UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE … · animais foram divididos em grupos, os quais...

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE BIOCIÊNCIAS

PROGRAMA DE PÓS-GRADUAÇÃO EM PSICOBIOLOGIA

EFEITO ANTICONVULSIVANTE DE FRAÇÕES ISOLADAS DA PEÇONHA

DA FORMIGA Dinoponera quadríceps (Formicidae: Ponerinae)

Aluna: Diana Aline Nôga Morais Ferreira

Orientadora: Profa. Dra. Alessandra Mussi Ribeiro

NATAL/RN 2015

DIANA ALINE NÔGA MORAIS FERREIRA

EFEITO ANTICONVULSIVANTE DE FRAÇÕES ISOLADAS DA PEÇONHA

DA FORMIGA Dinoponera quadríceps (Formicidae: Ponerinae)

Dissertação apresentada à

Universidade Federal do Rio Grande

do Norte para obtenção do título de

mestre em Psicobiologia.

Orientadora: Profa. Dra. Alessandra Mussi Ribeiro

Natal

2015

Título: Efeito Anticonvulsivante de Frações Isoladas da Peçonha da Formiga

Dinoponera quadríceps (Formicidae: Ponerinae)

Autora: Diana Aline Nôga Morais Ferreira

Data da defesa: 11/05/2015

Banca Examinadora:

Profª Dr. Alessandra Mussi Ribeiro

Universidade Federal de São Paulo, SP

Profª Dr. Regina Helena da Silva

Universidade Federal de São Paulo, SP

Prof. Dr. Wagner Ferreira dos Santos

Faculdade de Filosofia, Ciências e Letras de

Ribeirão Preto da USP, SP

AGRADECIMENTOS

Agradeço primeiramente a minha família. Aos meus pais, Sônia e

Ferreira, que investiram pessoal e financeiramente em mim, permitindo que

chegasse a esse momento. E ao meu irmão mais velho, David, pela amizade,

suporte e as inúmeras caronas.

Agradeço a todo grupo do LEME que sempre esteve presente para

ajudar, fosse nos experimentos ou apenas com aquele incentivo tão

necessário. Especialmente a Ramón Hypolito e Ywlliane Meurer que

seguraram as pontas no momento de maior necessidade, atuando como

administradores, pedreiros, pintores, mas principalmente como co-orientadores

e amigos, que não permitiram que nos deixássemos abater diante das

dificuldades.

A todos os meus amigos, que me apoiaram nos momentos de dúvida e

tristeza e proporcionaram diversos momentos de alegria. Especialmente a

Jéssica Damasceno, Alexandre Costa, Amanda Borges, Fernanda Cagni,

Ramón Hypolito e Ywlliane Meurer.

Ao meu namorado e grande companheiro, Luiz Eduardo, que me ajudou

em praticamente todos os experimentos e esteve sempre ao meu lado, me

apoiando e me fazendo uma pessoa melhor a cada dia.

Por fim, a minha orientadora, Alessandra Ribeiro, pela paciência, pelos

direcionamentos e ensinamentos durante esses cinco anos de orientação. E a

todos que de alguma forma contribuíram para minha formação e para

construção desse trabalho.

Muito obrigada!

RESUMO

A epilepsia é uma patologia crônica do sistema nervoso central que afeta

cerca de 65 milhões de indivíduos no mundo. Aproximadamente 30% desses

indivíduos desenvolvem crises convulsivas que persistem apesar do tratamento

monitorado com drogas antiepilépticas. Assim, há uma evidente necessidade

do desenvolvimento de novos fármacos antiepilépticos e as peçonhas podem

ser uma excelente fonte de modelos. Nesse contexto, enquanto já vários

estudos sobre peçonhas de serpentes, escorpiões e aranhas, pouco se sabe

sobre as peçonhas de formigas. Estudos prévios do nosso laboratório

demonstraram que a peçonha desnaturada da formiga Dinoponera quadríceps

protegeu camundongos de crises convulsivas e morte induzidas por bicuculina

(BIC). Nesse contexto, o objetivo desse trabalho foi investigar o potencial

anticonvulsivante de frações isoladas da peçonha de D. quadríceps em crises

convulsivas induzidas pela BIC, bem como uma análise dos efeitos dessas

frações no comportamento natural dos camundongos no campo aberto. Os

animais foram divididos em grupos, os quais receberam injeções (1 mg/ml

i.c.v.) de seis frações distintas e tiveram seu comportamento geral observado

no campo aberto durante 30 min. No segundo experimento, os animais

receberam as mesmas frações 20 min antes da administração de bicuculina

(10 mg/ml). Em seguida, foi analisado o comportamento motor convulsivo

desses animais durante 30 minutos no campo aberto. No primeiro experimento,

não foram observadas alterações comportamentais. Já no segundo

experimento, a administração prévia de DqTx1, DqTx3, DqTx4 e DqTx6

aumentou a latência para o desenvolvimento de crises tônico-clônicas. Além

disso, todas as frações, exceto DqTx5, aumentaram a latência para a morte

dos animais. Ainda, os melhores resultados foram obtidos com a fração DqTx6,

que protegeu 62,5% dos animais testados contra o desenvolvimento de crises

tônico-clônicas e 100% dos animais contra a morte.

Palavras-chave: Bicuculina, crises tônico-clônicas, design de fármacos,

compostos bioativos, antiepiléticos.

ABSTRACT

Epilepsy affects at least 65 million people worldwide and the available

treatment is associated with various side effects. Approximately 20-30% of the

patients develop seizures that persist despite of careful monitored treatment

with antiepileptic drugs. Thus, there is a clear need for the development of new

antiepileptic drugs and the venoms can be an excellent source of probes. In this

context, while there are studies on venoms from snakes, scorpions and spiders,

little is known regarding venom from ants. Previous studies from our group

showed that denatured venom from ant Dinoponera quadriceps protected mice

from seizures and death induced by bicuculline (BIC). In this context, the aim of

this study was to investigate the anticonvulsant activity of compounds isolated

from D. quadriceps venom on seizures induced by BIC, as well as an analysis

of its effects on spontaneous behavior in mice. Animals were divided into

groups, which received injections (1 mg/ml; i.c.v.) of six distinct venom fractions

and had their general behavior analyzed for 30 min in the open field. In the

second experiment, we carried out the same fractions injection protocol 20 min

before the administration of bicuculline (10 mg/ml). Immediately after, we

analyzed animals’ seizures behavior during 30 min in open field. In the first

experiment we did not observe behavioral alterations. Conversely, in the

second experiment, previous administration of DqTx1, DqTx3, DqTx4 and

DqTx6 increased latency for onset of tonic-clonic seizures. Moreover, all

fractions, except DqTx5, increased latency to animals’ death. Yet, we obtained

our best result with DqTx6 fraction, which protected 62.5% of tested animals

from development of tonic-clonic seizures. Further, this fraction protected all

tested animals from seizure episodes followed by death.

Keywords: bicuculline, tonic-clonic seizures, drug design, bioactive

compounds, antiepileptic drug.

SUMÁRIO

1. INTRODUÇÃO ................................................................................................................ 11

1.1. Epilepsia ......................................................................................................................... 11

1.2. Breve histórico dos produtos naturais: Os venenos como fontes de fármacos .. 12

1.3. Venenos e peçonhas: uma visão geral ..................................................................... 14

1.4. Peçonhas e toxinas de vertebrados .......................................................................... 17

1.5. Peçonhas e toxinas de invertebrados ....................................................................... 19

1.5.1 Dinoponera quadriceps.......................................................................................... 24

2. REFERÊNCIAS ............................................................................................................... 26

3. OBJETIVOS .................................................................................................................... 33

3.1 Objetivo Geral ................................................................................................................. 33

3.2 Objetivos Específicos .................................................................................................... 33

4. ARTIGO ........................................................................................................................... 34

Introduction ............................................................................................................................ 38

Material and Methods .......................................................................................................... 40

Ants collection and fraction obtainment ........................................................................ 40

Animals ............................................................................................................................... 41

Surgery ............................................................................................................................... 41

General Procedures ......................................................................................................... 42

Behavioral analysis .......................................................................................................... 42

Verification of the injection site ....................................................................................... 43

Statistical analysis ............................................................................................................ 44

Results .................................................................................................................................. 44

HPLC purification .............................................................................................................. 44

Primary screening ............................................................................................................. 45

Anticonvulsant assay ....................................................................................................... 46

Discussion ........................................................................................................................... 48

Acknowledgements ........................................................................................................... 52

References ........................................................................................................................... 52

5 ANEXO ............................................................................................................................. 70

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1. INTRODUÇÃO

1.1. Epilepsia

A Epilepsia é uma doença do cérebro caracterizada por uma contínua

predisposição para a geração de uma atividade neuronal excessiva ou

sincrônica, bem como pelas consequências neurobiológicas, cognitivas,

psicológicas e sociais resultantes dessa condição (Fisher et al., 2005, 2014).

As crises epilépticas podem ser classificadas como do tipo focal ou

generalizada. As crises focais são caracterizadas por se originarem e

permanecem em redes neurais limitadas a um hemisfério cerebral e pela

consistência com relação ao seu local de início. Já as crises generalizadas se

caracterizam por envolverem redes distribuídas nos dois hemisférios cerebrais,

sendo subclassificadas em ausência, ausência com características especiais,

mioclônica, clônica, tônica, atônica e tônico-clônica (Berg et al., 2010). Este

transtorno neurológico afeta aproximadamente 65 milhões de indivíduos no

mundo (Thurman et al., 2011) e aproximadamente 30% desses desenvolvem

uma epilepsia crônica que não responde a nenhum fármaco antiepiléptico

(Löscher, 1997; Rosillo-de la Torre, 2014).

No Brasil, são poucos os estudos que abordam o caráter epidemiológico

da epilepsia. O mais recente, que foi realizado por Kanashiro (2006) em

Campinas e São José do Rio Preto sugere que aproximadamente 0,88% da

população brasileira teria epilepsia e destes, aproximadamente 25,3% não

receberia o tratamento correto.

Os fármacos antiepilépticos atualmente utilizados atuam através de três

mecanismos principais: (1) pelo aumento da neurotransmissão inibitória

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mediada pelo ácido gama-aminobutírico (GABA), tendo como exemplos os

benzodiazepínicos, os barbitúricos, a tiagabina, a vigabatrina, dentre outros. (2)

através da modulação de canais iônicos voltagem-dependente de sódio, cálcio

e potássio, tendo como exemplos a fenitoína, a pregabalina, a carbamazepina,

dentre outros. (3) através da atenuação da transmissão excitatória

glutamatérgica, tendo como representantes o felbamato e, de forma parcial, o

topiramato.

O grande problema é que estes fármacos precisam ser utilizados de

forma crônica e estão associados a efeitos colaterais negativos, como

desconforto gástrico, sedação, diplopia, ataxia, nistagmo, hipertrofia gengival,

osteomalacia, hirsutismo, neuropatia periférica, aumento paradoxal de crises,

prejuízos cognitivos, distúrbios comportamentais, bem como reações

idiossincráticas como agranulocitose, pseudolinfoma, falência hepática, falência

múltipla e anemia aplástica (Kwan, et. al., 2001; Macdonald & Kelly, 1995;

Mortari et al., 2007b; Rogawski, 2006). Nesse contexto, é clara a necessidade

do desenvolvimento de novos fármacos que possam tratar os pacientes

resistentes e/ou apresentar menos efeitos colaterais. Então, uma fonte

potencial de novos fármacos para o tratamento da epilepsia são os compostos

isolados de produtos naturais.

1.2. Breve histórico dos produtos naturais: Os venenos como

fontes de fármacos

Os produtos naturais constituem uma fonte para obtenção de novas

substâncias utilizadas no tratamento de doenças humanas desde tempos

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remotos (Koehn & Carter, 2005). Por exemplo, desde o século VII a.C. povos

na Índia utilizavam venenos extraídos de serpentes para prolongar a vida e

para o tratamento de problemas gastrointestinais (Gomes et al., 2010). Na

medicina tradicional chinesa (desde a dinastia Song – 960 a 1279), tanto a

peçonha e/ou corpo do escorpião Buthus martensis são utilizados no

tratamento de doenças como epilepsia, acidente vascular cerebral e paralisia

facial (Zhao et al., 2008 and 2011). Ainda, em populações indígenas na

América Latina, as tarântulas são utilizadas no tratamento de diversas

doenças, como asma, câncer e erisipela (Machkour-M’Rabet et al., 2011).

As pesquisas na área de produtos naturais derivados de venenos

tiveram um aumento considerável entre os anos de 1970 a 1980, com o

desenvolvimento do anti-hipertensivo captopril a partir do veneno da serpente

Bothrops jararaca, porém passaram por um declínio durante os anos seguintes.

Esse declínio foi provavelmente resultado de diversos fatores, entre eles o

desenvolvimento da química combinatória e os avanços na biologia celular,

molecular e genômica, que aumentaram a quantidade de substâncias para

testes e de alvos moleculares, além de reduzirem o tempo para descoberta de

novos fármacos (Greene et al, 1972; King, 2011; Koehn & Carter, 2005).

Porém, com o desenvolvimento de técnicas mais eficientes para fracionamento

e caracterização de produtos naturais e o fato de técnicas concorrentes, como

a química combinatória, terem falhado na apresentação de novas substâncias

promissoras, a pesquisa baseada na prospecção de compostos bioativos

obtidos a partir de produtos naturais voltou a ganhar notoriedade, de forma

que, em 2010, 50% dos fármacos lançados no mercado foram desenvolvidos a

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partir de substancias obtidas a partir de produtos naturais (King, 2011; Molinski

et al., 2009; Newman & Cragg, 2012).

Atualmente, dentre os diversos produtos naturais pesquisados, seja para

o desenvolvimento de novos fármacos utilizados na clínica ou como

ferramentas para a pesquisa científica, grande parte são princípios ativos

isolados a partir de peçonhas de serpentes. Isso se deve provavelmente ao

fato desses animais apresentarem maior quantidade de veneno quando

comparado com as quantidades obtidas de invertebrados como aranhas,

escorpiões, dentre outros. Esse quadro começou a mudar com o

desenvolvimento de técnicas sofisticadas de fracionamento, avanços em

espectrometria de massa, miniaturização dos testes funcionais e o advento das

bibliotecas de cDNA, que permitiram a análise direta dos transcritos dos

venenos (King, 2011).

1.3. Venenos e peçonhas: uma visão geral

Os venenos são secreções tóxicas, que contem moléculas que

interferem na fisiologia e em processos bioquímicos de outros animais e são

produzidas em uma glândula especializada (King, 2011). Alguns animais

desenvolveram sistemas complexos para aplicação dessas secreções em suas

vítimas, como dentes modificados, arpões, ferrões, presas, probóscides, entre

outros (Fry et al., 2009). Esses animais são denominados peçonhentos e seus

venenos, peçonhas. As composições dos venenos são resultado de um

processo evolutivo de milhões de anos, o qual permitiu o desenvolvimento e

incorporação de uma grande quantidade de compostos bioativos com o

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objetivo de defesa, predação e de afastar indivíduos competidores (Fry et al.,

2009; Miijanich, 1997; Mortari et al., 2007b).

Em geral, os venenos são formados por uma combinação de proteínas,

peptídeos, poliaminas, sais, aminoácidos, minerais e alcaloides (Fry et al.,

2009; Gomes et al., 2010; Lewis & Garcia, 2003; Wong & Belov, 2012). As

proteínas presentes nesses compostos são resultantes de um processo de

recrutamento, o qual envolve a duplicação de um gene que codifica uma

proteína e a expressão seletiva desse gene na glândula (Fry et al., 2009).

Essas duplicações de genes podem promover o surgimento de novas funções

e a formação de “famílias multigênicas” (multigene family), que consistem numa

família de proteínas codificadas por genes similares, que são variações de um

gene ancestral. Dessa forma, essas proteínas preservam uma mesma estrutura

básica, que recebe alterações chaves, permitindo uma diversidade de funções

(Fry et al., 2009; Wong & Belov, 2012).

Apesar da diversidade na composição de venenos uma grande

diversidade de espécies apresenta proteínas como as cistatinas, defensinas,

hialuronidases, lectinas, peptidases, fosfolipases, proteínas secretórias ricas

em cisteína, esfingomielinases, dentre outras. Todas essas proteínas

apresentam precursores que possuem um peptídeo sinal na posição N-terminal

(Fry et al., 2009). Outra característica em comum, é que essas proteínas, bem

como os peptídeos que agem como toxinas, possuem uma maior quantidade

de cisteínas, o que permite a formação de pontes de sulfeto, as quais garantem

maior estabilidade e resistência à degradação por proteases. Essa estabilidade

é importante para permitir que as toxinas cheguem ainda ativas aos seus alvos

e, além disso, pode favorecer a produção de fármacos derivados de venenos

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em que a via de administração seja oral (Fry et al., 2009; King, 2011; Miijanich,

1997).

Por ter uma composição diversificada, os venenos possuem alvos e

ações diversas no organismo humano. Eles podem agir nos sistemas nervoso,

cardiovascular, respiratório, gastrointestinal, além de poderem atingir pele,

músculos e rins, seja por uma ação tecido-específica ou por ações citotóxicas.

Como consequência, podem causar dor, inchaço, necrose tecidual, náuseas,

vômitos, paralisias, diarreia, febre, dores de cabeça, visão borrada, tonturas,

fraqueza ou falta de coordenação muscular, hipotensão, hemorragia, efeitos

trombóticos e podem levar até a morte, seja por ação direta ou por reações

anafiláticas (Sitprija & Suteparak, 2008; Wong & Belov, 2012). Quase todos

esses efeitos podem ser resultados de enzimas presentes no veneno, ou da

ação de suas toxinas sobre diversos alvos, como canais iônicos,

transportadores e os mais diversos receptores celulares.

Como mencionado, as toxinas de venenos sofrem constante pressão

seletiva, já que é de sua eficácia que depende a aquisição de alimento e

defesa de muitos animais que as produzem. Assim, geralmente possuem alta

potência e especificidade de ação para seu alvo molecular. Em suma, essas

características são difíceis de serem replicadas por outras fontes, e fazem das

toxinas animais uma fonte única para o desenvolvimento de novos modelos de

ferramentas e tratamentos farmacológicos (King, 2011).

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1.4. Peçonhas e toxinas de vertebrados

Os vertebrados peçonhentos incluem principalmente serpentes, outros

répteis, peixes e anfíbios. Dentre esses como já mencionados, os mais

estudados com relação ao potencial de suas peçonhas são as serpentes, as

quais são divididas em quatro famílias, Elapidae (najas, cobras-coral, etc),

Viperidae (cascavel, jararaca, etc), Atractaspidinae e Colubridae (Warrell,

2012).

Várias substâncias com grande potencial terapêutico já foram extraídas

de peçonhas de serpentes. Como já descrito anteriormente, uma das primeiras

foi a base para o desenvolvimento do anti-hipertensivo Captopril®, um inibidor

da enzima conversora de angiotensina II, largamente utilizado na clínica para

tratamento de hipertensão, alguns tipos de patologias cardíacas congestivas e

na preservação da função renal em indivíduos com neuropatia diabética

(Izidoro et al., 2014; Liu et al., 2014; Rodrigues & Santos, 2012; Vogel et al.,

2014)

Outros exemplos de substâncias promissoras extraídas de serpentes

são a eptifibatida (Integrilin®) e o tirofiban (Aggrastat®), dois anticoagulantes

que bloqueiam o receptor de glicoproteína IIb/IIIa, que são sintetizados a partir

de proteínas extraídas do veneno das serpentes Sistrurus miliarus barbouri e

Echis carinatus, respectivamente (Kereiakes et al., 1996; Earl et al., 2012).

Esses fármacos estão sendo testados como adjuvantes para recanalização

vascular em situações como infarto agudo do miocárdio, acidentes vasculares

isquêmicos e complicações cirúrgicas tromboembolíticas, uma vez que podem

impedir a ativação plaquetária, impedindo assim a reoclusão e facilitando uma

destruição mais completa e rápida do trombo (Asadi et al., 2014; Eisenberg et

18

al., 1992; Sedat et al., 2014). Outra substância é o peptídeo TNP-c, isolado do

veneno da Oxyuranus microlepidotus, que apresenta similaridade com

peptídeos natriuréticos, possuindo atividade vasodilatadora e hipotensiva (Fry

et al., 2005); ou a textilinina-1 (Q8008), um inibidor de serina protease isolada

do veneno da Pseudonaja textilis, que é um potente e seletivo inibidor de

plasmina e tripsina e vem sendo testada como agente hemostático em cirurgias

cardiotorácicas (Earl et al., 2012; Flight et al., 2009).

Alguns peixes também podem possuir peçonhas, um exemplo é o

Thalassophryne nattereri, que possui dois espinhos laterais e dois espinhos

dorsais conectados a glândulas produtoras de peçonha. Essa peçonha quando

inoculada em humanos é capaz de causar edema e dor severa, seguida de

necrose tecidual (Lopes-Ferreira et al., 2001). Estudos prévios mostram que a

peçonha desse peixe pode causar dano a membrana de células musculares,

bem como alteração de todas as organelas dessas células, além de outras

propriedades miotóxicas (Lopes-Ferreira et al., 2001). Outras ações

relacionadas a essa peçonha são a alteração da estrutura da matriz

extracelular, do conteúdo de colágeno durante a fase de recuperação, da

organização do citoesqueleto e da formação de pseudópodes em células

epiteliais (Pareja-Santos et al., 2009). Algumas toxinas isoladas do veneno do

T. nattereri como as natterinas apresentam ação proteolítica sobre os

colágenos dos tipos I e IV e componentes da matriz extracelular, convertem

angiotensina I em angiotensina II, além de inibirem a adesão entre célula e

matriz extracelular e causarem morte celular. A nattectina é uma lectina do tipo

C, que apresenta a capacidade de aumentar a adesão celular mediada por

integrinas e a sobrevivência de células Hela, em um processo também

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mediado por sua interação com integrinas (Komegae et al., 2011; Tenório et al.,

2015).

Um dos poucos, se não o único mamífero peçonhento é o

Ornithorhynchus anatinus, que injeta sua peçonha através de esporas nas

patas traseiras, as quais estão ligadas a uma glândula produtora da peçonha

que surge durante o período de reprodução. O envenenamento de humanos

causa inchaço e dor intensa, que não é amenizada pela administração de

morfina (Fenner et al., 1992). A peçonha bruta deste animal foi capaz de

causar edema na pata de ratos, com pico entre 20 a 30 minutos e relaxamento

do útero pré-contraídos de ratos (De Plater et al., 1995). O isolamento da

peçonha desse animal demonstrou a presença de peptídeos semelhantes à e

a -defensinas e peptídeos natriuréticos (Whittington et al., 2008).

Apesar dos vertebrados, especialmente as serpentes, terem sido os

principais alvos dos estudos iniciais com peçonhas, como já mencionado

anteriormente, os avanços técnicos recentes e o desenvolvimento de um maior

conhecimento sobre o sistema nervoso, permitiram que as peçonhas de

invertebrados também fossem alvo de novas pesquisas.

1.5. Peçonhas e toxinas de invertebrados

Os invertebrados peçonhentos incluem, entre outros, aranhas,

escorpiões, caramujos, águas-vivas, vespas, abelhas e formigas. As peçonhas

desses animais contêm componentes químicos de diversas classes, mas os

peptídeos e as poliaminas constituem os compostos neuroativos mais

estudados (Mortari et al., 2007b).

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As peçonhas de escorpiões podem provocar em humanos febre,

agitação psicomotora, salivação, lacrimejamento, aumento da mobilidade

intestinal, arritmias cardíacas e respiratórias, hipertensão seguida de

hipotensão, edema pulmonar e choque, dentre outros (Ossanai et al., 2012).

Dentre as várias espécies existentes, apenas algumas possuem importância

médica, sendo distribuídas principalmente entre os gêneros Centruroides,

Tityus, Buthus, Androctonus, Buthotus, Leiurus e Parabuthus (Nencioni et al.,

2009). Em relação ao gênero Tityus, a maioria dos estudos investigou a

peçonha do T. serrulatus, onde se observou que a injeção da peçonha bruta no

hipocampo de ratos é capaz de causar comportamentos convulsivos, como

mioclonias, automatismos faciais e sacudidela de cachorro (wet dog shakes), e,

além disso, a injeção intraperitoneal resulta na indução de descargas neuronais

epileptiformes (Dorce & Sandoval, 1994; Nencioni et al., 2009). Esses efeitos

se devem, pelo menos em parte, às toxinas TS-8F e TsTX-I, que isoladamente

causam descargas epileptiformes e wet dog shakes, quando injetadas no

hipocampo de ratos (Carvalho el al., 1998; Teixeira et al., 2010).

Outra espécie de escorpião bastante estudada é o Buthus martensis. A

peçonha desse escorpião é bastante interessante, pois contém tanto toxinas

com potencial convulsivante como com potencial anticonvulsivante. Assim, a

toxina convulsivante BmK I, é uma -toxina moduladora de canais de sódio

voltagem-dependente que, quando injetada no hipocampo dorsal de ratos em

doses baixas, promove crises límbicas, automatismos faciais, wet dog shakes e

mioclonias, enquanto que na dose de 2 g causa crises tônico-clônicas

seguidas de morte (Bai et al., 2006). Por outro lado, com relação ao potencial

anticonvulsivante, podemos citar as toxinas isoladas BmK AS e a BmK IT2. A

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primeira, quando injetada previamente no hipocampo, reduz de forma dose-

dependente a duração e o número de crises convulsivas induzidas pelo

pentilenotetrazol (PTZ) e aumenta a latência para o início do estado epiléptico

induzido pela pilocarpina (Zhao et al., 2011). Já a segunda toxina, quando

injetada previamente em CA1, protege os animais da morte e reduz a

intensidade das crises induzidas pelo PTZ (Zhao et al., 2008).

Entre os invertebrados também podemos destacar os estudos com

peçonhas de aranhas. As aranhas existem há pelo menos 300 milhões de

anos, representadas por aproximadamente 40.000 espécies já descritas (Rash

& Hodgson, 2002).

A grande maioria das neurotoxinas extraídas de aranhas são proteínas,

peptídeos ou acilpoliaminas (McCormick & Meinwald, 1993). Na literatura

existem vários exemplos de neurotoxinas extraídas de aranhas, dentre eles

está a Ph1, isolada da peçonha da Phoneutria nigriventer, que é capaz de

reduzir, de forma duradoura, a alodinia no modelo de incisão plantar, podendo

ter uso potencial no controle de dores persistentes (de Souza et al., 2011).

Outro exemplo, é a toxina JZTX-XIII isolada da peçonha da Chilobrachys

jingzhao, que apresenta a capacidade de bloquear canais de potássio

dependentes de voltagem dos tipos Kv2.1, Kv4.1 e Kv4.2, com maior afinidade

pelo primeiro (Yuan et al., 2012).

Ainda, a -latrotoxina e -latroinsetotoxina, isoladas da peçonha da

viúva negra (Latrodectus sp), atuam sobre a membrana pré-sináptica formando

poros que facilitam a passagem de íons como o cálcio, gerando uma liberação

maciça de neurotransmissor. A diferença entre as duas é que a -latrotoxina

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atua em mamíferos e não em insetos, enquanto que para a -latroinsetotoxina

é ao contrário, apresentando assim potencial inseticida (Finkelstein et al., 1992;

McCormick & Meinwald, 1993).

A peçonha da aranha Scaptocosa raptoria quando administrada i.c.v.

promove alterações comportamentais em ratos, como crises pro-cursivas (wild

running), um comportamento que geralmente precede crises convulsivas

(Ribeiro et al., 2000). Entretanto, quando a peçonha é desnaturada e injetada

previamente na substância negra parte reticulada, é capaz de proteger ratos de

crises convulsivas induzidas pela injeção de bicuculina na Area tempestas.

Este efeito foi atribuído a toxina isolada SrTx 1.3 (Mussi-Ribeiro et al., 2004).

A aranha Parawixia bistriata, cuja peçonha bruta promove o surgimento

de crises convulsivas límbicas em ratos, com mioclonias e wet dog shakes

(Rodrigues et al., 2001), também quando desnaturada protege animais de

crises tônico-clônicas induzidas por bicuculina, picrotoxina e PTZ. A toxina

Parawixina 10, também apresentou potencial anticonvulsivante em modelos de

crises tônico-clônicas induzidas por ácido kaínico, NMDA e PTZ em ratos,

através de um mecanismo que envolve o aumento da recaptação de glutamato

e glicina (Fachim et al., 2011).

Outro grupo importante no estudo de venenos é o dos insetos

peçonhentos que fazem parte da ordem Hymenoptera, a qual compreende

abelhas, vespas e formigas. Esses insetos injetam suas peçonhas através de

um ovopositor modificado, que se encontra em seu abdômen (Fitzgerald &

Flood, 2006).

23

As peçonhas das abelhas são mais conhecidas pelas reações

anafiláticas provocadas em mamíferos. No entanto, essas apresentam toxinas

com grande potencial terapêutico, por exemplo, na medicina oriental são

utilizadas para o tratamento de doenças imunológicas, como a artrite

reumatoide. Essa capacidade de modular a resposta imune vem sendo

investigada como forma de tratamento complementar em outros tipos de

patologias. Um exemplo é a doença de Parkinson, Chung et al., (2012)

demonstraram que, em camundongos com administrações intraperitoneais da

toxina MPTP, a aplicação intraperitoneal da peçonha de abelha foi capaz de

reduzir a morte de neurônios dopaminérgicos na substância negra, reduzir

citocinas pró-inflamatória como IL-1 e TNF-, além de diminuir a formação de

espécies reativas de oxigênio e o infiltrado de linfócitos TCD4.

Da mesma forma que as aranhas, as vespas possuem peçonhas com

potencial terapêutico. Um exemplo é a toxina AvTx8 isolada da peçonha da

vespa Agelaia vicina, que quando injetada previamente na Substância negra

parte reticulada, reduz os comportamentos defensivos gerados pela

estimulação química de camadas profundas do colículo superior (de Oliveira et

al., 2005).

A peçonha desnaturada da vespa Polybia occidentalis, quando injetada

previamente no ventrículo lateral direito de ratos, é capaz de proteger animais

de crises convulsivas causadas pela administração de bicuculina, picrotoxina e

ácido kaínico, além de aumentar a latência para o aparecimento de crises

induzidas por PTZ (Mortari et al., 2005). Além disso, o peptídeo Thr6-BK,

isolado dessa mesma peçonha, apresentou efeito antinociceptivo duas vezes

24

mais potente que a morfina no teste de retirada de cauda (tail-flick) em ratos

(Mortari et al., 2007a).

As formigas também possuem peçonhas com grande potencial

farmacológico, porém, apesar de existirem mais de 35.000 espécies descritas

(“Taxonomic List - Ants of All Antweb (Species) - AntWeb,” n.d.), o estudo de

suas peçonhas é bastante reduzido. Um dos poucos exemplos já descritos é da

poneratoxina, isolada do veneno da peçonha da formiga Paraponera clavata,

que é capaz de bloquear a transmissão sináptica no sistema nervoso central de

baratas (Piek et al., 1991) e aumentar a capacidade do baculovírus em matar a

larva da lagarta-do-cartucho Spodoptera frugiperda (Szolajska et al., 2004).

Outros exemplos são da ectamotina, isolada da peçonha da formiga

Ectatomma tuberculatum, que inibe a corrente de cálcio em miócitos

ventriculares de ratos (Pluzhnikov et al., 1999) e as mirmexinas, isoladas da

peçonha da Pseudomyrmex triplarinus, que possuem atividade antiinflamatória,

demonstrada pela inibição dose-dependente do edema de pata induzido por

carragenina em ratos (Pan & Hink, 2000).

1.5.1 Dinoponera quadriceps

A subfamília Ponerinae, que inclui o gênero Dinoponera, apresenta as

formigas com maior tamanho, com picadas dolorosas e capazes de provocar

manifestações sistêmicas como febre, tremores, suor frio, náusea, vômito,

linfadenopatia e arritmias cardíacas (Haddad Junior et al., 2005). A Dinoponera

quadriceps, possui uma organização social atípica, não possuindo rainha. É

encontrada em regiões da caatinga, cerrado, brejo de altitude e floresta

25

atlântica, sendo endêmica no Nordeste brasileiro (Medeiros et al., 2012;

Vasconcellos et al., 2004).

Poucos são os estudos com a peçonha dessa formiga, Sousa e

colaboradores (2012) demonstraram o potencial antinociceptivo da peçonha em

teste com formalina, ácido acético e carragenina em ratos. Mais recentemente

Lopes e colaboradores (2013) demonstraram o efeito neuroprotetor da peçonha

quando injetada i.p. no modelo de crises convulsivas induzidas por PTZ. No

campo molecular, Cologna e colaboradores (2013) realizaram a identificação

dos peptídeos presentes na peçonha de formigas coletadas em quatro

diferentes regiões, demonstrando significativas diferenças entre as

composições dessas peçonhas, bem como a presença de peptídeos com ação

antimicrobiana de amplo espectro. Enquanto que Torres e colaboradores

(2014) realizaram uma análise de transcriptoma da glândula de veneno da D.

quadriceps, demonstrando a presença de polipeptídeos alergênicos, proteínas

tipo-letal, dinoponeratoxinas e esterases.

Um trabalho realizado no nosso laboratório demonstrou que a peçonha

bruta, quando injetada em altas doses no ventrículo lateral esquerdo de

camundongos Swiss, é capaz de promover alterações comportamentais,

caracterizadas por um período inicial de imobilidade, seguido de um intenso

comportamento motor semelhante a crises convulsivas tônico-clônicas. Por

outro lado, a injeção i.c.v. prévia da peçonha desnaturada é capaz de proteger

animais de crises tônico-clônicas e morte no modelo de crises induzidas pela

bicuculina (Nôga et al. 2015). Esses resultados fomentaram o fracionamento da

peçonha, através de cromatografia líquida de alto desempenho, na tentativa de

isolar os componentes responsáveis pelos efeitos anticonvulsivantes.

26

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3. OBJETIVOS

3.1 Objetivo Geral

O objetivo do presente trabalho foi verificar qual (is) fração (ões) isolada

(s) da peçonha da formiga Dinoponera quadriceps apresenta (m) potencial

efeito anticonvulsivante em modelo de crises induzidas por bicuculina quando

administrada em camundongos.

3.2 Objetivos Específicos

Verificar o efeito comportamental da administração intracerebral, em

camundongos, das frações isoladas da peçonha quando os animais são

expostos a um campo aberto.

Avaliar, em camundongos, o potencial efeito anticonvulsivante da

administração intracerebral das frações isoladas da peçonha através do

modelo de crises convulsivas induzidas por bicuculina.

34

4. ARTIGO

Título: Pro and anticonvulsant effects of fractions isolated from Dinoponera

quadriceps ant venom (Formicidae: Ponerinae)

Autores: Diana Aline Nôga Morais Ferreira1, Luiz Eduardo Mateus Brandão1,

Fernanda Carvalho Cagni1, Delano Silva1, Dina Lília Oliveira Azevedo1, Arrilton

Araújo1, Regina Helena da Silva2 e Alessandra Mussi Ribeiro3.

Filiações:

1Depto de Fisiologia, Universidade Federal do Rio Grande do Norte, Natal, RN,

Brasil.

2Depto de Farmacologia, 3Depto de Biociências, Universidade Federal de São

Paulo, São Paulo, SP, Brasil

Periódico: Journal of Ethnopharmacology – Qualis B1 (psicologia)

Status da publicação: a ser submetido

35

Pro and anticonvulsant effects of fractions isolated from Dinoponera

quadriceps ant venom (Formicidae: Ponerinae)

DAMF Nôga1, LEM Brandão1, FC Cagni1, D Silva1, DLO. Azevedo1, A Araújo1,

RH Silva2, AM Ribeiro3

1Physiology Department, Federal University of Rio Grande do Norte, Natal, RN,

Brazil.

2Pharmacology Departament, 3Biosciences Department, Federal University of

São Paulo, São Paulo, SP, Brazil

Correspondence to: Alessandra M. Ribeiro, Departamento de Biociências,

UNIFESP, Rua Silva Jardim, 136, CEP 11015-020 - Santos, SP, Brasil. Tel.:

+55 13 38783700

E-mail: [email protected]

36

Resumo

Relevância etnofarmacológica: As tocandiras são utilizadas em rituais de

passagem para idade adulta na tribo indígena amazônica Sataré-Mawé

Objetivo do estudo: investigar o potencial anticonvulsivante de frações

isoladas da peçonha de D. quadríceps em crises convulsivas induzidas pela

BIC, bem como uma análise dos efeitos dessas frações no comportamento

natural dos camundongos no campo aberto.

Materiais e Métodos: Os animais foram divididos em grupos, os quais

receberam injeções (1 mg/ml i.c.v.) de seis frações distintas e tiveram seu

comportamento geral observado no campo aberto durante 30 min. No segundo

experimento, Os animais receberam as mesmas frações 20 min antes da

administração de bicuculina (10 mg/ml). Em seguida, foi analisado o

comportamento motor convulsivo desses animais durante 30 minutos no campo

aberto.

Resultados: No primeiro experimento, não foram observadas alterações

comportamentais. Já no segundo experimento, a administração prévia de

DqTx1, DqTx3, DqTx4 e DqTx6 aumentou a latência para o desenvolvimento

de crises tônico-clônicas. Além disso, todas as frações, exceto DqTx5,

aumentaram a latência para a morte dos animais. Ainda, os melhores

resultados foram obtidos com a fração DqTx6, que protegeu 62,5% dos animais

testados contra o desenvolvimento de crises tônico-clônicas e 100% dos

animais contra a morte.

Conclusões: As frações peptídicas isoladas da peçonha de D. quadríceps

possuem um potencial terapêutico para o tratamento de convulsões. Porém, é

necessária a determinação da estrutura e mecanismo de ação dos

componentes ativos.

Palavras-chave: peçonha de formiga, bicuculina, crises tônico-clônicas, fração

peptídica, produto natural.

37

Abstract

Ethnophamacological relevance: The tocandiras ants are used in rituals of

passage for adult age in the Sataré-Mawé tribe of Amazonian Indians.

Aim of the study: investigate the anticonvulsant activity of compounds isolated

from D. quadriceps venom on seizures induced by BIC, as well as an analysis

of its effects on spontaneous behavior in mice

Material and Methods: Animals were divided into groups, which received

injections (1 mg/ml; i.c.v.) of six distinct venom fractions and had their general

behavior analyzed for 30 min in the open field. In the second experiment, we

carried out the same fractions injection protocol 20 min before the

administration of bicuculline (10 mg/ml). immediately after, we analyzed

animals’ seizures behavior during 30 min in open field

Results: In the first experiment we did not observe behavioral alterations.

Conversely, in the second experiment, previous administration of DqTx1,

DqTx3, DqTx4 and DqTx6 increased latency for onset of tonic-clonic seizures.

Moreover, all fractions, except DqTx5, increased latency to animals’ death. Yet,

we obtained our best result with DqTx6 fraction, which protected 62,5% of

tested animals from development of tonic-clonic seizures. Further, this fraction

protected all tested animals from seizure episodes followed by death

Conclusions: Peptidic fractions isolated from D. quadriceps venom have an

therapeutic potential for seizures treatment. However further work is needed to

determine the structure and mechanism of action of the active compounds.

Keywords: ant venom, bicuculline, tonic-clonic seizures, peptide fraction,

natural product.

38

Introduction

Natural products comprise a immense chemical diversity and

architectural complexity that cannot be matched by synthetic molecules (Clardy

& Walsh, 2004; King, 2011). From this perspective the animals venoms stand

out because of the high specificity and potency of their toxins in relation to their

molecular targets of mammalian biological systems (King, 2011). These

venoms can exert noxious effects on several systems such as cardiovascular,

nervous, respiratory, renal, as well as skin and muscles. As consequence,

poisoning victims may experience pain, swelling, tissue necrosis, vomiting,

paralysis, fever, diarrhea, headaches, blurred sight, dizziness, hypotension,

hemorrhage and even death (Sitprija & Suteparak, 2008; Wong & Belov, 2012).

From another standpoint, these venoms can present benefic effects (de Souza

et al., 2014; Flight et al., 2009; Nunes et al., 2013; Ondetti et al., 1971; Sheng-

ming et al., 2014; T. Wang et al., 2014) or be used as pharmacological tools for

probing biochemical pathways and mechanisms (Mellor & Usherwood, 2004;

Morabito et al., 2014; Wang & Chi, 2004). Despite the remarkable potential of

the venoms their investigation and characterization remains underexplored.

Invertebrates through evolutionary process have incorporated a vast

range of neurotoxins in their venoms, and some compounds show high affinity

to receptors, ionic channels and transporters in the central nervous system

(CNS) (Beleboni et al., 2004; Mellor & Usherwood, 2004; Mortari et al., 2007;

Wang & Chi, 2004). Previous studies have demonstrated anticonvulsant effects

of toxin isolated from invertebrate venoms. Peptide fraction isolated from the

venom of the wasp Polybia paulista protected, at the dose of 350 g/animal,

60% of the rats against generalized tonic-clonic seizures induced by

39

pentylenotetrazol (PTZ) (do Couto et al., 2012). Further, the parawixin 2,

isolated from the venom of the spider Parawixia bistriata, when injected in the

right lateral ventricle, protected animals from seizures induced by PTZ,

picrotoxin, pilocarpine and kainic acid (Gelfuso et al., 2007), as well as inhibited

PTZ-induced kindling of rats when chronically administered for 27 days (Gelfuso

et al., 2013).

Seizures are the hallmark of epilepsy, a neurological disorder

characterized by an enduring predisposition to generate transient abnormal

excessive or synchronous neuronal activity, and by the neurobiologic, cognitive,

psychological, and social consequences of this condition (Fisher et al., 2005;

2014). This disorder affects about 65 million people worldwide (Thurman et al.,

2011) and approximately 30% of patients are resistant to pharmacotherapy

(Löscher, 1997; Rosillo-de la Torre, 2014). Furthermore, the patients who use

antiepileptic drugs frequently suffer from collateral effects ranging from gastric

discomfort to hepatic failure and aplastic anemia (Mortari et al., 2007). In this

context, invertebrate venoms appear as a possible source for new

anticonvulsant probes.

Recently, the venom from the giant ant Dinoponera quadriceps has

shown anticonvulsant effects. Lopes et al. (2013) demonstrated that

intraperitoneal administration of the crude venom increased the latency for

onset of seizures induced by PTZ in mice. Additionally, in our lab, after the

injection of the crude venom in the lateral ventricle of mice, we observed

procursive behavior and tonic-clonic seizures. Conversely, the prior

administration of the denatured venom protected the animals against tonic-

clonic seizures (66.7%) and death (100%) induced by administration of

40

bicuculline. Taken together, the findings demonstrated that D. quadriceps

venom might be potential source of new pro- and anticonvulsants molecules. In

this context, the aim of present study was to investigate the anticonvulsant

activity of fractions isolated from D. quadriceps ant venom on seizures induced

by BIC, a GABAA antagonist, as well as an analysis of the effects on

spontaneous behavior in mice.

Material and Methods

Ants collection and fraction obtainment

D. quadriceps were collected in Nísia Floresta (6º5’S, 35º12’W), Rio

Grande do Norte state, Brazil. Firstly, to collect the venom the specimens were

frozen at - 20 ºC and venom reservoirs were dissected. Content of two hundred

venom reservoirs were lyophilized and diluted in 0,1%TFA/H2O V/V. This

solution submitted to high performance liquid chromatography (HPLC - Hitachi)

purification using a Phenomenex C18 reverse phase column (2,6 x 25 cm, 12

m, 300 Å). Eluation was carried out with 0,1% TFA/H2O at a 100% gradient for

the first 10 minutes, followed by a linear gradient from 0 to 100% acetonitrile

(ACN) containing 0,1% TFA for 50 minutes. Eluates were monitored at 210 and

280 nm and the main fractions collected were lyophilized and resuspended in

1mL of distilled water. Six major fractions were obtained and named DqTx1,

DqTx2, DqTx3, DqTx4, DqTx5 and DqTx6.

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Animals

Three-month-old male Swiss mice (30-50 g) were housed with free

access to food and water, in a number of 5-6 animals in plastic cages (20 x 30 x

13 cm), under conditions of controlled temperature (25 ± 1 ºC) and a 12 h

light/12 h dark cycle (lights on 6:30 a.m.). Animals were handled in accordance

to Brazilian law for the use of animals in research (Law Number 11.794), and all

the procedures were approved by the local ethics committee (protocol

035/2010). All efforts were made to minimize animal potential pain, suffering or

discomfort.

Surgery

Prior to surgery mice were anesthetized with intraperitoneal injection of

ketamine (100 mg/kg) plus xylazine (50 mg/kg). Afterwards, the animals were

positioned in the stereotaxic frame (Insight, Brazil) and the skull was exposed.

Stainless steel guide cannula (25 gauge, 8mm length) was implanted in the

lateral ventricle, and the stereotaxic coordinates were anterior-posterior = - 0.6

mm, medial-lateral = 1.1 mm, and dorsal-ventral = 1.0 mm from bregma

(Paxinos & Franklin, 2008). Guide cannula was anchored to the skull with dental

acrylic. At the end of the surgery the cannula was temporarily sealed with a

stainless-steel wire to avoid obstruction. Animals were given one week of post-

operative recovery prior to the start of the experimental proceedings.

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General Procedures

Drug, vehicle and chromatography fractions were injected i.c.v. at a rate

of 0.5 µL/min to a final volume of 1 L via a microsyringe pump (Insight, Brazil)

with a 10 µl syringe (Hamilton Co., USA) connected to an injection needle. After

infusion, the needle was left in the guide cannula for additional 60 s to allow

drug to diffuse from the needle tip. Afterwards, mice were placed in a circular

open field (30 cm in diameter with wall height of 60 cm) located in an

experimental room illuminated by a 40 W fluorescent lamp (at the arena floor

level) for 30 min. Behavior session was recorded by a digital camera placed

above the apparatus and the behavioral parameters were registered. The

apparatus was cleaned with a 5% alcohol solution after each session.

Behavioral analysis

General screening: The animals were randomly assigned into groups:

Control, animals received 1 µL of distillate water (vehicle) (CTR, n = 9), and six

experimental groups that received 1 mg/mL of each fraction (DqTx1 n = 7;

DqTx2 n = 7; DqTx3 n = 8; DqTx4 n = 8; DqTx5 n = 8; DqTx6 = 8). We

quantified the time spent in the following behavioral clusters: exploration

(exploration activities involve behaviors such as exploratory sniffing, walking,

scanning, and erect posture); grooming (comprises grooming of head, snout,

claws, and back); and immobility (animals do not present movements, except

respiratory movements).

Anticonvulsant assay: Chemical convulsant (GABAA receptor antagonist

bicuculline, 10 mg/mL, Sigma, USA) was standardized in order to provoke tonic-

clonic seizures in 100% of injected animals in less than 30 min. Animals were

43

randomly assigned into groups: control (CTR, n = 8), animals were

microinjected with vehicle 20 minutes prior to bicuculline administration, and

animals were injected with DqTx1 - 6 20 minutes prior bicuculline administration

in the left lateral ventricle (DqTx1 n = 7; DqTx2 n = 7; DqTx3 n = 7; DqTx4 n =

7; DqTx5 n = 7; DqTx 6 n = 8). Immediately after the administration of

bicuculline, animals were placed in the open field and behavior was registered

for 30 minutes.

The severity of seizures was evaluated using an adapted (removal of the

0.5 score) Racine’s scale (Racine, 1972), as following: 1 – myoclonic jerks of

contralateral paw; 2 – mild paw clonus lasting at least 5 s; 3 – severe paw

clonus lasting at least 15 s; 4 – rearing in addition to severe paw clonus; 5 –

rearing and falling in addition to severe paw clonus. Moreover, latency to de

onset of tonic-clonic seizures (score 5) and death, as well as percentage of

protection against tonic-clonic seizures were evaluated.

Verification of the injection site

Upon completion of the behavioral procedures, mice were euthanized

with intraperitoneal injection of sodium thiopental (70 mg/kg) and microinjected

into left lateral ventricle with 1µl of methylene blue stain to mark the correct site

on injection. Brains were removed and manually cut to check the position of the

cannula. Only animals with correct injection sites were included in the analysis.

The same procedure was held if the death occurred before the end of the

experiments.

44

Statistical analysis

Data normality and homogeneity of variances were respectively tested by

Shapiro-Wilk and Levene’s tests. Comparisons among different fractions of the

ant venom in relation to behavioral clusters were analyzed using one-way

analysis of variance (ANOVA) followed by Dunnett’s post hoc (one tailed). The

same tests were used to compare the latencies for onset of seizure and death.

The number of protected animals in the anticonvulsant assays was analyzed

using χ2 test followed by residual analysis and the difference between means of

score in these assays were analyzed using Mann-Whitney test with Bonferroni

correction. We considered p < 0.05 as significant values. All statistical analyses

were conducted with PASW Statistics 22 software (IBM, USA).

Results

HPLC purification

Fractionation of the venom resulted in six major fractions, referred to as

DqTx1 to DqTx6 (Figure 1).

45

Figure 1. Reverse-phase high performance liquid chromatography of Dinoponera

quadriceps venom showing six major fractions at 210 and 280 nm in an acetonitrile

gradient.

Primary screening

One-way ANOVA did not reveal effect of treatment for the exploratory

activity [F(6,54) = 0.908, p = 0.498], grooming [F(6,54) = 2.075, p = 0.074], or

immobility [F(6,54) = 0.675, p = 0.679] (table I). These fractions did not induce

motor and behavioral alterations in the animals.

46

Table I. Effects of intracerebral injection of fractions isolated from Dinoponera

quadriceps ant venom (DqTx) in mice on total of time spent in exploratory activities,

grooming cluster and immobility.

Treatment

Behavioral cluster

Exploration Grooming Immobility

Control 1395.48 ± 56.38 293,14 ± 55.36 111.36 ± 36.47

DqTx1 1281.37 ± 110.25 352,47 ± 104.24 166.15 ± 93.05

DqTx2 1250.36 ± 128.08 267.05 ± 58.79 282.58 ± 119.69

DqTx3 1367.91 ± 58.9 294.31 ± 37.89 137.77 ± 53.78

DqTx4 1193.58 ± 49.32 493.80 ± 50.81 112.61 ± 44.11

DqTx5 1250.40 ± 87.55 406 ± 38.47 143.59 ± 62.27

DqTx6 1378.41 ± 84.41 280.44 ± 43.68 141.14 ± 64.03

Data expressed as the mean ± SEM. p > 0.05 (One-way ANOVA).

Anticonvulsant assay

As expected, all animals that received vehicle prior to bicuculline

administration showed tonic-clonic seizures (level 5) followed by death (table II).

One-way ANOVA revealed effects in relation to latency to onset of

seizures [F(6,50) = 2.617, p = 0.029] and latency to death [F(6,50) = 3.719, p =

0.004] (Fig. 2). Dunnett’s post hoc analysis detected that groups pretreated with

DqTx1 (p = 0.08), DqTx4 (p = 0.044) and DqTx6 (p = 0.002) showed an

increase in the latency for the onset of seizures, and tendency for DqTxF3 (p =

0.057) (fig. 2A). Similar results occurred regarding the latency for death i.e.

groups that received pretreatment with DqTx1 (p = 0.005), DqTx2 (p = 0.009),

DqTx3 (p = 0.01), DqTx4 (p = 0.025) and DqTx6 (p < 0.001) showed an

increase of the latency for death (fig. 2B).

The analysis of seizures score showed that pretreatment with DqTx1,

DqTx4 and DqTx5 fractions prevented the development of tonic-clonic seizures

(level 5) in 42.6% of animals. DqTx3 and DqTx 6 fractions prevented 28.6% and

47

62.5% of animals, respectively. Regarding survival, the pretreatment with

DqTx2 prevented the death of 42.8% of animals, whereas DqTx4 and DqTx5

prevented the death of 57.1% of animals, DqTx1 and DqTx3 protected 71.4%

and DqTx6 protected all the animals treated (table II).

Figure 2. Effects of intracerebral injection of fractions isolated from ant venom of

Dinoponera quadriceps in mice. (A) Latency for the onset of tonic-clonic seizures; (B)

48

Latency for death. Data expressed as the mean ± SEM. #p = 0.057, *p< 0.05

compared to control (One-way ANOVA followed by Dunnett’s post hoc test).

Table II. Effects of injection into ventricle lateral of Dinoponera quadriceps venom

fractions against seizures elicited by bicuculline model in mice.

TREATMENT

Vehicle DqTx1 DqTx2 DqTx3 DqTx4 DqTx5 DqTx6

Median seizure score 5 5 5 5 5 5 4

Incidence of seizures

Stage 1 0/8 6/7 4/7 3/7 5/7 3/7 5/8

Stage 2 0/8 4/7 4/7 3/7 5/7 3/7 5/8

Stage 3 0/8 2/7 5/7 2/7 3/7 2/7 4/8

Stage 4 0/8 1/7 2/7 2/7 0/7 1/7 1/8

Stage 5 8/8 4/7 7/7 5/7 4/7 4/7 3/8

Percentage of protection 0* 42.8 0* 28.6 42.8 42.8 62.5*

Percentage of survival 0* 71.4 42.8 71.4 57.1 57.1 100*

Incidence of death 8/8 2/7 4/7 2/7 3/7 3/7 0/8

Bicuculline was injected into the lateral ventricle at a dose of 1 mg/ml following pre-treatment with vehicle and fractions. *p < 0.05 compared to control (Chi-square, followed by residual analysis).

Discussion

Results of the present study showed that the Dinoponera quadriceps ant

venom contains six major fractions detectable by 210 and 280 nm in a reverse-

phase HPLC (Fig. 1). Prior administration of the isolated fractions prevented the

development of tonic-clonic seizures and death induced by the administration of

bicuculline into lateral ventricle of mice. Administration of the DqTx6 fraction

protected 62.5% of animals against seizures induced by bicuculline and 100%

of mice survived (table II).

Reverse-phase HPLC in the conditions utilized in the present study

separated the mixture based on hydrophobicity of its compounds. Wavelength

49

of 210 nm detects the peptide bond, while the wavelength of 280 nm detects the

aromatic amino acids tryptophan and tyrosine (Aguilar, 2003). Therefore, the

fractions tested are most likely composed of peptides, with the DqTx1 being the

most hydrophilic fraction whereas the DqTx6 is the most hydrophobic one (Fig.

1).

Peptides are the most common compounds found in animal venoms,

representing a main source of bioactive toxins with high affinity and selectivity

for a range of targets (Aili et al., 2014; Mortari et al., 2007). The venoms from

the ant subfamilies Paraponerinae and Ponerinae have been shown to be

especially rich in peptides, these have antimicrobial properties like the

ponericins (Orivel et al., 2001), pilosulins (Inagaki et al., 2004; Zelezetsky et al.

2005) and certain dinoponeratoxins (Cologna et al., 2013). Moreover, others

peptides from ant venoms, like the Poneratoxin, a V-shaped peptide with two -

helices connected by a -turn that modulates voltage-gated sodium channels

and blocks synaptic transmission in the insect CNS (Aili et al., 2014; Duval et

al., 1992; Hendrich & Mozrzymas, 2002; Piek et al., 1991; Szolajska et al.,

2004).

There are several peptides isolated from invertebrates’ venoms with

anticonvulsant properties. The BmK IT2 and BmK AS are -type toxins isolated

from the scorpion Buthus martensis venom, which when injected in the rat

hippocampus were able to increase the latency for onset of seizures and reduce

de mortality induced by the administration of PTZ (Zhao et al., 2008, 2011). A

similar approach was taken with the CGX-1007 isolated from the cone snail

Conus geographus venom that, when injected in the lateral ventricle of mice,

blocks seizures induced by maximal electroshock, audiogenic, threshold tonic

50

extension, PTZ, picrotoxin and BIC induced seizures (Armstrong et al., 1998),

and reduces the score of seizures in amygdala kindled rats (Barton & White,

2004). Moreover, the -agatoxin IVA is a voltage-sensitive calcium channel

blocker isolated from the spider Agelenopsis aperta venom, which when

injected in the lateral ventricle of mice blocked audiogenic seizures (Jackson &

Scheideler, 1996). Therefore, as shown by do Couto et al. (2012) a peptide

fraction isolated from the Polybia paulista wasp venom increased the latency for

the onset of seizures and protected 60% of the animals from tonic-clonic

seizures induced by PTZ in rats.

Bicuculline is a competitive antagonist of the GABAA receptor that is well

established as a model for the study of seizures in rodents and mainly for the

scanning of new anticonvulsant drugs (Capasso & Gallo, 2009; Cunha et al.,

2005; Faggion et al., 2011; Mussi-Ribeiro et al., 2004).

Antiepileptic drugs act basically by reducing the neuronal excitability

through three main mechanisms: modulation of voltage-dependent ion

channels, decreasing the excitatory transmission, or increasing of inhibitory

neurotransmission mediated by GABA or glycin (Kwan et al., 2001; Mortari et

al., 2007). Voltage-dependent ion channels (Na+, Ca2+ or K+) have a crucial role

in the neuronal action potential and consequently in neurotransmission, thus the

modulation on these channels may reduce neurotransmission. Hence, some

venom toxins with anticonvulsant potential have voltage-dependent channels as

targets. For example, the -conotoxins MVIIA and GVIA are N-type calcium

channels antagonists (Gasior et al., 2007), while the -type neurotoxins BmK

AS and BmK IT2 are sodium channels modulators (Zhao et al., 2008 and 2011).

51

Considering that L-glutamate mediates the majority of the excitatory

synapses in human brain, its receptors and transporters are a rational target for

anticonvulsant drug design. An example of venom toxin with anticonvulsant

potential whose mechanism of action involves modulation of glutamatergic

transmission is the Parawixin 10 which increases the glutamate uptake (Fachim

et al., 2011). However, this mechanism is not common in antiepileptic drugs due

to the wide range of side effects (Kwan et al., 2001; Macdonald & Kelly, 1995;

Mortari et al., 2007b).

There are several antiepileptic drugs that act on the GABAA receptor

acting as direct agonists or increasing its availability in the synaptic cleft by

uptake blockade (Dalby, 2000; Duncan et al., 2006; Rogawski, 2006). The

toxins SrTx1 and FrPbA2, isolated from the venom of the spiders Scaptocosa

raptoria and Parawixia bistritata, respectively act by GABAergic mechanism

(Cairrão et al., 2002).

Considering that the DqTx fractions protected animals from seizures and

death elicited by a GABAA antagonist, moreover the fact that Lopes et al. (2013)

showed that the intraperitoneal administration of the D. quadriceps venom

protected animals from seizures elicited by the another GABAA antagonist

(PTZ), but not from seizures elicited by other drugs that act through others

pathways (pilocarpine and strychnine), we can suggest that the action

mechanism of DqTx fractions involves the GABAergic transmission. Indeed,

recent transcriptome (Torres et al., 2014) and peptidomic (Cologna et al., 2013)

analysis of the D. quadriceps venom revealed the presence of peptides with a

similar structure to toxins that interact with ion channels. However, the

mechanism underlying the anticonvulsant effects of the DqTx fractions is

52

noteworthy of further investigation. So far, the results obtained indicate that ant

venom and its components are promising tools for experimental pharmacology,

and further studies will be conducted in order to improve understanding of their

effects and exploit their potential.

Acknowledgements

This research was supported by fellowships from Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPQ); Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Pró-reitoria de

Pesquisa da Universidade Federal do Rio Grande do Norte (PROPESQ/UFRN)

and Fundação de Apoio à Pesquisa do Estado do Rio Grande do Norte

(FAPERN).

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6. Commentaries - invited, peer-reviewed, critical discussion about crucial aspects of the field but most importantly methodological and conceptual-theoretical developments in the field and should also provide a standard, for example, for pharmacological methods to be used in papers in the Journal of Ethnopharmacology. The scientific dialogue differs greatly in the social / cultural and natural sciences, the discussions about the common foundations of the field are ongoing and the papers published should contribute to a transdisciplinary and multidisciplinary discussion. The length should be a maximum of 2-3 printed pages or 2500 words. Please contact the Reviews Editor [email protected] with an outline.

7. Conference announcements and news.

Before you begin

Ethics in publishing

For information on Ethics in publishing and Ethical guidelines for journal publication

seehttp://www.elsevier.com/publishingethics and http://www.elsevier.com/journal-authors/ethics.

Policy and ethics

In the covering letter, the author must also declare that the study was performed according to the

international, national and institutional rules considering animal experiments, clinical studies and

biodiversity rights. See below for further information. The ethnopharmacological importance of the study

must also be explained in the cover letter.

Animal and clinical studies Investigations using experimental animals must state in the Methods

section that the research was conducted in accordance with the internationally accepted principles for

laboratory animal use and care as found in for example the European Community guidelines (EEC

Directive of 1986; 86/609/EEC) or the US guidelines (NIH publication #85-23, revised in 1985).

Investigations with human subjects must state in the Methods section that the research followed guidelines

59

of the Declaration of Helsinki and Tokyo for humans, and was approved by the institutional human

experimentation committee or equivalent, and that informed consent was obtained. The Editors will reject

papers if there is any doubt about the suitability of the animal or human procedures used.

Biodiversity rights - Each country has its own rights on its biodiversity. Consequently for studying plants

one needs to follow the international, national and institutional rules concerning the biodiversity rights.

Author contributions

For each author the contribution to the publication should be mentioned.

Conflict of interest

All authors are requested to disclose any actual or potential conflict of interest including any financial,

personal or other relationships with other people or organizations within three years of beginning the

submitted work that could inappropriately influence, or be perceived to influence, their work. See

alsohttp://www.elsevier.com/conflictsofinterest. Further information and an example of a Conflict of Interest

form can be found at: http://help.elsevier.com/app/answers/detail/a_id/286/p/7923.

Submission declaration and verification

Submission of an article implies that the work described has not been published previously (except in the

form of an abstract or as part of a published lecture or academic thesis or as an electronic preprint,

seehttp://www.elsevier.com/sharingpolicy), that it is not under consideration for publication elsewhere, that

its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the

work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English

or in any other language, including electronically without the written consent of the copyright-holder. To

verify originality, your article may be checked by the originality detection service

CrossCheck http://www.elsevier.com/editors/plagdetect.

Changes to authorship

This policy concerns the addition, deletion, or rearrangement of author names in the authorship of

accepted manuscripts

Before the accepted manuscript is published in an online issue: Requests to add or remove an author, or

to rearrange the author names, must be sent to the Journal Manager from the corresponding author of the

accepted manuscript and must include: (a) the reason the name should be added or removed, or the

author names rearranged and (b) written confirmation (e-mail, fax, letter) from all authors that they agree

with the addition, removal or rearrangement. In the case of addition or removal of authors, this includes

confirmation from the author being added or removed. Requests that are not sent by the corresponding

author will be forwarded by the Journal Manager to the corresponding author, who must follow the

procedure as described above. Note that: (1) Journal Managers will inform the Journal Editors of any such

requests and (2) publication of the accepted manuscript in an online issue is suspended until authorship

has been agreed.

After the accepted manuscript is published in an online issue: Any requests to add, delete, or rearrange

author names in an article published in an online issue will follow the same policies as noted above and

result in a corrigendum.

Article transfer service

This journal is part of our Article Transfer Service. This means that if the Editor feels your article is more

suitable in one of our other participating journals, then you may be asked to consider transferring the

article to one of those. If you agree, your article will be transferred automatically on your behalf with no

need to reformat. Please note that your article will be reviewed again by the new journal. More information

about this can be found here:http://www.elsevier.com/authors/article-transfer-service.

Copyright

Upon acceptance of an article, authors will be asked to complete a 'Journal Publishing Agreement' (for

more information on this and copyright, see http://www.elsevier.com/copyright). An e-mail will be sent to

60

the corresponding author confirming receipt of the manuscript together with a 'Journal Publishing

Agreement' form or a link to the online version of this agreement.

Subscribers may reproduce tables of contents or prepare lists of articles including abstracts for internal

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must obtain written permission from the copyright owners and credit the source(s) in the article. Elsevier

has preprinted forms for use by authors in these cases: please

consult http://www.elsevier.com/permissions.

For open access articles: Upon acceptance of an article, authors will be asked to complete an 'Exclusive

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Author rights

As an author you (or your employer or institution) have certain rights to reuse your work. For more

information seehttp://www.elsevier.com/copyright.

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You are requested to identify who provided financial support for the conduct of the research and/or

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Funding body agreements and policies

Elsevier has established a number of agreements with funding bodies which allow authors to comply with

their funder's open access policies. Some authors may also be reimbursed for associated publication fees.

To learn more about existing agreements please visit http://www.elsevier.com/fundingbodies.

Open access

This journal offers authors a choice in publishing their research:

Open access

• Articles are freely available to both subscribers and the wider public with permitted reuse

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institution

Subscription

• Articles are made available to subscribers as well as developing countries and patient groups through our

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• No open access publication fee payable by authors.

Regardless of how you choose to publish your article, the journal will apply the same peer review criteria

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For open access articles, permitted third party (re)use is defined by the following Creative Commons user

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Creative Commons Attribution (CC BY)

Lets others distribute and copy the article, create extracts, abstracts, and other revised versions,

adaptations or derivative works of or from an article (such as a translation), include in a collective work

(such as an anthology), text or data mine the article, even for commercial purposes, as long as they credit

the author(s), do not represent the author as endorsing their adaptation of the article, and do not modify

the article in such a way as to damage the author's honor or reputation.

61

Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND)

For non-commercial purposes, lets others distribute and copy the article, and to include in a collective work

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The open access publication fee for this journal is USD 3000, excluding taxes. Learn more about Elsevier's

pricing policy: http://www.elsevier.com/openaccesspricing.

Language (usage and editing services)

Please write your text in good English (American or British usage is accepted, but not a mixture of these).

Authors who feel their English language manuscript may require editing to eliminate possible grammatical

or spelling errors and to conform to correct scientific English may wish to use the English Language

Editing service available from Elsevier's WebShop (http://webshop.elsevier.com/languageediting/) or visit

our customer support site (http://support.elsevier.com) for more information.

Submission

Our online submission system guides you stepwise through the process of entering your article details and

uploading your files. The system converts your article files to a single PDF file used in the peer-review

process. Editable files (e.g., Word, LaTeX) are required to typeset your article for final publication. All

correspondence, including notification of the Editor's decision and requests for revision, is sent by e-mail.

Additional information

Authors who want to submit a manuscript should consult and peruse carefully recent issues of the journal

for format and style. Authors must include the following contact details on the title page of their submitted

manuscript: full postal address; fax; e-mail. All manuscripts submitted are subject to peer review. The

minimum requirements for a manuscript to qualify for peer review are that it has been prepared by strictly

following the format and style of the journal as mentioned, that it is written in good English, and that it is

complete. Manuscripts that have not fulfilled these requirements will be returned to the author(s).

In addition, you are recommended to adhere to the research standards described in the following articles:

Cos P., Vlietinck A.J., Berghe D.V., et al. (2006) Anti-infective potential of natural products: how to develop

a stronger in vitro 'proof-of-concept'. Journal of Ethnopharmacology, 106: 290-302.

Matteucci, E., Giampietro, O. (2008) Proposal open for discussion: defining agreed diagnostic procedures

in experimental diabetes research. Journal of Ethnopharmacology,115: 163-172.

Froede, T.SA. and Y.S. Medeiros, Y.S. (2008) Animal models to test drugs with potential antidiabetic

activity. Journal of Ethnopharmacology 115: 173-183. Gertsch J. (2009) How scientific is the science in

ethnopharmacology? Historical perspectives and epistemological problems. Journal of

Ethnopharmacology, 122: 177-183.

Chan K., et al. (2012) Good practice in reviewing and publishing studies on herbal medicine, with special

emphasis on traditional Chinese medicine and Chinese Materia Medica. Journal of Ethnopharmacology

140: 469-475.

Heinrich, M., Edwards. S., Moerman. D.E.. and Leonti. M. (2009), Ethnopharmacological field studies: a

critical assessment of their conceptual basis and methods. J. Ethnopharmacol, 124: 1-17.

Preparation

Use of word processing software

It is important that the file be saved in the native format of the word processor used. The text should be in

single-column format. Keep the layout of the text as simple as possible. Most formatting codes will be

removed and replaced on processing the article. In particular, do not use the word processor's options to

justify text or to hyphenate words. However, do use bold face, italics, subscripts, superscripts etc. When

preparing tables, if you are using a table grid, use only one grid for each individual table and not a grid for

each row. If no grid is used, use tabs, not spaces, to align columns. The electronic text should be prepared

in a way very similar to that of conventional manuscripts (see also the Guide to Publishing with

62

Elsevier: http://www.elsevier.com/guidepublication). Note that source files of figures, tables and text

graphics will be required whether or not you embed your figures in the text. See also the section on

Electronic artwork.

To avoid unnecessary errors you are strongly advised to use the 'spell-check' and 'grammar-check'

functions of your word processor.

Article structure

Subdivision - numbered sections

Divide your article into clearly defined and numbered sections. Subsections should be numbered 1.1 (then

1.1.1, 1.1.2, ...), 1.2, etc. (the abstract is not included in section numbering). Use this numbering also for

internal cross-referencing: do not just refer to 'the text'. Any subsection may be given a brief heading. Each

heading should appear on its own separate line.

Introduction

State the objectives of the work and provide an adequate background, avoiding a detailed literature survey

or a summary of the results.

Material and methods

Provide sufficient detail to allow the work to be reproduced. Methods already published should be

indicated by a reference: only relevant modifications should be described.

Theory/calculation

A Theory section should extend, not repeat, the background to the article already dealt with in the

Introduction and lay the foundation for further work. In contrast, a Calculation section represents a practical

development from a theoretical basis.

Results

Results should be clear and concise.

Discussio

This should explore the significance of the results of the work, not repeat them. A combined Results and

Discussion section is often appropriate. Avoid extensive citations and discussion of published literature.

Conclusions

The main conclusions of the study may be presented in a short Conclusions section, which may stand

alone or form a subsection of a Discussion or Results and Discussion section.

Glossary

Please supply, as a separate list, the definitions of field-specific terms used in your article.

Appendices

If there is more than one appendix, they should be identified as A, B, etc. Formulae and equations in

appendices should be given separate numbering: Eq. (A.1), Eq. (A.2), etc.; in a subsequent appendix, Eq.

(B.1) and so on. Similarly for tables and figures: Table A.1; Fig. A.1, etc.

Essential title page information

• Title. Concise and informative. Titles are often used in information-retrieval systems. Avoid abbreviations

and formulae where possible.

• Author names and affiliations. Please clearly indicate the given name(s) and family name(s) of each

author and check that all names are accurately spelled. Present the authors' affiliation addresses (where

the actual work was done) below the names. Indicate all affiliations with a lower-case superscript letter

immediately after the author's name and in front of the appropriate address. Provide the full postal address

of each affiliation, including the country name and, if available, the e-mail address of each author.

63

• Corresponding author. Clearly indicate who will handle correspondence at all stages of refereeing and

publication, also post-publication. Ensure that the e-mail address is given and that contact details are

kept up to date by the corresponding author.

• Present/permanent address. If an author has moved since the work described in the article was done,

or was visiting at the time, a 'Present address' (or 'Permanent address') may be indicated as a footnote to

that author's name. The address at which the author actually did the work must be retained as the main,

affiliation address. Superscript Arabic numerals are used for such footnotes.

Abstract

A concise and factual abstract is required. The abstract should state briefly the purpose of the research,

the principal results and major conclusions. An abstract is often presented separately from the article, so it

must be able to stand alone. For this reason, References should be avoided, but if essential, then cite the

author(s) and year(s). Also, non-standard or uncommon abbreviations should be avoided, but if essential

they must be defined at their first mention in the abstract itself.

The author should divide the abstract with the headings Ethnopharmacological relevance, Aim of the

study ,Materials and Methods, Results, and Conclusions.

Click here to see an example.

Graphical abstract

A Graphical abstract is mandatory for this journal. It should summarize the contents of the article in a

concise, pictorial form designed to capture the attention of a wide readership online. Authors must provide

images that clearly represent the work described in the article. Graphical abstracts should be submitted as

a separate file in the online submission system. Image size: please provide an image with a minimum of

531 × 1328 pixels (h × w) or proportionally more. The image should be readable at a size of 5 × 13 cm

using a regular screen resolution of 96 dpi. Preferred file types: TIFF, EPS, PDF or MS Office files.

See http://www.elsevier.com/graphicalabstracts for examples.

Authors can make use of Elsevier's Illustration and Enhancement service to ensure the best presentation

of their images also in accordance with all technical requirements: Illustration Service.

Keywords

After having selected a classification in the submission system, authors must in the same step select 5

keywords. These keywords will help the Editors to categorize your article accurately and process it more

quickly. A list of the classifications and set keywords can be found here.

In addition, you can provide a maximum of 6 specific keywords, using American spelling and avoiding

general and plural terms and multiple concepts (avoid, for example, "and", "of"). Be sparing with

abbreviations: only abbreviations firmly established in the field may be eligible. These keywords will be

used for indexing purposes.

Chemical compounds

You can enrich your article by providing a list of chemical compounds studied in the article. The list of

compounds will be used to extract relevant information from the NCBI PubChem Compound database and

display it next to the online version of the article on ScienceDirect. You can include up to 10 names of

chemical compounds in the article. For each compound, please provide the PubChem CID of the most

relevant record as in the following example: Glutamic acid (PubChem CID:611). The PubChem CIDs can

be found viahttp://www.ncbi.nlm.nih.gov/pccompound. Please position the list of compounds immediately

below the 'Keywords' section. It is strongly recommended to follow the exact text formatting as in the

example below:

Chemical compounds studied in this article

Ethylene glycol (PubChem CID: 174); Plitidepsin (PubChem CID: 44152164); Benzalkonium chloride

(PubChem CID: 15865)

More information is available at: http://www.elsevier.com/PubChem.

64

Plant names

In the Materials and Methods section there must be a separate heading for describing the material used.

That includes official name, local name, English name (if known), GPS position in case of collection in the

wild or cultivation, a voucher specimen must be deposited in an official herbarium for possible future

comparison. In the text it should be stated that the plant name has been checked

with www.theplantlist.org mentioning the data of accessing that website.

In case of commercially procured material should mention the source, batch number, quality control data.

Data on chemical characterization (metabolomics, chromatographic methods) should also be presented, in

case of known active compounds their quantitative analysis should be presented.

Acknowledgements

Collate acknowledgements in a separate section at the end of the article before the references and do not,

therefore, include them on the title page, as a footnote to the title or otherwise. List here those individuals

who provided help during the research (e.g., providing language help, writing assistance or proof reading

the article, etc.).

Math formulae

Please submit math equations as editable text and not as images. Present simple formulae in line with

normal text where possible and use the solidus (/) instead of a horizontal line for small fractional terms,

e.g., X/Y. In principle, variables are to be presented in italics. Powers of e are often more conveniently

denoted by exp. Number consecutively any equations that have to be displayed separately from the text (if

referred to explicitly in the text).

Footnotes

Footnotes should be used sparingly. Number them consecutively throughout the article. Many word

processors can build footnotes into the text, and this feature may be used. Otherwise, please indicate the

position of footnotes in the text and list the footnotes themselves separately at the end of the article. Do

not include footnotes in the Reference list.

Artwork

Electronic artwork

General points

• Make sure you use uniform lettering and sizing of your original artwork.

• Embed the used fonts if the application provides that option.

• Aim to use the following fonts in your illustrations: Arial, Courier, Times New Roman, Symbol, or use

fonts that look similar.

• Number the illustrations according to their sequence in the text.

• Use a logical naming convention for your artwork files.

• Provide captions to illustrations separately.

• Size the illustrations close to the desired dimensions of the published version.

• Submit each illustration as a separate file.

A detailed guide on electronic artwork is available on our website:

http://www.elsevier.com/artworkinstructions.

You are urged to visit this site; some excerpts from the detailed information are given here

65

Formats

If your electronic artwork is created in a Microsoft Office application (Word, PowerPoint, Excel) then please

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Regardless of the application used other than Microsoft Office, when your electronic artwork is finalized,

please 'Save as' or convert the images to one of the following formats (note the resolution requirements for

line drawings, halftones, and line/halftone combinations given below):

EPS (or PDF): Vector drawings, embed all used fonts.

TIFF (or JPEG): Color or grayscale photographs (halftones), keep to a minimum of 300 dpi.

TIFF (or JPEG): Bitmapped (pure black & white pixels) line drawings, keep to a minimum of 1000 dpi.

TIFF (or JPEG): Combinations bitmapped line/half-tone (color or grayscale), keep to a minimum of 500 dpi.

Please do not:

• Supply files that are optimized for screen use (e.g., GIF, BMP, PICT, WPG); these typically have a low

number of pixels and limited set of colors;

• Supply files that are too low in resolution;

• Submit graphics that are disproportionately large for the content.

Please note that figures and tables should be embedded in the text as close as possible to where they are

initially cited. It is also mandatory to upload separate graphic and table files as these will be required if

your manuscript is accepted for publication.

Color artwork

Please make sure that artwork files are in an acceptable format (TIFF (or JPEG), EPS (or PDF), or MS

Office files) and with the correct resolution. If, together with your accepted article, you submit usable color

figures then Elsevier will ensure, at no additional charge, that these figures will appear in color online (e.g.,

ScienceDirect and other sites) regardless of whether or not these illustrations are reproduced in color in

the printed version. For color reproduction in print, you will receive information regarding the costs

from Elsevier after receipt of your accepted article. Please indicate your preference for color: in print or

online only. For further information on the preparation of electronic artwork, please

see http://www.elsevier.com/artworkinstructions.

Please note: Because of technical complications that can arise by converting color figures to 'gray scale'

(for the printed version should you not opt for color in print) please submit in addition usable black and

white versions of all the color illustrations.

Figure captions

Ensure that each illustration has a caption. Supply captions separately, not attached to the figure. A

caption should comprise a brief title (not on the figure itself) and a description of the illustration. Keep text

in the illustrations themselves to a minimum but explain all symbols and abbreviations used.

Tables

Please submit tables as editable text and not as images. Tables can be placed either next to the relevant

text in the article, or on separate page(s) at the end. Number tables consecutively in accordance with their

appearance in the text and place any table notes below the table body. Be sparing in the use of tables and

ensure that the data presented in them do not duplicate results described elsewhere in the article. Please

avoid using vertical rules.

References

66

Citation in text

Please ensure that every reference cited in the text is also present in the reference list (and vice versa).

Any references cited in the abstract must be given in full. Unpublished results and personal

communications are not recommended in the reference list, but may be mentioned in the text. If these

references are included in the reference list they should follow the standard reference style of the journal

and should include a substitution of the publication date with "Unpublished results". "Personal

communication" will not be accepted as a reference. Citation of a reference as "in press" implies that the

item has been accepted for publication.

Reference links

Increased discoverability of research and high quality peer review are ensured by online links to the

sources cited. In order to allow us to create links to abstracting and indexing services, such as Scopus,

CrossRef and PubMed, please ensure that data provided in the references are correct. Please note that

incorrect surnames, journal/book titles, publication year and pagination may prevent link creation. When

copying references, please be careful as they may already contain errors. Use of the DOI is encouraged.

Reference management software

Most Elsevier journals have a standard template available in key reference management packages. This

covers packages using the Citation Style Language, such as Mendeley

(http://www.mendeley.com/features/reference-manager) and also others like EndNote

(http://www.endnote.com/support/enstyles.asp) and Reference Manager

(http://refman.com/support/rmstyles.asp). Using plug-ins to word processing packages which are available

from the above sites, authors only need to select the appropriate journal template when preparing their

article and the list of references and citations to these will be formatted according to the journal style as

described in this Guide. The process of including templates in these packages is constantly ongoing. If the

journal you are looking for does not have a template available yet, please see the list of sample references

and citations provided in this Guide to help you format these according to the journal style.

If you manage your research with Mendeley Desktop, you can easily install the reference style for this

journal by clicking the link below:

http://open.mendeley.com/use-citation-style/journal-of-ethnopharmacology

When preparing your manuscript, you will then be able to select this style using the Mendeley plug-ins for

Microsoft Word or LibreOffice. For more information about the Citation Style Language,

visit http://citationstyles.org.

Reference style

Text: All citations in the text should refer to:

1. Single author: the author's name (without initials, unless there is ambiguity) and the year of publication;

2. Two authors: both authors' names and the year of publication;

3. Three or more authors: first author's name followed by 'et al.' and the year of publication.

Citations may be made directly (or parenthetically). Groups of references should be listed first

alphabetically, then chronologically.

Examples: 'as demonstrated (Allan, 2000a, 2000b, 1999; Allan and Jones, 1999). Kramer et al. (2010)

have recently shown ....'

List: References should be arranged first alphabetically and then further sorted chronologically if

necessary. More than one reference from the same author(s) in the same year must be identified by the

letters 'a', 'b', 'c', etc., placed after the year of publication.

Examples:

Reference to a journal publication:

Van der Geer, J., Hanraads, J.A.J., Lupton, R.A., 2010. The art of writing a scientific article. J. Sci.

Commun. 163, 51–59.

67

Reference to a book:

Strunk Jr., W., White, E.B., 2000. The Elements of Style, fourth ed. Longman, New York.

Reference to a chapter in an edited book:

Mettam, G.R., Adams, L.B., 2009. How to prepare an electronic version of your article, in: Jones, B.S.,

Smith , R.Z. (Eds.), Introduction to the Electronic Age. E-Publishing Inc., New York, pp. 281–304.

Video data

Elsevier accepts video material and animation sequences to support and enhance your scientific research.

Authors who have video or animation files that they wish to submit with their article are strongly

encouraged to include links to these within the body of the article. This can be done in the same way as a

figure or table by referring to the video or animation content and noting in the body text where it should be

placed. All submitted files should be properly labeled so that they directly relate to the video file's content.

In order to ensure that your video or animation material is directly usable, please provide the files in one of

our recommended file formats with a preferred maximum size of 150 MB. Video and animation files

supplied will be published online in the electronic version of your article in Elsevier Web products, including

ScienceDirect: http://www.sciencedirect.com. Please supply 'stills' with your files: you can choose any

frame from the video or animation or make a separate image. These will be used instead of standard icons

and will personalize the link to your video data. For more detailed instructions please visit our video

instruction pages at http://www.elsevier.com/artworkinstructions. Note: since video and animation cannot

be embedded in the print version of the journal, please provide text for both the electronic and the print

version for the portions of the article that refer to this content.

AudioSlides

The journal encourages authors to create an AudioSlides presentation with their published article.

AudioSlides are brief, webinar-style presentations that are shown next to the online article on

ScienceDirect. This gives authors the opportunity to summarize their research in their own words and to

help readers understand what the paper is about. More information and examples are available at

http://www.elsevier.com/audioslides. Authors of this journal will automatically receive an invitation e-mail to

create an AudioSlides presentation after acceptance of their paper.

Supplementary material

Elsevier accepts electronic supplementary material to support and enhance your scientific research.

Supplementary files offer the author additional possibilities to publish supporting applications, high-

resolution images, background datasets, sound clips and more. Supplementary files supplied will be

published online alongside the electronic version of your article in Elsevier Web products, including

ScienceDirect: http://www.sciencedirect.com. In order to ensure that your submitted material is directly

usable, please provide the data in one of our recommended file formats. Authors should submit the

material in electronic format together with the article and supply a concise and descriptive caption for each

file. For more detailed instructions please visit our artwork instruction pages at

http://www.elsevier.com/artworkinstructions.

Database linking

Elsevier encourages authors to connect articles with external databases, giving readers access to relevant

databases that help to build a better understanding of the described research. Please refer to relevant

database identifiers using the following format in your article: Database: xxxx (e.g., TAIR: AT1G01020;

CCDC: 734053; PDB: 1XFN). See http://www.elsevier.com/databaselinking for more information and a full

list of supported databases.

Submission checklist

The following list will be useful during the final checking of an article prior to sending it to the journal for

review. Please consult this Guide for Authors for further details of any item.

Ensure that the following items are present:

One author has been designated as the corresponding author with contact details:

68

• E-mail address

• Full postal address

All necessary files have been uploaded, and contain:

• Keywords

• All figure captions

• All tables (including title, description, footnotes)

Further considerations

• Manuscript has been 'spell-checked' and 'grammar-checked'

• References are in the correct format for this journal

• All references mentioned in the Reference list are cited in the text, and vice versa

• Permission has been obtained for use of copyrighted material from other sources (including the Internet)

Printed version of figures (if applicable) in color or black-and-white

• Indicate clearly whether or not color or black-and-white in print is required.

• For reproduction in black-and-white, please supply black-and-white versions of the figures for printing

purposes.

For any further information please visit our customer support site at http://support.elsevier.com.

After Acceptance:

Use of the Digital Object Identifier

The Digital Object Identifier (DOI) may be used to cite and link to electronic documents. The DOI consists

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5 ANEXO

Este artigo foi aceito para publicação na Neotropical Entomology

Correspondence to: Alessandra M. Ribeiro, Departamento de Biociências, UNIFESP,

Rua Silva Jardim, 136, CEP 11015-020 - Santos, SP, Brasil. Tel.: +55 13 3783700

E-mail: [email protected]

Public Health

Pro- and Anticonvulsant Effects of the Ant Dinoponera quadriceps Venom in Mice

DAMF. Nôga1, FC Cagni1, JR Santos2, D Silva1, DLO Azevedo1, A Araújo1, RH Silva3,

AM Ribeiro4

1Depto de Fisiologia, Univ Federal do Rio Grande do Norte, Natal, RN, Brasil

2Depto de Biologia, Univ Federal de Sergipe, Aracaju, SE, Brasil

3Depto de Farmacologia, 4Depto de Biociências, Univ Federal de São Paulo, São Paulo,

SP, Brasil

Edited by Fernando L Cônsoli – ESALQ/USP

Received 10 September 2014 and accepted XX March 2015

Running title: Pro- and anticonvulsant effects of the venom of a neotropical ant

mailto:[email protected]

71

Abstract

Epilepsy affects at least 50 million people worldwide and the available treatment is

associated with various side effects. Approximately 20-30% of the patients develop

seizures that persist despite of careful monitored treatment with antiepileptic drugs.

Thus, there is a clear need for the development of new antiepileptic drugs and the

venoms can be an excellent source of probes. In this context, while there are studies on

venoms from snakes, scorpions and spiders, little is known regarding venom from ants.

The aim of this study was to investigate the potential pro- and anticonvulsant effects of

the venom from the ant Dinoponera quadriceps (Kempf) in Swiss mice. After the

injection of the crude venom (DqTx - 5, 50 and 500 mg/ml) in the lateral ventricle of

mice, we observed a reduction of exploration and grooming behaviors, as well as an

increase in immobility duration. In addition, the crude venom induced procursive

behavior and tonic-clonic seizures at the highest concentration. Conversely, the

preadministration of the denatured venom (AbDq) at the concentration of 2 mg/ml

protected the animals against tonic-clonic seizures (66.7%) and death (100%) induced

by administration of bicuculline. Taken together, the findings demonstrate that D.

quadriceps venom might be potential source of new pro- and anticonvulsants

molecules.

Keywords

Bicuculline, Freezing, Tonic-clonic seizures; Wild running

72

Introduction

Epilepsy is an episodic disorder of the nervous system result of the excessive

synchronous and sustained discharge of a group of neurons (Bai et al 2006). This

disorder affects at least 50 million people worldwide and approximately 20 - 30% of the

patients develop a chronic epilepsy, in which the seizures persist despite of careful

monitored treatment with antiepileptic drugs (Löscher 1997). Further, patients who use

antiepileptic drugs frequently suffer from collateral effects such as gastric discomfort,

sedation, diplopy, ataxia, nystagmus, gingival hypertrophy, hirsutism, cognitive

impairment, behavior disturbances, as well as idiosyncratic reactions such as rash,

agranulocytosis, hepatic failure and aplastic anemia (Mortari et al 2007). Thus, there is

a clear need for the development of new antiepileptic drugs that could treat the

pharmacoresistant cases and/or induce less adverse effects.

Possible sources of substances with this profile are invertebrate venoms (Mortari

et al 2007). They have a considerable amount of bioactive compounds, which have

showed pharmacological effects on several biological systems (Rodrigues et al 2001,

Beleboni et al 2004, Rajendra et al 2004). It is known that toxins isolated from animal

venoms can exert neurophysiological effects. Some of them present high affinity to

receptors, ionic channels, and transporters in the central nervous system (Beleboni et al

2004, Mellor & Usherwood 2004, Wang & Chi 2004). It is estimated that only a small

part of this biodiversity has been scientifically explored, and most studies have focused

on the structure-function relationship of these neurotoxins in vitro (Mortari et al 2007).

Some studies have focused in the possible pro and anti-convulsant action of

these venoms and/or their active compounds. The crude venom of the spider Parawixia

bistriata Rengger induced the appearance of limbic seizures (Rodrigues et al 2001)

when administered intracerebroventricularly in rats. Oliveira et al. (2013) showed the

73

intraperitoneal (i.p.) injection of Tityus serrulatus Lutz & Mello venom in mice elicited

spasms and convulsions preceded by intense salivation. Further, compounds isolated

from venoms can also elicit seizures in rodents. For example, the BmK I, an alpha-like

scorpion neurotoxin isolated from the venom of Buthus martensi Karsch induced

seizures in rats by intrahippocampal administration (Bai et al 2006). Moreover, the

TsTX-I, a scorpion neurotoxin isolated from T. serrulatus venom promoted short and

long epileptic-like discharges when injected in the hippocampus of rats (Teixeira et al

2010).

On the other hand, conversely to the pro-convulsant action of the P. bistriata

spider crude venom, toxins isolated from this venom, such as Parawixin 10, blocked

generalized seizures induced by kainic acid, N-methyl-D-aspartate and

pentylenetetrazole in rats (Fachim et al 2011). In addition, Parawixin 2 is a potent

anticonvulsant against chemically induced acute seizures (Liberato et al 2006, Gelfuso

et al 2007), and showed similar effects against PTZ-induced kindling (Gelfuso et al

2013). Further, besides the effects of their isolated toxins, the denatured crude venoms,

i.e., venom free of high molecular weight proteins, have also shown anticonvulsant

actions. The denatured crude venom of the solitary spider Scaptocosa raptoria Roewer

protected rats against seizures evoked by the administration of bicuculline in the area

tempestas when injected bilaterally into substantia nigra pars reticulate (SNpr). The

SrTx1.3 toxin isolated from this venom was also capable of protecting rats against

seizures induced by the administration of bicuculline in the area tempestas when

administrated in the SNpr (Mussi-Ribeiro et al 2004). Likewise, Cunha et al (2005)

showed that lower doses of the crude venom of the wasp Polybia ignobilis Haliday via

intracerebroventricular (i.c.v.) provoked severe generalized tonic-clonic seizures. In

contrast, the denatured venom protected against acute seizures induced by

74

microinjections of bicuculline, picrotoxin and kainic acid. In this context, it is extremely

important to know the effects of animal venoms on the central nervous system because

venom neurotoxins appear as potential candidates for new anticonvulsant drugs.

The ants are members of the Hymenoptera, which also comprises bees, wasps,

and hornets. Despite the more than 35,000 species of ants known (Fitzgerald & Flood

2006, Taxonomic List - Ants of All Antweb (Species) - AntWeb), few studies have

explored the potential biological effects of ant venoms. One example is the Poneratoxin,

a neuropeptide isolated from the venom of the ant Paraponera clavata Fabricius. This

toxin blocked the synaptic transmission in central nervous system of cockroachs (Piek

et al 1991) and sodium channels in frog skeletal muscle fibers (Duval et al 1992). In

addition, the Ectamotin toxin isolated from the venom of the ant Ectatomma

tuberculatum Olivier can inhibit calcium currents in ventricular myocites of rats

(Pluzhnikov et al 1999).

Relevant to the present study, the Ponerinae sub-family includes the genus

Dinoponera Roger a primitive group of large ants that can provoke extremely painful

stings and potential systemic manifestations such as fever, tremor, cold sweating,

nausea, vomiting, lymphadenophaty and cardiac arrhythmias (Haddad Junior et al

2005). Dinoponera quadriceps (Kempf) is a queenless Ponerinae ant found in

northeastern Brazil (Medeiros et al 2012). Because very few studies have focused on the

venoms of ants as sources of excitatory and inhibitory neuronal modulators, the aim of

this study was to investigate the effects of the intracerebroventricular injection of the D.

quadriceps ant venom on mice behavior in the open field. Considering the fact that the

crude venom from invertebrates sometimes induce seizures when tested in rodents

whereas the denatured venom causes the opposite effect (Cairrão et al 2002, Cunha et al

75

2005), we also evaluated the anticonvulsant potential of the denatured venom on

seizures induced by the administration of bicuculline in mice.

Material and Methods

Ant collection and venom preparation

Ants were collected in an area of Atlantic Forest (6°5’S, 35°12’W) located in Nísia

Floresta city, state of Rio Grande do Norte (Northeasthern Brazil). To collect the

venom, ants were first euthanized by freezing at - 20ºC and venom reservoirs were

dissected out. Two hundred venom reservoirs were homogenized in 2 mL of distilled

water and centrifuged at 5,000 g for 10 min. The supernatant (crude venom - DqTx) was

lyophilized overnight and stored at -20ºC. The crude extract was freshly dissolved in

saline solution at different concentrations (5, 50 and 500 mg/mL). Afterwards, 1:1

acetonitrile-water (ACN/H2O) was added to the crude venom to prepare the acetonitrile

boiled D. quadriceps crude venom (AbDq). This solution was centrifuged at 5,000 g for

10 min. The supernatant was boiled for 10 min and centrifuged. The final supernatant

was dissolved in saline solution at different concentrations (2, 4 and 6 mg/mL).

Subjects

Male and female Swiss mice (3-5 months, 30-50 g) were used. Groups of 5-6 animals

were kept in plastic cages (20 x 30 x 13 cm) under conditions of acoustic isolation and

controlled temperature (25 ± 1 ºC), with a 12:12 h photoperiod (lights on 6:30 a.m.).

Food and water were available ad libitum. Mice were handled in accordance with the

guidelines of the Brazilian law for the use of animals in research (Law Number 11.794),

and all procedures were approved by the local ethics committee. All efforts were made

to minimize animal potential pain, suffering or discomfort.

76

Surgery

Mice were anesthetized with ketamine (100 mg/kg i.p.) plus xylazine (10 mg/kg i.p.)

and fixed in the stereotaxic frame (Insight, Brazil). A stainless steel guide cannula (25

gauge, 8 mm length) was implanted in the lateral ventricle. The stereotaxic coordinates

for the guide cannula placement were 0.6 mm posterior from bregma, - 1.1 mm lateral

from midline and 1.0 mm ventral from surface of the brain according to the mice atlas

(Paxinos & Franklin 2008). Before surgical incision, 0.1 mL of 2% lidocaine was

injected percutaneously. The guide cannula was anchored to the skull with screws and

dental acrylic. At the end of the surgery, the cannula was temporarily sealed with a

stainless-steel wire to avoid obstruction. The animals were given 5-7 days of post-

operative recovery prior to the start of the experimental proceedings.

Behavioral analysis

Primary screening with crude venom. The animals received an intraventricular injection

of saline (CRT) or the crude venom at a rate of 0.5 µL/min to a final volume of 1 L via

a microsyringe pump (Insight, Brazil) with a 10 µL syringe (Hamilton Co., USA)

connected to an injection needle. The injection needle was left in the guide cannula for

additional 60 s following infusion to allow drug to diffuse from the needle tip.

Immediately after the administration, animals were placed in a circular arena (30 cm

diameter and 60 cm high) located in an experimental room illuminated by a 40 W

fluorescent lamp (at the arena floor level) for 20 min. The behavioral session was

77

recorded by a digital camera placed above the apparatus and the behavioral parameters

were registered.

The animals were randomly assigned to control (CRT, n = 9) or one of three

experimental groups that received 5 (n = 9), 50 (n = 10), or 500 mg/mL (n = 10) of the

DqTx. We quantified the time spent in the following behavioral clusters: exploration

(behaviors such as exploratory sniffing, walking, scanning, and erect posture);

grooming (grooming of head, snout, claws, and back); immobility (time of complete

absence of movements, except breathing); procursive seizures (myoclonic jerks of paws,

running and gyrating) and tonic-clonic seizures (behaviors such as jumping, rearing and

atonic falling).

Anticonvulsant assay. The concentration of the chemical convulsant GABAA receptor

antagonist bicuculline, 10 mg/mL (Sigma, USA), was standardized in order to provoke

tonic-clonic seizures in 100% of injected animals in less than 30 min (BIC group).

Animals were randomly divided into the following groups: CRT (n= 8), animals that

were injected with saline solution 20 min prior to bicuculline administration, animals

that were injected with AbDq2 (n=6), AbDq4 (n = 6) or AbDq6 (n = 6), and animals

that were injected with AbDq 2 (n = 8), AbDq 4 (n = 9), or AbDq 6 mg/mL (n = 8) 20

min prior bicuculline administration in the left lateral ventricle. Drugs and venom

concentrations were injected as explained in the previous section (see Surgery session).

Immediately after the administration of bicuculline, animals were placed in the open

field and behavior was registered for 30 min.

The severity of seizures was evaluated using an adapted Racine’s scale (Racine

1972), as following: 1 – myoclonic jerks of contralateral paw; 2 – mild paw clonus

78

lasting at least 5 s; 3 – severe paw clonus lasting at least 15 s; 4 – rearing in addition to

severe paw clonus; 5- rearing and falling in addition to severe paw clonus. Moreover,

percentage of tonic-clonic seizures and deaths was evaluated.

Verification of the injection site. Upon completion of the behavioral procedures, mice

were euthanized with and intraperitoneal injection of sodium thiopental (50 mg/kg) and

injected i.c.v. with 1 µL of methylene blue stain to mark the correct site on injection.

Brains were removed and manually cut to check the position of the cannula. Only

animals with correct injection sites were included in the analysis. The same procedure

was held if the death occurred before the end of the experiments.

Statistical analysis

Data normality and the homogeneity of variances were respectively tested by the

Shapiro-Wilk and Levene’s tests. Comparisons among different concentrations of the

ant venom in relation to behavioral clusters were analyzed using one-way analysis of

variance (ANOVA) followed by Tukey’s post hoc test. The number of protected

animals in the anticonvulsant assays was analyzed using χ2 test followed by residual

analysis and the difference between means of score in these assays were analyzed using

Mann-Whitney test with Bonferroni correction. We considered p < 0.05 as significant

values. All statistical analyses were conducted with PASW Statistics 18 software (IBM,

USA).

79

Results

Primary screening with crude venom

One-way ANOVA revealed a significant effect of treatment for the exploratory activity

(F3, 37 = 17.43, p < 0.001). The groups that received different concentrations of ant

venom (DqTx) showed reduction of the time spent in exploratory activities, with post

hoc analysis detecting a significant difference at the highest concentration (DqTx 500

mg/mL) (p < 0.001) (Fig 1A).

Significant effects of treatment were also observed for the grooming cluster (F3,

37 = 11.74, p < 0.001). There was a reduction of these behaviors in groups that received

the DqTx, reaching significant values at the highest concentration according to the

Tukey’s post hoc test (p = 0.006) (Fig 1B).

One way ANOVA also revealed a treatment effect for the immobility (F3, 37 =

9.54, p < 0.001). The group that received saline barely displayed immobility, whereas

the animals treated with DqTx 50 and 500 mg/mL showed increased immobility

duration. Tukey’s post hoc test showed a tendency for the DqTx 50 (p = 0.07) and a

significant difference for the DqTx 500 (p < 0.001) (Fig 1C).

The analysis of percentage of time spent in procursive seizures revealed

significant effects for treatment (F3, 37 = 4.32, p < 0.05), but this behavioral cluster only

appeared at 50 and 500 mg/mL, and the Tukey´s post hoc test showed a significant

difference between control and DqTx 500 (p < 0.05) (Fig 1D). Moreover, the highest

concentration of the venom (DqTx 500) promoted tonic-clonic seizures (6.23 ± 3.9%, p

< 0.05) (Fig 1E).

Procursive seizures appeared in three mice after administration of the DqTx 50

whereas the DqTx 500 provoked these seizures in eight animals (Table 1). In addition,

the intermediate concentration (DqTx 50) promoted the death of two animals, while

80

four animals died as a consequence of the administration of DqTx 500 mg/mL (Table

1).

Anticonvulsant assay

All animals that received saline solution prior to bicuculline administration presented

tonic-clonic seizures (level 5) followed by death. Conversely, animals that received

different concentrations of AbDq did not present seizures or other seizure-related

behaviors (Table 2).

The administration of 2 mg/mL AbDq prior to bicuculline significantly reduced

the median score of the seizures from 5 to 3 (U = 12, p < 0.008) (Table 2) and also

protected 66.7% of the animals against tonic-clonic seizures (χ23 = 10.943, p < 0.05,

residual p < 0.05) (Fig 2A) as well as 100% of the animals against death (χ23 = 16.985, p

= 0.001, residual p < 0.05] (Fig 2B). The other concentrations of the AbDq did not

change the median score, but these treatments decreased the number of animals showing

higher scores seizures (Table 2). Furthermore, 4 and 6 mg/mL AbDq protected 12.5 and

25.0% of the animals against tonic-clonic seizures, respectively. Moreover, 4 and 6

mg/mL AbDq protected 50% of the animals against death (Fig 2B).

Discussion

We demonstrated that the crude venom of the D. quadriceps promotes behavioral

alterations in mice, such as reduction of exploration and grooming behaviors and

increasing of immobility. Furthermore, at the highest concentrations (50 and 500

mg/mL), the venom induced procursive and tonic-clonic seizures. However, the

81

previous administration of the denatured venom protected the animals against tonic-

clonic seizures and death induced by administration of the convulsant bicuculline.

In general, the chemoconvulsants cause seizures through two basic mechanisms,

i.e. the facilitation of excitatory neurotransmission or the inhibition of the inhibitory

neurotransmission. The convulsants pentylenetetrazol (PTZ), bicuculline and picrotoxin

block the inhibitory neurotransmission mediated by GABA, acting on the same site of

the neurotransmitter (bicuculline and PTZ) or through action on a modulatory biding

site at the receptor (picrotoxin) (Ramanjaneyulu & Ticku 1984). Conversely, the kainic

acid and NMDA agonists increase the excitatory neurotransmission by acting on their

specific receptors, leading to massive neuronal depolarization, and increase in inward

ion currents, such as Na+ and Ca2+.

Studies have showed that venoms from invertebrates can elicit epileptic-like

seizures in rodents. As mentioned, the BmK I and TsTX-I neurotoxins from scorpions

induce seizures in rats, both acting in sodium channels. In this context, it is possible that

the crude venom of D. quadriceps ant contains neurotoxins that can act in some of these

mechanisms.

The crude venom of the spider S. raptoria caused changes in animal behavior

characterized by a initial period of freezing followed by an explosion of a behavior

similar to wild running (similar the procursive seizures), which can precede tonic-clonic

seizures, when administered in the lateral ventricle of rats (Ribeiro et al 2000).

However, the denatured venom from the same spider was capable of protecting animals

from seizures induced by PTZ and bicuculline when injected in the lateral ventricle of

rats (Cairrão et al 2002). Similar results were observed for the venom of the spider P.

bistriata, which induced limbic seizures when injected in the lateral ventricle of rats

(Rodrigues et al 2001), but protected animals against seizures induced by PTZ,

82

picrotoxin and bicuculline when denatured (Cairrão et al 2002). Similar to the pattern

observed with the venoms of S. raptoria and P. bistriatra, we showed that the venom of

D. quadriceps also promoted an increased period of immobility, followed by procursive

and/or tonic-clonic seizures. Thus, we can propose that convulsive activity induced by

D. quadriceps venom is due to high weight molecules, such as protein or enzymes,

which probably lose their three-dimensional conformation and, consequently, their

activity when the venom is denatured.

From another standpoint, the classic antiepileptic drugs act through three basic

mechanisms: modulation of voltage-dependent ion channels, decreasing the excitatory

transmission, or increasing the GABA-mediated inhibitory neurotransmission (Kwan et

al 2001, Porter et al 2012).

Voltage-dependent ion channels (Na+, Ca2+ or K+) are important in the

neurotransmission by participating of neuronal action potential, thus modulating the

opening of these channels may reduce the excess of excitatory transmission. The

sodium channels are responsible for the upstroke of neuronal action potential, thus

blocking these channels may reduce the excess of excitatory transmission in epileptic

patients. The calcium channels are involved in the neurotransmitter release, and their

blocking would reduce the neurotransmission. Lastly, the potassium channels are

responsible for repolarization of the membrane and its direct activation hyperpolarizes

the neuronal membrane, consequently limiting the firing of action potentials (Kwan et

al 2001, Duncan et al 2006, Porter et al 2012). Some venom toxins with anticonvulsant

potential act on these voltage-dependent ion channels. The alpha-type neurotoxins BmK

IT2 and BmK AS are sodium channels modulators (Zhao et al 2008, Zhao et al 2011)

and the -conotoxins GVIA and MVIIA are N-type calcium channels antagonists

(Gasior et al 2007).

83

The majority of the excitatory synapses in human brain are mediated by l-

glutamate; thereby, it is a rational target for anticonvulsant drug design. However, none

of the commonly used antiepileptic drugs exerts their effects solely by action on the

glutamate system given the wide range of side effects. Notwithstanding, this mechanism

is believed to be involved in the activity of some antiepileptic drugs (Macdonald &

Kelly 1995, Kwan et al 2001, Mortari et al 2007). An example of venom toxin with

anticonvulsant potential whose mechanism of action includes modulation of

glutamatergic transmission is the Parawixin 10, which increases glutamate uptake

(Fachim et al 2011).

There are several antiepileptic drugs that act on the GABAA receptor, enhancing

the action of GABA or acting as agonists. Moreover, other drugs inhibit the uptake of

GABA, increasing its availability in the synaptic cleft (Dalby 2000, Duncan et al 2006,

Rogawski 2006). This last mechanism is the case of the aforementioned toxins SrTx1

and FrPbA2, isolated from the venom of the spiders S. raptoria and P. bistritata,

respectively (Cairrão et al 2002).

Several other studies on invertebrate venoms have shown the anticonvulsant and

neuroprotective activities of compounds in several animal models of seizures induction

and neuronal damage (see Rajendra et al 2004, Mortari et al 2007). This inhibitory

activity might be attributed to the selective antagonism of glutamatergic receptors

(conantoxin-L; Jimenez et al 2002), blockage of Na+ channels (BmK AS toxin; Zhao et

al 2011), blockage of Ca2+ channels (omega-conotoxin MVIIC and omega-agatoxin

IVA; Jackson and Scheideler 1996), inhibition of glutamate release (PnTx3-6 toxin;

Vieira et al 2003), enhancement of glutamate transporters (Fontana et al 2003, Fontana

et al 2007) or the inhibition of GABA and glycine transporters (Parawixin1; Beleboni et

al 2006).

84

Given the pro-convulsant action of bicuculline is due to GABAA receptor

antagonism, one possible explanation to our data could be the increased availability of

GABA due to an effective blockage of neuronal or glial GABA uptake. Despite the

large relative amount of proteins usually present in invertebrate venoms, the majority of

neurobiological effects are related to small peptides and low molecular weight

compounds, such as acylpolyamines (Beleboni et al 2004, Mortari et al 2007). These

compounds are not removed in the denaturation process; thereby they may be

responsible for the neuroprotective effects showed by the venom of D. quadriceps.

Despite the evidence of possible mode of mechanisms of the molecules in this venom,

isolation and purification of fractions and activity testing at the cellular level are still

required to obtain conclusive data on their mode of action.

Recently, Lopes et al (2013) reported the neuroprotective effect of the venom of

D. quadriceps through i.p. injection in the model of seizures induced by PTZ and an

opposite effect when the venom was injected e.v. The differences observed could be

related to differences in the experimental procedures when compared with our data.

They used i.p. and e.v. as administration routes, while we opted to investigate the

effects of the venom injected directly into the brain. Moreover, we tested both the crude

and the denatured forms of the venom, and added an overall analysis of behavioral

changes caused by the venom administration in mice. Finally, we used a different

pharmacological model to induce the seizures. In this respect, that study showed that the

venom was effective in reducing seizures induced by PTZ (a GABAA receptor

antagonist), but not by pilocarpine and strychnine. These findings corroborate our

results, strengthening the idea that the mode of action of the venom involves the

GABAergic transmission.

85

In summary, this work is one of the few in vivo studies on the effects of ant

venoms on the neurological system of vertebrates. Although a GABA-related

mechanism is suggested, further research is needed to understand the basis of the

anticonvulsant effect of the D. quadriceps venom. Thus, further work will deal with the

isolation and determination of the structure of the active components from this ant

venom as well as clarify their mode of action.

Acknowledgments This research was supported by fellowships from Conselho

Nacional de Desenvolvimento Científico e Tecnológico (CNPQ); Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Pró-reitoria de Pesquisa da

Universidade Federal do Rio Grande do Norte (PROPESQ/UFRN) and Fundação de

Apoio à Pesquisa do Estado do Rio Grande do Norte (FAPERN).

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Figure captions

Fig 1 Effects of intracerebral injection of the venom of Dinoponera quadriceps (DqTx)

in mice. (A) Percentage of time spent in exploratory activities; (B) Grooming; (C)

Immobility; (D) Procursive seizures; and (E) Tonic-clonic seizures. Data expressed as

the mean ± SEM. *p < 0.05 and #p = 0.07 compared to control (One way ANOVA

followed by Tukey’s post hoc test).

Fig 2 (A) Percentage of protection against tonic-clonic seizures in mice microinjected

with different concentrations of AbDq prior to the administration of bicuculline; (B)

Percentage of protection against death. *p < 0.05 (χ2 test followed by residual analysis

compared to CRT group).

91

Table 1 Effects of i.c.v. injection of Dinoponera quadriceps crude venom in mice.

Treatment Saline 5 mg/mL 50 mg/mL 500 mg/mL

Number of Animals 9 9 10 10

Animals with procursive seizures 0 % 0 % 30 % 80 %

Time in procursive seizures 0 % 0 % 2.07 ± 1.9 % 13.31 ± 5.91

Animal with tonic-clonic seizures 0 % 0 % 0 % 50 %

Time in tonic-clonic seizures 0 % 0 % 0 % 49.24 ± 10.17 %

Death 0 % 0 % 20 % 40 %

92

Table 2 Effects of i.c.v. injection of Dinoponera quadriceps denatured venom on BIC seizure model in

mice.

Treatment

Saline

×

BIC

AbDq2 AbDq4 AbDq6

AbDq2

×

BIC

AbDq4

×

BIC

AbDq6

×

BIC

Number of animals 8 8 6 6 9 8 8

Mortality 8/8 0/8 0/6 0/6 0/9 4/8 4/8

Median of scores 5 0 0 0 3 5 5

Incidence of seizures 8/8 0/8 0/6 0/6 3/9 7/8 6/8

Stage 1 2/8 0/8 0/6 0/6 9/9 6/8 6/8

Stage 2 4/8 0/8 0/6 0/6 6/9 2/8 1/8

Stage 3 2/8 0/8 0/6 0/6 2/9 3/8 2/8

Stage 4 3/8 0/8 0/6 0/6 3/9 3/8 5/8

Stage 5 8/8 0/8 0/6 0/6 3/9 7/8 6/8

93

0

10

20

30

40

50

CRT 5 mg/ml 50 mg/ml 500 mg/ml

Gro

om

ing

(%

)

DqTx

0

20

40

60

80

100


Exp

lora

tio

n (

%)

DqTx

0

20

40

60

80

100


Imm

ob

ilit

y (%

)

DqTx

0

5

10

15

20

25

30


Pro

cu

rsiv

e S

eiz

ure

s (

%)

DqTx

0

5

10

15

20

25

30


To

nic

-clo

nic

seiz

ure

s (

%)

DqTx

A B

C D

E

*

*

* *

*

#

94

0

20

40

60

80

100

2 mg/ml 4 mg/ml 6 mg/ml

Pro

tec

tio

n a

ga

ins

t s

eiz

ure

s (

%)

AbDq

0

20

40

60

80

100

2 mg/ml 4 mg/ml 6 mg/ml

Pro

tec

tio

n a

ga

ins

t d

ea

th (%

)

AbDq

A B*

*

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