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Toxicon 125 (2017) 74e83

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journal homepage: www.elsevier .com/locate/ toxicon

Inhibition of local effects induced by Bothrops erythromelas snakevenom: Assessment of the effectiveness of Brazilian polyvalentbothropic antivenom and aqueous leaf extract of Jatropha gossypiifolia

Juliana Felix-Silva a, Jacyra A.S. Gomes a, Jacinthia B. Xavier-Santos a, Jlia G.R. Passos a,Arnobio A. Silva-Junior a, Denise V. Tambourgi b, Matheus F. Fernandes-Pedrosa a, *

a Laboratorio de Tecnologia & Biotecnologia, Faculdade de Farmacia, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazilb Laboratorio de Imunoqumica, Instituto Butantan, S~ao Paulo, SP, Brazil

a r t i c l e i n f o

Article history:Received 12 September 2016Received in revised form31 October 2016Accepted 23 November 2016Available online 25 November 2016

Keywords:Bothrops erythromelasCaatinga's lanceheadJatropha gossypiifoliaAntiophidic activityBothropic antivenom

Abbreviations: AE, aqueous leaf extract of Jatrophaof variance; AUC0e2h, area under the time-course curantivenom; BeV, Bothrops erythromelas venom; BjV, BoBrazilian Genetic Heritage Management Council; CIOOrganizations of Medical Sciences; CONCEA, NationaAnimal Experimentation of Brazil; DAMP, damage-aECM, extracellular matrix; ELISA, enzyme-linked immhuman embryonic kidney 293 cells; IL, interleukin;phosphate buffered saline; PLA2, phospholipase A2; Selectrophoresis with sodium dodecil sulphate; SEM, stBrazilian Biodiversity Authorization and Informationmetalloproteinase; TNF-a, tumor necrosis factor alph* Corresponding author. Av. Gal. Cordeiro de Farias

570, Natal, RN, Brazil.E-mail address: (M.F. Fernand 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

Bothrops erythromelas is a snake of medical importance responsible for most of the venomous incidentsin Northeastern Brazil. However, this species is not included in the pool of venoms that are used in theBrazilian polyvalent bothropic antivenom (BAv) production. Furthermore, it is well known that anti-venom therapy has limited efficacy against venom-induced local effects, making the search for com-plementary alternatives to treat snakebites an important task. Jatropha gossypiifolia is a medicinal plantwidely indicated in folk medicine as an antidote for snakebites, whose effectiveness against Bothropsjararaca venom (BjV) has been previously demonstrated in mice. In this context, this study assessed theeffectiveness of the aqueous extract (AE) of this plant and of the BAv against local effects induced byB. erythromelas venom (BeV). Inhibition of BeV-induced edematogenic and hemorrhagic local effects wasassayed in mice in pre-treatment (treatment prior to BeV injection) and post-treatment (treatment post-envenomation) protocols. Inhibition of proteolytic, phospholipase A2 (PLA2) and hyaluronidase enzy-matic activities of BeV were evaluated in vitro. BAv cross-reactivity and estimation of antibody titersagainst BeV and BjV were assessed by Ouchterlony double diffusion test. The results show that in pre-treatment protocol AE and BAv presented very similar effects (about 70% of inhibition for edemato-genic and 40% for hemorrhagic activities). However, BAv poorly inhibited edema and hemorrhage inpost-envenomation protocol, whilst, in contrast, AE was significantly active even when used after BeVinjection. AE was able to inhibit all the tested enzymatic activities of BeV, while BAv was active onlyagainst hyaluronidase activity, which could justify the low effectiveness of BAv against BeV-induced localeffects in vivo. Ouchterlony's test showed positive cross-reactivity against BeV, but the antibody titerswere slightly higher against BjV. Together, these data indicate that despite the presence of immuno-logical cross-reactivity, Brazilian polyvalent bothropic antivenom presented low inhibitory potentialagainst biological and enzymatic effects of BeV, illustrating the need for new strategies in the productionof antivenom with broad neutralizing potential in the treatment of Bothrops spp. envenomationthroughout the country. Together, the results highlight the antiophidic potential of J. gossypiifolia,

gossypiifolia; ANOVA, analysisves after 2 h; BAv, bothropicthrops jararaca venom; CGEN,MS, Council of Internationall Council for the Control ofssociated molecular pattern;unosorbent assay; HEK-293,MPO, myeloperoxidase; PBS,DS-PAGE, polyacrylamide gelandard error of mean; SISBIO,System; SVMP, snake venoma., s/n, Petropolis, CEP 59012-



J. Felix-Silva et al. / Toxicon 125 (2017) 74e83 75

suggesting that it can be considered a potential adjuvant in the treatment of bothropic envenomationlocal effects.

2016 Elsevier Ltd. All rights reserved.

1. Introduction

Snake envenoming is a serious public health problem in manyregions around the world. In 2013e2015, the Bothrops snakescaused about 87% of snakebites that occurred in Brazil (Brasil,2016). Bothropic envenomation is characterized by immediateand intense local effects (edema, hemorrhage and necrosis, whichcan often lead to permanent disabilities), cardiovascular alterations(especially systemic bleeding and hypovolemic shock), coagulop-athy and renal alterations (which could evolve into acute kidneyinjury) (Brasil, 2010; Gutierrez et al., 2009a; Gutierrez andLomonte, 1989). The specific treatment for bothropic envenom-ation is the intravenous administration of bothropic antivenom(BAv). This antivenom is produced in Brazil by hyperimmunizationof horses with a pool of venoms from Bothrops jararaca, Bothropsjararacussu, Bothrops moojeni, Bothrops alternatus and Bothropsneuwiedi snakes, with the aim of neutralizing all the Bothropsvenoms distributed in this country (Brasil, 2010). However, someprevious studies have shown pre-clinical evidence that the anti-venom used in Brazil may not fully neutralize the toxic activitiesinduced by all bothropic venoms, suggesting that other venomsshould be included in the immunization pool for the preparation ofa universal bothropic antivenom (Muniz et al., 2000; Queiroz et al.,2008). Many factors including phylogeny, sex, geographic origin,season, age and prey preference may influence the venomcomposition, which could affect the neutralizing capacity of anti-venom (Chippaux et al., 1991). Bothrops erythromelas (Amaral,1923), commonly known as Caatinga lancehead, jararaca daseca or jararaca malha de cascavel, is a small terrestrial venomoussnake responsible for most of the snakebites in Northeastern Brazil(Lira-da-Silva et al., 2009). Therefore, since B. erythromelas is notincluded in the pool of venoms that are used in the Brazilianpolyvalent bothropic antivenom production, this calls into questionthe efficacy of BAv.

Snake venom toxins such as metalloproteinases (SVMPs),phospholipases A2 (PLA2) and hyaluronidases are mostly respon-sible for the local tissue damage frequently observed in bothropicenvenomation. The action of these toxins are, in general, poorlyneutralized by antibodies present in antivenom, so it is well knownthat antivenom therapy has limited efficacy against venom-induced local effects, making the search for complementary alter-natives to treat snakebites an important task (Gutierrez et al.,2006). In addition to poor inhibition of local damage, there is arisk of development of immunological reactions, the high cost ofproduction and very difficult access in some regions (Gutierrezet al., 2011; Silva et al., 2015). This inability to treat local effects,as well the increased time between accident and treatment, are themain reasons for the temporary or permanent disability observedin many victims, which can lead to serious negative social, eco-nomic, and health impacts, given that most victims live in ruralareas (Gutierrez et al., 2013).

The use of medicinal plants against snakebites is a historicalpractice carried out throughout human history, with this knowl-edge being transferred down among the rural communities fromgeneration to generation (Butt et al., 2015). Nowadays, these herbalantidotes used in traditional folk medicine have gained muchattention by toxinologists worldwide as a tool for designing potent

inhibitors against snake venom toxins (Sulochana et al., 2015). Thepossible advantages of a potential application of medicinal plants asantiophidic agents are that, in general, they would be cheap, easilyavailable, stable at room temperature and could be able toneutralize a broad spectrum of toxins, including those related to thelocal tissue damage (Gomes et al., 2010; Santhosh et al., 2013).

Jatropha gossypiifolia is a medicinal plant popularly known inBrazil as pinh~ao-roxo or worldwide as bellyache-bush, and itbelongs to the Euphorbiaceae family. It is largely used in folkmedicine for various purposes, especially as antiophidic, anti-inflammatory, anti-hemorrhagic, hemostatic and healing,amongst others (Felix-Silva et al., 2014a). The effectiveness of theaqueous extract of this plant against enzymatic and biological ef-fects of Bothrops jararaca snake venom has been previouslydemonstrated, including significant inhibitory potential against thelocal tissue damage induced by this venom in mice (Felix-Silvaet al., 2014d).

In this context, this study was carried out aiming two maingoals: (a) to evaluatewhether the polyspecific bothropic antivenom(BAv) manufactured in