum assay (referred as total antioxidant activity) (Blois 1958, Prieto et al. 1999, Re et al. 1999. Total antioxidant activity (%) was expressed as ascorbic acid equivalent, relative to 1mg/mL ascorbic acid to 1mg/mL EGEO.

Statistical analysis
The experiments were performed in triplicate with at least two independent repetition, data were presented as means ± standard variation (SD) or percentages.
Statistical analyses were performed using the software GraphPad Prism version 5.0. Data were analyzed by one-way analysis of variance (ANOVA) and Tukey test. A p-value of <0.05 was considered as statistically significant. IC 50 values were determined by linear