Journal of Ethnopharmacology 138 (2011) 513–522 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm In vitro and in vivo toxicological evaluation of extract and fractions from Baccharis trimera with anti-inflammatory activity N.P.A. Nogueira a , P.A. Reis a , G.A.T. Laranja a , A.C. Pinto a , C.A.F. Aiub b , I. Felzenszwalb b , M.C. Paes a , F.F. Bastos a , V.L.F.C. Bastos a , K.C.C. Sabino a , M.G.P. Coelho a,∗ a Departamento de Bioquímica, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Av. Professor Manoel de Abreu, 444, PAPC, 4o andar, CEP 20550-170, Rio de Janeiro, RJ, Brazil b Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Av. Professor Manoel de Abreu, 444, PAPC, 4o andar, CEP 20550-170, Rio de Janeiro, RJ, Brazil a r t i c l e i n f o Article history: Received 11 May 2011 Received in revised form 5 September 2011 Accepted 8 September 2011 Available online 12 October 2011 Keywords: Baccharis trimera Cytotoxicity Genotoxicity Mutagenicity a b s t r a c t Ethnopharmacological relevance: Baccharis trimera (Less) DC. (Asteraceae), popularly known in Brazil as “carqueja”, have been used in folk medicine to treat gastrointestinal, hepatic and renal diseases, and inflammatory processes as rheumatism. Aim of the study: To evaluate the in vitro and in vivo toxicological effects of anti-inflammatory Baccharis trimera aqueous extract and fractions. Materials and methods: Aqueous extract of Baccharis trimera (AEBt) was produced by infusion in boiling water. After lyophylization AEBt was extracted with 80% ethanol, originating the ethanolic supernatant fraction (EFBt) and the aqueous sediment fraction (AFBt). Anti-inflammatory properties of AEBt, EFBt or AFBt (3, 30 or 300 ␮g/kg b.w.) were evaluated by the carrageenan-induced mouse paw edema using indomethacin (10 mg/kg) as positive control. The growth of rat hepatoma cells (HTC) and human embryo kidney epithelial cells (HEK) was determined by protein staining assay. Cytotoxicity was assayed by the tetrazolium salt (MTT) reduction. Cyclosporin was used as reference cytotoxic drug for spleen cells and doxorubicin for HTC and HEK cells. For in vivo toxicological evaluation SW male mice were daily and oral (gavage) treated with extract/fractions at 4.2 mg/kg or 42 mg/kg during 15 days. After treatment liver or kidney cells were submitted to comet assay to determine the DNA damage index, and the glutathione S-transferase activity was assayed towards ETHA (class Pi) and CDNB (several classes). Mutagenicity was evaluated by the Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102. Results: The anti-inflammatory effects of EFBt were higher than those of AEBt or AFBt. Mice treatment (3–300 ␮g/kg) with AFBt reduced the paw edema (3 h) at lower levels (29.2–37.3%; P < 0.01), than those observed for AEBt (44.7–54.2%; P < 0.001), EFBt (49.3–58.2%; P < 0.001) or indomethacin (64.6%, P < 0.001, 10 mg/kg). The growth of kidney cells (HEK) was inhibited by AEBt (IC50 182.6 ␮g/ml), EFBt (IC50 78.1 ␮g/ml) and AFBt (IC50 86.2 ␮g/ml), with lower effects on HTC hepatic cell (IC50 308.8 ␮g/ml, 396.5 ␮g/ml and 167.9 ␮g/ml, respectively). As evaluated by MTT test, AFBt exhibited cytotoxicity for HEK cells (IC50 372.5 ␮g/ml), but none for HTC ones; by the way, AFBt stimulated spleen cells (EC50 2.2 ␮g/ml) while cyclosporine, a cytotoxic reference drug inhibited them with IC50 of 0.42 ␮g/ml; the IC50 for doxorubicin for HEK and HTC cells was 0.28 ␮g/ml and 14.4 ␮g/ml, respectively, at 96 h. No mutagenic potential was observed. Mice treatment with AEBt or AFBt at 42 mg/kg for 15 days altered the kidney relative weight, but not at 4.2 mg/kg. Baccharis trimera did not change liver, spleen or popliteal lymph node relative weight. DNA damage index of kidney cells was observed on mice treated with AEBt/AFBt, but not on animals treated with EFBt, while DNA lesions were detected on liver cells only after AFBt treatment. The general activities of hepatic GST and Pi GST were reduced by EFBt and AFBt treatment, respectively. Conclusions: Baccharis trimera did not show mutagenicity, inhibited the GST activity, a hepatic detoxification enzyme, and induced in vivo (genotoxicity) and in vitro toxicological effects to kidney cells. © 2011 Elsevier Ireland Ltd. All rights reserved. ∗ Corresponding author. Tel.: +55 21 2587 6143; fax: +55 21 2587 6136. E-mail addresses: marsengpc@hotmail.com, marsen@uerj.br (M.G.P. Coelho). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.09.051 514 N.P.A. Nogueira et al. / Journal of Ethnopharmacology 138 (2011) 513–522 1. Introduction Baccharis trimera (Less) DC. (Asteraceae), a widespread South American woody perennial shrub, 0.5–4.0 m in height is popularly known in Brazil as “carqueja”. Flower and leave teas of Baccharis trimera and Baccharis genistelloides, have been used in folk medicine to treat gastrointestinal, liver and renal diseases, and inflammatory processes as rheumatism (Pio Correa, 1984; Sousa et al., 1991; Verdi et al., 2005). The popular use of Baccharis trimera consists of drinking 50–200 ml/day of an aqueous infusion (4–5 g) of the dried herb (Coimbra, 1942). Several species of the Baccharis genus as Baccharis articulate and Baccharis crispa (Gené et al., 1992), Baccharis trinervis (De las Heras et al., 1998), Baccharis calliprinos and Baccharis rethinoides (Gianello et al., 1999), Baccharis grisebachii, Baccharis incarum and Baccharis latifolia (Perez-García et al., 2001), Baccharis medullosa and B. rufescens (Cifuente et al., 2001), Baccharis pentlandii (Parejo et al., 2003), Baccharis grisebachii (Tapia et al., 2004), Baccharis genistelloides (Coelho et al., 2004), Baccharis obtusifolia, Baccharis latifolia, Baccharis pentlandii and Baccharis subulata (Abad et al., 2006), as well as Baccharis trimera (Gené et al., 1992, 1996) have been investigated for their chemical composition and anti-inflammatory properties. On the other hand, toxic effects to animals have been reported for Baccharis halimifolia, Baccharis cordifolia DC. and Baccharis megapotamica Sprengel (Manley et al., 1982; Jarvis et al., 1996; Rizzo et al., 1997) and toxicological studies have been evaluated for Baccharis trinervis (Sanchez-Palomino et al., 2002), Baccharis illinita (Baggio et al., 2003), and Baccharis genistelloides (Coelho et al., 2004). Baccharis trimera toxicological effects have been reported for the hydroethanolic extract of the plant (Grance et al., 2008), which induced in vivo toxicity (8.4 mg/kg) for kidney and liver cells of pregnant rats; although such alterations are reversible once administration is discontinued. No genotoxic effects for blood cells or liver were observed after treatment of mice with Baccharis trimera aqueous extract, but the frequency of micronucleus in bone marrow cells increased, indicating a chromosomal mutagenic activity (Rodrigues et al., 2009). Nevertheless, antimutagenic effect of Baccharis trimera has been described and attributed for the flavones genkwanin, cirsimaritin, hispidulin, and apigenin (Nakasugi and Komai, 1998). The multigene family of glutathione S-transferases (GST) isoenzymes acts by binding together reduced glutathione with electrophilic metabolites formed by biotransformation, producing conjugates generally more water soluble and less cytotoxic. This enzyme activity helps cells to detoxify harmful compounds, including those sourced from endogenous reactive oxygen species (Nordberg and Arn’er, 2001) and environmental exogenous toxicants (Hayes and Pulford, 1995). The activity of different classes of GST isoenzymes may be assayed using different substrates, such as 2,4 dichloro-nitrobenzene (CDNB), a rather nonspecific substrate, for several GST classes (general activity); while GST class Pi (GSTPi), the major class in the mouse liver (Raza et al., 1991), has a comparatively high activity with ethacrynic acid (ETHA) (McLellan and Hayes, 1987; Egaas et al., 1995). Although aqueous preparations of Baccharis trimera, as decoction and infusion, have been widely used to treat different pathologies in Brazil, there are still few reports of their toxicological potential. Thus, the aim of this work was to study the in vitro and in vivo toxicological effects of Baccharis trimera aqueous extract and fractions, both presenting anti-inflammatory properties. The in vivo toxicity of Baccharis trimera was evaluated by its genotoxic potential for kidney and liver cells and by the level of general and Pi class cytosolic GST activities in the liver after oral and daily treatment of mice with the Baccharis trimera samples for 15 days. The samples effects on mutagenicity (Salmonella/mammalian microsome and survival assays) and on liver, kidney and immune system cells in vitro toxicity were also evaluated. The effects of these samples on mutagenic test (Salmonella microsome/mammals and tests of survival) and in vitro toxicity with cells from liver, kidneys and immune system were also evaluated. 2. Materials and methods 2.1. Chemicals and reagents (3-[4,5-dimethylthiazole-2-il]-2,5-dipheniltetrazolium MTT bromide), 2-anthramine (2-AA), 2-aminofluorene (2AF), mitomycin C, 4-nitroquinoline 1-oxide (4NQNO), Benzo [a]pyren (BaP), 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid (ETHA) and Dulbecco’s modified Eagle’s medium (DMEM), were purchased from Sigma Chemical Co. (USA) and dimethyl sulfoxide (DMSO) from Merck Indústria Química (Brazil).Vogel-Bonner E medium and Bacto Nutrient Broth were purchased from Difco, BD (USA). Fetal bovine serum (FBS) was obtained from Gibco BRL (USA) and 1,2-dichloro-4-nitrobenzene (DCNB) was purchased from Fluka. All other chemicals and reagents were of the highest grade available. 2.2. Preparation of plant extract and fractions The Baccharis trimera specie was collected on August, 2001, in Friburgo, Rio de Janeiro, Brazil. A voucher sample was deposited (HB 86447) at the herbarium Bradeanum (Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil). Aerial parts of the dry plant were extracted by infusion (100 g/3 l) in boiling water for 45 min with agitation. Then, it was filtered and lyophilized (Heto Drywinner, Denmark), yielding about 8% of the initial mass (AEBt). This procedure was repeated five times. After homogeneity analysis of individual extracts by HPLC (Shim-pack C-8 column), they were polled and stored at −20 ◦ C. AEBt was submitted to extractions with 80% ethanol, originating the ethanolic supernatant fraction (EFBt) and the aqueous sediment fraction (AFBt), yielding 76% and 24% of AEBt initial mass, respectively. 2.3. Animals Male Swiss Webster (SW) mice, 29–35 g body weight (b.w.), fed ad libitum with a commercial rodent diet (Nuvilab Ltda., Curitiba, Brazil) and free access to drinking water were used in all experiments. For each experiment, mice were randomly selected into groups (n = 6/group). All experiments were performed under the consent and surveillance of the Ethical Committee for animals use in research of Biomedical Center, Universidade do Estado do Rio de Janeiro, Brazil (CEA-IBRAG/protocol 05/2009). 2.4. Carrageenan-induced mouse paw edema The carrageenan assay was carried out according to Levy (1969) with modifications. Male SW mice, fasted for 1 h with free access to water, were randomly selected to perform the study groups: control (vehicle); indomethacin (10 mg/kg b.w.); AEBt, EFBt or AFBt (3, 30 or 300 ␮g/kg b.w.). The extracts were dissolved in NaCl 0.9% (AEBt and AFBt) or ethanol 15% containing 1.25% Tween 20 (EFBt). One hour after the intraperitoneal (i.p.) administration of test solutions or of the appropriated vehicle (control with NaCl 0.9% or ethanol 15% with 1.25% Tween 20), edema was induced by a sub plantar injection of 50 ␮l of 0.6 g% (w/v) carrageenan suspension in NaCl 0.9% into the right hindpaw of each mouse. The swelling of the hindpaw was measured after 3 h (peak of edema) in a plethysmometer (7150 Ugo Basile), being compared with the volume of N.P.A. Nogueira et al. / Journal of Ethnopharmacology 138 (2011) 513–522 the same foot prior to the carrageenan challenge (edema index). The results were analyzed using the mean of edema index ± SD of each group and expressed as percentage of paw edema inhibition related to control group. 2.5. In vitro toxicological evaluations 2.5.1. Mammalian cell lines and murine splenocytes The rat hepatoma cells (HTC), and human embryo kidney epithelial cells (HEK), were purchased from Rio de Janeiro Cell Bank (BCRJ/UFRJ, Rio de Janeiro, Brazil). These adherent lines were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 ␮g/ml streptomycin (supplemented DMEM), being maintained at 37 ◦ C in 7% CO2 by standard cell culture techniques. Murine lymphocytes were isolated from SW male mice after euthanize with a CO2 chamber. The spleen was aseptically removed and the isolated splenocytes suspended in 2 ml of RPMI-1640 medium, treated with hypotonic ammonium chloride buffer (0.16 M NH4 Cl, 0.1 mM EDTA and 10 mM KHCO3 , pH 7.4) for haemolysis, washed with RPMI 1640 containing 2 mM EDTA, centrifuged (400 × g, 5 min), and then, resuspended in supplemented RPMI medium (5% FBS, 2 mM l-glutamine, 5 × 10−5 M 2-mercaptoethanol and antibiotics) and cultured at 37 ◦ C with 5% CO2 . Cell viability was determined by Trypan Blue dye (0.2%) exclusion. 2.5.2. Cytotoxicity by MTT reduction assay After culture of HEK, HTC (5.0 × 105 cells/ml) or spleen cells (2.0 × 106 cells/ml; activated with Con A 10 ␮g/ml) in 96 flatbottom well plates (Costar 3596, Cambridge, MA) in supplemented media (200 ␮l) for 72 h (splenocytes) or 96 h (HEK and HTC), in the presence or not (controls) of different concentrations of plant preparations, the tetrazolium salt (MTT) reduction by cells was determined according to Mossman (1983). After cell culture, 20 ␮l/well of MTT solution (5 mg/ml in phosphate buffered saline (PBS), pH 7.4) were added to cells and then incubated for additional 2 h at 37 ◦ C under CO2 atmosphere. DMSO (200 ␮l/well) were added to plate, after removal of 150 ␮l of culture supernatant, for formazan crystals dissolution and the absorbance determined at 570 nm using a microplate reader (␮Quant, Bio-Tek Instruments Inc., USA). Doxorubicin was used as cytotoxic drug for tumoral line cells, and cyclosporine for spleen cells. The mitochondrial reduction activity (MRA) was calculated as percent of control (no addition of plant sample). Incubation of AEBt, EFBt or AFBt with MTT in supplemented media without cells showed no significant MTT reduction. The formazan production is proportional to the total mitochondrial dehydrogenase activity in the well, which in turn correlates with the total number of viable cells. 515 animals in the control group received only the vehicle. At the end of experiment all animals were submitted to autopsy for examination of the anatomical localization and pathological changes of the organs. Selected organs like spleen, popliteal lymph node, liver and kidney were excised, trimmed of fat and connective tissue, and had the organ-to-body weight ratio calculated (g organ or mg for popliteal lymph node/g body weight × 100). 2.6.2. Single cell gel electrophoresis assay (comet assay) The alkaline version of the comet assay detects single and double strand breaks in DNA (Fairbairn et al., 1995). To detect these lesions, 10 ␮l of liver or kidney cell suspension from SW male mice were mixed with 120 ml of 0.5% low-melting-point agarose in PBS and added to microscope slides pre-coated with 1.5% normal-meltingpoint agarose in PBS. Slides were covered with a microscope cover slip and keep at 4 ◦ C for 5 min to gel, followed by immersion in icecold alkaline lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 20% DMSO, 2% Triton X-100, pH 10.0) for 1 h. Slides were then incubated for 20 min in ice-cold electrophoresis solution (0.2 M NaOH, 1 mM EDTA), followed by electrophoresis at 25 V/300 mA, for 25 min. After electrophoresis, slides were rinsed with water, allowed to dry at 37 ◦ C and was stained with ethidium bromide 20 ␮g/ml. DNA of individual cells was viewed using an epifluorescence microscope (Olympus), with 516–560 nm emission from a 50-W mercury light source, and image magnification of 200×. Quantification of DNA breakage was achieved by visual scoring of 50 randomly-selected cells per slide, classifying them into five categories based on the migration length and/or the perceived relative proportion of DNA in the tail. These five categories represent different degrees of DNA damage, allowing for qualitative evaluation, ranging from the absence of comet (type 0, undamaged cells) to maximum length comet (type 4, maximally damaged cells). Comets of type 1 are representative of cells with a minimal detectable frequency of DNA lesions while comets of types 2 and 3 are representative of cells with moderate-low and moderatehigh frequency of DNA lesions, respectively. Cellular comets were classified by investigators blinded to the animal experimental conditions from which the tissue samples were obtained. The results are expressed as DNA damage index, calculated as [(cell number of score 0 × 0) + (cell number of score 1 × 1) + (cell number of score 2 × 2) + (cell number of score 3 × 3) + (cell number of score 4 × 4)]; the results can run between 0 and 200. 2.5.3. Cell growth determination After culture of HTC (9.0 × 105 ) or HEK (6.75 × 105 ) cells onto 35 mm diameter culture dishes (Nunc) in supplemented DMEM for 24 h, medium was renewed and AEBt, EFBt or AFBt, solubilized in the same medium, added at different concentrations. Cell growth was determined daily by cell fixation with 5% TCA and staining with 1% bromophenol blue (BPB) in 1% acetic acid for 30 min (Lopes et al., 2000). Samples of 200 ␮l were then transferred to 96 flat-bottom well plates (Costar 3596, Cambridge, MA) and the absorbance determined at 570 nm by a microplate reader. Doxorubicin was also used as reference drug. 2.6.3. Glutathione S-transferase (GST) assays Considering that GST-Pi is the major class in mouse liver and a number of GST isoenzymes from liver are well known for conjugating CDNB, we assayed GST activities towards ETHA (class Pi) and CDNB (several classes). Activities of GST were determined in the hepatic cytosolic fraction (a 100,000 × g supernatant from homogenized liver) in 0.1 M Na-phosphate buffer containing 1 mM EDTA, following Habig et al. (1974) and Egaas et al. (1995) with minor modifications. The optimum pH for CDNB was 7.5, whereas ETHA was best conjugated at pH 6.5. The general GST activity was determined using 10 ␮g of protein, 1 mM CDNB in ethanol 4% and 1 mM GSH. The activity towards ETHA was determined with 500 mg of protein, 0.25 mM GSH and 0.2 mM ETHA. Every assay was carried out in triplicate with correction for non-enzymatic conjugation (Egaas et al., 1995). Protein concentration was determined according to Peterson (1977) using bovine serum albumin as standard (10–100 ␮g). 2.6. In vivo toxicological evaluation 2.7. Salmonella/mammalian microsome and survival assays 2.6.1. Treatment schedule and organ weights determinations Groups of six animals were daily treated by gavage (100 ␮l) with AEBt/AFBt/EFBt at 4.2 mg/kg or 42 mg/kg during 15 days. The The Salmonella typhimurium strains TA97, TA98, TA100, and TA102 described by Maron and Ames (1983) were provided by Dr. B.N. Ames (University of California, Berkeley, CA, USA) and their N.P.A. Nogueira et al. / Journal of Ethnopharmacology 138 (2011) 513–522 516 Table 1 Salmonella typhimurium strains used in this study. Strain Relevant genotype Kind of effect observed References TA97 TA98 TA100 TA102 hisD 6610 pKM101 uvrB rfa hisD 3052 pKM101 uvrB rfa hisG 46 pKM101 uvrB rfa hisG 428 (pAQ1) pKM101 rfa Frameshift mutation Frameshift mutation Base-pair substitution mutation Base-pair substitution mutation Levin et al. (1982) Ames et al. (1975) Ames et al. (1975) Levin et al. (1982) The multicopy plasmid pAQ1 carries the hisG428 mutation and a tetracycline resistance gene; rfa mutations cause partial loss of the lipopolysaccharide barrier and an increase in permeability to large molecules. The deletion () through uvrB gene also includes the nitrate reductase (chl) and biotin (bio) genes. genetic characteristics are listed in Table 1. The S9 fraction, prepared from the liver of Sprague-Dawley rats pretreated with a polychlorinated biphenyl mixture (Aroclor 1254), was purchased from Molecular Toxicology Inc. (Moltox TM, USA). The S9 metabolic activation mixture (S9 mix) was prepared according to Maron and Ames (1983) and was used in Salmonella/microsome tests and survival assays as described elsewhere (Lopes et al., 2000), with modifications. 2.7.1. Salmonella/mammalian microsome assay Mutagenicity was measured using the procedure described by Maron and Ames (1983) with Salmonella typhimurium strains TA97, TA98, TA100, and TA102 preincubated with different concentrations of extract/fractions Baccharis trimera with or without S9 mix. The assay mixture consisted of 100 ␮l extract/fractions Baccharis trimera (final concentration of 1, 10 or 100 ␮g/ml), 500 ␮l S9 mix (2.4, 4.78, 9.55, and 19.1 mg total protein/ml) or the same volume of 0.2 M sodium phosphate buffer, pH 7.4, in experiments without S9 mix, and 100 ␮l bacterial suspension (2 × 109 cells/ml). The mixture was preincubated at 37 ◦ C with shaking. After 20, 60, 90 and 120 min, 2 ml top agar (0.6% agar, 0.6% NaCl w/v, 50 ␮M lhistidine, 50 ␮M biotin, pH 7.4, 45 ◦ C) was added to the test tube and the final mixture was poured onto a Petri dish with minimal agar (1.5% agar, Vogel-Bonner E medium, containing 2% glucose). The plates were incubated at 37 ◦ C for 72 h, and the His + revertant colonies were counted. The results were expressed as a mutagenic index (MI = number of His + induced in the sample/number of spontaneous His + in the negative control). Positive controls were as described elsewhere (Aiub et al., 2003). Mutagenicity was considered positive when: (a) the number of revertant colonies in the assay was at least twice the number of spontaneous revertants (MI ≥ 2), (b) analysis of variance (ANOVA) revealed a significant response (P ≤ 0.05), and (c) a reproducible positive dose–response curve (P ≤ 0.01) was present. This evaluation was based on previously established criteria (Vargas et al., 1993, 1995). 2.7.2. Survival experiments To determine the cytotoxicity, cultures of each bacterial strain were preincubated in a final volume of 700 ␮l (100 ␮l of 2 × 109 cells/ml; 100 ␮l of 0.9% NaCl or extract/fractions Baccharis trimera for final concentration of 1, 10 or 100 ␮g/ml; and 500 ␮l of each concentration of S9 mix or 0.2 M sodium phosphate buffer, pH 7.4). After 60, 90 and 120 min, the cells were washed and diluted in 0.9% NaCl to obtain a suspension containing 2 × 103 cells/ml. A suitable aliquot (100 ␮l) of this suspension was plated on nutrient agar (0.8% Bacto nutrient broth, 0.5% NaCl and 1.5% agar), after which the plates were incubated at 37 ◦ C for 24 h and the colonies then counted. Each experiment was repeated at least twice, with triplicate. 2.8. Statistics The results were reported as mean ± SD. Variance analysis was established by one-way ANOVA. Significant differences between pairs of groups were calculated using Tukey’s multiple comparison Fig. 1. Effects of AEBt, its fractions and indomethacin (INDO) on carrageenaninduced mouse paw edema. Hindpaw edema was induced 1 h after i.p. administration of test solutions or vehicle by sub plantar injection of carrageenan (0.6%, w/v in NaCl 0.9%). Foot paw volume was measured at the peak of edema (3 h) by plethysmography. Each point represents the mean inhibition index of hindpaw edema of each group (n = 6/group) calculated as described in Section 2. The edema index of control groups were: 100.6 ± 5.9 (NaCl 0.9%) and 104.1 ± 16.2 (ethanol 15% with 1.25% Tween 20). b P < 0.01, and c P < 0.001, related to control group (vehicle) by Tukey’s test. tests or Student’s t-test, with significance level reported at P ≤ 0.05, as indicated. When appropriate, the mean effective concentration IC50 values (concentration that reduces 50% of control group response) or EC50 (concentration that stimulate 50% of control group response) were determined by non-linear regression using Graph Pad Prism, 5.0 (GraphPad Software Inc., San Diego, CA, USA). 3. Results 3.1. Antiedematogenic activity The aqueous extract of Baccharis trimera (AEBt), and its ethanolic (EFBt) and aqueous (AFBt) fractions, exhibited significantly inhibition of the paw edema induced by carrageenan for all doses tested, related to the control group (Fig. 1). The treatment with different doses (3–300 ␮g/kg) of AFBt reduced the paw edema at 3 h (peak of edema in the control group, vehicle) at lower levels (29.2–37.3%; P < 0.01), than that observed for AEBt (44.7–54.2%; P < 0.001) and EFBt (49.3–58.2%; P < 0.001). Indomethacin (10 mg/kg), used as a control drug, reduced (P < 0.001) 64.6% of the edema at 3 h (data not shown). 3.2. In vitro toxicological effects on mammalian cells The toxicological evaluations were initiated by the in vitro assays. The effects of AEBt, EFBt or AFBt on human embryo kidney epithelial cells (HEK) and rat hepatoma cells (HTC) growth were studied (Figs. 2 and 3). Different behavior of cell growth was observed for the cell lines along the culture. No cytotoxic effect for HEK cells was shown with AEBt up to 100 ␮g/ml (Fig. 2A), while N.P.A. Nogueira et al. / Journal of Ethnopharmacology 138 (2011) 513–522 A AEBt 40 30 20 10 0 0 AEBt 20 Control 1 μg/ml 10 μg/ml 100 μg/ml 500 μg/ml HTC cell growth (Abs 570 nm) HEK cell growth (Abs 570 nm) A c c c c 24 48 72 96 517 15 10 5 b b 0 120 a 0 24 Time (h) 1 μg/ ml 10 μg /ml 50 μg/ ml 100 μg/ ml B Control 5 a 24 c c 48 72 Time (h) c 96 EFBt 20 HTC cell growth (Abs 570 nm) 10 0 HEK cell growth (Abs 570 nm) 48 72 96 120 96 120 EFBt 15 0 C a Time (h) 15 10 5 0 24 1 μg/ ml 10 μg/ ml 50 μg/ ml 100 μg/ ml 10 C c c 0 24 72 Control 5 0 48 Time (h) AFBt 15 Control 1 μg/ ml 10 μg/ ml 50 μg/ ml 100 μg/ml 0 120 HTC cell growth (Abs 570 nm) HEK cell growth (Abs 570 nm) B Control 1 μg/ ml 10 μg/ ml 100 μg/ ml 500 μg/ml c 48 72 Time (h) 96 120 AFBt 20 1 μg/ ml 10 μg/ ml 50 μg/ ml 100 μg/ml 15 10 5 a 0 0 Fig. 2. Growth kinetics of HEK cells in the presence of AEBt (A), EFBt (B) and AFBt (C). Exponentially growing cells were cultured in the absence (control cells) or in the presence of different concentrations of extracts/fractions. At the indicated times, the total cells number was determined by bromophenol blue staining (BPB) of proteins, as described in Section 2. Each point represents the mean absorbance at 570 nm ± SD of culture duplicates (representative of three experiments). The calculated IC50 at 96 h were 182.6 ␮g/ml (AEBt), 78.1 ␮g/ml (EFBt) and 86.2 ␮g/ml (AFBt). a P < 0.05, c P < 0.001, related to control culture by Tukey’s test. significant inhibition can be observed at 500 ␮g/ml, reaching inhibition rates of 94.3% just after 24 h of culture (P < 0.001), with IC50 of 182.6 ␮g/ml. EFBt (Fig. 2B) and AFBt (Fig. 2C) inhibited cell proliferation at the higher tested concentration (100 ␮g/ml) just after 24 h of culture, with reduction indexes (P < 0.001) after 96 h of culture of 94.9% and 79.9% and IC50 of 78.1 ␮g/ml and 86.2 ␮g/ml, respectively. The AEBt (Fig. 3A) has inhibited HTC cell line proliferation only at 500 ␮g/ml, exhibiting cell growth inhibition of 57.3% (P < 0.001), after 96 h of culture and IC50 308.8 ␮g/ml. Different from HEK cells, EFBt induced no significant difference on HTC cells proliferation (Fig. 3B) with IC50 396.5 ␮g/ml while AFBt inhibited HTC cell growth only at 96 h of culture (Fig. 3C) with reduction of 53.9% (P < 0.05) and IC50 167.9 ␮g/ml. The EFBt or AFBt cytotoxicity was also studied on HEK, HTC and mice spleen cells (Fig. 4). Only the AFBt fraction induced cytotoxic effect (viability inhibition of 30%) and only HEK cells were sensitive to it (Fig. 4C), with IC50 372.5 ␮g/ml. Doxorubicin, used as positive control drug, exhibited at 96 h IC50 14.4 ␮g/ml and 0.28 ␮g/ml for HTC cells and HEK cells, respectively. By the way, AFBt treatment stimulated mitochondrial reduction activity of spleen cells in a concentration dependent Control 24 48 72 96 120 Time (h) Fig. 3. Growth kinetics of HTC cells in the presence of AEBt (A), EFBt (B) and AFBt (C). Exponentially growing cells in DMEM with 10% FBS were cultured in the absence (control cells) or in the presence of different concentrations of extracts/fractions. At the indicated times, the total number of cells were determined by bromophenol blue staining (BPB) of proteins, as described in methods. Each point represents the mean absorbance at 570 nm ± SD of culture duplicates (representative of three experiments). The calculated IC50 at 96 h were 308.8 ␮g/ml (AEBt), 396.5 ␮g/ml (EFBt) and 167.9 ␮g/ml (AFBt). a P < 0.05, b P < 0.01, related to control culture by Tukey’s test. manner (Fig. 4A), with EC50 2.2 ␮g/ml, reaching 104% of stimulation with 100 ␮g/ml, while no significant difference was observed with any dose of EFBt. The IC50 of cyclosporine for spleen cells was 0.42 ␮g/ml. 3.3. In vivo toxicological studies 3.3.1. Effects on the body and organ weight Oral and daily administration of AEBt or its fractions to healthy mice for 15 days did not induce clinical symptoms of toxicity or death (data not shown). All groups increased the body weight about 3.2 ± 1.6 g during the treatment. Table 2 shows no significant difference in the spleen or liver relative weights after treatment with 4.2 mg/kg of AEBt or its fractions AFBt and EFBt related to the control group. At the higher dose (42 mg/kg), slight variation on the kidney relative weights (P < 0.05) was observed in mice treated with AEBt or AFBt. N.P.A. Nogueira et al. / Journal of Ethnopharmacology 138 (2011) 513–522 518 Table 2 Effects of the AEBt/fractions treatment on relative organ weights of healthy mice. Group Relative weight (%) Liver Control AEBt 4.2 mg/kg AEBt 42 mg/kg Control AFBt 4.2 mg/kg AFBt 42 mg/kg Control EFBt 4.2 mg/kg EFBt 42 mg/kg 6.7 6.7 5.9 5.8 5.4 5.4 5.4 5.8 5.5 ± ± ± ± ± ± ± ± ± 0.8 0.5 0.6 0.3 0.5 0.4 0.8 0.2 0.2 Kidneys Spleen 0.62 0.59 0.67 0.55 0.50 0.46 0.56 0.58 0.59 0.40 0.37 0.43 0.56 0.51 0.44 0.57 0.53 0.64 ± ± ± ± ± ± ± ± ± 0.03 0.02 0.04a,d 0.07 0.03 0.02a 0.03 0.02 0.05 ± ± ± ± ± ± ± ± ± a 0.05 0.07 0.04 0.05 0.10 0.07 0.07 0.07 0.10 Popliteal lymph node ND ND ND 7.17 ± 2.67 13.46 ± 3.07 10.88 ± 4.42 6.90 ± 4.80 7.62 ± 2.23 10.26 ± 0.11 Animals have been treated orally and daily for 15 days. Relative weight was calculated as organ weight (g or a mg)/body weight (g) × 100 and are expressed as means ± SD (n = 6 per group). ND – not determined. a P < 0.05 vs. control group (vehicle) and d P < 0.01 vs. AEBt 4.2 mg group, by Student’s t-test. MTT reduction (%) Spleen cells A 250 AFBt EFBt Cyclosporin 200 c 150 c c a 100 c 50 c c c c 0 0.1 1 10 100 Concentration (μg/ml) MTT reduction (%) HTC cells B 200 AFBt EFBt Doxorubicin 150 100 50 c a c c 0 0.1 1 10 100 Concentration (μg/ml) MTT reduction (%) HEK cells C 200 AFBt Doxorubicin EFBt 150 100 c 50 Fig. 5. GST activity towards: (A) 1-chloro-2,4-dinitrobenzene (CDNB, total GST activity); (B) ethachrynic acid (ETHA, GST class Pi activity), in hepatic microsomes from SW male healthy mice treated during 15 days with oral doses of AFBt and EFBt (n = 5/group). All analysis was performed in duplicate. a P < 0.05 vs. control; # P < 0.05 vs. 4.2 mg/kg group by Tukey’s test. a Fig. 4. Mitochondrial reduction activity (MRA) of spleen cells (A), HTC cells (B), or HEK cells (C) in the presence of EFBt or AFBt. The cells were incubated in the absence (control) or presence of different concentrations of AEBt fractions for 72 h (splenocytes) or 96 h (HEK and HTC). MRA was determined by the MTT assay, as described in Section 2. EC50 AFBt 2.2 ␮g/ml and IC50 cyclosporine 0.42 ␮g/ml for spleen cells. IC50 doxorubicin 14.4 ␮g/ml and 0.28 ␮g/ml for HTC and HEK cells, respectivelly. No inhibition was observed for EFBt and AFBt for HTC or HEK cells. The results express the mean percentage of control ± S.D. of three experiments with duplicates. a P < 0.05, c P < 0.001, related to control culture by Tukey’s test. animals treated with vehicle (control), AEBt, AFBt and EFBt were examined by the comet assay. We did not observe significant difference in liver DNA damage index between groups treated with AEBt (Table 3), with predominance of undamaged cells (score 0) and low levels of maximal DNA lesions (score 4). However, AEBt treatment induced kidney DNA damage at 4.2 and 42 mg/kg. The treatment of healthy mice with AFBt at both doses increased the number of liver and kidney cell lesions when related to control and 4.2 mg/kg treated group (P < 0.05), as can be observed by their higher DNA damage index and the increase in the number of liver cells with score 4. No significant differences in the kidney and liver DNA damage were observed on mice groups treated with EFBt in relation to untreated groups. 3.3.2. Single cell gel electrophoresis assay To detect potential in vivo genotoxic effects of AEBt/fractions to liver and kidney cells, the DNA of these tissues from healthy 3.3.3. Glutathione S-transferase levels As can be seen in Fig. 5A, the general GST activity (assayed with CDNB) was reduced 37.6% with the treatment of mice with 42 mg EFBt/kg (P < 0.05). Class Pi GST activity was reduced 47.8% in mice c c c 0 0.1 1 10 100 Concentration (μg/ml) N.P.A. Nogueira et al. / Journal of Ethnopharmacology 138 (2011) 513–522 519 Table 3 Effects of AEBt/fractions on DNA damage of healthy mice. Group Kidney Control AEBt 4.2 mg/kg AEBt 42 mg/kg Control AFBt 4.2 mg/kg AFBt 42 mg/kg Control EFBt 4.2 mg/kg EFBt 42 mg/kg Liver Damage index % score 0 % score 4 Damage index % score 0 % score 4 14.0 21.5 24.0 19.3 23.7 30.3 22.2 18.5 24.3 86.5 74.5 78.3 80.7 73.3 68.5 84.0 84.0 80.7 3.5 4.0 6.0 3.3 4.8 5.5 4.9 3.3 6.3 13.5 22.2 22.7 17.8 24.8 43.0 34.6 35.8 45.3 89.0 81.0 77.0 89.5 73.6 69.0 9.0 64.3 57.2 3.5 5.5 5.0 2.0 3.2 13.0 71.1 6.1 8.2 ± ± ± ± ± ± ± ± ± 7.1 3.4a 2.7 b 2.9 7.4a 8.5c,d 7.6 6.8 4.3 ± ± ± ± ± ± ± ± ± 5.3 11.3 16.8 4.3 4.5 a 7 .5 c,d 13.0 15.2 The comet assay (n = 6/group) was evaluated by visual scoring of 50 randomly selected cells per slide, scored from 0 to 4. The values are expressed as DNA damage index (mean ± SD), calculated as  (cell number of scoren × scoren ), where subscript “n” can turn among 0 (without DNA lesion)–4 (total DNA lesion) and the damage index from 0 to 200. Control group was treated (p.o.) with the vehicle. a P < 0.05; b P < 0.01; c P < 0.001 vs. control group and d P < 0.05 vs. 4.2 mg group by Student’s t-test. treated with AFBt (42 mg/kg) in comparison to the groups control or treated with 4.2 mg/kg (Fig. 5B). On the other hand, Pi activity (Fig. 5B) was slightly higher in animals treated with 4.2 mg EFBt/kg (P < 0.05) or 42 mg EFBt/kg (statistically not significant). 3.3.4. Mutagenicity Tables 4–6 show the mutagenicity induction (M.I.) and survival indexes of Baccharis trimera aqueous extract or fractions, after 60 min of pre-incubation, with or without 19.1 mg protein of S9 mixture/plate. For all tested strains, AEBt (Table 4), EFBt (Table 5) and AFBt (Table 6) were not mutagenic either in the presence or in the absence of S9 mix. However, toxicity was detected in TA97 and TA98 strains for AEBt (Table 4) and in TA97 for EFBt (Table 5), in the absence of S9 mix (cell survival lower than 70% in comparison to the control group), for all used concentrations of AEBt in TA98 and at 100 ␮g/ml of AEBt and EFBt in TA97. In the presence of S9 mix, cytotoxic response was observed only for 100 ␮g/ml AEBt in TA97. 4. Discussion and conclusion This work evaluated the toxicological potential of the Baccharis trimera aqueous extract and fractions because it is the most popular preparation of this herb. The anti-inflammatory potential of AEBt, AFBt and EFBt was also demonstrated, by the acute inflammation model in mice induced with carrageenan injection into hindpaw using indomethacin as positive control. This procedure evokes a potent local acute inflammatory reaction (Levy, 1969) with a biphasic profile (Henriques et al., 1987). The paw edema evaluation was made at 3 h, during the first phase of response in mice (maximal edema development at 2–4 h). The 2–5 h interval of the first phase in this model, in which neutrophils are the predominant homing cells, is very sensitive to cyclooxygenase inhibitors (COX) (Sugishita et al., 1981; Nishikori et al., 2002). Therefore, as AEBt and fractions reduced significantly the paw edema at 3 h, their antiedematogenic properties could be related with the synthesis inhibition of arachidonic acid metabolites. Similar results were demonstrated by Gené et al. (1992, 1996) with Baccharis trimera extracts. The toxicological studies of Baccharis trimera were initiated by the in vitro evaluation of mammalian cell cytotoxicity performed with rat hepatoma cells (HTC cell line), as liver cells are pivotal in detoxification reactions and general metabolism control; with human embryo epithelial kidney cells (HEK cell line), as kidney cells are potentially targeted by substances present in Baccharis trimera; and with splenocytes, as representative of immune system cells. Mitochondrial reduction activity evaluation (MTT assay) indicated cytotoxicity only for treatment with AFBt and only for HEK cells (Fig. 2). On the other hand, stimulatory effects of spleens Table 4 Salmonella/mammalian microsome assay with aqueous extract of Baccharis trimera. Strain AEBt M.I.a TA97 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 1.0 0.8 0.9 0.8 2.3 1.0 2.1 2.1 1.5 15 1.0 1.0 1.0 1.0 4.9 1.0 1.2 1.2 1.2 3.2 TA98 TA100 TA102 His+b 339 278 311 267 440 13 27 27 20 201 267 273 263 277 1320 183 223 222 217 591 % survivalc M.I.a His+b 100 100 100 69* 1.0 0.9 0.9 1.0 4.3 1.0 1.1 1.1 1.1 7.7 1.0 1.0 1.2 1.2 3.5 1.0 0.8 0.8 0.9 3.8 151 140 138 151 648 27 34 29 30 210 233 238 274 271 812 285 242 237 265 1090 100 43* 43* 30* 100 99 97 71 100 97 97 91 % survivalc 100 89 71 62* 100 96 80 73 100 99 87 87 100 82 80 70 a Mutagenic index (number of His + induced in the sample/number of spontaneous His + in the negative control); b revertant colonies/plate; c percent survival calculated in relation to negative control. The dose was considered toxic when percent survival < 70%. Positive controls (P.C.): 4-nitroquinoline 1-oxide (1.0 ␮g/ml) for Salmonella typhimurium TA97, TA98, TA100 and Mitomycin C (0.2 nl/ml) for TA102, without S9 mix; 2-aminofluorene (10.0 ␮g/ml) for TA97, TA98, TA102 and Benzo [a]pyrene (5.0 |ig/ml) for TA100, with S9 mix. Bold numbers denotes toxic effects (*P < 0.05). N.P.A. Nogueira et al. / Journal of Ethnopharmacology 138 (2011) 513–522 520 Table 5 Salmonella/mammalian microsome assay with ethanolic fraction of Baccharis trimera. Strain EFBt −S9 mix M.I.a TA97 TA98 TA100 TA102 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 1.0 1.0 1.0 1.0 2.3 1.0 0.9 1.0 1.0 8.0 1.0 0.9 0.9 1.0 4.9 1.0 1.1 1.1 1.2 3.3 +S9 mix His+b 272 290 290 285 440 25 25 25 23 201 267 252 251 282 1320 177 188 201 209 591 % survivalc M.I.a His+b % survivalc 100 76 70 68* 1.0 0.8 0.7 0.8 3.4 1.0 0.8 0.7 0.9 5.9 1.0 1.0 1.1 1.0 5.8 1.0 1.0 1.1 1.0 3.8 190 152 140 145 648 55 44 40 52 325 139 141 155 144 812 53 51 51 51 202 100 93 81 94 100 97 91 72 100 99 87 89 100 99 91 91 100 100 100 100 100 94 92 92 100 100 97 97 a Mutagenic index (number of His + induced in the sample/number of spontaneous His + in the negative control); b revertant colonies/plate; c percent survival calculated in relation to negative control. The dose was considered toxic when percent survival < 70%. Positive controls (P.C.): 4-nitroquinoline 1-oxide (1.0 ␮g/ml) for Salmonella typhimurium TA97, TA98, TA100 and Mitomycin C (0.2 nl/ml) for TA102, without S9 mix; 2-aminofluorene (10.0 ␮g/ml) for TA97, TA98, TA102 and Benzo [a]pyrene (5.0 ␮g/ml) for TA100, with S9 mix. Bold number denotes toxic effects (*P < 0.05). cells were observed for AFBt while no effect was observed for EFBt (Fig. 2A). The results of cell growth also showed higher sensitivity of HEK cells to Baccharis trimera samples than HTC cells, presenting severe reduction of cell proliferation by EFBt or AFBt treatment (100 ␮g/ml), which was not observed with HTC cells (Fig. 2). These results suggest specific toxic effect of Baccharis trimera on kidney cells. No mutagenic potential was observed for any Baccharis trimera samples in the Salmonella typhimurium microsomal activation assay using different strains (TA97a, TA98, TA100 and TA102), in the presence or absence of S9 mixture (Tables 4–6). However, cytotoxicity was observed for TA97 with AEBt and EFBt at 100 ␮g/ml and for TA98 with all AEBt concentrations tested. The in vivo toxicological evaluation after 15 days of oral and daily treatment with AEBt, AFBt or EFBt was performed in male SW mice. This evaluation was performed with a dose equivalent to the daily AEBt dose recommended to humans (4.2 mg of AEBt dried extract/kg b. w.) and with 42 mg/kg b.w. (ten times higher than the human dose). This higher dose is important for overdose conditions and long-term treatments, resulting in the herb cumulative buildup in the organism. Mice treated with AEBt or AFBt exhibited significant alteration only on kidney relative weight (Table 2) at 42 mg/kg b.w. Although AFBt has stimulated mitochondrial activity of spleen cells (Fig. 4A), which is generally proportional to cell viability or proliferation, no alteration was observed in spleen relative weight, as observed by Grance et al. (2008). The high percentage of red Table 6 Salmonella/mammalian microsome assay with aqueous fraction of B.trimera. Strain AFBt −S9 mix a TA97 TA98 TA100 TA102 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. 0 1 ␮g/ml 10 ␮g/ml 100 ␮g/ml P.C. +S9 mix +b M.I. His 1.0 0.9 0.9 1.0 2.1 1.0 1.1 0.8 0.8 6.1 1.0 0.8 0.9 0.9 5.3 1.0 0.9 1.0 0.9 3.0 207 189 186 201 440 33 37 27 27 201 249 216 240 210 1320 192 177 189 178 591 c % survival 100 89 88 85 100 98 98 80 100 100 100 84 100 93 93 93 M.I.a His+b % survivalc 1.0 1.0 1.1 1.1 3.6 1.0 1.0 0.8 0.8 7.5 1.0 0.9 1.0 1.1 8.6 1.0 1.0 1.0 1.0 3.8 180 188 201 197 648 43 43 35 37 325 70 65 74 75 812 52 51 51 49 202 100 91 91 87 100 100 88 88 100 99 96 91 100 99 97 92 a Mutagenic index (number of His + induced in the sample/number of spontaneous His + in the negative control); b revertant colonies/plate; c percent survival calculated in relation to negative control. The dose was considered toxic when percent survival < 70%.Positive controls (P.C.): 4-nitroquinoline 1-oxide (1.0 ␮g/ml) for Salmonella typhimurium TA97, TA98, TA100 and Mitomycin C (0.2 ␮l/ml) for TA102, without S9 mix; 2-aminofluorene (10.0 ␮g/ml) for TA97, TA98, TA102 and Benzo [a]pyrene (5.0 ␮g/ml) for TA100, with S9 mix. N.P.A. Nogueira et al. / Journal of Ethnopharmacology 138 (2011) 513–522 blood cells in spleen, compared to lymphocytes and macrophages, can be contributing for the non altered spleen relative weight. The tendency of lymph node to increase its relative weight reinforces this hypothesis, since lymphocytes and antigen presenting cells are the major cells in this lymphoid organ. To evaluate if Baccharis trimera could contain substances potentially genotoxic to mammalian cells, DNA of liver and kidney cells of the treated animals was examined by the comet assay. In the alkaline version of the comet assay, which detects a broad spectrum of DNA lesions, including double and single strand breaks (BrendlerSchwaab et al., 2005), DNA evaluation of kidney cells indicated the presence of higher damage index in animals treated with both doses of AEBt and AFBt, but none on those treated with EFBt. Liver cells showed no detectable DNA lesions after treatment of animals with AEBt or EFBt, while AFBt treatment induced genotoxic effect on both doses. Thus, kidney cells showed higher in vivo sensitivity to AEBT genotoxic effects than liver ones. These results are reinforced by the absence of genotoxic effect (comet assay) in liver from mice treated orally for three consecutive days with 500, 1000 or 2000 mg/kg of Baccharis trimera aqueous extract, and presence of toxic effects for kidney cells of pregnant rats orally treated with hydroethanolic extract of Baccharis trimera (8.4 mg/kg) for 19 days (Grance et al., 2008). The results also indicate that AFBt seems to concentrate the genotoxic agents of AEBt for both liver and kidneys cells. As the conjugation activity of xenobiotics with glutathione is an important detoxifying reaction, the Baccharis trimera effects on general and specific GST activities in the liver were evaluated in this work. Although reduction in the hepatic GST-Pi activity has been observed after treatment of mice with 42 mg AFBt/kg (Fig. 5B), no change was observed in the GST general activity with the same treatment (Fig. 5A). This finding indicates that the AFBt might be weakening cytosolic GST antioxidant activity in the liver of mice. On the other hand, as hepatic GST-Pi activity was slightly increased in mice treated with EFBt (Fig. 5B) and general GST activity was diminished after mice treatment with 42 mg EFBt/kg (Fig. 5A), one may conjecture that GST activities other than Pi were reduced by EFBt. Furthermore, the increase of GST-Pi activity after EFBt treatment might be indicating that this Baccharis trimera fraction stimulates detoxification and antioxidant activities. In this regard, it has been published that extracts from leaves of Ginkgo biloba, which has been used for their high antioxidant property, also induced GST-Pi activity in human and mice hepatoma cell lines (Liu et al., 2009). The flavonoids represent a large and important group of polyphenolic compounds. They have been reported to have therapeutic potential effects by their antioxidant (Chen et al., 1990), anti-inflammatory, antihepatotoxic, antiulcer (Ferrandiz and Alcaraz, 1991), and antimutagenic (Edenhardder et al., 1993) activities. Furthermore, several works have been reporting that plant polyphenols inhibit the GST activity (Kawabata et al., 2000; Gyamfi et al., 2004; Krajka-Kuzniak et al., 2008). Notably, a plant polyphenol, the tannic acid, injected i.p. in mice, decreased the general liver GST activity (Krajka-Kuzniak and Baer-Dubowska, 2003). As flavonoids have been reported in several species of the Baccharis genus (Weimann et al., 2002; Akaike et al., 2003; Feresin et al., 2003; Rodrigues et al., 2009), the anti-inflammatory action and the inhibitory effects of Baccharis trimera on GST activities (Fig. 2) could be at least partially attributed to these substances. However, other polyphenolic compounds (tannins, saponins), alkaloids and isoprenoids (diterpenes, triterpenes) have also been described in this specie (Simões-Pires et al., 2005; Verdi et al., 2005; Silva et al., 2007; Lago et al., 2008). Current studies are on the way to identify these substances. In conclusion, the Baccharis trimera samples showed similar anti-inflammatory potential as indomethacin at lower doses, did not show mutagenicity, inhibited the activity of an important 521 detoxification hepatic enzyme, the glutathione S-transferase, and induced in vivo (genotoxicity) and in vitro toxicological effect for kidney cells. These results suggest that care should be taken in terms of overdose and prolonged treatments, which result in accumulation of the medicinal herb in the organism. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments We would like to thank the technical assistance from LIABPPN Laboratory. This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Universidade do Estado do Rio de Janeiro (UERJ). 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