Journal of Ethnopharmacology 144 (2012) 802–805 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep Ethnopharmacological communication Anti-inflammatory effects and acute toxicity of hydroethanolic extract of Jacaranda decurrens roots in adult male rats Joyce Alencar Santos a, Aline Arruda a, Magaiver Andrade Silva a, Claudia Andrea Lima Cardoso b, Maria do Carmo Vieira c, Cândida Aparecida Leite Kassuya a, Arielle Cristina Arena d,n a School of Health Sciences, Federal University of Grande Dourados, Dourados-MS, Brazil Mato Grosso do Sul State University, Dourados-MS, Brazil c School of Agrarian Sciences, Federal University of Grande Dourados, Dourados-MS, Brazil d ~ Junior, S/N, Caixa Postal 510, CEP 18618970, Department of Morphology, Institute of Biosciences of Botucatu, Sa~ o Paulo State University (UNESP), Distrito de Rubiao Botucatu-SP, Brazil b a r t i c l e i n f o a b s t r a c t Article history: Received 14 June 2012 Received in revised form 14 September 2012 Accepted 12 October 2012 Available online 23 October 2012 Ethnopharmacological relevance: Jacaranda decurrens subsp. symmetrifoliolata Farias and Proenc- a (Bignoniaceae) is a species traditionally used for the treatment of inflammatory diseases. However, until this moment, there is no scientific evidence of these effects. Aim of study: To evaluate the anti-inflammatory effects of hydroethanolic root extract of Jacaranda decurrens in rats and to determine the safe of this plant after acute exposure. Materials and methods: The acute toxicity of Jacaranda decurrens root extract (EJD) was evaluated by oral administration to male rats as single doses of 0; 500; 1000 or 2000 mg/kg body weight. General behavior and toxic symptoms were observed for 14 days. The anti-inflammatory activity was evaluated in carrageenan-induced inflammatory paw edema and myeloperoxidase activity in male rats. Results: No signs of acute toxicity were observed, indicating that the LD50 is greater than 2000 mg/kg. EJD (100 and 300 mg/kg) significantly reduced edema formation and at higher dose, the reduction was similar to dexamethasone. A significant decrease in myeloperoxidase activity was also observed. Conclusions: The present study shows that Jacaranda decurrens extract has anti-inflammatory properties in rats without causing acute toxicity. These properties observed may be due to the presence of bioactive constituents such as ursolic acid. & 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license. Keywords: Jacaranda decurrens Inflammation Paw edema Carrageenan Toxicity 1. Introduction Steroidal or nonsteroidal anti-inflammatory drugs (NSAIDs) have a number of adverse effects (Batlouni, 2010) and, there is considerable interest in identifying new anti-inflammatory agents obtained from plants used in popular medicine. Jacaranda decurrens subsp. symmetrifoliolata Farias and Proenc- a (Bignoniaceae), traditional known as ‘‘carobinha-docampo’’, ‘‘carobinha’’ or ‘‘caroba’’, is an endemic species found in the Brazilian states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais and Sa~ o Paulo (Bertoni et al., 2010). According to folk medicine, the leaves and/or the roots were prepared in form of infusion, decoction and ‘‘garrafadas’’ against inflammatory diseases and infections (Tresvenzol et al., 2006). Pharmacological evaluations have revealed that species from Jacaranda genus possess antioxidants (Carvalho et al., 2009), antimicrobials (Zatta et al., 2009), chemopreventives properties n Corresponding author. Tel.: þ55 14 3880 0495. E-mail addresses: ariellearena@ibb.unesp.br, ariellearena@yahoo.com (A.C. Arena). 0378-8741 & 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.jep.2012.10.024 (Subbaramaiah et al., 2000). It is suggested that the responsible for these activities are compounds such as ursolic acid. Phytochemical analyses of the leaves of Jacaranda decurrens indicated the presence of triterpenes, ursolic acid, oleanolic, flavonoid, saponins and coumarins (Carvalho et al., 2009; Zatta et al., 2009). Due to the use of Jacaranda decurrens roots in folk medicine to combat inflammatory diseases, without scientific evidence of this potential therapeutic application, the aim of the study was to evaluate the anti-inflammatory effects of hydroethanolic extract of Jacaranda decurrens (EJD) roots in male rats and to determine the toxicity of this plant after acute exposure. 2. Materials and methods 2.1. Plant material, preparation and isolation of extract Jacaranda decurrens subsp. symmetrifoliolata roots were collected (April 2010) in the Medicinal Plant Garden of the Federal University of Grande Dourados. A voucher specimen was identified by Dr. Rosana Farias Singer and deposited (register: W.G. J.A. Santos et al. / Journal of Ethnopharmacology 144 (2012) 802–805 Garcia 14.008) in the Herbarium of the Department of Botany at the Biology Institute of the State University of Campinas. The dry roots (560 g) of Jacaranda decurrens were extracted with 0.7 L of ethanol:water (70:30) at room temperature. Extracts were united, filtered and concentrated under vacuum and lyophilizer. During the treatment, the extract was dissolved in a hydroethanolic solution. The EJD (1.14 g) was dissolved in water (0.2 L) and fractionated by XAD-2 (Supelco, Bellefonte, PA, USA) resin column chromatography (30  3 cm) eluted with water (0.3 L), followed by methanol (0.2 L) and with ethyl acetate (0.2 L). The methanolic fraction (0.59 g) was dissolved in methanol (10 mL) and fractionated by Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden), column chromatography (100  3 cm) eluted with methanol (0.3 mL/min). In total, 29 fractions of 5 mL were collected. The fractions were combined according to their behavior by thin-layer chromatography (silica gel plates, ethyl acetate/npropanol/water, 140:8:80 by volume, upper phase). Fractions 17 (12.3 mg) resulted in the isolation of the compound oleanolic acid (3.9 mg). Fractions 18 was purified using polyvinypolypyrrolidone (Sigma, St. Louis, MO, USA) column chromatography (10  1 cm) eluted with methanol, leading to the identification of the compounds oleanolic and ursolic acids (9.8 mg). 2.2. Animals Adult male Wistar rats (90 days old, weighing approximately 340 g) from the Federal University of Mato Grosso do Sul, were maintained under controlled temperature (231 C), with a constant 12 h light–dark cycle and free access to food and water. The experimental procedures were in accordance with the Ethical Principles in Animal Research and approved by the Committee for Ethics in Animal Experimentation at the University Center of Grande Dourados (Protocol no. 334/10). 2.3. Acute oral toxicity The acute toxicity studies were conducted using the OECD (Organization for Economic Cooperation and Development)—Guideline 425 and ANVISA guidelines (Brazilian Health Surveillance Agency) (Brazilian Health Surveillance Agency (ANVISA), 2010; Organisation for Economic Co-operation and Development (OECD), 2008). After 12 h of fasting, the animals were divided into four groups. The treatments were performed by single oral administration at doses 0; 500; 1000 or 2000 mg/kg of body weight of EJD. Animals were observed for signs of toxicity during the first 0.5, 1, 2, 4, 8, and 12 h and at every 24 h for 14 days each. Behaviors parameters, death, the weight, the amount of water and feed were analyzed. After 14 days of treatment, the animals were weighed and anesthetized (ketamine and xylazine, 25 and 10 mg/kg, respectively). Next, blood samples were collected from the renal vein, with and without anticoagulant (Heparin sodium, Cristália). The blood samples were used to determine the hematology parameters (total and differential leukocyte count, hematocrit, hemoglobin and erythrocyte count), and the non-anticoagulated serum samples were used for biochemical analysis (aspartate aminotransferase—AST, alanine aminotransferase—ALT, gammaglutamyl transferase—d-GT, creatinine and urea) (Balani et al., 2011; Organisation for Economic Co-operation and Development (OECD), 2008). The biochemical parameters were determined using the semi-automatic Bioplus Bio200 equipment (Gold Analysis kits). After that, the animals were euthanised and the vital organs (liver, lung and right kidney) were removed, weighed (absolute and relative to body weight) were determined. For the histopathological 803 evaluation of these organs, the samples were fixed in 10% buffered formalin and processed for histological study by light microscopy. The parameters investigated were: reversible (degeneration) and irreversible cell damage (necrosis and apoptosis), leukocyte infiltration, congestion, extravasation of blood and fibrosis. 2.4. Carrageenan-induced rat paw edema Different groups of rats were orally treated with EJD (100 and 300 mg/kg), or vehicle. Another group was treated subcutaneously with dexamethasone (1 mg/kg). After 1 h, the animals received a solution of 50 mL carrageenan injection (300 mg/paw) in one of the hind paws. The other paw received the same volume of sterile 0.9% saline. The thickness of the paw edema was measured using a digital micrometer, before the treatment and at 0.5; 1; 2 and 4 h after the carrageenan. Results were expressed as micrometer and the difference between basal and postinjection values quantified as edema (Kassuya et al., 2009). 2.5. Determination of myeloperoxidase activity In this experiment, a carrageenan non-injected group was inserted and was called as naı̈ve (N) group. Six hours after carrageenan, the skins of paws were removed after euthanasia and the methodology of De Young et al. (1989) was performed with modifications. The tissue was homogenized in phosphate buffer (80 mM, pH 5.4) containing hexadecyltrimethylammonium bromide (0.5%). The homogenate was centrifuged at 12,000g (4 1C, 20 min). Thirty microliter of each supernatant were mixed with 100 ml of buffer (80 mM), 85 ml of phosphate buffer (0.22 M) and 15 ml of H2O2 (0.017%) in a 96-well plate. The reaction was initiated with 20 ml of 3,3,3-tetramethylbenzidine (dissolved in N,N-dimethylformamide). The plate was kept at 37 1C for 3 min and the reaction stopped by adding sodium acetate (30 ml, 1.46 M, pH 3.0). The enzymatic activity was determined by measuring the optical density (630 nm) and expressed as OD/mg of protein. Protein levels were measured in the supernatants (Bradford, 1976) using a mixture of each sample with Bradford’s reactant and after the absorbance was measured. 2.6. Statistical analyses Data are presented as mean7SEM. Difference between groups was evaluated by analyses of variance (one-way ANOVA) followed by Newman–Keuls test. Statistical differences were considered to be significant at P o0.05. 3. Results and discussion Jacaranda decurrens roots are empirically used for the treatment of inflammatory disorders (Nunes et al., 2003); however, its toxic and pharmacological effects on inflammatory experimental models have not been scientifically investigated. To our knowledge, the present study represents the first research into the anti-inflammatory effects and toxicity study of extract of Jacaranda decurrens roots. Several pharmaceutical products currently used to treat inflammation are not completely efficient in chronic disease and produce many adverse side effects. Therefore, it is necessary to develop more effective agents that are also less toxic. No changes in food consumption, water uptake or behavior (irritability, contortion, tremors, convulsions, lacrimation and piloerection) were observed in the animals after acute treatment with EJD. Additionally, the absolute and relative weight of the vital organs, the hematological parameters, biochemical 804 J.A. Santos et al. / Journal of Ethnopharmacology 144 (2012) 802–805 parameters (Table 1) and the histopathological analysis of the vital organs (data not shown) showed no statistically significant differences in any of the doses tested. These results confirm those reported by Zatta et al. (2009), which used the leaf ethanol extract of Jacaranda decurrens at doses of 2000 and 5000 mg/kg and also did not observe any signs of toxicity. Thus, it is suggested that the oral lethal dose (LD50) of Jacaranda decurrens is greater than 2000 mg/kg, and can be classified as a low toxicity extract according to Organisation for Economic Co-operation and Development (OECD) (2008). Table 1 Body weight, relative organs weights and hematological and biochemical parameters of animals exposed to Jacaranda decurrens in the acute toxicity study. Parameter Control (vehicle) 500 mg/kg 1000 mg/kg 2000 mg/kg Body weight (g) Liver (g/100 g) Lung (g/100 g) Creatinine (mg/dL) Urea (mg/dL) AST (U/L) ALT (U/L) d-GT (U/L) Erythrocyte count (  106) mm3 Hematocrit (%) Platelet count (  103/lL) Lynphocytes (%) Eosinophils (%) Monocyte (%) Neutrophils (%) 370.00 714.28 2.71 70.08 0.44 70.16 0.90 70.10 35.50 71.20 96.50 721.20 20.90 72.20 11.50 71.30 8.50 70.30 38.20 73.20 1048.00 724.00 73.10 74.10 1.10 70.10 3.60 70.30 22.20 73.20 388.00 7 19.21 2.70 7 0.07 0.47 7 0.03 1.10 7 0.00 35.50 7 1.20 104.20 7 16.90 23.40 7 3.90 11.80 7 2.40 8.60 7 0.40 40.107 3.30 1034.00 7 36.00 75.80 7 2.30 1.10 7 0.20 4.10 7 0.40 19.0 7 1.20 402.007 9.23 2.66 7 0.17 0.457 0.02 1.007 0.10 35.10 7 0.90 113.70 7 19.20 19.50 7 2.40 13.20 7 1.80 8.607 1.10 43.60 7 1.20 1053.00 7 36.00 78.50 7 1.20 1.207 0.20 3.307 0.30 19.40 7 4.20 391.00 7 13.21 2.73 7 0.15 0.44 7 0.02 1.007 0.00 35.00 7 0.80 108.30 7 16.20 22.70 7 2.20 12.20 7 1.30 8.70 7 0.50 42.90 7 0.50 1089.00 7 45.00 76.70 7 2.10 1.20 7 0.20 3.10 7 0.20 21.00 7 1.50 Values expressed as mean 7 SEM, n¼8 animals/group. P 40.05 by ANOVA. Fig. 1. Effect of Jacaranda decurrens on carrageenan-induced paw edema in rats. Animals received subcutaneously EJD (100 or 300 mg/kg), vehicle (V) or dexamethasone (Dex—1 mg/kg) and after 1 h, an intraplantar injection of carrageenan (300 mg/paw) was performed. In (A), the time-course of the inhibition induced by EJD and dexamethasone is shown. In (B), bars show the effect of different doses of EJD and Dex in paw edema (mm) 2 h after carrageenan injection. In (C), bars show the effect of different doses of EJD and Dex in activity of MPO (D.O) 6 h after carrageenan injection. Each bar represents the mean 7 SEM of 6 animals. # carrageenan naı̈ve (N) group. *Po 0.05, **P o0.01, ***P o 0.001, compared with the vehicle-treated group. Difference between groups were analyzed by analysis of variance (one-way ANOVA) followed by Newman–Keuls test. J.A. Santos et al. / Journal of Ethnopharmacology 144 (2012) 802–805 Acute inflammation is characterized by edema, fever, redness and pain. The edema is an effective measure of inflammation and is useful for quantifying induced cutaneous inflammation (Cabrini et al., 2011). Carrageenan induced edema is a biphasic model with multiple mediators acting in sequence to produce an inflammatory response. In the early phase (0–1 h) the release of histamine, serotonin and bradykinin occurs. The later stage (1–6 h) is correlated with an increased production of prostaglandins, COX-2 activation and NO release in the inflammatory response (Di Rosa et al., 1971). It was observed that EJD caused a reduction in paw edema induced by carrageenan. Fig. 1 shows that EJD at doses of 100 and 300 mg/kg (P o0.05) and (P o0.001) significantly reduced the edema of the hind paw after 2 h, and the 300 mg/kg extract showed the same decrease as the group treated with dexamethasone, and after 4 h it was observed that the groups treated with EJD showed a statistical significant reduction when compared to controls. According to the results of this study, it can be seen that EJD appears to act mainly on the initial phase of the carrageenan induced inflammatory response. This activity is dose-dependent, because with the increasing dose there was increased edema inhibition. Myeloperoxidase is effective in killing microorganisms and is abundant in primary azurophilic granules of neutrophils and monocytes after the activation by a variety of stimuli. Because of these characteristics, it has been used to quantify tissue MPO as an inflammatory marker (Kassuya et al., 2009). As for the treatment with EJD, only the group treated with 300 mg/kg showed a significant decrease in MPO activity, compared to the control group, as well as the positive control, dexamethasone (Fig. 1). The protocol used for measuring MPO evaluates the presence of active enzyme in the tissue, thus, a treatment that can reduce this parameter can act in two ways: by inhibiting the migration of neutrophils into the inflammatory site, or by preventing the enzyme activity (Uzkeser et al., 2012). Analyses performed using high performance liquid chromatography (HPLC) indicated the presence of ursolic acid in fractions of the Jacaranda decurrens extract (Carvalho et al., 2009), a compound whose molecule is similar to dexamethasone. In the present study, the oleanolic and ursolic acids were isolated of the EJD roots. The identification of these compounds was achieved by the experimental data (infrared, nuclear magnetic resonance, mass spectrometry). Their structures were also confirmed by comparing with the previously reported respective literature data (Mahato and Kundu, 1994; Taketa et al., 2004). Thus, ursolic acid could explain the anti-inflammatory action of the Jacaranda decurrens extract in the adult male rats in this study (Subbaramaiah et al., 2000). The present study shows that Jacaranda decurrens extract has anti-inflammatory properties in rats without causing acute toxicity. The properties observed may be due to the presence of bioactive components such as ursolic acid. 805 Acknowledgments The authors thank CAPES (Coordination for the Improvement of Higher Education Personnel) and FUNDECT (Foundation to Support the Development of Education, Science and Technology of the State of Mato Grosso do Sul) for the financial assistance. References Balani, T., Agrawal, S., Thaker, A.M., 2011. Hematological and biochemical changes due to short-term oral administration of imidacloprid. Toxicology International 18, 2–6. Batlouni, M., 2010. Anti-inflamatórios na~ o esteroides: efeitos cardiovasculares, cérebro-vasculares e renais. Arquivos Brasileiros de Cardiologia 94, 556–563. Bertoni, B.W., Telles, M.P.C., Malosso, M.G., Torres, S.C.Z., Pereira, J.O., Lourenc- o, M.V., Franc- a, S.C., Pereira, A.M., 2010. Genetic diversity in natural populations of Jacaranda decurrens Cham. determined using RAPD and AFLP markers. Genetics and Molecular Biology 33, 532–540. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Brazilian Health Surveillance Agency (ANVISA), 2010. Guide for Conducting Non-Clinical Safety Studies Required for Drug Development, vol. 1; p:37: Cabrini, D.A., Moresco, H.H., Imazu, P., Silva, C.D., Pietrovski, E.F., 2011. Analysis of the potential topical anti-inflammatory activity of Averrhoa carambola L. in mice. Evidence-based Complementary Alternative Medicine, 1–7. Carvalho, C.A.D., Lourenc- o, M.V., Bertoni, B.W., Franc- a, S.C., Pereira, P.S., Fachin, A.L., Pereira, A.M.S., 2009. Atividade antioxidante de Jacaranda decurrens Cham., Bignoniaceae. Revista Brasileira de Farmacognosia 19, 592–598. De Young, L.M., Kheifets, J.B., Ballaron, S.J., Young, J.M., 1989. Edema and cell infiltration in the phorbol ester-treated mouse ear are temporally separate and can be differentially modulated by pharmacologic agents. Agents and Actions 26, 335–376. Di Rosa, M., Giroud, J.P., Willoughby, D.A., 1971. Studies on the mediators of the acute inflammatory response induced in rats in different sites by carrageenan and turpentine. Journal of Pathology 104, 15–44. Kassuya, C.A., Cremoneze, A., Barros, L.F., Simas, A.S., Lapa, F.R., Mello-Silva, R., Stefanello, M.E., Zampronio, A.R., 2009. Antipyretic and anti-inflammatory properties of the ethanolic extract, dichloromethane fraction and costunolide from Magnolia ovata (Magnoliaceae). Journal of Ethnopharmacology 124, 369–376. Mahato, S.B., Kundu, A.P., 1994. Spectra of pentacyclic triterpenoids-a complication and some salient features. Phytochemistry 37, 1517. Nunes, G.P., Silva, M.F., Resende, U.M., Siqueira, J.M., 2003. Plantas medicinais comercializadas por raizeiros no Centro de Campo Grande, Mato Grosso do Sul. Revista Brasileira de Farmacognosia 13, 83–92. Organisation for Economic Co-operation and Development (OECD), 2008. Guideline 425:Acute Oral Toxicity–Up-and-Down-Procedure, 4. Head of Publications Service, Paris, p. 27. Subbaramaiah, K., Michaluart, P., Sporn, M.B., Dannenberg, A.J., 2000. Ursolic acid inhibits cyclooxygenase-2 transcription in human mammary epithelial cells. Cancer Research 60, 2399–2453. Taketa, A.T.C., Breitmaier, E., Schenkel, E.P., 2004. Tritepenos de Hyptis fasciculata Benth. Journal of the Brazilian Chemical Society 15, 205. Tresvenzol, L.M.F., Paula, J.R., Ricardo, A.F., Ferreira, H.D., Zatta, D.T., 2006. Study about informal trade of medicinal plants in Goiania and neighboring cities. Brazil. Revista Eletrônica de Farmácia 3, 23–28. Uzkeser, H., Cadirci, E., Halici, Z., Odabasoglu, F., Polat, B., Yuksel, T.N., Ozaltin, S., Atalay, F., 2012. Anti-inflammatory and antinociceptive effects of salbutamol on acute and chronic models of inflammation in rats: involvement of an antioxidant mechanism. Mediators of Inflammation 2012, 1–10. Zatta, D.T., Pimenta, F.C., Tresvenzol, L.M.F., Fiuza, T.S., Bara, M.T.F., Cunha, L.C., Pucci, L.L., Garrote, C.F.D., Oliveira, F.N.M., Paula, J.R., 2009. Study of antibacterial activity against Pseudomonas aeruginosa strains and acute toxicity of Jacaranda decurrens leaves. Latin American Journal of Pharmacy 28, 485–494.