Rev Bras Farmacogn 24(2014): 644-650 Original article Nitric oxide production, inhibitory, antioxidant and antimycobacterial activities of the fruits extract and flavonoid content of Schinus terebinthifolius Natalia R. Bernardesa, Marlon Heggdorne-Araújoa,b, Isabela F. J. C. Borgesb,c, Fabricio M. Almeidab, Eduardo P. Amaralb, Elena B. Lasunskaiab, Michelle F. Muzitanob,c,*, Daniela B. Oliveiraa,* aLaboratório de Tecnologia de Alimentos, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil bLaboratório de Biologia do Reconhecer, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil cLaboratório de Produtos Naturais, Curso de Farmácia, Universidade Federal do Rio de Janeiro, Campus Macaé, Polo Novo Cavaleiro, Instituto Macaé de Metrologia e Tecnologia, Macaé, RJ, Brazil ARTICLE INFO ABSTRACT Article history: The extract of the fruits from Schinus terebinthifolius Raddi, Anacardiaceae, was obtained by Received 28 May 2014 exhaustive extraction with methanol. Its fractions and isolated compounds were collected Accepted 16 October 2014 by fractionation with RP-2 column chromatography. The crude extract, the flavonoid frac- tion and the isolated compound identified as apigenin (1), were investigated regarding its Keywords: inhibitory action of nitric oxide production by LPS-stimulated macrophages, antioxidant Anacardiaceae activity by DPPH and the antimycobacterial activity against Mycobacterium bovis BCG. The Apigenin samples exhibited a significant inhibitory effect on the nitric oxide production (e.g., 1, IC50 Inflammation 19.23 ± 1.64 μg/ml) and also showed antioxidant activity. In addition, S. terebinthifolius sam- Mycobacterium ples inhibited the mycobacterial growth (e.g., 1, IC50 14.53 ± 1.25 μg/ml). The necessary con- Nitric oxide centration to produce 50% of the maximum response (IC50) of these activities did not elicit Schinus terebinthifolius a significant cytotoxic effect when compared with the positive control (100% of lysis). The antioxidant and nitric oxide inhibition activity displayed by S. terebinthifolius corroborates its ethnopharmacological use of this specie as an anti-inflammatory. In addition, our results suggest that the flavonoids of S. terebinthifolius are responsible for the activities found. We, describe for the first time the activity against Mycobacterium bovis BCG and the inhibition of nitric oxide production for S. terebinthifolius. © 2014 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved. * Corresponding author. E-mail: [email protected] (D.B. Oliveira); [email protected] (M.F. Muzitano). 0102-695X/$ - see front matter © 2014 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved. http://dx.doi.org/10.1016/j.bjp.2014.10.012 Natalia R. Bernardes et al. / Rev Bras Farmacogn 24(2014): 644-650 645 from 0% to 15%, 5 min, 15% to 20%, 5 min, 20% to 30%, 5 min, Introduction 30% to 40%, 5 min, 40% to 41%, 5 min, 41% to 42%, 5 min and 42% to 50% for 10 min, 40 min as total time of analysis. Flow elution The species Schinus terebinthifolius Raddi, Anacardiaceae, was 1 ml min-1; 20 μl of the samples were injected. popularly known as pepper tree, is a native species of the Brazilian flora (Carvalho et al., 2006). Its bark, leaves and fruits Botanical material have medicinal properties as febrifuge, antioxidant and anti- inflammatory (Degáspari et al., 2005; Ceruks et al., 2007). Fruits of pepper tree, identified as Schinus terebinthifolius Raddi, Inflammation is a protective mechanism mediated by Anacardiaceae, were collected at Campos dos Goytacazes, Rio various chemical factors, and is comprised by complex de Janeiro, Brazil (Latitude 21°44’S and 41°18’W; Altitude 12 sequential changes in order to eliminate the initial cause m above sea level). A voucher specimen was identified and (Iwasaki and Medzhitov, 2010). Many diseases are followed deposited at the UENF’s herbarium under the code H5073. by acute and/or chronic inflammatory processes with a high production of chemical mediators, such as atherosclerosis, Preparation and fractionation of methanol extract Alzheimer's disease, cancer, asthma and infections, like tuberculosis (Gaestel et al., 2009). Currently non-steroidal The fruits were cleaned and dried at room temperature for anti-inflammatory drugs (NSAID) are the main drugs used to one day. Then the peel from the fruits (50 g) were subjected treat inflammation; however, these are frequently associated to exhaustive extraction with methanol (10% w/v) by static with gastric and cardiovascular side effects. Thus, there is maceration for 30 days and filtered twice per week. The extract a continuous need for discovery of new and less toxic anti- was evaporated at 35°C in a water bath in the dark. The yield of inflammatory drugs (Rang et al., 2007). the crude extract was 12.5 g. The fractionation was performed by The physiological NO production is extremely important to open column chromatography using a RP-2 column (50.0 × 5.0 defend the body; however, its overproduction and metabolites cm; H2O/MeOH gradient) affording three fractions A1 (5.0 g), A2 have been implicated in the development of pathologies, such (3.0 g) and A3 (1.0 g). The fraction A3 (0.5 g), the only one rich in as bacterial septic shock and chronic inflammation (Wadsworth flavonoids, was subject to further fractionation with RP-2 column and Koop, 2001). Therefore, NO production blocking agents (25.0 × 2.5 cm; H2O/MeOH gradient) resulting in two other sub- might be beneficial for the treatment of the inflammatory fractions (133.7 mg and 247.2 mg, respectively). The first (133.7 response. In addition, free radical species are also responsible mg), with the compounds of interest, was again fractionated by for activating several pro-inflammatory transcription factors, RP-2 column (25.0 × 2.5 cm; H2O/MeOH gradient) resulting in the involved with the promotion of inflammatory diseases isolated compound 1 (12.0 mg) which was analyzed by NMR (1H, (Reynertson et al., 2008). 13C, COSY, HSQC and HMBC) for structural elucidation. 1H NMR Thus, the aim of this work was to investigate the ability (DMSO-d6): G (ppm) 5.90; s (H-3), 6.05; d; J 1.3 Hz (H-6), 6.69; d; J 1.3 of the Schinus terebinthifolius fruit extract, its fractions and Hz (H-8), 6.85; d; J 8.6 Hz (H-3', 5') and 7.20; d; J 8.6 Hz (H-2', 6'). 13C apigenin (1) to inhibit the nitric oxide (NO) production by LPS- NMR (DMSO-d6): G (ppm) 167.4 (C-2), 102.7 (C-3), 181.7(C-4), 161.7 stimulated macrophages and its antioxidant activity, as well as (C-5), 99.7 (C-6), 162.9 (C-7), 94.7 (C-8), 157.9 (C-9), 108.0 (C-10), 122.0 evaluating its cytotoxic effect, contributing to justify its popular (C-1’), 128.4(C-2’), 115.9(C-3’), 161.2(C-4’), 115.9(C-5’) and 128.4(C-6’). use as anti-inflammatory, and enabling the use of this species to study the reduction of exacerbated inflammatory process. Inhibition of NO production by LPS-stimulated macrophages Besides the anti-inflammatory activity, the antimycobacterial and cytotoxicity activity against Mycobacterium bovis BCG was evaluated in order to contribute to the discovery of new antituberculosis agents. The murine macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (ATCC), grown at 37°C and 5% CO2 in DMEM/F-12 supplemented with 10% Materials and methods fetal calf serum. Macrophages (1×105 cells/well) were seeded in 96-well plates in the presence or absence of samples at General different concentrations and/or LPS [1 μg/ml] (Escherichia coli 055:B5). After 24 h incubation, supernatants were collected 13C and 1H NMR data were obtained using a Varian 400 MHz and the nitrite concentration was measured as indicator of spectrometer at the LAMAR/NPPN - UFRJ (Laboratory of Multi- NO production, according to the Griess test (Chi et al., 2001). User Analyses by NMR) and on a Brucker 400 MHz spectrometer Positive control: macrophages stimulated with LPS and treated at the National Center for Nuclear Magnetic Resonance Jiri Jones with L-NMMA (Sigma-Aldrich-98% purity), a nonspecific NO (Department of Biochemistry UFRJ). High Performance Liquid synthase inhibitor at 20 μg/ml. Negative control: macrophages Chromatography analyses were performed using a Shimadzu stimulated with LPS at 1 μg/ml and untreated. Prominence HPLC system with two LC10AT pumps, a scanning ultraviolet SPD-M10A photodiode array detector and a Rheodyne Cytotoxic effect 7725i injector. The reverse-phase column used was an RP-18 (5 μm, 250 mm, 4.5 mm i.d., Macherey-Nagel). The eluent used The LDH release (cytoplasmic enzyme lactate dehydrogenase) was purified water adjusted to pH 3.2 with phosphoric acid and was determined using the culture supernatant. The LDH acetonitrile. The following acetonitrile gradients were applied: release, which represents an indirect indication of cytotoxicity, 646 Natalia R. Bernardes et al. / Rev Bras Farmacogn 24(2014): 644-650 was determined using a commercial kit (Raso et al., 2001; Moraes et al., 2011). The specific release was calculated as percentage of the controls: untreated macrophages and 1% Triton X-100 (Vetec Chem.) treated macrophages. Antioxidant activity of extracts and fractions The DPPH (1,1-diphenyl-2-picrylhydrazyl), free radical scavenging activity of the samples were determined as described below. Samples were prepared in methanol at 2, 0.2 and 0.02 mg/ml. Samples (500 μl) were added to 500 μl of DPPH stock solution at 0.1 mM, to final concentrations of 1, 0.1 and 0.01 mg/ml. The reaction was carried at room temperature. After 1 h, absorbance values were measured at 515 nm.