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Acute and chronic toxicity and antimicrobial activity of the extract of Stryphnodendron adstringens (Mart.) Coville

Toxicidade aguda e crônica e atividade antimicrobiana do extrato de Stryphnodendron adstringens (Mart.) Coville

ABSTRACT:

This study evaluated the antimicrobial activity and acute or chronic toxicity of the extract of Stryphnodendron adstringens. The stem bark dry extract was obtained by static maceration with ethanol. Quantification of tannins was performed by the Folin-Denis method, which indicated a total tannin content of 32.7%. The antimicrobial activity of the dry extract of S. adstringens was evaluated by agar-based disk diffusion assay with Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) in the concentration of 200, 400 and 600μL/mL. The results indicated that 600μL/mL inhibited microbial growth, i.e. had antimicrobial activity against these species. Acute and chronic toxic effects of S. adstringens was evaluated in Wistar rats treated with 200, 400, 600 and 800mg/kg of extract, administrated by gavage. Liver degeneration was observed in the group of rats receiving 800mg/kg in chronic exposure, what may indicate some degree of toxicity at this concentration. However, no systemic toxicity was observed at lower doses. Considering the broad use of S. adstringens as a phytotherapeutic agent for various human and animal diseases and the livertoxicity observed at high concentrations, attention should be paid to the possible adverse effect of using the extract from this plant at high concentration.

INDEX TERMS:
Toxicity; antimicrobial activity; extract; Stryphnodendron adstringens; tannins; liver toxicity; “barbatimão”; hematological parameters; biochemical parameters

RESUMO:

Avaliou-se neste estudo a atividade antimicrobiana e a toxicidades aguda e crônica do extrato da entrecasca de Stryphnodendron adstringens. A partir do extrato seco, obtido através da maceração estática da casca do caule em etanol de cereais, foi realizada a quantificação de taninos totais (32,7%) pelo método de Folin-Denis. A atividade antimicrobiana do extrato seco extraído de cascas do caule de S. adstringens foi avaliada pela técnica de disco-difusão para os micro-organismos Escherichia coli (ATCC 25922) e Staphylococcus aureus (ATCC 25923) nas concentrações de 200, 400 e 600μL/mL. Os testes de concentração inibitória mínima revelaram que 600μL/mL inibiu o crescimento dos dois micro-organismos testados; o mesmo resultado foi observado para atividade bactericida na concentração de 600μL/mL sobre essas espécies. Efeitos tóxicos sistêmicos agudos e crônicos do extrato seco de S. adstringens administrados por gavagem foram estudados em ratos Wistar, utilizando as doses de 400, 600 e 800mg/kg. Foi observada degeneração hepática no grupo de animais que receberam 800mg/kg tanto no estudo da toxicidade aguda quanto crônica, que pode indicar algum grau de toxicidade de S. adstringens nessa concentração. Considerando o amplo uso de S. adstringens como fitoterápico para humanos e animais, atenção deve ser dispensada para ingestão em altas doses mediante os efeitos tóxicos observados neste estudo.

TERMOS DE INDEXAÇÃO:
Toxicidade; atividade antimicrobiana; extrato; Stryphnodendron adstringens; taninos; toxicidade hepática; “barbatimão”; parâmetros hematológicos; parâmetros bioquímicos

Introduction

Tannins are water-soluble polyphenols that are present in many plants. There is a series of previous studies describing the biological action of tannins including their pharmacological, and toxic activities. The dosage and type of tannins determine these effects (Macáková et al. 2014Macáková K., Kolečkář V., Cahlíková L., Chlebek J., Hošťálková A., Kuča K., Jun D. & Opletal L. 2014. Tannins and their Influence on health, p.159-208. In: Rahman A. , Choudhary M.I. (Eds), Recent Advances in Medicinal Chemistry: tannins_and_their_influence_on_health. Available in <Available in https://www.researchgate.net/publication/282796040 > Accessed on: January 22, 2016.
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).

Stryphnodendron adstringens, popularly known as barbatimão, is a medicinal plant abundant in central Brazil, which has been traditionally used as a popular herbal medicine (Melo et al. 2007Melo J.O., Endo T.H., Bersani-Amado L.E., Estivalet S.A., Silmara B., Mello J.C.P. & Bersani-Amado C.A. 2007. Effect of Stryphnodendron adstringens (barbatimão) bark on animal models of nociception. Braz. J. Med. Biol. Res. 43(3):465-469.), which is endemic to the savannah, and its bark has a high tannin content (25-37%). Macáková et al. (2014)Macáková K., Kolečkář V., Cahlíková L., Chlebek J., Hošťálková A., Kuča K., Jun D. & Opletal L. 2014. Tannins and their Influence on health, p.159-208. In: Rahman A. , Choudhary M.I. (Eds), Recent Advances in Medicinal Chemistry: tannins_and_their_influence_on_health. Available in <Available in https://www.researchgate.net/publication/282796040 > Accessed on: January 22, 2016.
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and Okuda & Ito (2011)Okuda T. & Ito H. 2011. Tannins of constant structure in medicinal and food plants-hydrolyzable tannins and polyphenols related to tannins. Molecules 16:2191-2217. described a wide pharmacological spectrum of activity of polyphenols including hydrolysable tannins. However, Ferreira et al. (2009)Ferreira E.V., Boabaid F.M., Arruda L.P., Lemos R.A.A., Souza M.A., Nakazato L. & Colodel E.M. 2009. Intoxicação por Stryphnodendron fissuratum (Mimosoideae) em bovinos. Pesq. Vet. Bras. 29(11):951-957. and Aguiar-Filho (2013)Aguiar-Filho C.R., Albuquerque R.F., Rocha B.P., Colodel E.M., Lemos R.A.A., Riet-Correa F., Evêncio-Neto J. & Mendonça F.S. 2013. Avaliação da toxicidade das favas de Stryphnodendron fissuratum (Mimosoideae) em vacas gestantes. Pesq. Vet. Bras. 33(5):607-612. reported intoxication of ruminants with plants that have tannins.

Local populations in the savannah regions of Brazil use infusion or tea of leaves or stem bark from S. adstringens as astringent, antimicrobial, homeostatic agent, antidiarrheal, antihypertensive or blood clotting agent. In addition, it is used for treating leucorrhoea, hemorrhoids, hemorrhages, and inflammation. Various studies have suggested the use of infusions and decoctions of this plant in Brazil for the treatment of animal diseases (Confessor et al. 2009Confessor M.V.A., Mendonça L.V., Mourão J.S. & Alves R.R.N. 2009. Animals to heal animals: ethnoveterinary practices in semiarid region, northeastern Brazil. J. Ethnobiol. Ethnomed. 5(37). Available in <Available in http://ethnobiomed.biomedcentral.com/articles/10.1186/1746-4269-5-37 > Accessed on December 10, 2013.
http://ethnobiomed.biomedcentral.com/art...
, Viu & Viu 2011Viu A.F.M. & Viu M.A.O. 2011. Cerrado e etnoveterinária: o que se sabe em Jataí, GO. Revta Bras. Agroecol. 6(3):49-61, Monteiro et al. 2011Monteiro M.V.B., Bevilaqua C.M.L., Palha M.D.C., Braga R.R., Schwanke K., Rodrigues S.T. & Lameira O.A. 2011. Ethnoveterinary knowledge of the inhabitants of Marajó Island, eastern Amazonia, Brazil. Acta Amaz. 41(2):233-242.).

Several in vitro and in vivo studies on the activity of Strynodendrum sp. tannins showed that the crude and purified extracts have anti-inflammatory, antiulcerogenic, antiprotozoal, anthelmintic, antiviral, and antifungal activities (Martins et al. 2003Martins P.S., Alves A.L.G., Hussni J.L., Sequeira J.L., Nicoletti J.L.M. & Thomassian A. 2003. Comparação entre fitoterápicos de uso tópico na cicatrização de pele em equinos. Arch. Vet. Sci. 8(2):1-7., Silva et al. 2009Silva L.A.F., Moura M.I., Persiano C.B., Helou J.B., Lima C.R.O., Freitas S.L.R., Silva J.A., Goulart D.S. & Castro L.T.S. 2009. Extrato da casca do barbatimão (Stryphnodendron barbatiman Martius) associado ao tratamento cirúrgico e toalete dos cascos na recuperação de bovinos da raça nelore com dermatite digital. Ciênc. Anim. Bras. (Supl.1):373-378., Cansi & Demo 2011Cansi E.R. & Demo C. 2011. Ocorrência de miíases em animais de companhia no Distrito Federal, Brasil. Acta Scient. Vet. 39(3):1-5., Moura 2011Moura M.I. 2011. Enfermidades cutâneas digitais bovina: aspectos genéticos e terapêuticos. Tese de Doutorado, Escola de Veterinária e Zootecnia, Universidade Federal de Goiás, Goiânia, Go. 111p.). Its use in the clinical treatment of bacterial and parasitic diseases and for skin lesions in animals have also been reported (Confessor et al. 2009Confessor M.V.A., Mendonça L.V., Mourão J.S. & Alves R.R.N. 2009. Animals to heal animals: ethnoveterinary practices in semiarid region, northeastern Brazil. J. Ethnobiol. Ethnomed. 5(37). Available in <Available in http://ethnobiomed.biomedcentral.com/articles/10.1186/1746-4269-5-37 > Accessed on December 10, 2013.
http://ethnobiomed.biomedcentral.com/art...
, Monteiro et al. 2011Monteiro M.V.B., Bevilaqua C.M.L., Palha M.D.C., Braga R.R., Schwanke K., Rodrigues S.T. & Lameira O.A. 2011. Ethnoveterinary knowledge of the inhabitants of Marajó Island, eastern Amazonia, Brazil. Acta Amaz. 41(2):233-242., Viu & Viu 2011Viu A.F.M. & Viu M.A.O. 2011. Cerrado e etnoveterinária: o que se sabe em Jataí, GO. Revta Bras. Agroecol. 6(3):49-61).

Studies on the acute and chronic toxicity of Strynodendrum sp. extracts showed variable results depending on the method and extract concentration studied. Some researchers have evaluated the acute toxicity of extract of stem bark from S. adstringens orally ingested (Lima et al. 1998Lima J.C.S., Martins D.T.O. & Souza Júnior P.T. 1998. Experimental evaluation of stem bark of Stryphnodendron adstringens (Mart.) Coville for anti-inflammatory activity. Phytother. Res. 12:212-220., Audi et al. 1999Audi E.A., Toledo D.P., Perez P.G., Kimura E., Pereira W.K., Mello J.C., Nakamura C., Alves-do-Prado W., Cuman R.K. & Bersani-Amado C.A. 1999. Gastric antiulcerogenic effects of Stryphnodendron adstringens in rats. Phytother. Res. 13(3):264-266., Rebecca et al. 2002Rebecca M.A., Ishii-Iwamoto E.L., Grespan R., Cuman R.K., Caparroz-Assef S.M., Mello J.C. & Bersani-Amado C.A. 2002. Toxicological studies on Stryphnodendron adstringens. J. Ethnopharmacol. 83(1/2):101-104.). They demonstrated that LD50% ranged from 400 to 2699mg/kg. An initial preclinical toxicological assay conducted by Almeida et al. (2010)Almeida A.C., Sobrinho E.M., Pinho L., Souza P.N.S., Martins E.R., Duarte E.R., Santos H.O., Brandi I.V., Cangussu A.S. & Costa J.P.R. 2010. Toxicidade aguda de extratos hidroalcóolicos de folhas de alecrim-pimenta, Stryphnodendron adstringens, aroeira e farelo da casca de pequi administrados intraperitonealmente. Ciência Rural 40(1):200-203. demonstrated that S. adstringens leaf extract was most toxic among the tested (leaf hydroalcoholic extracts of Lippia sidoides, Myracroduon urundeuva and Caryocar brasilliense), however, limited studies have been conducted to verify its toxic potential.

The National Policy for Medicinal Plants and Herbal Medicines (PNPMF), and the Brazilian National Program for Medicinal Plants and Phytotherapics published the Brazilian National List of Medicinal Plants of Interest to the SUS (RENISUS) in January 2009. This list contains medicinal plants that exhibit potential for generating products of interest to Brazilian Unified Health System (SUS), including S. adstringens (Brasil 2006Brasil 2006. Ministério da Saúde. Agência Nacional de Vigilância Sanitária. Decreto no.5813, de 22 de junho de 2006. Aprova a Política Nacional de Plantas Medicinais e Fitoterápicos e de Outras Providências. Ministério da Saúde. Brasíla, DF. Available in <Available in http://bvsms.saude.gov.br/bvs/publicacoes/politica_nacional_fitoterapicos.pdf > Accessed on December 10, 2013.
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). This study aimed to evaluate the antimicrobial activity and acute and chronic toxicity of hydroalcoholic extracts of stem bark from S. adstringens from the northern region of Minas Gerais, Brazil.

Materials and Methods

Obtaining and preparing theStryphnodendron adstringens extract.S. adstringens stem bark was harvested at Bela Vista farm, in Botumirim, State of Minas Gerais, Brazil (16° 57.847’ S, 43° 04.308’ W, elevation: 890m). The plant was identified by Rubens Teixeira de Queiroz at the Federal University of Paraíba, Brazil. To obtain the extract, stem bark of S. adstringens was dried in a forced air circulation chamber under an average temperature of 50°C, followed by daily weighing until the material reached a constant weight. The dry S. adstringens bark was ground using a Willey knife mill in a continuous system. The ground material was subjected to extraction by static soaking in cereal ethanol for eight days (bark:ethanol ratio of 1:2). The mixture was then filtered, and the solvent was evaporated from the extract in an air-circulated oven at 45°C until completely dry. The dried S. adstringens extract obtained was stored in an amber vial away from moisture and light.

Phenols and tannins content. Initially, 0.75g of dry extract of S. adstringens was dissolved in 150ml of distilled water to obtain a solution 1 which was used to determine total phenolic by the Folin-Denis method using tannic acid (0.1, 0.5, 1.0, 2.5, and 3.75mg/mL) as the standard for the calibration curve (Pansera et al. 2003Pansera M.R., Santos A.C.A., Paese K., Wasum R., Rossato M., Rota L.D., Pauletti G.F. & Serafini L.A. 2003. Análise de taninos totais em plantas aromáticas e medicinais cultivadas no Nordeste do Rio Grande do Sul. Revista Bras. Farmacogn. 13(1):17-22.). The system was maintained at agitation for 30 minutes at 90°C. Next, the system was cooled, the content was transferred to a volumetric flask and the volume was completed to 250mL with distilled water. The flask was maintained at rest for 30 minutes, followed by filtration, and the first 50mL of the filtrate was discarded. The Folin-Denis method comprised addition of the solution 1 (5mL) to 2mL of Folin-Denis reagent (2.50g sodium tungstate, 0.5g phosphomolybdic acid, 1.25mL phosphoric acid, and distilled water for a final volume of 25mL) and 50mL of 20% (w/v) sodium carbonate solution. The solution 1 also was used to calculate the content of tannin. This calculation was carried out using a reference solution containing Folin-Denis reagent, pyrogallol and sodium carbonate. The absorbance was measured at wavelength of 715nm. The parameters was used in the expressions below Samples were analyzed using a spectrophotometer 3 minutes after adding the last reagent. The analyses were performed in triplicate, and the tannin content was expressed in percentage (w/w) according to the equation proposed by Dôres & Casali (2007)Dôres R.G.R. & Casali V.W.D. 2007. Plantas Medicinais e Aromáticas: controle de qualidade. Universidade Federal de Viçosa, Viçosa, MG, p.106-107.:

A 1 % = A 3 × 10 C

T T = F D × A 1 ( m p ) × A 1 %

Where: A1% = specific absorbance of the reference solution, A3 = measured absorbance of the reference substance, c = concentration in mg/mL, TT = total tannins in % (m/m), FD = 50 (dilution factor of the sample), A1 = measured absorbance of the total tannins, m = water determination, p = drug mass (g) considering the water determination.

Microbial sensibility test. The microbial sensitivity test of S. adstringens extract was performed using the disk-diffusion technique. The minimum-inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were defined according to the National Committee for Clinical Laboratory Standards (CLSI 2012CLSI 2012. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard-Ninth Edition. CLSI document M07-A9. Clinical and Laboratory Standards Institute, Wayne, PA. Avaiable in < Avaiable in http://antimicrobianos.com.ar/ATB/wp-content/uploads/2012/11/03-CLSI-M07-A9-2012.pdf > Accessed on January 22, 2016.
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). Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were prepared and standardized to 0.5 on the MacFarland scale. Based on results of previous research (Pinho et al. 2012Pinho L., Souza P.N.S., Sobrinho Macedo E., Almeida A.C. & Martins E.R. 2012. Atividade antimicrobiana de extratos hidroalcóolicos das folhas de alecrim-pimenta, aroeira, barbatimão, erva baleeira e do farelo da casca de pequi. Ciênc. Rural 42(2):326-331.). Concentrations of 200, 400, and 600μL/mL were used for the disk-diffusion tests, and the MIC test. Further, 600 μL/mL was used for the MBC test. Tests were performed in duplicate for each of the extract concentrations used.

Rats (in vivotoxicity). The acute toxicity classification method established by the OECD 420 (2001)OECD 2001. Guidelines for the Testing of Chemicals, n. 420: acute oral toxicity, fixed dose procedure. Avaiable in < Avaiable in http://www.oecd-ilibrary.org/docserver/download/9742001e.pdf?expires=1455306679&id=id&accname=guest&checksum=635B12BD5729564AEC4136E85BC32D12 > Accessed on January 22, 2016.
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was adopted to evaluate acute and OECD 452 (2008)OECD 2008. Guidelines for the Testing of Chemicals, n. 452: chronic toxicity studies. Avaiable in < Avaiable in http://www.oecd-ilibrary.org/docserver/download/9745201e.pdf?expires=1455306478&id=id&accname=guest&checksum=417154C7DA531CDAB6D1BD24D2DDA885 > Accessed on January 22, 2016.
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with adaptations was employed to evaluate chronic toxicity. For this procedure, female Wistar rats, weighing between 180 and 200g, were used. The experimental procedures using rats were approved by the Animal Experimentation Ethics Committee of the UFMG, under protocol no. 034/2008.

Acute toxicity. After obtaining the microbiological test results, 200, 400, and 600mg/kg concentrations were tested. These doses were also defined by considering previously published data that reported concentrations up to 600mg/kg of S. adstringens bark extract administered orally that did not induce animal death (Rebecca et al. 2002Rebecca M.A., Ishii-Iwamoto E.L., Grespan R., Cuman R.K., Caparroz-Assef S.M., Mello J.C. & Bersani-Amado C.A. 2002. Toxicological studies on Stryphnodendron adstringens. J. Ethnopharmacol. 83(1/2):101-104.). For evaluation of acute systemic toxicity, a single dose of each concentration prepared in sterile saline solution was administered by gavage. The rats were divided into four groups with five rats in each group: group 1 (control with sterile saline solution), group 2 (200mg/kg), group 3 (400mg/kg), and group 4 (600mg/kg). After treatment, all rats received food and water and a 12-h light/dark circadian cycle at 22±2°C. The rats were weighed every other day. At the end of the experimental period (14 days), the surviving rats were sedated with a combination of ketamine (80mg) and xylazine (15mg/kg) administered intraperitoneally and killed. Blood samples (approximately 4mL) were collected by cardiac puncture to determine the hematological (erythrocyte, hemoglobin, hematocrit (PCV), MCV, MCH, MCHC, platelet, and lymphocyte count) and biochemical parameters (ALT, AST urea and creatinine). Macroscopic analyses were performed, and the liver, heart, and kidneys were weighed to determine their relative weights. Tissue samples were collected for histopathological analyses. These tissues were fixed by immersion in 10% buffered formalin for 24 hours, followed by dehydration in increasing ethanol concentrations, diaphonization in xylene, and paraffin embedding. Five-μm thick sections were cut and stained with hematoxylin and eosin (HE). Lesions were evaluated according to the intensity, and a score was given to each lesion, where 0 = absence of lesions, 1 = mild lesion, 2 = moderate lesion, and 3 = severe lesion. The analyses were performed at the Molecular Pathology Laboratory of the Veterinary School of the UFMG.

Chronic toxicity. Rats were kept under the same conditions described for the acute toxicity tests. The experiment was divided into two phases, which had either 15 or 30 days of duration. For each of these phases, two groups of five rats each were formed. One group received sterile saline solution (control) and the other received 800mg/kg of the S. adstringens extract, daily, once a day. At the end of each phase the rats were killed and fragments of the kidneys, liver, and heart were collected. Animal weight, hematological and biochemical parameters, macroscopic evaluation of the organs and histopathological analyses were performed according to the procedure described for acute toxicity evaluation.

Statistical analysis. Data were analyzed using analysis of variance (ANOVA) followed by Bonfferroni post test or Dunn’s multiple comparison test. When appropriate, the Student t-test with the Bonferroni adjustment was used for pairwise comparisons. A value of p<0.05 was used to indicate a statistically significant difference. For histological analyses, the Kruskal-Wallis non-parametric test was performed followed by Dunn’s multiple comparison test. All statistical tests were performed using the software Graph Pad Prism (San Diego, CA).

Results and Discussion

Through the determination of phenolic compounds, it was possible to infer the total tannin content (32.7%) of the of S. adstringens stem bark. The result agrees with the Brazilian Pharmacopeia recommends minimum 8.0% of total tannins (Brasil 2010Brasil 2010. Farmacopeia Brasileira. Vol.2. Agência Nacional de Vigilância Sanitária, Brasília. 904p.) and was similar to that reported in studies conducted in the same species (20-40%) (Castro et al. 2009Castro A.H.F., Paiva R., Alvarenga A.A. & Vitor S.M.M. 2009. Calogênese e teores de fenóis e taninos totais em barbatimão [Stryphnodendron adstringens (Mart.) Coville]. Ciênc. Agrotec. 33(2):385-390., Corrêa et al. 2012Corrêa V.S., Cerdeira A.L., Fachin A.L., Bertoni B.W., Pereira P.S., França P.S., Momm H.G., Moraes R.M. & Pereira A.M.S. 2012. Geographical variation and quality assessment of Stryphnodendron adstringens (Mart.) Coville within Brazil. Genet. Resour. Crop Evolution 59(7):1349-1356.). Tannins are the major compounds responsible for pharmacological activity of this particular tree and they exhibit various biological functions. Antimicrobial activity of tannins is well documented; however the mechanisms of this activity, as well as their bioavailability have not been satisfactorily clarified (Macáková et al. 2014Macáková K., Kolečkář V., Cahlíková L., Chlebek J., Hošťálková A., Kuča K., Jun D. & Opletal L. 2014. Tannins and their Influence on health, p.159-208. In: Rahman A. , Choudhary M.I. (Eds), Recent Advances in Medicinal Chemistry: tannins_and_their_influence_on_health. Available in <Available in https://www.researchgate.net/publication/282796040 > Accessed on: January 22, 2016.
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).

Results of the sensitivity tests using the S. adstringens extract disks (600μL/mL) exhibited a significant inhibition zone against S. aureus; however, there was no significant difference against Escherichia coli (Fig.1). This same concentration inhibited Staphylococcus aureus and E. coli growth in the test tube. This was confirmed by the absence of microbial growth in Petri dishes, according to the recommendations of NCCLS (2003). Costa et al. (2011)Costa J.P.R., Almeida A.C., Martins E. R., Rodrigues M.N., Santos C.A. & Menezes I.R. 2011. Atividade antimicrobiana do óleo essencial de alecrim-pimenta e do extrato bruto seco do barbatimão diante de bactérias isoladas do leite. Biotemas 24(4):1-6. used extract obtained by methodology different from the one adopted in this study to evaluate growth of E. coli and S. aureus isolated from bovine milk, and Pinho et al. (2012)Pinho L., Souza P.N.S., Sobrinho Macedo E., Almeida A.C. & Martins E.R. 2012. Atividade antimicrobiana de extratos hidroalcóolicos das folhas de alecrim-pimenta, aroeira, barbatimão, erva baleeira e do farelo da casca de pequi. Ciênc. Rural 42(2):326-331. evaluated the activity of S. adstringens extract obtained from leaves in these same microorganisms. These studies showed that concentrations ranging from 25 to 500mg/mL of dried crude S. adstringens extract did not inhibit E. coli growth (Costa et al. 2011Costa J.P.R., Almeida A.C., Martins E. R., Rodrigues M.N., Santos C.A. & Menezes I.R. 2011. Atividade antimicrobiana do óleo essencial de alecrim-pimenta e do extrato bruto seco do barbatimão diante de bactérias isoladas do leite. Biotemas 24(4):1-6., Pinho et al. 2012Pinho L., Souza P.N.S., Sobrinho Macedo E., Almeida A.C. & Martins E.R. 2012. Atividade antimicrobiana de extratos hidroalcóolicos das folhas de alecrim-pimenta, aroeira, barbatimão, erva baleeira e do farelo da casca de pequi. Ciênc. Rural 42(2):326-331.). Concentrations higher than or equal to 300mg/mL can inhibit S. aureus growth (Pinho et al. 2012Pinho L., Souza P.N.S., Sobrinho Macedo E., Almeida A.C. & Martins E.R. 2012. Atividade antimicrobiana de extratos hidroalcóolicos das folhas de alecrim-pimenta, aroeira, barbatimão, erva baleeira e do farelo da casca de pequi. Ciênc. Rural 42(2):326-331.). Although it is not possible to make a comparison of our results with the former ones, as they used methodology and sources of different extracts, our results corroborate with those findings and suggest that S. adstringens extract also inhibits microbial growth.

Fig.1.
Sensitivity test for Escherichia coli and Staphylococcus aureus to the Stryphnodendron adstringens extract. Data represent mean and standard deviation. * Indicates a significant difference between the treatments and the control (p<0.05).

The rat weights (Fig.2A) and relative organ weights (Fig.2B) of the groups treated with the different concentrations of S. adstringens extract did not statistically differ compared to the control group, in contrast to the study of Rebecca et al. (2002)Rebecca M.A., Ishii-Iwamoto E.L., Grespan R., Cuman R.K., Caparroz-Assef S.M., Mello J.C. & Bersani-Amado C.A. 2002. Toxicological studies on Stryphnodendron adstringens. J. Ethnopharmacol. 83(1/2):101-104. and Costa et al. (2013)Costa M.A., Mello J.C., Kaneshima E.N., Ueda-Nakamura T., Dias Filho B.P., Audi E.A. & Nakamura C.V. 2013. Acute and chronic toxicity of an aqueous fraction of the stem bark of Stryphnodendron adstringens (barbatimão) in rodents. Evid. Based Complement. Alternat. Med. Available in: <Available in: http://dx.doi.org/10.1155/2013/841580 > Accessed on April 14, 2014.
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that observed impairment in animal development at the dosages tested.

Fig.2.
Evaluation of the animal and organ weights. (A) Animal weight of the control and groups treated with different concentrations of Stryphnodendron adstringens extract; the values show a variation in the animal weight during 14 days of treatment. (B) Relative organ weight (p<0.05).

Hematological parameters were within the reference ranges, whereas no significant changes were observed in the complete blood count analyses and biochemical parameters, indicating that the rats did not develop any clinical abnormalities throughout the experiment.

Biochemical analysis of blood taken from rats treated with S. adstringens showed that there were changes only in concentration of aspartate aminotransferase (AST) in rats treated with 200mg/kg of S. adstringens, indicating a possible hepatic alteration in this group (Table 1). However, results of the histopathological analyses of the organs collected for the acute toxicity tests (based on histopathology scores) revealed that only the livers of the group treated with 600mg/kg of S. adstringens extract exhibited significant microscopic changes compared with the control group (p<0.001) (Fig.3). This alteration was characterized by mild centrilobular degeneration, similar to lesions found in chronic toxicity tests as presented below. Pathological alterations were observed in high concentration, while physiological production of AST was altered only in lower concentration. This finding suggests that the S. adstringens extract may exhibit some liver toxicity. S. adstringens extract has been shown to be toxic in other studies in rats. Rebecca et al. (2002)Rebecca M.A., Ishii-Iwamoto E.L., Grespan R., Cuman R.K., Caparroz-Assef S.M., Mello J.C. & Bersani-Amado C.A. 2002. Toxicological studies on Stryphnodendron adstringens. J. Ethnopharmacol. 83(1/2):101-104. demonstrated that repeated 800mg/kg and 1600mg/kg doses of the extract administered orally caused chronic toxicity in rats, where an LD50% of 2669mg/kg was obtained. Rebecca et al. (2003)Rebecca M.A., Ishii-Iwamoto E.L., Kelmer-Bratch A.M., Caparroz-Assef S.M., Cuman R.K., Pagadigorria C.L., Mello J.C., Bracht A. & Bersani-Amado C.A. 2003. Effect of Strphnodendron adstringens (barbatimão) on energy metabolism in the rat liver. Toxicol. Lett. 143:55-63. evaluated the toxicity of the same methanol extract on mitochondria obtained from liver cells and reported altered energy metabolism. Costa et al. (2013)Costa M.A., Mello J.C., Kaneshima E.N., Ueda-Nakamura T., Dias Filho B.P., Audi E.A. & Nakamura C.V. 2013. Acute and chronic toxicity of an aqueous fraction of the stem bark of Stryphnodendron adstringens (barbatimão) in rodents. Evid. Based Complement. Alternat. Med. Available in: <Available in: http://dx.doi.org/10.1155/2013/841580 > Accessed on April 14, 2014.
http://dx.doi.org/10.1155/2013/841580...
evaluated acute and chronic toxicity of the same orally ingested fraction and reported no acute toxicity, thereby defining an LD50%of 3015mg/kg. Furthermore, toxicity was not observed at the orally ingested doses of 10, 100, and 200mg/kg during the 90 days of chronic toxicity tests. Oliveira et al. (2013)Oliveira J.R., Castro V.C., Vilela P.G.F., Camargo S.E.A., Carvalho A.T., Jorge A.O.C. & Oliveira L.D. 2013. Cytotoxicity of Brazilian plant extracts against oral microorganisms of interest to dentistry. BMC Complement. Altern. Med. Avaiable in < Avaiable in http://www.biomedcentral.com/1472-6882/13/208 > Accessed on April 14, 2014.
http://www.biomedcentral.com/1472-6882/1...
evaluated the cytotoxicity of aqueous fractions of S. adstringens on rat macrophages at 50mg/mL doses and did not observe any decrease in cell viability.

Table 1.
Biochemical parameters of Wistar rats treated with a single oral dose of the Stryphnodendron adstringens bark extract

Fig.3.
Histopathological analysis of acute toxicity of the orally ingested Stryphnodendron adstringens extract in rats treated with 200, 400, and 600mg/mL for 14 days. *Indicates a significant difference between the treatments compared and the control (p<0.001).

The values obtained in this study were lower than those previously described (Rebecca et al. 2002Rebecca M.A., Ishii-Iwamoto E.L., Grespan R., Cuman R.K., Caparroz-Assef S.M., Mello J.C. & Bersani-Amado C.A. 2002. Toxicological studies on Stryphnodendron adstringens. J. Ethnopharmacol. 83(1/2):101-104., Costa et al. 2013Costa M.A., Mello J.C., Kaneshima E.N., Ueda-Nakamura T., Dias Filho B.P., Audi E.A. & Nakamura C.V. 2013. Acute and chronic toxicity of an aqueous fraction of the stem bark of Stryphnodendron adstringens (barbatimão) in rodents. Evid. Based Complement. Alternat. Med. Available in: <Available in: http://dx.doi.org/10.1155/2013/841580 > Accessed on April 14, 2014.
http://dx.doi.org/10.1155/2013/841580...
), indicating that 400mg/kg of S. adstringens extract orally do not result in acute toxicity and can be safe and do not cause acute toxicity. Although this dose has not shown toxicity, was defined the dose of 800mg/kg for chronic toxicity experiments.

The results of the chronic toxicity experiments revealed no changes in body weight during 30 days of the experiment (Fig.4A). No significant changes in the organ weights were observed in treated rats when compared with the control group (Fig.4B). Regarding hematological and biochemical parameters, significant variation was not observed in any of the parameters evaluated (neither at 15 days nor at 30 days) relative to the control group, suggesting that there was no chronic toxicity in the rats. After 30 days of treatment only the MCV (mean corpuscular volume) and MCH (mean corpuscular concentration) had significantly decreased (Table 2). MCH indicates the quantity of hemoglobin in a sample of blood and MHCH indicates the concentration of hemoglobin; reduction in hemoglobin is associated with anemia, so these results suggested a possible anemia of rats treated with 800mg/mL of S. adstringens, when compared with the control group. Also a decrease in urea concentration after 30 days was observed, which can be the result of a deregulation of renal and hepatic function when rats were treated with 800mg/mL of S. adstringens (Table 2). Similar data were obtained by Costa et al. (2013)Costa M.A., Mello J.C., Kaneshima E.N., Ueda-Nakamura T., Dias Filho B.P., Audi E.A. & Nakamura C.V. 2013. Acute and chronic toxicity of an aqueous fraction of the stem bark of Stryphnodendron adstringens (barbatimão) in rodents. Evid. Based Complement. Alternat. Med. Available in: <Available in: http://dx.doi.org/10.1155/2013/841580 > Accessed on April 14, 2014.
http://dx.doi.org/10.1155/2013/841580...
who evaluated chronic toxicity with repeated doses for 90 days. They did not report any toxicity following the oral administration of the proanthocyanidin fraction to mice. However, Rebecca et al. (2002)Rebecca M.A., Ishii-Iwamoto E.L., Grespan R., Cuman R.K., Caparroz-Assef S.M., Mello J.C. & Bersani-Amado C.A. 2002. Toxicological studies on Stryphnodendron adstringens. J. Ethnopharmacol. 83(1/2):101-104. found different results while using 800 or 1,600mg/kg for prolonged periods. The authors reported animal weight loss, increased blood glucose levels, and increased AST levels at the end of the 30-day treatment period.

Fig.4.
Evaluation of the animal weight. The animal weight of the control and groups treated with 800mg/mL of Stryphnodendron adstringens extract. The values show a variation in the rat weight after 30 days of treatment. (A) Relative animal weight. (B) Relative organ weight.

Table 2.
Hematological and biochemical parameters of Wistar rats treated with oral doses of 800mg/kg of the Stryphnodendron adstringens bark extract for 30 days

Chronic toxicity was assessed by histopathological examination, and the results did not indicate any significant microscopic changes in heart and kidneys, neither at 15 days nor at 30 days after administering 800mg/kg of S. adstringens extract. However, in the liver, there was significant microscopic alteration in the group treated with 800mg/kg at 30 days compared to the control group (p<0.05) (Fig.5A,B) as demonstrated by histopathology scores. This analysis revealed that the livers of the group treated with of S. adstringens extract exhibited minimal or mild to moderate centrilobular degeneration (hydropic hepatocellular degeneration) (Fig.6).

Fig.5.
Histopathological analysis of the chronic toxicity of the Stryphnodendron adstringens extract. (A) Histopathological results of rats treated with 800mg/mL for 15 days, and (B) for 30 days. *Indicates a significant difference between the treatments and the control (p<0.001).

Fig.6.
Representative micrographs of the liver from a control rat (with no histological changes), or treated with 800mg/kg of Stryphnodendron adstringens extract for 15 or 30 days, with minimal or mild to moderate centrilobular degeneration (hydropic hepatocellular degeneration) respectively.

Rebecca et al. (2002Rebecca M.A., Ishii-Iwamoto E.L., Grespan R., Cuman R.K., Caparroz-Assef S.M., Mello J.C. & Bersani-Amado C.A. 2002. Toxicological studies on Stryphnodendron adstringens. J. Ethnopharmacol. 83(1/2):101-104., 2003)Rebecca M.A., Ishii-Iwamoto E.L., Kelmer-Bratch A.M., Caparroz-Assef S.M., Cuman R.K., Pagadigorria C.L., Mello J.C., Bracht A. & Bersani-Amado C.A. 2003. Effect of Strphnodendron adstringens (barbatimão) on energy metabolism in the rat liver. Toxicol. Lett. 143:55-63. observed altered plasma glucose and AST levels combined with a decline in weight of the rats. The effect of the extracts on energy metabolism in the liver was investigated and the results showed that the toxic action may be correlated with interference in the oxidative phosphorylation or electron transport chain or with inhibition ATP-synthase. The authors concluded that such injuries were associated with toxic activity of the tannin present in the extracts when administered at doses above 600mg/kg. Costa et al. (2013)Costa M.A., Mello J.C., Kaneshima E.N., Ueda-Nakamura T., Dias Filho B.P., Audi E.A. & Nakamura C.V. 2013. Acute and chronic toxicity of an aqueous fraction of the stem bark of Stryphnodendron adstringens (barbatimão) in rodents. Evid. Based Complement. Alternat. Med. Available in: <Available in: http://dx.doi.org/10.1155/2013/841580 > Accessed on April 14, 2014.
http://dx.doi.org/10.1155/2013/841580...
found lesions indicative of toxicity, while analyzing the organs from rats subjected to acute and chronic toxicity evaluation of the proanthocyanidin fraction, where only 4000 and 5000mg/kg doses were used, thereby indicating that the fraction exhibited lower toxicity at the tested concentrations than those used by Rebecca et al. (2002Rebecca M.A., Ishii-Iwamoto E.L., Grespan R., Cuman R.K., Caparroz-Assef S.M., Mello J.C. & Bersani-Amado C.A. 2002. Toxicological studies on Stryphnodendron adstringens. J. Ethnopharmacol. 83(1/2):101-104., 2003)Rebecca M.A., Ishii-Iwamoto E.L., Kelmer-Bratch A.M., Caparroz-Assef S.M., Cuman R.K., Pagadigorria C.L., Mello J.C., Bracht A. & Bersani-Amado C.A. 2003. Effect of Strphnodendron adstringens (barbatimão) on energy metabolism in the rat liver. Toxicol. Lett. 143:55-63. and those employed in this study.

Liver lesions have been described in ruminants after intake of mashed beans from Stryphnodendron fissuratum (Ferreira et al. 2009Ferreira E.V., Boabaid F.M., Arruda L.P., Lemos R.A.A., Souza M.A., Nakazato L. & Colodel E.M. 2009. Intoxicação por Stryphnodendron fissuratum (Mimosoideae) em bovinos. Pesq. Vet. Bras. 29(11):951-957., Aguiar-Filho 2013Aguiar-Filho C.R., Albuquerque R.F., Rocha B.P., Colodel E.M., Lemos R.A.A., Riet-Correa F., Evêncio-Neto J. & Mendonça F.S. 2013. Avaliação da toxicidade das favas de Stryphnodendron fissuratum (Mimosoideae) em vacas gestantes. Pesq. Vet. Bras. 33(5):607-612.). Fonseca & Linbradi (2008)Fonseca P. & Librandi A.P.L. 2008. Avaliação das características físico-químicas e fitoquímicas de diferentes tinturas de barbatimão (Stryphnodendron adstringens barbatiman). Revta Bras. Ciênc. Farm. 44(2):271-277. reported that possible toxicity of tannins in dye prepared with bark of Stryphnodendron barbatiman occurred in mammalian cells. Regarding the cytotoxicity in other mammalian cells, Oliveira et al. (2013)Oliveira J.R., Castro V.C., Vilela P.G.F., Camargo S.E.A., Carvalho A.T., Jorge A.O.C. & Oliveira L.D. 2013. Cytotoxicity of Brazilian plant extracts against oral microorganisms of interest to dentistry. BMC Complement. Altern. Med. Avaiable in < Avaiable in http://www.biomedcentral.com/1472-6882/13/208 > Accessed on April 14, 2014.
http://www.biomedcentral.com/1472-6882/1...
found mild toxicity of the S. barbatiman Mart. plant extract at 50mg/mL concentration on rat macrophages and significantly reduced the production of TNF-α when compared to the control group.

Conclusions

According to the sensitivity tests, the Stryphnodendron adstringens extract obtained from northern Minas Gerais exhibited antimicrobial activity at 600μL/mL.

Liver degeneration, although minimal, was observed 30 days after ingestion of the bark extract of S. adstringens in the groups of rats that received 800mg/kg, what may indicate some degree of toxicity.

No systemic toxicity was observed at lower doses.

Considering the broad use of S. adstringens as a phytotherapeutic agent for various human and animal diseases and liver toxicity observed at high concentrations, attention should be paid to the possible adverse effects of using the extract from this tree at high concentrations

Acknowledgements

To FAPEMIG, CNPq, CAPES, the Dean of Research of the UFMG (PRPq/UFMG), and to the Laboratory of Instrumental Chemistry-ICA/UFMG

References

  • Aguiar-Filho C.R., Albuquerque R.F., Rocha B.P., Colodel E.M., Lemos R.A.A., Riet-Correa F., Evêncio-Neto J. & Mendonça F.S. 2013. Avaliação da toxicidade das favas de Stryphnodendron fissuratum (Mimosoideae) em vacas gestantes. Pesq. Vet. Bras. 33(5):607-612.
  • Almeida A.C., Sobrinho E.M., Pinho L., Souza P.N.S., Martins E.R., Duarte E.R., Santos H.O., Brandi I.V., Cangussu A.S. & Costa J.P.R. 2010. Toxicidade aguda de extratos hidroalcóolicos de folhas de alecrim-pimenta, Stryphnodendron adstringens, aroeira e farelo da casca de pequi administrados intraperitonealmente. Ciência Rural 40(1):200-203.
  • Audi E.A., Toledo D.P., Perez P.G., Kimura E., Pereira W.K., Mello J.C., Nakamura C., Alves-do-Prado W., Cuman R.K. & Bersani-Amado C.A. 1999. Gastric antiulcerogenic effects of Stryphnodendron adstringens in rats. Phytother. Res. 13(3):264-266.
  • Brasil 2010. Farmacopeia Brasileira. Vol.2. Agência Nacional de Vigilância Sanitária, Brasília. 904p.
  • Brasil 2006. Ministério da Saúde. Agência Nacional de Vigilância Sanitária. Decreto no.5813, de 22 de junho de 2006. Aprova a Política Nacional de Plantas Medicinais e Fitoterápicos e de Outras Providências. Ministério da Saúde. Brasíla, DF. Available in <Available in http://bvsms.saude.gov.br/bvs/publicacoes/politica_nacional_fitoterapicos.pdf > Accessed on December 10, 2013.
    » http://bvsms.saude.gov.br/bvs/publicacoes/politica_nacional_fitoterapicos.pdf
  • Cansi E.R. & Demo C. 2011. Ocorrência de miíases em animais de companhia no Distrito Federal, Brasil. Acta Scient. Vet. 39(3):1-5.
  • Castro A.H.F., Paiva R., Alvarenga A.A. & Vitor S.M.M. 2009. Calogênese e teores de fenóis e taninos totais em barbatimão [Stryphnodendron adstringens (Mart.) Coville]. Ciênc. Agrotec. 33(2):385-390.
  • CLSI 2012. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard-Ninth Edition. CLSI document M07-A9. Clinical and Laboratory Standards Institute, Wayne, PA. Avaiable in < Avaiable in http://antimicrobianos.com.ar/ATB/wp-content/uploads/2012/11/03-CLSI-M07-A9-2012.pdf > Accessed on January 22, 2016.
    » http://antimicrobianos.com.ar/ATB/wp-content/uploads/2012/11/03-CLSI-M07-A9-2012.pdf
  • Confessor M.V.A., Mendonça L.V., Mourão J.S. & Alves R.R.N. 2009. Animals to heal animals: ethnoveterinary practices in semiarid region, northeastern Brazil. J. Ethnobiol. Ethnomed. 5(37). Available in <Available in http://ethnobiomed.biomedcentral.com/articles/10.1186/1746-4269-5-37 > Accessed on December 10, 2013.
    » http://ethnobiomed.biomedcentral.com/articles/10.1186/1746-4269-5-37
  • Corrêa V.S., Cerdeira A.L., Fachin A.L., Bertoni B.W., Pereira P.S., França P.S., Momm H.G., Moraes R.M. & Pereira A.M.S. 2012. Geographical variation and quality assessment of Stryphnodendron adstringens (Mart.) Coville within Brazil. Genet. Resour. Crop Evolution 59(7):1349-1356.
  • Costa J.P.R., Almeida A.C., Martins E. R., Rodrigues M.N., Santos C.A. & Menezes I.R. 2011. Atividade antimicrobiana do óleo essencial de alecrim-pimenta e do extrato bruto seco do barbatimão diante de bactérias isoladas do leite. Biotemas 24(4):1-6.
  • Costa M.A., Mello J.C., Kaneshima E.N., Ueda-Nakamura T., Dias Filho B.P., Audi E.A. & Nakamura C.V. 2013. Acute and chronic toxicity of an aqueous fraction of the stem bark of Stryphnodendron adstringens (barbatimão) in rodents. Evid. Based Complement. Alternat. Med. Available in: <Available in: http://dx.doi.org/10.1155/2013/841580 > Accessed on April 14, 2014.
    » http://dx.doi.org/10.1155/2013/841580
  • Dôres R.G.R. & Casali V.W.D. 2007. Plantas Medicinais e Aromáticas: controle de qualidade. Universidade Federal de Viçosa, Viçosa, MG, p.106-107.
  • Ferreira E.V., Boabaid F.M., Arruda L.P., Lemos R.A.A., Souza M.A., Nakazato L. & Colodel E.M. 2009. Intoxicação por Stryphnodendron fissuratum (Mimosoideae) em bovinos. Pesq. Vet. Bras. 29(11):951-957.
  • Fonseca P. & Librandi A.P.L. 2008. Avaliação das características físico-químicas e fitoquímicas de diferentes tinturas de barbatimão (Stryphnodendron adstringens barbatiman). Revta Bras. Ciênc. Farm. 44(2):271-277.
  • Lima J.C.S., Martins D.T.O. & Souza Júnior P.T. 1998. Experimental evaluation of stem bark of Stryphnodendron adstringens (Mart.) Coville for anti-inflammatory activity. Phytother. Res. 12:212-220.
  • Macáková K., Kolečkář V., Cahlíková L., Chlebek J., Hošťálková A., Kuča K., Jun D. & Opletal L. 2014. Tannins and their Influence on health, p.159-208. In: Rahman A. , Choudhary M.I. (Eds), Recent Advances in Medicinal Chemistry: tannins_and_their_influence_on_health. Available in <Available in https://www.researchgate.net/publication/282796040 > Accessed on: January 22, 2016.
    » https://www.researchgate.net/publication/282796040
  • Martins P.S., Alves A.L.G., Hussni J.L., Sequeira J.L., Nicoletti J.L.M. & Thomassian A. 2003. Comparação entre fitoterápicos de uso tópico na cicatrização de pele em equinos. Arch. Vet. Sci. 8(2):1-7.
  • Melo J.O., Endo T.H., Bersani-Amado L.E., Estivalet S.A., Silmara B., Mello J.C.P. & Bersani-Amado C.A. 2007. Effect of Stryphnodendron adstringens (barbatimão) bark on animal models of nociception. Braz. J. Med. Biol. Res. 43(3):465-469.
  • Monteiro M.V.B., Bevilaqua C.M.L., Palha M.D.C., Braga R.R., Schwanke K., Rodrigues S.T. & Lameira O.A. 2011. Ethnoveterinary knowledge of the inhabitants of Marajó Island, eastern Amazonia, Brazil. Acta Amaz. 41(2):233-242.
  • Moura M.I. 2011. Enfermidades cutâneas digitais bovina: aspectos genéticos e terapêuticos. Tese de Doutorado, Escola de Veterinária e Zootecnia, Universidade Federal de Goiás, Goiânia, Go. 111p.
  • OECD 2001. Guidelines for the Testing of Chemicals, n. 420: acute oral toxicity, fixed dose procedure. Avaiable in < Avaiable in http://www.oecd-ilibrary.org/docserver/download/9742001e.pdf?expires=1455306679&id=id&accname=guest&checksum=635B12BD5729564AEC4136E85BC32D12 > Accessed on January 22, 2016.
    » http://www.oecd-ilibrary.org/docserver/download/9742001e.pdf?expires=1455306679&id=id&accname=guest&checksum=635B12BD5729564AEC4136E85BC32D12
  • OECD 2008. Guidelines for the Testing of Chemicals, n. 452: chronic toxicity studies. Avaiable in < Avaiable in http://www.oecd-ilibrary.org/docserver/download/9745201e.pdf?expires=1455306478&id=id&accname=guest&checksum=417154C7DA531CDAB6D1BD24D2DDA885 > Accessed on January 22, 2016.
    » http://www.oecd-ilibrary.org/docserver/download/9745201e.pdf?expires=1455306478&id=id&accname=guest&checksum=417154C7DA531CDAB6D1BD24D2DDA885
  • Okuda T. & Ito H. 2011. Tannins of constant structure in medicinal and food plants-hydrolyzable tannins and polyphenols related to tannins. Molecules 16:2191-2217.
  • Oliveira J.R., Castro V.C., Vilela P.G.F., Camargo S.E.A., Carvalho A.T., Jorge A.O.C. & Oliveira L.D. 2013. Cytotoxicity of Brazilian plant extracts against oral microorganisms of interest to dentistry. BMC Complement. Altern. Med. Avaiable in < Avaiable in http://www.biomedcentral.com/1472-6882/13/208 > Accessed on April 14, 2014.
    » http://www.biomedcentral.com/1472-6882/13/208
  • Pansera M.R., Santos A.C.A., Paese K., Wasum R., Rossato M., Rota L.D., Pauletti G.F. & Serafini L.A. 2003. Análise de taninos totais em plantas aromáticas e medicinais cultivadas no Nordeste do Rio Grande do Sul. Revista Bras. Farmacogn. 13(1):17-22.
  • Pinho L., Souza P.N.S., Sobrinho Macedo E., Almeida A.C. & Martins E.R. 2012. Atividade antimicrobiana de extratos hidroalcóolicos das folhas de alecrim-pimenta, aroeira, barbatimão, erva baleeira e do farelo da casca de pequi. Ciênc. Rural 42(2):326-331.
  • Rebecca M.A., Ishii-Iwamoto E.L., Kelmer-Bratch A.M., Caparroz-Assef S.M., Cuman R.K., Pagadigorria C.L., Mello J.C., Bracht A. & Bersani-Amado C.A. 2003. Effect of Strphnodendron adstringens (barbatimão) on energy metabolism in the rat liver. Toxicol. Lett. 143:55-63.
  • Rebecca M.A., Ishii-Iwamoto E.L., Grespan R., Cuman R.K., Caparroz-Assef S.M., Mello J.C. & Bersani-Amado C.A. 2002. Toxicological studies on Stryphnodendron adstringens J. Ethnopharmacol. 83(1/2):101-104.
  • Silva L.A.F., Moura M.I., Persiano C.B., Helou J.B., Lima C.R.O., Freitas S.L.R., Silva J.A., Goulart D.S. & Castro L.T.S. 2009. Extrato da casca do barbatimão (Stryphnodendron barbatiman Martius) associado ao tratamento cirúrgico e toalete dos cascos na recuperação de bovinos da raça nelore com dermatite digital. Ciênc. Anim. Bras. (Supl.1):373-378.
  • Viu A.F.M. & Viu M.A.O. 2011. Cerrado e etnoveterinária: o que se sabe em Jataí, GO. Revta Bras. Agroecol. 6(3):49-61

Publication Dates

  • Publication in this collection
    Aug 2017

History

  • Received
    27 Nov 2015
  • Accepted
    04 Sept 2016
Colégio Brasileiro de Patologia Animal - CBPA Pesquisa Veterinária Brasileira, Caixa Postal 74.591, 23890-000 Rio de Janeiro, RJ, Brasil, Tel./Fax: (55 21) 2682-1081 - Rio de Janeiro - RJ - Brazil
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