Evaluation of genotoxicity and subchronic toxicity of the standardized leaves infusion extract of Copaifera malmei Harms in experimental models

Journal of Ethnopharmacology 211 (2018) 70–77

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Evaluation of genotoxicity and subchronic toxicity of the standardized leaves infusion extract of Copaifera malmei Harms in experimental models

MARK

Eduarda Pavana, Amilcar Sabino Damazob, Larissa Maria Scalon Lemosc, Bulus Adzua,d, Sikiru Olaitan Baloguna,e, Karuppusamy Arunachalama, ⁎ Domingos Tabajara de Oliveira Martinsa, a

Área de Farmacologia, Departamento de Ciências Básicas em Saúde, Faculdade de Medicina, Universidade Federal de Mato Grosso (UFMT), Cuiabá, Brazil Área de Histologia e Biologia Celular, Departamento de Ciências Básicas em Saúde, Faculdade de Medicina, Universidade Federal de Mato Grosso (UFMT), Cuiabá, Brazil c Faculdade de Ciências da Saúde, Universidade do Estado de Mato Grosso (UNEMAT), Cáceres, Brazil d Department of Pharmacology and Toxicology, National Institute for Pharmaceutical Research and Development (NIPRD), Abuja, Nigeria e Curso de Farmácia, Faculdade Noroeste do Mato Grosso, Associação Juinense de Ensino Superior (AJES), Juína, Mato Grosso, Brazil b

A R T I C L E I N F O

A B S T R A C T

Keywords: Copaifera malmei Leaves Safety Genotoxicity Subchronic toxicity NOAEL

Ethnopharmacological relevance: Copaifera malmei Harms (Fabaceae), known mainly as óleo-mirim, is a native and endemic plant found in the states of Mato Grosso and Goiás of Brazil. The plant's leaves infusion is popularly used by riverine communities of the northern Araguaia microregion, Mato Grosso, Brazil, for the treatment of gastric ulcers and inflammatory diseases of the respiratory tract. The gastric antiulcer activity of the standardized leaves infusion extract of Copaifera malmei (SIECm) in rodents has been reported. The objective of this study was to advance the investigation of the safety profile of SIECm by evaluating the genotoxicity and subchronic toxicity using in vitro and in vivo experimental models. Materials and methods: SIECm was prepared by infusion, by incubating the powdered dried leaves material in boiled water for 15 min. In vitro genotoxicity of SIECm (10, 30 or 100 μg/mL) was assessed by micronucleus and comet tests using Chinese hamster ovary (CHO-k1) epithelial cells. The evaluation of subchronic toxicity profile was performed by daily oral administration of SIECm (100, 400 or 1000 mg/kg) to Wistar rats for 30 days. Clinical observations of toxicological related parameters were done every 6 days. After the treatment period, blood was collected for hematological and biochemical analysis, and some organs were removed for macroscopic and histopathological analysis. Results: In the micronucleus assay, SIECm demonstrated anti-mutagenic activity. In the comet assay, SIECm presented anti-genotoxic effect preventing DNA damage at all the three concentrations tested with pre-treatment, while the same effect was only observed in the co-treatment at the lowest concentration. Post-treatment with SIECm increased the genetic damage induced by hydrogen peroxide (H2O2) at the highest concentration. In the subchronic toxicity test, few changes were observed, such as increase in feed consumption in the group of animals treated with 100 mg/kg of the SIECm, which reversed after 6 days. There were no macroscopic, histological and relative weights changes in the organs of animals treated with SIECm. No toxicologically relevant changes were observed in the hematological analysis. Subchronic administration of SIECm reduced levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in animals treated with 100 mg/kg and serum triglyceride levels at 400 and 1000 mg/kg. However, the hematological and biochemical changes observed are within the physiological ranges for this animal species. Conclusion: The results demonstrate that SIECm is not genotoxic, and does not present toxicity when used orally for up to 30 days. In addition, it showed protection to the genetic damage induced by H2O2. The SIECm therefore has a high safety margin for therapeutic use.

⁎

Corresponding author. E-mail address: [email protected] (D.T.d.O. Martins).

http://dx.doi.org/10.1016/j.jep.2017.09.027 Received 24 June 2017; Received in revised form 4 September 2017; Accepted 18 September 2017 Available online 21 September 2017 0378-8741/ © 2017 Elsevier B.V. All rights reserved.

Journal of Ethnopharmacology 211 (2018) 70–77

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1. Introduction

2. Materials and methods

Medicinal plants have a long history of use for the prevention, mitigation and cure of various diseases, and have been important sources for the discovery of new drugs or use as herbal medicines. Most of these plants have been used for long periods of time based on traditional knowledge; and because of this, they are often considered safe. Despite their claimed beneficial effects and proven scientific values, many of these medicinal plants have not been subjected to exhaustive toxicological tests to establish their safety profile (Wiesner, 2014). In vivo preclinical toxicological tests begin with the acute toxicity assessment. When there is indication of continuous use of the drug for up to six months in a year in humans, the subchronic toxicity test in animals is carried out (OECD, 2008). The subchronic test allows for the characterization of the toxicological profile of the drug on several parameters that include hematological, biochemical, anatomical and histopathological on target organs; as well as indication of no-observedeffect level (NOEL) and the no-observed-adverse-effect-level (NOAEL) to support clinical phases 1, 2 and 3. In the case of prolonged exposure time (> 6 months) to cumulative doses of the drug in humans, chronic toxicity tests are performed in animals (OECD, 2009). The commonly used cytogenetic assays for in vitro genotoxicity evaluation are the micronucleus (OECD, 2016a) and comet (OECD, 2016b) tests. The micronucleus test is based on the presence of an additional nucleus separated from the main nucleus of a cell and consists of chromosomes or fragment of chromosomes that are not included in the main nucleus during mitosis. The comet assay is technique for detecting the presence of single strand breaks (SSB) of DNA lesions at alkali-sensitive sites and SSB at sites of incomplete excision repair in mammalian cells (OECD, 2016b). Copaifera malmei Harms (C. malmei), Fabaceae, belongs to the Copaifera genus, which comprises of about 72 species, of which 28 have been described in the literature due to their medicinal and economic importance (Albuquerque et al., 2017). It is an endemic shrub that grows wild in the states of Mato Grosso and Goiás, Brazil. It is locally known as “copaíba-mirim”, “óleo-mirim”, “podoinho”, “pau-d'óleo”, “guaranazinho”, “pau-d′inho” and “copaibinha” (Adzu et al., 2015; Ribeiro et al., 2017). The plant has as heterotypic synonym: Copaifera bulbotricha Rizzini and Heringer (www.theplantlist.org). Infusion of the powdered leaves is used by the riverine communities of the North Araguaia microregion of the State of Mato Grosso for the treatment of gastric ulcer, inflammation, bronchitis, asthma and pneumonia (Adzu et al., 2015; Ribeiro et al., 2017). In the locals' ethnopharmacy, it is recommended to take a cup of the infusion (150 mL) twice a day until symptoms disappear. We have previously described the anti-gastric ulcer activity of the standardized leaves infusion extract of C. malmei (SIECm) in experimentally induced gastric ulcers in rodents. The study also demonstrated that SIECm has no acute oral toxicity in mice and CHOk1 cytotoxicity (Adzu et al., 2015). In the same work by Adzu et al. (2015), we standardized SIECm by qualitative evaluation with thin layer chromatography, where phenolics, flavonoids and phytosterols were detected as the major constituents. We thereafter performed HPLC analysis of SIECm and demonstrated the presence of gallic acid, rutin, ellagic acid, catechin, and quercetin. Using spectrophotometric quantification, we were able to show that the total phenolics, flavonoids and phytosterol in SIECm were 148.117 ± 1.5 mg gallic acid equivalent (GAE)/g, 67.077 ± 0.28 mg rutin equivalent (RE)/g and 192.107 ± 2.26 mg stigmasterol equivalent (SE)/g, respectively. Despite the popular and widespread ethnomedical uses of C. malmei (Ribeiro et al., 2017), little information is known about the safety level of preparations made from this plant. The present study was carried out to address this gap by assessing the genotoxicity and subchronic toxicity profile of SIECm using in vitro and in vivo experimental models.

2.1. Plant material C. malmei were collected in the municipality of Serra Nova Dourada (11°58′41′′ S, 51°34′57′′W), Mato Grosso, Brazil, in April 2014. Authorization to access the associated traditional knowledge (No. 135/ 2013) and genetic heritage (no. 199/2014) was granted by the Genetic Heritage Management Council, Ministry of Environment (CGEN/MMA) of Brazil. The plant was identified by the taxonomist Prof. Dr. Germano Guarim Neto of the Department of Botany and Ecology, Federal University of Mato Grosso (UFMT), Cuiabá, Brazil. A voucher specimen of the flowering plant is deposited (No. 40,754) at “Herbário UFMT”. 2.2. Drugs, dyes and reagents Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), doxorubicin, cytochalasin B, Trizma® Base, Tris-HCl, Dimethyl sulfoxide (DMSO), low melting agarose, normal melting point agarose, trypan blue and trypsin were purchased from Sigma Aldrich Co., Missouri, USA. Hydrogen peroxide, sodium chloride, ethylenediamine tetra-acetic acid (EDTA), sodium citrate and formaldehyde were obtained from Dinâmica®, Química Contemporânea Ltda, Diadema, São Paulo, Brazil. Some reagents used were: Panor Dye (Newprov, Paraná, Brazil), hematoxylin and eosin (Easypath, São Paulo, Brazil), GelRed® (Biotium, California, USA), ketamine and xylazine (Syntec Pharmaceuticals Ltd., Brasília, Brazil). All other drugs and reagents used were of analytical purity. 2.3. Cell lines Chinese hamster ovary epithelial cells (CHO-k1, code: 0067) was purchased from the Cell Bank of Rio de Janeiro (BCRJ), Brazil. After thawing, the cells were cultured in DMEM, supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL), in an oven (Quimis® Aparelhos Científicos, Diadema – SP, Brazil) maintained at 37 °C in a humidified atmosphere with 5% CO2 and 90% air. Cell viability was assessed by the trypan blue exclusion method. 2.4. Animals Wistar albino female rats (Rattus novergicus), weighing 160–180 g, and 45–50 days old, were obtained from the Animal House of the Universidade Federal de Mato Grosso (UFMT). They were kept in a room maintained at a temperature of 23 ± 1 °C, under cycle of 12 h light/dark with free access to treated water and standard feeds (Purina©, Labina, Goiás, Brazil). Procedures involving animals and their care were approved by the Institutional Committee for Ethics in the Use of Animals (CEUA- UFMT, no. 23108.110009/2015-10) and the studies conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals (CIOMS/ICLAS). 2.5. Preparation of the extract The extract used in the present work was sourced from that standardized by Adzu et al. (2015). The physicochemical parameters of SIECm reported are: yield, 7.68% w/w; pH, 4.93 (1 mg/mL at 25 °C); and color, brown (Adzu et al., 2015). SIECm was packed in an amber bottle and kept in a refrigerator (Brastemp 350L, São Paulo, Brazil) maintained at 4 °C. Prior to use, the SIECm was diluted in distilled water (in vivo test) or DMSO (in vitro assays) to the desired concentration. 2.6. Phytochemical fingerprints The fingerprints profiles of SIECm were determined using High Performance Liquid Chromatography (HPLC) technique as reported elsewhere (Adzu et al., 2015). 71

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then covered with coverslip and kept in refrigerator at 4 °C for 5 min to solidify. Two slides were prepared for each sample. They were then incubated in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris) at 4 °C overnight. After lysis, the slides were placed in running buffer (300 mM NaOH, 1 mM EDTA, pH > 13) for 20 min and subjected to electrophoresis run at 25 V, 30 mA for 30 min. At the end of the electrophoresis, the slides were incubated in neutralization solution (0.4 M Tris, pH 7.5) for 15 min, fixed in ethanol for 5 min, stained with GelRed™ and read under a fluorescence microscope (AxioScope.A1, Carl Zeiss, Germany) at 400× magnification. The genetic damage was identified by the presence of a tail similar to that of a comet made up of fragments of DNA. They were quantified by the ratio of comet tail intensity to total comet intensity, multiplied by 100 and expressed as % DNA in the tail. One hundred (100) nucleoids were counted per slide, photographed and evaluated using TriTek Comet Score™ Freeware software (Virginia, USA).

2.7. Cell viability Cell viability test was performed before the genotoxicity assays using the trypan blue exclusion method (Strober, 2001). CHO-k1 cells were removed from the bottles, centrifuged at 400×g for 5 min, and resuspended in 1 mL of complete medium. Then 10 μL of the substance was added on to an eppendorf tube containing 390 μL trypan blue. Cells were counted in Neubauer's chamber and primed to the required density in each assay. Cells that remained colorless and those that were stained in blue were considered viable. 2.8. Genotoxicity assays The micronucleus and comet assays were used. In both, CHO-k1 cells cultured to the fourth passage were plated at the density of 1 × 106 cells/well in the micronucleus assay; and 5 × 105 cells/well for the comet assay, and incubated overnight at 37 °C in 5% CO2. For each group, two wells containing cells were incubated in 6 well microplates containing DMEM, which served as sterility control or DMEM containing 0.02% DMSO, which served as the negative control. For the micronucleus test, the cells were pretreated with SIECm (10, 30 or 100 μg/mL) and doxorubicin (0.03 μg/mL). In the comet test, the cells received H2O2 (50 μM) for a period of 4 h, after 24 h of pre-treatment with SIECm (10, 30 or 100 μg/mL). The co-treated cells simultaneously received the SIECm and H2O2 for 4 h; and for post-treatment, the cells were incubated for 4 h with H2O2. The genotoxic agent was then removed and incubated with SIECm for a period of 24 h.

2.9. Subchronic toxicity The subchronic toxicity profile of SIECm was evaluated using the method described by Chan et al. (1982) using metabolic cages (Model 41800, Ugo Basile, Varese, Italy). Rats were grouped into four (n = 6) were treated oral (p.o.) with vehicle (distilled water, 10 mL/kg) and SIECm (100, 400 or 1000 mg/kg) for thirty days. The animals’ weight changes, water consumption, feed intake, excretion of feces and urine were measured every 3 days, and grouped every 6 days (D6, D12, D18, D24 and D30) during the treatment period. Changes in clinical signs and behavioral symptoms, including changes observed in the skin, hair, eyes, gastrointestinal, respiratory and nervous systems were each noted. At the end of the 30 days duration of treatment, the animals were anesthetized intraperitoneally (i.p.) with ketamine/xylazine (100/ 10 mg/kg) and blood were collected (in Vacutainer® tubes containing EDTA) for hematological analyzes, and without anticoagulant for biochemical analysis through inferior vena cava puncture. The animals were then sacrificed to remove the major organs for analysis.

2.8.1. Micronucleus (MN) test with cytokinesis block For the MN test, the technique described by Fenech (2000), and modified by Oliveira et al. (2014) was used. After 24 h of pre-treatment, the cells were treated with cytochalasin B (4.5 μg/mL), a cytokinesisblocking drug and incubated for another 24 h. After trypsinization, the cells were centrifuged (Fanem Excelsa II, São Paulo, Brazil) at 400×g (4 °C) for 5 min. The supernatant was discarded, 5 mL of 1% sodium citrate was added and the cells resuspended. After 15 s, 5 mL of fixative solution (methanol/acetic acid, 3:1) and 4 drops of formaldehyde were added. The cell suspension was centrifuged at 400×g for 5 min. The supernatant was again discarded, and the pellet fixed in methanol/ acetic acid (3:1) for two more times, without the addition of formaldehyde. After the third fixation step, part of the supernatant was discarded, 1 mL of it was retained to permit resuspension of the cells; and the suspension was dripped onto clean glass slides that had previously been frozen, to dry. After drying, the slides were stained with panotic dye for 4 min. One thousand binucleated cells per slide with intact nuclei of equal size, similar pattern of cytoplasm staining, intact membrane, and distinguishable from adjacent cells, excluding apoptotic and necrotic cells were analyzed. The MN that had the same morphology and color, with 1/16 to 1/3 of the major nucleus diameter, non-refringent, unbound or connected and not superimposed on one of the major nuclei were considered. The presence of dicentric bridges (DB) and nuclear buds (NB) in the binucleated cells were also quantified. The nuclear division index (NDI) was calculated as: NDI = [(1 × mononuclear cells) + (2 × binucleate cells) + (3 × multinucleated cells)]/N, where N = total cells number.

2.9.1. Determinations of the relative organ weights, macroscopic and histopathological analysis The rats were necropsied, and the relative weights of the heart, lungs, liver, spleen, kidneys, brain and stomach were determined; followed by macroscopic analysis of all external surfaces of the organs and also the pelvic, thoracic and cranial cavities. Fragments of each organ were taken and fixed in 4% formalin solution for histopathological analysis. They were dehydrated in increasing concentrations of ethanol, clarified in xylol using a histological processor (MTP 100 Slee, Mainz, Germany), embedded in paraffin and sectioned (3 µm) using a manual microtom (Hyrax M60 Carl Zeiss, Oberkochen, Germany). The tissues were then stained by hematoxylin and eosin, and histopathological examinations performed using light-microscopic (Axio Scope.A1, Carl Zeiss, Oberkochen, Germany). For all organs, the presence of necrosis/ degeneration, leukocyte infiltrate and vascularization were analyzed. 2.9.2. Hematological and biochemical analyzes The bloods collected from the rats were used to perform hematological tests. Hematocrit (Ht), hemoglobin (Hb), platelets, erythrocytes, total leukocytes, neutrophils, lymphocytes, monocytes and eosinophils were quantified in automated cell counters (Cell Dyn 3700, Abott Laboratories, Minnesota, USA). For biochemical analysis, blood (collected without anticoagulant) were centrifuged at 3000×g at 4 °C for 10 min. Serum was separated and quantification of the biochemical parameters: glucose, urea, creatinine, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total cholesterol, triglycerides, total proteins and gammaglutamyltranspeptidase (Gamma GT) were performed by colorimetric assays using Labtest® kits.

2.8.2. Comet assay For the comet test, the technique developed by Collins (2004), was used with little modification. Briefly, after the treatment protocols, the cells were removed, 1% trypsin added, and centrifuged at 400×g for 5 min. The supernatant was discarded, the cells were resuspended and the viable cells counted by the addition of trypan blue, until at least 2 × 104 cells in 200 μL of 0.5% low melting agarose gel were obtained, then preheated in a water bath at 37 °C. Samples (80 μL) were dripped onto slides covered with a thin layer of normal melting point agarose, 72

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the animals presented altered behavioral changes or clinical signs and symptoms of toxicity, such as alterations in the coloration of the skin, eyes, respiratory system, digestive system and the central and autonomic nervous system (tremors, convulsions, abnormal gait, ptosis and salivation).

2.10. Data analysis The results of parametric data were expressed in terms of mean ± standard error (SEM). One-way analysis of variance (ANOVA) was used for comparisons of groups, followed by the Student-Newman-Keuls test for multiple comparisons using GraphPad Prism Version 6.07 software for Windows (San Diego California, USA). Non-parametric tests were expressed in terms of the median (Q1;Q3), using the Kruskal-Wallis test, followed by Dunn's test.

3.3.2. Determination of body weight, weight gain, water and feed intake and excretions of feces and urine The daily dosing of SIECm (100, 400 or 1000 mg/kg) to rats for 30 days did not cause significant changes in the following parameters: body weight, cumulative weight gain, feed and water consumption and excretion of feces and urine when compared to the vehicle group (Table 2). There was an increase in feed intake in the group that received 100 mg/kg of SIECm by the 18th day (D18), by 11.7% (p < 0.05) when compared to the vehicle group. However, feed consumption normalized by the 24th day (D24).

3. Results 3.1. Phytochemical fingerprints The analysis by HPLC revealed the presence of gallic acid, ellagic acid, catechin, and quercetin.

3.3.3. Gross and histopathological analyzes and determinations of relative organ weights Subchronic treatment of rats with SIECm (100, 400 or 1000 mg/kg) did not cause macroscopic and histopathological changes in the kidneys, stomach, liver, lungs, heart and brain. In addition, no significant changes were observed in the weight of the animal's organs, relative to the vehicle group.

3.2. Genotoxicity tests 3.2.1. Micronucleus test The mean values of MN, DB, NB and NDI in the vehicle group were 24.75 ± 2.59, 7.75 ± 2.29, 6.00 ± 0.71 and 1.74, respectively. Treatment of CHO-k1 cells with SIECm (10, 30 or 100 μg/mL) did not significantly alter any of the evaluated parameters. Doxorubicin, a standard for this assay, induced an increase (p < 0.001) of 266.1% in the number of MN, 385.2% in the number of DB and 413.3% in the number of NB, with a NDI of 1.11.

3.3.4. Hematological and biochemical analyzes Treatment of the animals with SIECm at a dose of 100 mg/kg resulted in an increase (p < 0.05) of 7.4% in the hematocrit and 15.4% in the absolute value of platelets when compared to the vehicle group. There was a decrease (p < 0.01) in the total number of leukocytes by 41.2% in the animals treated with SIECm at the dose of 1000 mg/kg, as well as the absolute lymphocyte count (43.1%), relative to the vehicle group. Treatment of the rats with SIECm at a dose of 100 mg/kg for 30 days reduced (p < 0.05) the serum levels of ALT and AST by 57.8% and 74.4%, respectively, compared to the vehicle group. There was a decrease (p < 0.05) in triglyceride serum levels in animals treated with 400 (49.4%) and 1000 mg/kg (46.5%) compared to the vehicle (Table 3).

3.2.2. Comet test Table 1 shows the percentage of DNA damage at the comet tail in CHO-k1 cells for the medium, SIECm (10, 30 or 100 μg/mL) and H2O2 (50 μM). Incubation of the cells with H2O2 for 4 h caused an increase of about 3170 fold (p < 0.001) in DNA damage. Pre-treatment with SIECm protected (p < 0.001) the CHO-k1 cells from the H2O2-induced genetic damage at all concentrations, with their highest effect (3170 times) at concentrations of 10 and 30 μg/mL, comparable with the values of the medium group (negative control). Co-treatment of the cells with SIECm decreased the DNA damage at the concentration of 10 μg/mL (429.7 times, p < 0.001), a value comparable to the group receiving only the medium. Post-treatment of the cells with the higher concentration of SIECm (100 μg/mL) resulted in a 1.4-fold increase (p < 0.001) in the genotoxicity induced by H2O2.

4. Discussion The World Health Organization postulate that approximately 64% of the world's population depends on plants or plant extract and allied products as herbal medicines for primary health care (WHO, 2011). However, one of the dangers associated with the use of such plants is their potential to cause unexpected effects, including toxicity (Staines, 2011). Therefore, scientific evaluation of the efficacy as well as the safety of herbal medicines is crucial. Previous study shows that SIECm does not exhibit cytotoxicity in

3.3. Subchronic toxicity 3.3.1. General and behavioral parameters There were no deaths recorded throughout the 30 days duration treatment of rats with SIECm (100, 400 or 1000 mg/kg, p.o.). None of

Table 1 DNA damage values in Chinese hamster ovary epithelial cells (CHO-k1) treated with standardized leaves infusion extract of Copaifera malmei (SIECm) and hydrogen peroxide (H2O2). a

Medium SIECm (µg/mL)

H2O2(µM)

10 30 100 50

DNA at the comet tail (%)

Pre-treatment

Co-treatment

Post-treatment

0.0009 (0.0007; 0.0015) 0.0009 (0.0007; 0.0011)*** 0.0009 (0.0006; 0.0057)*** 0.0013 (0.0008; 6.3580)*** 2.8540 (0.0010; 9.6360) †††

0.0005 (0.0004; 0.0006) 0.0006 (0.0006; 0.0008)*** 0.0008 (0.0005; 5.0990) 0.0026 (0.0006; 5.2030) 0.2578 (0.0006; 3.8730) †††

0.0005 0.0011 0.0012 0.0016 0.0011

a

The damage was quantified at 100 nucleoids per slide. The results were expressed as median (Q1;Q3). Kruskal-Wallis analysis, followed by the Dunn test. p < 0.001 vs medium. *** p < 0.001 vs H2O2. †††

73

(0.0004; (0.0008; (0.0008; (0.0011; (0.0007;

0.0006) 0.0014) 0.0016) 0.0021)*** 0.0022) †††

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Table 2 Effect of subchronic oraled leaves infusion extract of Copaifera malmei (SIECm) for 30 days on body weight, cumulative weight gain, water and feed intake, and excretion of feces and urine in rats. Parameter

Period of treatment (Days) D0

D6

D12

D18

D24

D30

Vehicle (10 mL/kg) Body weight (g) Cumulative weight gain (g) Water intake (mL) Feed intake (g) Feces output (g) Urine output (mL)

161.9 ± 4.45 0.00 0.00 0.00 0.00 0.00

178.10 ± 4.15 16.20 ± 0.99 57.83 ± 4.26 31.80 ± 1.02 15.60 ± 1.05 23.30 ± 1.50

180.10 ± 3.91 18.20 ± 3.31 64.00 ± 4.55 24.20 ± 0.85 13.60 ± 1.67 25.70 ± 3.56

202.30 ± 3.79 40.40 ± 2.58 61.00 ± 2.57 31.50 ± 0.90 17.20 ± 1.44 21.00 ± 0.86

218.30 ± 5.43 56.40 ± 1.81 65.60 ± 2.65 32.30 ± 0.66 19.00 ± 1.32 27.60 ± 1.67

230.80 ± 5.54 68.80 ± 1.56 55.60 ± 2.70 29.20 ± 1.11 13.30 ± 1.33 22.70 ± 1.84

SIECm (100 mg/kg) Body weight (g) Cumulative weight gain (g) Water intake (mL) Feed intake (g) Feces output (g) Urine output (mL)

156.8 ± 5.18 0.00 0.00 0.00 0.00 0.00

177.00 ± 4.81 20.90 ± 1.40 58.70 ± 1.60 31.60 ± 0.98 15.30 ± 1.07 19.50 ± 0.50

184.20 ± 7.08 27.30 ± 3.40 62.50 ± 6.99 26.60 ± 2.43 14.60 ± 1.89 25.00 ± 5.17

205.00 ± 6.11 48.10 ± 1.80 66.70 ± 2.91 35.20 ± 1.16* 18.20 ± 1.42 23.30 ± 3.45

220.80 ± 5.54 64.00 ± 2.50 71.60 ± 2.60 32.70 ± 1.42 18.60 ± 1.34 31.00 ± 3.13

230.00 ± 5.78 73.10 ± 2.57 57.70 ± 3.94 27.60 ± 0.79 14.50 ± 0.83 23.80 ± 3.61

SIECm (400 mg/kg) Body weight (g) Cumulative weight gain (g) Water intake (mL) Feed intake (g) Feces output (g) Urine output (mL)

159 ± 3.76 0.00 0.00 0.00 0.00 0.00

175.00 ± 5.64 15.90 ± 3.70 62.70 ± 4.99 29.60 ± 1.75 15.50 ± 1.72 24.00 ± 2.31

179.40 ± 3.61 20.30 ± 3.10 54.20 ± 10.01 24.80 ± 1.86 11.80 ± 1.43 28.00 ± 5.84

200.40 ± 4.12 41.30 ± 3.03 68.70 ± 3.37 34.20 ± 0.79 18.90 ± 1.71 17.30 ± 3.64

210.00 ± 6.05 50.90 ± 5.28 72.20 ± 3.58 29.60 ± 1.89 18.70 ± 1.15 30.30 ± 2.89

225.00 ± 5.78 65.10 ± 5.05 49.00 ± 3.34 24.70 ± 2.25 10.90 ± 1.26 17.20 ± 3.51

SIECm (1000 mg/kg) Body weight (g) Cumulative weight gain (g) Water intake (mL) Feed intake (g) Feces output (g) Urine output (mL)

154.1 ± 2.10 0.00 0.00 0.00 0.00 0.00

173.80 ± 2.72 18.90 ± 2.81 53.00 ± 1.98 29.20 ± 1.28 14.90 ± 1.39 28.00 ± 3.09

180.30 ± 2.99 25.50 ± 1.22 67.30 ± 7.50 24.50 ± 1.10 11.00 ± 0.77 31.80 ± 3.25

199.70 ± 1.56 44.80 ± 2.49 70.50 ± 5.25 31.30 ± 0.97 18.90 ± 0.71 24.50 ± 2.63

210.00 ± 4.47 55.20 ± 4.53 67.70 ± 3.52 28.30 ± 2.51 20.20 ± 1.38 31.30 ± 3.41

219.20 ± 4.36 64.30 ± 4.06 51.00 ± 3.68 24.10 ± 1.18 11.30 ± 2.08 22.30 ± 2.45

The values represent the mean ± SEM (n = 6/group). One-way ANOVA, followed by the Student-Newman-Keuls test. * p < 0.05 vs vehicle.

mitochondria. The breakdown of DNA occurs, both by attacking the nitrogen bases and deoxyribose. The H2O2 generated in vivo is partially eliminated by catalases, glutathione peroxidase and peroxidases bound to thioredoxin, but since this elimination has low efficiency much of the H2O2 is released into the cell (Barreiros et al., 2006). The SIECm completely attenuated the DNA damage induced by H2O2 in the comet test, both in the pre-treatment and in the co-treatment. In the latter case, only the lower concentration was active, indicating that the SIECm has partial antigenotoxic activity. Adzu et al. (2015) demonstrated that SIECm exhibits antioxidant activity in vivo by increasing the activity of the catalase enzyme, which converts H2O2 to H2O and O2. SIECm also reduced the activity of myeloperoxidase, an enzyme that normally catalyzes oxidation reactions involving H2O2, which together with the neutrophil membrane NADPH oxidase is involved in the generation of ROS (Arnhold, 2004). This indicates that the reduction of ROS is responsible for the antigenotoxic activity of the SIECm. Glutathione (GSH), which acts together with the glutathione peroxidase (GPx) and glutathione reductase (GR) enzymes to catalyze the disruption of H2O2 in H2O and O2, cannot be ruled out in the genoprotective effect of SIECm. On the other hand, post-treatment with SIECm resulted in a significant increase in DNA damage at the highest concentration tested. The exact mechanism responsible for this observation is not yet clear. It is possible that one or more compounds from the extract may inhibit repair enzymes or that of the regulation of thioredoxin expression enzyme implicated in the regulation of the cellular redox balance (Sghaier et al., 2016). However, this result was not confirmed in the MN test, indicating that the observation of increased DNA damage of SIECm does not seem to have great relevance in relation to genotoxicity.

CHO-k1 cells using alamar blue oxy-reduction indicator (Adzu et al., 2015). However, such single in vitro toxicity study cannot be used as conclusive evidence that the agent is nontoxic. Thus, the present study is to advance the pre-clinical safety evaluation of SIECm by including genotoxicity tests in CHO-k1 cells and subchronic toxicity in rats. The genotoxicity of SIECm was evaluated by micronucleus (MN) and comet tests. The two assays are some of the most commonly employed for genotoxicity assessment of medicinal plant extracts. The MN assay allows for the study of DNA damage at the level of chromosome, which is an essential part of genetic toxicology. In this sense, the MN assay is useful in the monitoring of genetic damage and screening of chemicals for genotoxic potential. While, the comet assay detects repairable single- and double-stranded DNA breaks (Cavalcanti et al., 2006). Both techniques detect reversible and irreversible damage, aneugenic and clastogenic effects, as well as nonspecific damages (Sponchiado et al., 2016) as an alternative to animal testing. The micronucleus test was used to evaluate chromosomal DNA damage by measuring MN, DB, NB and NDI in CHO-k1 cells using a cytokinesis blocker. Mutations that occur in dividing cells may be related to processes of oncogenesis, suppression or even production of defective genes, leading to number of genetic diseases (Fenech, 2000). No changes in the MN, DB, NB and NDI were observed in cells treated with SIECm, demonstrating that it is potentially non-mutagenic and thus has high safety margin. The action of SIECm on CHO-k1 cells exposed to H2O2 was investigated using the comet assay. H2O2 is poorly reactive in the absence of transition metals, but can diffuse easily through cell membranes (including nuclear membranes) and generate the most deleterious of reactive oxygen specie (ROS). This is capable of causing damage to DNA, RNA, proteins, lipids and cell membranes of the nucleus and 74

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Table 3 Effect of subchronic oral administration of the standardized leaves infusion extract Copaifera malmei (SIECm) on hematological and biochemical parameters in rats after 30 days of treatment. Parameter

Hematological parameters Red blood cells (106/mm3) Hemoglobin (g/dL) Hematocrit (%) MCV (fl)a MCH (pg)b MCHC (%)c Platelets (103/mm3) Total leukocytes (103/mm3) Neutrophiles Relative (%) Absolute (103/mm3) Lymphocytes Relative (%) Absolute (103/mm3) Eosinophil Relative (%) Absolute (103/mm3) Monocytes Relative (%) Absolute (103/mm3) Biochemical parameters Glucose (mg/dL) Urea (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Alanine amino transferase (UI/L) Aspartate amino transferase (UI/L) Alkaline phosphate (UI/L) Total Cholesterol (mg/dL) Triglycerides (mg/dL) Total proteins (mg/dL) Gama GTd

Vehicle

SIECm (mg/kg) 100

400

1000

7.59 ± 0.15 14.60 ± 0.17 43.40 ± 0.62 57.20 ± 0.55 19.20 ± 0.24 33.60 ± 0.15 1126 ± 32.3 6.82 ± 0.59

8.09 ± 0.10 15.40 ± 0.20 46.60 ± 0.73* 57.6 ± 0.39 19.10 ± 0.17 33.00 ± 0.22 1299 ± 47.5* 6.02 ± 0.53

7.55 ± 0.17 14.70 ± 0.35 43.90 ± 1.01 58.20 ± 0.80 19.40 ± 0.18 33.40 ± 0.19 1210 ± 31.8 5.50 ± 0.63

7.41 ± 0.14 14.30 ± 0.79 42.10 ± 0.95 56.70 ± 0.50 19.20 ± 0.19 33.90 ± 0.14 1241 ± 48.6 4.01 ± 0.28**

24.05 ± 2.17 1.45 ± 0.06

31.00 ± 2.50 1.82 ± 0.15

29.10 ± 4.03 1.26 ± 0.16

26.50 ± 2.54 1.06 ± 0.12

73.50 ± 2.21 5.01 ± 0.45

66.20 ± 2.83 4.03 ± 0.49

66.60 ± 4.16 3.64 ± 0.30

71.00 ± 2.59 2.85 ± 0.23**

0.67 ± 0.21 0.04 ± 0.02

1.16 ± 0.30 0.06 ± 0.01

1.16 ± 0.47 0.05 ± 0.02

0.17 ± 0.17 0.01 ± 0.01

1.33 ± 0.21 0.09 ± 0.01

1.66 ± 0.33 0.09 ± 0.01

2.00 ± 0.25 0.11 ± 0.02

2.16 ± 0.34 0.08 ± 0.02

171.00 ± 6.48 48.80 ± 2.19 0.60 ± 0.00 2.81 ± 0.44 40.30 ± 6.76 68.40 ± 6.18 268.00 ± 12.41 119.00 ± 10.56 127.30 ± 21.61 8.28 ± 0.13 1.00 ± 0.00

165.00 ± 11.29 53.60 ± 3.63 0.63 ± 0.02 3.88 ± 0.69 17.00 ± 5.99* 17.50 ± 7.37* 226.00 ± 10.87 148.80 ± 8.50 139.30 ± 14.11 8.66 ± 0.10 1.17 ± 0.16

148.00 ± 14.22 48.80 ± 3.92 0.66 ± 0.07 2.35 ± 0.21 29.30 ± 7.36 71.00 ± 16.41 233.80 ± 23.25 137.70 ± 8.17 64.40 ± 8.59* 8.46 ± 0.27 1.00 ± 0.00

148.00 ± 14.22 47.50 ± 3.04 0.56 ± 0.03 2.58 ± 0.28 34.60 ± 4.80 67.00 ± 12.6 223.00 ± 24.25 139.30 ± 9.23 68.10 ± 8.36* 8.46 ± 0.06 1.00 ± 0.00

The values represent the mean ± SEM (n = 6/group). One-way ANOVA, followed by Student-Newman-Keuls test. a MCV: mean corpuscular volume. b MCH: mean corpuscular hemoglobin. c MCHC: mean corpuscular hemoglobin concentration. d Gama GT: gamaglutamil transferase. * p < 0.05 vs vehicle. ** p < 0.01 vs vehicle.

concentration of total leukocytes and absolute lymphocytes at the highest concentration tested. Despite the significance, these values are within the physiological ranges for Wistar rats (Melo et al., 2012), and therefore are of no toxicological relevance. In addition, the effect was not dose-dependent and was not accompanied by any clinical signs or symptoms, thus reducing the clinical importance of this finding (Balogun et al., 2014). SIECm did not alter the serum levels of glucose, urea, creatinine, uric acid, alkaline phosphatase, cholesterol, total proteins and gamma GT in all the three doses tested. However, at the lowest dose of SIECm, reduced the levels of AST and ALT enzymes were observed. The basis for this lowering effect in the activities of both enzymes is not yet understood, however, there are reports of high correlation between the deficiency of pyridoxal-5′-phosphate and low activities of these two aminotransferases, particularly in patients on hemodialysis (Lum, 1995; Ono et al., 1995). We are of the opinion that this result of AST and ALT is unrelated to toxicological effect of the extract for the following reasons: the observation was not dose-dependent and besides no other biochemical parameters related to the kidney or liver, nor were histopathological alterations seen in the organs of these animals (Balogun et al., 2014; Mu et al., 2011). In addition, the leukogram did not show any increase in leukocyte counts that suggests tissue damage or infection, indicating that the observed effect has no clinical relevance (Awounfack et al., 2016).

Despite the high sensitivity of in vitro bioassays, the extrapolation of the results to in vivo models and use in humans may not be very predictive of adverse effects. When a drug is being used for an extended period, the subchronic toxicity test is essential to assess the safety of the drug. Several parameters were evaluated during and after the 30 days of treatment regimen. We employed limitrophic values, obtained from analysis of control group (higher and lower values), to explain the significant difference among groups. And when the normal range of control group did not explain the significant difference, reference values published in the literature were utilized (Palmeiro et al., 2003). The subchronic administration of SIECm did not alter the parameters related to weight changes, water consumption and excretion of feces and urine, except for increase in feed intake in the group treated with the 100 mg/kg, which reverses 6 days after the manifestation of the effect. This shows that the extract does not alter the normal growth and development of the animals. The subchronic administration of SIECm in rats, at the lowest dose, resulted in marginal increases in the platelets (15.36%) and hematocrit (7.37%) values. Physiological alterations, where the concomitant increase of both parameters occurs may be related to several diseases, but usually accompanied by biochemical changes, as well as behavioral and histopathological alterations of the organs, none of which were observed in animals treated with SIECm. The SIECm reduced the absolute 75

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human myeloperoxidase. Biochemistry 69, 4–9. Awounfack, C.F., Ateba, S.B., Zingue, S., Mouchili, O.R., Safety, D.N., 2016. Evaluation (acute and sub-acute studies) of the aqueous extract of the leaves of Myrianthus arboreus P. Beauv. (Cecropiaceae) in Wistar rats. J. Ethnopharmacol. 194, 169–178. Balogun, S.O., da Silva, I.F., Colodel, E.M., de Oliveira, R.G., Ascêncio, S.D., Martins, D.T., de, O., 2014. Toxicological evaluation of hydroethanolic extract of Helicteres sacarolha A. St.- Hil. et al. J. Ethnopharmacol. 157, 285–291. http://dx.doi.org/10.1016/j. jep.2014.09.013. Barreiros, L.B.S., David, J.M., David, J.P., 2006. Estresse oxidativo: relação entre geração de espécies reativas e defesas do organismo. Quím. Nova 29, 113–123. Cavalcanti, B.C., Costa-Lotufo, L.V., Moraes, M.O., Burbano, R.R., Silveira, E.R., Cunha, K.M.A., Rao, V.S.N., Moura, D.J., Rosa, R.M., Henriques, J.A.P., Pessoa, C.C., 2006. Genotoxicity evaluation of kaurenoic acid, a bioactive diterpenoid present in Copaíba oil. Food Chem. Toxicol. 44, 388–392. Chan, P., O'Hara, G., Hayes, A.W., 1982. Principles and methods for acute and subchronic toxicity. Princ. Method Toxicol. 12, 17–19. Collins, A.R., 2004. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol. Biotechnol. 3, 249–261. Fenech, M., 2000. The in vitro micronucleus technique. Mutat. Res./Fundam. Mol. Mech. Mutagen. 455, 81–95. Kushner, P.A., Cobble, M.E., 2016. Hypertriglyceridemia: the importance of identifying patients at risk. Postgrad. Med. 128. http://dx.doi.org/10.1080/00325481.2016. 1243005. Lewis, R.W., Billington, R., Debryune, E., Gamer, A., Lang, B., Carpanini, F., 2002. Recognition of adverse and nonadverse effects in toxicity studies. Toxicol. Pathol. 30, 66–74. Lum, G., 1995. Low activities of aspartate and alanine aminotransferase: their significance in alcoholic liver disease. Lab. Med. 26, 273–276. http://dx.doi.org/10. 1093/labmed/26.4.273. Melo, M.G.D., Dória, G.A.A., Serafini, M.R., Araújo, A.A.S., 2012. Valores de referência hematológicos e bioquímicos de ratos (Rattus novergicus linhagem Wistar) provenientes do biotério central da Universidade Federal de Sergipe. Sci. Plena 8, 1–6. Mu, L.-H., Huang, Z.-X., Liu, P., Hu, Y., Gao, Y., 2011. Acute and subchronic oral toxicity assessment of the herbal formula Kai-Xin-San. J. Ethnopharmacol. 138, 351–357. http://dx.doi.org/10.1016/j.jep.2011.08.033. Niho, N., Shibutani, M., Tamura, T., Toyoda, K., Uneyama, C., Takahashi, N., Hirose, M., 2001. Subchronic toxicity study of gallic acid by oral administration in F344 rats. Food Chem. Toxicol. 39, 1063–1070. OECD/OCDE, 2008. Guideline for the Testing of Chemicals, Test No. 425. Acute Oral Toxicity – Up-and-Down-Procedure (UDP). Organization for Economic Cooperation and Development (OECD), Paris, 27pp. 〈http://doi.org/10.1787/ 9789264071049-en〉. OECD/OCDE, 2009. Guideline for the Testing of Chemicals, Test No. 452: Chronic Toxicity Studies. Organization for Economic Cooperation and Development (OECD), Paris, 16pp. 〈http://doi.org/10.1787/9789264071209-en〉. OECD/OCDE, 2016a. Guideline for theTesting of Chemicals, No. 487: In Vitro Mammalian Cell Micronucleus Test, Organization for Economic Cooperation and Development (OECD), Paris, 29pp. 〈http://doi.org/10.1787/9789264224438-en〉. OECD/OCDE, 2016b. Guideline for the Testing of Chemicals Test No. 489: In Vivo Mammalian Alkaline Comet Assay. Organization for Economic Cooperation and Development (OECD), Paris, 27pp. 〈http://doi.org/10.1787/9789264264885-en〉. Oliveira, M.C., Lemos, L.M.S., Oliveira, R.G., Dall‫׳‬Oglio, E.L., Sousa Júnior, P.T., Martins, D.T.O., 2014. Evaluation of toxicity of Calophyllum brasiliense stem bark extract by in vivo and in vitro assays. J. Ethnopharmacol. 155, 30–38. Ono, K., Ono, T., Matsumata, T., 1995. The pathogenesis of decreased aspartate aminotransferase and alanine aminotransferase activity in the plasma of hemodialysis patients: the role of vitamin B6 deficiency. Clin. Nephrol. 43, 405–408. Palmeiro, N.M.S., Almeida, C.E., Ghedini, P.C., Goulart, L.S., Pereira, M.C.F., Huber, S., da Silva, J.E.P., Lopes, S., 2003. Oral subchronic toxicity of aqueous crude extract of Plantago australis leaves. J. Ethnopharmacol. 88, 15–18. http://dx.doi.org/10.1016/ S0378-8741(03)00137-5. Patil, S.L., Rao, N.B., Somashekarappa, H.M., Rajashekhar, K.P., 2014. Antigenotoxic potential of rutin and quercetin in Swiss mice exposed to gamma radiation. Biomed. J. 37, 305–313. Ribeiro, R.V., Bieski, I.G.C., Balogun, S.O., Martins, D.T.O., 2017. Ethnobotanical study of medicinal plants used by Ribeirinhos in the North Araguaia microregion, Mato Grosso, Brazil. J. Ethnopharmacol. 205, 69–102. Reagan-Shaw, S., Nihal, M., Ahmad, N., 2007. Dose translation from animal to human studies revisited. FASEB J. 22, 659–661. Rehman, M.U., Tahir, M., Ali, F., Qamar, W., Lateef, A., Khan, R., Quaiyoom, A., Oday-OHamiza., Sultana, S., 2012. Cyclophosphamide-induced nephrotoxicity, genotoxicity, and damage in kidney genomic DNA of Swiss albino mice: the protective effect of ellagic acid. Mol. Cell Biochem. 365, 119–127. Sghaier, M. Ben, Ismail, M., Ben, Bouhlel, I., Ghedira, K., Chekir-Ghedira, L., 2016. Leaf extracts from Teucrium ramosissimum protect against DNA damage in human lymphoblast cell K562 and enhance antioxidant, antigenotoxic and antiproliferative activity. Environ. Toxicol. Pharmacol. 44, 44–52. http://dx.doi.org/10.1016/j.etap. 2016.04.006. Silva, I.C., Polaquini, C.R., Regasini, L.O., Ferreira, H., Pavan, F.R., 2017. Evaluation of cytotoxic, apoptotic, mutagenic, and chemopreventive activities of semi-synthetic esters of gallic acid. Food Chem. Toxicol. 105, 300–307. Sponchiado, G., Adam, M.L., Silva, C.D., Soley, B.S., Mello-Sampayo, C., Cabrini, D.A., Correr, C.J., Otuki, M.F., 2016. Quantitative genotoxicity assays for analysis of medicinal plants: a systematic review. J. Ethnopharmacol. 178, 289–296. Staines, S.S., 2011. Herbal medicines: adverse effects and drug-herb interactions. J. Malta Coll. Pharm. Pract. 17, 32–42.

The SIECm reduced plasma triglyceride levels in the groups treated with the two higher doses. Although the values are within the physiological range, it is interesting to note that high levels of triglycerides are directly associated with increased risk of developing cardiovascular diseases, accumulation of fat in the liver and pancreatitis thereby leading to a decline in the quality of life of the patients and even morbidities. An agent capable of significantly reducing triglyceride levels may be of great interest and further research is needed to better characterize this effect of SIECm (Kushner and Cobble, 2016). In general, the observed changes in some hematological and biochemical parameters appear to be of no toxicological importance, since the mean values of the altered laboratory parameters are within the physiological ranges (Melo et al., 2012) and were not dose-dependent. The riverine communities of northern Araguaia prepare the infusion by incubating approximately 40 g of dry leaves of C. malmei in 1 L of boiled water that corresponds to 5.04 mg/mL in terms of extractives. Therefore, for an adult of 70 kg, the daily intake is 21.6 mg/kg. The highest dose used in the rats was 1000 mg/kg indicating that the dose used in the animal was 46.3 times greater than in humans. Taking into account the body absorption surface (Reagan-Shaw et al., 2007), the NOAEL dose (Lewis et al., 2002) was 1000 mg/kg for rats which is equivalents to the dose of 162.16 mg/kg in the human species, indicating that it is safe to use doses of SIECm up to 7.5 times greater than the dose usually used. Using HPLC technique, the presence of gallic acid, ellagic acid, rutin, quercetin and catechin were identified in SIECm (Adzu et al., 2015). Among these substances, ellagic acid and gallic acid were previously shown to have antigenotoxic activity in comet and micronucleus models (Rehman et al., 2012; Silva et al., 2017), as well as having no subchronic toxicity (Niho et al., 2001; Tasaki et al., 2008). Patil et al. (2014) described the genoprotective activity of quercetin and rutin in in vitro experimental models. 5. Conclusion It is concluded from this study that the SIECm has a high margin of safety in rats with NOAEL of up to 1000 mg/kg/day for 30 days in rats. SIECm showed genoprotective activity at concentrations up to 30 μg/ mL, and was non-genotoxic in vitro. Acknowledgments We would like to thank the ‘Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)’, Brazil for granting master's degree grant to E. Pavan (No. 132286/015-7); the ‘Instituto Nacional de Áreas Úmidas (INAU/CNPq/MCTI)’, Brazil for granting a postdoctoral fellowship to Bulus Adzu (No. 151135/2014-2); and the ‘Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/Pró-Amazônia)’, Brazil for financial support (No. 23038.000731/2013-56). We are also grateful to the Lunicéia Oliveira Pharmacy of the Bioseg Laboratory, Cuiabá, Mato Grosso, Brazil for their assistance in performing the biochemical tests. References Adzu, B., Balogun, S.O., Pavan, E., Ascêncio, S.D., Soares, I.M., Aguiar, R.W.S., Ribeiro, R.V., Beserra, Â.M.S.E.S., De Oliveira, R.G., Da Silva, L.I., Damazo, A.S., Martins, D.T.D.O., 2015. Evaluation of the safety, gastroprotective activity and mechanism of action of standardised leaves infusion extract of Copaifera malmei Harms. J. Ethnopharmacol. 175, 378–389. http://dx.doi.org/10.1016/j.jep.2015.09.027. Albuquerque, K.C.O., Veiga, A.S.S., Silva, J.V.S., Brigido, H.P.C., Ferreira, E.P.R., Costa, E.V.S., Marinho, A.M.R., Percário, S., Dolabela, M.F., 2017. Brazilian Amazon traditional medicine and the treatment of difficult to heal leishmaniasis wounds with Copaifera. Evid.-Based Complement. Altern. Med. http://dx.doi.org/10.1155/2017/ 83503202017. Arnhold, J., 2004. Free radicals – friends or foes? Properties, functions, and secretion of

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Journal of Ethnopharmacology 211 (2018) 70–77

E. Pavan et al.

a subchronic toxicity study using F344 rats. Food Chem. Toxicol. 46, 1119–1124. WHO, 2011. Herbal Medicine Research and Global Health: An Ethical Analysis. World Health Organisation, 20 Avenue Appia, 1211 Geneva 27, Switzerland. Wiesner, J., 2014. Challenges of safety evaluation. J. Ethnopharmacol. 158, 467–470.

Strober, W., 2001. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. http://dx.doi.org/10.1002/0471142735.ima03bs21. (A.3B.1–A.3B.2). Tasaki, M., Umemura, T., Maeda, M., Ishii, Y., Okamura, T., Inoue, T., Kuroiwa, Y., Hirose, M., Nishikawa, A., 2008. Safety assessment of ellagic acid, a food additive, in

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