Toxic Effects of the Pericarp of the Enterolobium contortisiliquum (Vell.) Morong Fruit on Chicks Ivana Cristina Nunes Gadelha1 Antônio Carlos Lopes Câmara1 Idalécio Pacífico-da-Silva1 Jael Soares Batista1 Marília Martins Melo2 Benito Soto-Blanco2,*

1 Programa de Pós-graduação em Ciência Animal, Universidade Federal Rural do Semiárido, BR 110 Km 47, Mossoró, RN 59628-360, Brazil 2 Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG 30123-970, Brazil * Corresponding author. Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG 30123-970, Brazil. E-mail address: [email protected].

KEY WORDS: toxic plants, Mimosoideae, intestinal crypts, villous atrophy, and mono- hepatotoxicity, saponins, chicken nuclear inflammatory infiltrate in the lamina ABSTRACT propria. In the liver, apparent and diffuse hepatocellular necrosis was observed along The aim of this study was to determine with disarray of the hepatic architecture and whether the pericarp of the Enterolobium sinus congestion. The results of this study contortisiliquum fruit, which contain sapo- revealed that chicks provide a good experi- nins, may cause damage to the gastrointes- mental model for evaluating the toxic effects tinal tract and liver of Gallus gallus domes- of E contortisiliquum pods. The observed ticus chicks. Chicks were fed the seedless effects may be attributed to the presence of pods of E contortisiliquum at concentrations triterpenic saponins in the pericarp of broad of 0% (control), 5%, or 10% of their total beans. daily food intake for 5 consecutive days. On day 6, the animals underwent pathologi- INTRODUCTION cal examination. Necropsy revealed that Enterolobium contortisiliquum (Vell) Mor is all birds fed with the seedless pods of E a plant of the Fabaceae-Mimosoideae (Legu- contortisiliquum at a concentration of 10% minosae-Mimosoideae) family, commonly showed congested livers and enlarged ceca. known as the pacara earpod tree (timbaúba, Lesions of the digestive tract were char- timbaúva, ximbaúva, timburi, orelha-de- acterized by necrosis of the epithelium of negro, orelha-de-macaco, tamboril, and Intern J Appl Res Vet Med • Vol. 13, No. 2, 2015. 135 Table 1: Body weight, weight gain, and feed intake by chicks fed with 0% (control), 5%, and 10% seedless E contortisiliquum fruit in the feed. E contortisiliquum concentration in feed Parameters 0% 5% 10% Weight (day 1), g 106.3 ± 6.82 110.0 ± 8.37 105.5 ± 4.80 Weight (day 5), g 152.7 ± 9.75 152.3 ± 9.08 147.5 ± 4.92 Weight gain, g 46.3 ± 4.93 42.3 ± 3.07 42.0 ± 3.62 Feed intake (group), g 1402 1016 707 tamboril-da-mata). The tree can reach up to bromelain,17,18 and a 35-kDa protein with 30 m in height, is widely distributed across arylamidase activity.19 Moreover, the seeds South America, being found in Uruguay, also show phospholipase D activity.10 The Argentina, Brazil, and Venezuela.1 Natural phytochemical analysis of the pericarps of cases of cattle poisoning by E contortisiliqu- the E contortisiliquum fruit, on the other um have been reported.2-4 Suspected toxicity hand, revealed the presence of several of the broad beans has also been reported triterpenic saponins, including 3β-O-β-D- in two goats.5 Clinical signs of this toxicity glucopyranosyl-21β-E-cinamoiloxiolean-12- include anorexia, depression, hepatogenous en-28-oic acid,20 enterolosaponins A and photosensitization, and miscarriages.2-4 The B,21 and contortisiliosides A-G.22 experimental replication of cattle poisoning The aim of this study was to determine by broad beans resulted in photosensitiza- whether the pericarp of the E contortisiliqu- tion in some studies,3 but only in severe um fruit, which contain saponins, might diarrhea in other experiments conducted in cause damage to the gastrointestinal tract both cattle,2,4,6,7 and sheep.8 The abortive ac- and liver, and whether Gallus gallus domes- tivity of the broad beans was experimentally ticus (Linnaeus, 1758) chicks can be used as replicated in guinea pigs only.9 an experimental model for the evaluation of The toxic compound of E contortisiliqu- this toxicity. um broad beans has not yet been identi- MATERIALS AND METHODS fied. A compound named enterolobin was isolated from its seeds, and some authors Ripe Enterolobium contortisiliquum (Vell.) consider this the responsible component Morong fruits were collected from five trees for the plant’s toxic activity.1 Enterolobin is present in the Toxic Plant Bed at the Veteri- a protein with a molecular weight of 51.3 nary School of the UFMG, in Belo Hori- to 59.8 kDa, depending on the extraction zonte, MG. The fruits were cleaned and cut process, and is responsible for hemolytic,10 open with knives specific for seed removal. cytolytic,11,12 and pro-inflammatory activ- Following drying, the seedless pods were ity.13 ground. Saponin concentration in the pods was assayed using a spectrophotometric Several other compounds have been technique,23 and using Saponin from quil- isolated from E contortisiliquum seeds, laja bark (Sigma-Aldrich, Saint Louis, MO, including a Kunitz-type trypsin inhibitor USA) as a standard. Saponin concentration with two polypeptide chains,14,15 a 60-kDa was determined to be 21.9% in the ground endopeptidase that is not inhibited by the pods used in this study. trypsin inhibitor or by cysteine prote- ases,16 a 23-kDa serine protease inhibitor The ground pods of E contortisiliquum which inhibits trypsin, chymotrypsin, and were mixed into the feed (Bichos & Sítios, plasma kallikrein,17 a 60-kDa thiol prote- IRCA Nutrição e Avicultura S.A., Carpina, ase inhibitor, which inactivates papain and PE). Eighteen (18) chicks were randomly

136 Vol. 13, No.2, 2015 • Intern J Appl Res Vet Med. distributed across three groups Figure 1: Liver of chick that received feed with 10% of six animals each, and were seedless E. contortisiliquum pods, showing apparent and fed the E contortisiliquum pods diffuse hepatocellular necrosis and sinus congestion. H&E at concentrations of 0% (con- staining, 40× magnification. trol), 5%, and 10% total daily dietary intake. The experimental feeds were supplied for 5 con- secutive days. Throughout the experimental period, water and feed were supplied ad libitum. On day six, the animals were euthanized by cervical dislo- cation. Each bird underwent necroscopic evaluation, and fragments were collected from the liver, heart, spleen, kidneys, proventriculus, gizzard, cecum, rectum, and brain for evaluation by microscopy. The collected fragments were fixed in forma- Figure 2: Large intestine of chick that received feed con- lin for subsequent routine histolog- taining 10% pericarp of E. contortisiliquum pods, show- ical processing and staining with ing necrosis of the epithelium of the crypts and villous hematoxylin and eosin (H&E). atrophy. H&E staining, 10× magnification. Animal weight and weight gain results are shown as the mean plus the standard er- ror (SEM). The comparisons between the groups were performed using analysis of variance (ANOVA), followed by Tukey’s test with a preset statistical significance level of P<0.05. Analysis of the data was carried out with the help of R software, version 3.0.0. RESULTS The body weight, weight gain, Necropsy revealed that all birds fed with and feed intake are shown in Table 1. There E contortisiliquum at a concentration of 10% was no statistically significant difference in showed congested livers and enlarged ceca. body weight and weight gain between the None of the animals from the control group birds of the different groups. On the other or those fed with E contortisiliquum at a hand, feed intake was lower in the groups concentration of 5% showed macroscopic treated with E contortisiliquum than in the lesions. control group. Microscopic evaluation of the livers of Throughout the experimental period, no birds that received E contortisiliquum (at birds showed clinical signs of toxicity, ex- either concentration) revealed apparent and cept for increased water intake demonstrated diffuse hepatocellular necrosis with hepa- by the animals fed with the plant pods at tocyte strand breaks, disarray of the hepatic either concentration. architecture, and sinus congestion (Fig 1).

Intern J Appl Res Vet Med • Vol. 13, No. 2, 2015. 137 These lesions were more severe in birds that Another compound isolated from this plant received 10% pods in the feed than in those is a Kunitz-type trypsin inhibitor,14,15 which that received 5% pods. During microscopic combines with the active sites of bovine evaluation of the digestive tract, necrosis of trypsin25 and could, therefore, potentially the epithelium of the intestinal crypts and be responsible for the effects on the diges- villous atrophy was observed (Fig 2). These tive tract in poisoned animals. However, lesions were accompanied by mononuclear since enterolobin, the trypsin inhibitor, and inflammatory infiltrate in the lamina propria. other compounds with enzymatic activity Lesions in the digestive tract were moderate are present only in the seeds of E contor- in birds that received 10% E contortisiliqu- tisiliquum,10,14-19 the occurrence of lesions um and mild in those that received 5%. No caused by the pericarp of the fruit demon- lesion was observed in any other organs. strates that the toxic ingredient is likely the DISCUSSION triterpenic saponins. Furthermore, at least a dozen different triterpenic saponins have The main factor influencing intake of the been isolated and identified in the pericarps broad beans in large quantities is food of pods of E contortisiliquum.20-22 scarcity, since apparently the beans have Saponins are glycosides often found low palatability and animals will ingest in plants, and are known to be responsible them only when they are hungry.3,24 In this study, feed intake was lower when the pod for causing digestive and hepatic lesions 26,27 According bran was added to the feed. This observation in several different species. to their chemical structure, they are clas- strengthens the theory that broad beans have sified as steroidal or triterpenic saponins. low palatability. Photosensitizing plants that contain sapo- The main site for lesions caused by E nins include Panicum spp, Brachiaria spp, contortisiliquum is the digestive tract. These Tribulus terrestris, Narthecium ossifragum, lesions were characterized by necrosis of Agave techeguilla, and Nolina texana. In the epithelium of intestinal crypts, villous all of these plants, the saponins are steroi- atrophy, and mononuclear inflammatory in- dal.26 Only one experimental study was able filtrate in the lamina propria. Similar lesions to replicate the photosensitization effect in the digestive tract have also been found after administration of E contortisiliquum,3 in both natural and experimental cases in 2-4,6,7 8 whereas several other studies were unsuc- cattle, and experimentally in sheep and 2,4,6,7 9 cessful. It is possible that significant guinea pigs. variation exists in the saponin composition The liver is an important target organ of the pods, and they may even contain other for lesions caused by E contortisiliquum. unidentified steroidal saponins. Experimental replication of pod poison- The results of this study revealed that ing in cattle resulted in areas of necrosis chicks could serve as a good experimental induced by coagulation of hepatocytes.6,7 model for studying toxicity induced by E Microscopic analysis of the liver in experi- contortisiliquum pods. The observed toxic ments conducted with guinea pigs, revealed effects may likely be attributed to the pres- periportal hemorrhagic necrosis induced ence of triterpenic saponins in the pericarp by ingestion of broad beans.9 In this study, of broad beans. chicks showed apparent and diffuse hepato- cellular necrosis with disarray of the hepatic ACKNOWLEDGMENTS architecture and sinus congestion. This research was supported in part by the Enterolobin is a protein isolated from grant of the CNPq (grant #305761/2013-7) E contortisiliquum with hemolytic,10 and supported for language editing of the cytolytic,11,12 and pro-inflammatory activ- Pró-Reitoria de Pesquisa of the Universi- ity,13 which some authors consider to be dade Federal de Minas Gerais (Edital PRP- responsible for the plant’s toxic activity.1 UFMG 01/2014).

138 Vol. 13, No.2, 2015 • Intern J Appl Res Vet Med. REFERENCES enterolobin and bacterial aerolysins,” Journal of Protein Chemistry, vol. 13, pp. 659-667, 1994. 1 F. J. Abreu de Matos, H. Lorenzi, L. F. L. Santos, M. E. O. Matos, M. G. V. Silva, and M. P. Sousa, 12 W. Fontes, M. V. Sousa, J. B. Aragão, and L. Plantas Tóxicas – Estudo de fitotoxicologia quími- Morhy, “Determination of the amino acid sequence ca de plantas brasileiras, pp. 106-107, Instituto of the plant cytolysin enterolobin,” Archives of Plantarum de Estudos da Flora, São Paulo, Brazil, Biochemistry and Biophysics, vol. 347, pp. 201- 2011. 207, 1997. 2 C. H. Tokarnia, J. Döbereiner, I. S. Dutra, I. S. 13 H. C. Castro-Faria-Neto, M. A. Martins, P. T. Brito, B. R. Chagas, T. N. França, and L. A. G. Bozza, S. A. Perez, A. C. Correa-da-Silva, M. C. Brust, “Experimentos em bovinos com as favas Lima, H. N. Cruz, R. S. Cordeiro, M. V. Sousa, and de Enterolobium contortisiliquum e E. timbouva L. Morhy, “Pro-inflammatory activity of enterolo- para verificar propriedades fotossensibilizantes e/ bin: a haemolytic protein purified from seeds of ou abortivas,” Pesquisa Veterinária Brasileira, vol. the Brazilian tree Enterolobium contortisiliquum,” 19, pp. 39-45, 1999. Toxicon, vol. 29, pp. 1143-1150, 1991. 3 F. B. Grecco, A. F. Dantas, F. Riet-Correa, C. G. 14 I. F. Batista, M. L. Oliva, M. S. Araujo, M. U. Leite, and J. B. Raposo, “Cattle intoxication from Sampaio, M. Richardson, H. Fritz, and C. A. Sam- Enterolobium contortisiliquum pods,” Veterinary paio, “Primary structure of a Kunitz-type trypsin and Human Toxicology, vol. 44, pp. 160-162, inhibitor from Enterolobium contortisiliquum 2002. seeds,” Phytochemistry, vol. 41, pp. 1017-1022, 1996. 4 F. S. Mendonça, J. Evêncio-Neto, L. Baratella- Evêncio, R. G. S. Dória, S. H. Freitas, L. F. 15 I. F. C. Batista, M. C. Nonato, M. R. Bonfadini, L. Pelegrini, R. A. S. Cruz, E. V. Ferreira, and E. M. M. Beltramini, M. L. V. Oliva, M. U. Sampaio, C. Colodel, “Natural and experimental poisoning of A. M. Sampaio, and R. C. Garratt, “Preliminary cattle by Enterolobium contortisiliquum pods (Fa- crystallographic studies of EcTI, a serine protein- baceae Mimosoideae) in Central-Western Brazil,” ase inhibitor from Enterolobium contortisiliquum Acta Veterinaria Brno, vol. 78, pp. 621-625, 2009. seeds,” Acta Crystallographica Section D: Biologi- cal Crystallography, vol. 57, pp. 602-604, 2001. 5 T. M. A. Benício, M. J. Nardelli, F. R. B. Nogueira, J. A. S. Araújo, and F. Riet-Correa, “Intoxication 16 G. A. Silva, M. L. Oliva, M. U. Sampaio, M. by the pods of Enterolobium contortisiliquum in S. Araujo, and C. A. Sampaio, “Isolation and goats,” in Poisonous Plants: global research and partial characterization of an endopeptidase from solutions, K. E. Panter, T. L. Wierenga, and J. A. Enterolobium contortisiliquum seeds,” Brazilian Pfister, Eds., pp. 67-71, CABI Publishing, Walling- Journal of Medical and Biological Research, vol. ford, UK, 2007. 27, pp. 1299-1310, 1994. 6 C. H. Tokarnia, C. F. C. Canella, and J. Dö- 17 M. L. Oliva, M. U. Sampaio, and C. A. Sampaio, bereiner, “Intoxicação experimental pela fava da “Serine- and SH-proteinase inhibitors from En- “timbaúba”(Enterolobium contortisiliquum (Vell.) terolobium contortisiliquum beans. Purification and Morong.) em bovinos,” Arquivos do Instituto de preliminary characterization,” Brazilian Journal Biologia Animal, vol. 3, pp. 73-81, 1960. of Medical and Biological Research, vol. 20, pp. 767-770, 1987. 7 D. C. Marques, M. I. Santos, E. S. Couto, M. A. Mello, R. M. P. Ribeiro, and P. M. Ferreira, “In- 18 M. L. Oliva, M. U. Sampaio, and C. A. Sampaio, toxicação experimental pelo tamboril Enterolobium “Purification and partial characterization of a thiol contortisiliquum (Vell.) Morong. em bovinos,” proteinase inhibitor from Enterolobium contor- Arquivos da Escola de Veterinária da Universidade tisiliquum beans,” Biological Chemistry Hoppe- Federal de Minas Gerais, vol. 26, pp. 283-286, Seyler, vol. 369, Suppl., pp. 229-232, 1988. 1974. 19 R. H. Costa, R. C. Stella, and K. B. Alves, “Purifi- 8 F. B. Bacha, A. C. Santos, T. C. Faccin, P. V. Leal, cation and partial characterization of Enterolobium R. C. Pupin, A. Pott, C. C. B. F. Itavo, and R. A. A. contortisiliquum seed arylamidase,” Brazilian Lemos, “Intoxicação por Enterolobium contor- Journal of Medical and Biological Research, vol. tisiliquum em ovinos,” in VII Encontro Nacional 24, pp. 337-344, 1991. de Diagnóstico Veterinário, Porto Alegre, Brazil, 20 M. C. C. Delgado, M. S. Silva, and R. Braz 2012. Filho, “Ácido 3β-O-β-D-glicopiranol-21β-E- 9 J. Bonel-Raposo, F. Riet-Correa, T. N. Guim, I. D. cinamoiloxiolean-12-en-28-óico, um novo triter- Schuch, F. B. Grecco, and C. G. Fernandes, “In- peno glicosídico de Enterolobium contortisiliquum toxicação aguda e abortos em cobaias pelas favas (Vell.) Morong,” Química Nova, vol. 9, pp. 119- de Enterolobium contortisiliquum (Leg. Mimosoi- 122, 1986. deae),” Pesquisa Veterinária Brasileira, vol. 28, 21 Y. Mimaki, H. Harada, C. Sakuma, M. Haraguchi, pp. 593-596, 2008. S. Yui, T. Kudo, M. Yamazaki, and Y. Sashida, 10 M. V. Sousa and L. Morhy, “Enterolobin, a hemo- “Enterolosaponins A and B, novel triterpene bis- lytic protein from Enterolobium contortisiliquum desmosides from Enterolobium contortisiliquum, seeds (Leguminosae - Mimosoideae): purifica- and evaluation for their macrophage-oriented cyto- tion and characterization,” Anais da Academia toxic activity,” Bioorganic & Medicinal Chemistry Brasileira de Ciências, vol. 61, pp. 405-412, 1989. Letters, vol. 13, pp. 623-627, 2003. 11 M. V. Sousa, M. Richardson, W. Fontes, and L. 22 Y. Mimaki, H. Harada, C. Sakuma, M. Haraguchi, Morhy, “Homology between the seed cytolysin S. Yui, T. Kudo, M. Yamasaki, and Y. Sashida,

Intern J Appl Res Vet Med • Vol. 13, No. 2, 2015. 139 “Contortisiliosides A-G: isolation of seven new tri- 25 D. Zhou, Y. A. Lobo, I. F. Batista, R. Marques-Por- terpene bisdesmosides from Enterolobium contor- to, A. Gustchina, M. L. Oliva, and A. Wlodawer, tisiliquum and their cytotoxic activity,” Helvetica “Crystal structures of a plant trypsin inhibitor from Chimica Acta, vol. 87, pp. 851-865, 2004. Enterolobium contortisiliquum (EcTI) and of its 23 C. L. S. Vigo, E. Narita, L. C. Marques, “Validação complex with bovine trypsin,” PLoS One, vol. 8, da metodologia de quantificação espectrofotomé- Article ID e62252, 2013. trica das saponinas de Pfaffia glomerata (Speng.) [26] P.R. Cheeke, “Toxins intrinsic to forages,” in Natu- Pedersen – Amaranthaceae,” Revista Brasileira de ral Toxicants in Feeds, Forages, and Poisonous Farmacognosia, vol. 13, Suppl. 2, pp. 46-49, 2003. Plants, 2nd ed., pp. 275-324, Interstate Publishers, 24 C. H. Tokarnia, M. F. Brito, J. D. Barbosa, P. V. Danville, USA, 1998. Peixoto, and J. Döbereiner, “Plantas que afetam o 27 G. Francis, Z. Kerem, H. P. S. Makkar, and K. trato digestório,” in Plantas Tóxicas do Brasil, pp. Becker, “The biological action of saponins in 95-146, Editora Helianthus, Rio de Janeiro, Brazil, animal systems: a review,” British Journal of 2012. Nutrition, vol. 88, pp. 587-605, 2002.

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