Desenvolvimento de uma nanoemulsão bioinseticida preparada
com extrato de Manilkara subsericea
CAIO PINHO FERNANDES
Tese apresentada ao Programa de Pós-
graduação em Biotecnologia Vegetal da
Universidade Federal do Rio de Janeiro,
como requisito parcial para obtenção do
título de doutor em Biotecnologia.
Orientadores:
Prof. Dr. Leandro Machado Rocha
Profª Drª Deborah Quintanilha Falcão
Rio de Janeiro – RJ
2014
Caio Pinho Fernandes
Desenvolvimento de uma nanoemulsão bioinseticida preparada com extrato de Manilkara
subsericea
_________________________________________
Prof. Dr. Leandro Machado Rocha (UFF)
____________________________________________
Profª. Drª. Deborah Quintanilha Falcão (UFF)
____________________________________________
Profª. Drª. Adriana Passos Oliveira (UFRJ)
____________________________________________
Profª. Drª. Alice Sato (UNIRIO)
____________________________________________
Profª. Drª. Carla Holandino Quaresma (UFRJ)
____________________________________________
Prof. Dr. José Carlos Tavares Carvalho (UNIFAP)
____________________________________________
Prof. Dr. Marcelo Guerra Santos (UERJ)
____________________________________________
Profª. phD Fernanda Reinert Thomé Macrae (UFRJ)
Aprovada em 18 de fevereiro de 2014, Rio de Janeiro
Dedico este trabalho aos meus
pais, minha irmã e minha esposa,
que são meu alicerce.
Não há vegetal algum que não
mereça ocupar a atenção de um
verdadeiro sábio; nenhum há, por
mais desprezível que pareça, de
que não se possa esperar alguma
utilidade.
(Frei Veloso)
AGRADECIMENTOS
À minha esposa Fernanda, que me acompanha em todos os momentos com seu sorriso,
apoio, amizade, paciência, tornando minha vida mais feliz.
Aos meus pais Luiz Carlos Fernandes e Luizaura Pinho Fernandes, pelo apoio irrestrito
e amor incondicional.
À minha irmã Laís Pinho Fernandes, pelo caráter, amizade e exemplo que sempre
demonstrou.
Às minhas vós Aurora e Lacy, minha tia Laurinha e demais familiares, por demonstrar o
significado mais puro de uma família unida, tão presente em minha vida.
Aos meus orientadores Leandro Machado Rocha e Deborah Quintanilha Falcão, pelo
incentivo e ensinamentos que consolidaram a vontade de seguir a carreira acadêmica, além de
serem verdadeiramente amigos.
Aos amigos do Laboratório de Tecnologia de Produtos Naturais (LTPN-UFF), Jonathas,
Rodrigo, Ricardo (Paulo Betti), Luis Armando (Gaúcho), Rafael, Hildegardo, Iraí, Arthur,
Bruno, Henrique (Iow), Francisco (Kiko), Diogo (Che Guevara), Adriana, Raquel, Bárbara,
Jeane, Amanda, Gisele, pelo apoio, amizade, cafés, lanches, músicas, longos dias/noite de
risadas, além de contribuírem firmemente no meu caminho de aprendizado sobre as plantas.
Ao Prof Marcelo Guerra, pela amizade e grande ajuda durante as coletas na restinga.
Ao Jorge (Mateiro), por nos receber na sua casa (Restinga de Jurubatiba) e
ensinamentos valiosos.
À Professora Denise Feder e demais professores e alunos do Laboratório de Biologia de
Insetos do GBG/UFF, pelo grande auxílio e ensinamentos.
Aos Professores e funcionários da Faculdade de Farmácia – UFF.
Aos Professores e funcionários do Curso de Farmácia – UNIFAP, em especial ao Prof.
José Carlos Tavares Carvalho, por terem proporcionado um ambiente tão acolhedor.
Aos membros da banca, por aceitarem avaliar esse trabalho e contribuir com seus
conhecimentos e experiência.
Ao Programa de Pós-Graduação em Biotecnologia Vegetal, em especial a Prof.
Fernanda Reinert, por ter depositado tanta confiança em mim e pelos bate-papos sempre
produtivos, e a Regina, pela paciência e carinho com que sempre me ajudou
Ao CNPQ pelo suporte financeiro.
Muito Obrigado
RESUMO
FERNANDES, Caio Pinho. Desenvolvimento de uma nanoemulsão bioinseticida preparada
com extrato de Manilkara subsericea Rio de Janeiro, 2014. Tese (Doutorado em
Biotecnologia) – Programa de Pós-graduação em Biotecnologia Vegetal - Centro de Ciências
da Saúde - Universidade Federal do Rio de Janeiro.
Manilkara subsericea (Mart.) Dubard (Sapotaceae) é uma espécie brasileira endêmica,
amplamente distribuída no Parque Nacional da Restinga de Jurubatiba e popularmente
conhecida nessa localidade como “guracica”. Estudos fitoquímicos com essa espécie
indicaram a predominância de triterpenos pentacíclicos. Além disso, foi verificada a atividade
inseticida modulada por seus extratos, principalmente a fração apolar de frutos. O presente
trabalho descreve o desenvolvimento de uma nanoemulsão inseticida contendo uma fração
solúvel em hexano obtida de frutos de M. subsericea. Também foi realizada a identificação e
elucidação estrutural de metabólitos secundários desta espécie, incluindo a descrição pela
primeira vez dos caproatos de alfa- e beta-amirina, caprilatos de alfa- e beta- amirina, ácido
pomólico, miricetina, quercetina, kaempferol, miricitrina, quercitrina, óxido de linalol, linalol,
terpineol, safranal, beta-ciclocitral, geraniol, entre outros. A nanoemulsão contendo a fração
apolar de frutos de M. subsericea apresentou baixo tamanho médio de partícula (155,2 ± 3,8
nm) e reflexo azulado característico. A nanoemulsão obtida foi capaz de induzir mortalidade
sobre Dysdercus peruvianus, espécie que causa sérios danos no cultivo do algodão. Além
disso, essa formulação apresentou atividade inibitória sobre a acetilcolinesterase de peixe
elétrico e não provocou efeitos letais em camundongos (Mus musculus). Este trabalho
permitiu a obtenção de uma nanoemulsão inseticida, indicando seu potencial como pesticida
biodegradável e sem efeitos indesejáveis sobre outros organismos.
Palavras-chave: Dysdercus peruvianus, insecticida, óleo essencial, flavovóides, Manilkara
subsericea, nanoemulsão, triterpenos.
ABSTRACT
FERNANDES, Caio Pinho. Development of a bioinsecticide nanoemulsion prepared with
Manilkara subsericea extract. Rio de Janeiro, 2014. Tese (Doutorado em Biotecnologia) –
Programa de Pós-graduação em Biotecnologia Vegetal - Centro de Ciências da Saúde -
Universidade Federal do Rio de Janeiro.
Manilkara subsericea (Mart.) Dubard (Sapotaceae) is a Brazilian endemic species widely
distributed at “Restinga de Jurubatiba National Park”, being popularly known in this locality
as “guracica”. Studies with this species indicated the predominance of pentacyclic triterpenes.
Moreover, it was observed an insecticidal activity modulated by its extracts, mainly apolar
fraction from fruits. The present study describes development of an insecticide nanoemulsion
containing hexane-soluble fraction obtained from fruits of M. subsericea. Identification and
structural elucidation of secondary metabolites from this species was also performed,
includind first reports of alpha- and beta-amyrin caproates, alpha- and beta-amyrin caprylates,
pomolic acid, myricetin, quercetin, kaempferol, myricitrin, quercitrin, linalool oxide, linalool,
terpineol, safranal, beta-cyclocitra, geraniol, among others. Nanoemulsion containing apolar
fraction from M. subsericea fruits presented small mean droplet size (155.2 ± 3.8 nm) and
characteristic bluish reflect. It was observed that this nanoemulsion was able to induce
mortality of Dysdercus peruvianus, a species that causes damages in cotton crops. Moreover,
this formulation did not interfere with acetylcholinesterase from electric eel and did not cause
any lethal effects in mice (Mus musculus). The present study allowed achievement of a
insecticide nanoemulsion from Manilkara subsericea, indicating its potential as biodegradable
pesticide, without harmful effects over non-target species.
Keywords: Dysdercus peruvianus, insecticide, essential oil, flavonoids, Manilkara
subsericea, nanoemulsion, triterpenes.
LISTA DE FIGURAS
Capítulo 1
Figura 1. Estruturas químicas do acetato de beta-amirina (esquerda)
e acetato de alfa-amirina (direita). 24
Figura 2. Espécime em frutificação de Manilkara subserica (Mart.) Dubard encontrado
no Parque Nacional da Restinga de Jurubatiba (RJ). 25
Figura 3. Ilustração botânica de Manilkara subsericea (Mart.) Dubard. publicada
na Flora Brasiliensis 26
Figura 4. Macroemulsão (esquerda) e nanoemulsão (direita). 30
Capítulo 2
Figura 1. Esquema ilustrativo do isolamento de miricetina, quercetina,
kaempferol, miricetrina, quercitrina e ácido pomólico da fração solúvel
em acetato de etila, proveniente do extrato etanólico de folhas de
Manilkara subsericea. 42
Figura 2. Representação das reações de hidrólise do substrato enzimático
(acetiltiocolina) pela enzima acetilcolinesterase. 49
Capítulo 3
Figure 1. Manilkara subsericea (Mart.) Dubard, Sapotaceae,
at Restinga de Jurubatiba National Park (Rio de Janeiro, Brazil). 58
Figure 2. Fragmentation pattern for Δ12-oleane/Δ12-ursane series.
A: beta-amyrin acetate. B: alpha-amyrin caproate. C: alpha-amyrin caprylate. 63
Figure 3. Chemical structures of the amyrin esters: beta-amyrin acetate (E),
alpha-amyrin acetate (F), beta-amyrin caproate (G), alpha-amyrin caproate (H),
beta-amyrin caprylate (I) and alpha-amyrin caprylate (J ) from the hexanic extract
from fruits of Manilkara subsericea. 66
Figure 4. GC-FID chromatogram of the hexanic extract from fruits of
Manilkara subsericea, Sapotaceae. 67
Figure 5. Vero cell viability in the presence of extracts for 24 h measured
by LDH assay 70
Capítulo 4
Figure 1. Structures of flavonoids and triterpene from leaves
of Manilkara subsericea 84
Figure 2. ESI(-)-FT-ICR mass spectra for (a) FL4 and (b) FL5 samples
and CID experiments for ion of m/z 463 and 447 corresponding to (c) myricitrin
and (d) quercitrin. 88
Figure 3. ESI(-)-FT-ICR mass spectrum of triterpene acids present in the
leaves of M. subsericea 90
Capítulo 5
Figura 1. Pseudo-ternary phase diagram constructed with water, MOD
and surfactants (sorbitan monoleate/polysorbate 80, HLB =10.75) at different
compositions. Nanoemulsion region is delimited in blue. 110
Figure 2. Nanoemulsions obtained by low energy method. HFNE shown in left side and blank
nanoemulsio shown in right side of the picture. 113
Figure 3. Particle size distribution of (a) negative control (57.0 ± 0.3 nm)
and (b) nanoemulsion with hexane-soluble fraction from fruits of M. subsericea
(155.2 ± 3.8 nm). Polidispersity was 0.270 ± 0.006 for blank nanoemulsion and
0.150 ± 0.050 for nanoemulsion with hexane-soluble fraction from fruits
of M. subsericea. 113
Figure 4. Analysis of mortality after topical treatment of Dysdercus peruvianus
with nanoemulsion containing hexane-soluble fraction from fruits of
Manilkara subsericea (HFNE) (filled column). Negative control group was
topically applied with blank nanoemulsion (crosshatched columns). Untreated group
is represented by open columns. Each group represents mean
of three experiments. 115
Figure 5. Linear regression between AchE activity (mU) x natural logarithm of
(a) effective concentration of eserine (p<0.05) and (b) effective concentration
of hexane-soluble fraction from fruits of Manilkara subsericea (p>0.05). 117
LISTA DE TABELAS
Capítulo 1
Capítulo 2
Capítulo 3
Table 1. Means of the inhibition halos (mm + SD) for Staphylococcus aureus
and Escherichia coli tested with extracts (100 mg/mL) from Manilkara subsericea. 68
Capítulo 4
Table 1. Relative abundance of essential oil constituents from leaves
of Manilkara subsericea. 92
Capítulo 5
Table 1. Composition, mean droplet size and polydispersity of each formulation
prepared during construction of pseudo-ternary phase diagram for
delimitation of nanoemulsion region. 108
Table 2. Weight variation in adult female and male Swiss albino mice (Mus musculus)
treated with HFNE (5% of MOD, 5 % of surfactants (HLB of 10.75), 5% of
hexane-soluble fraction from fruits of M. subsericea and 85% of water)
by oral route, corresponding to 3g/kg of extract. Control groups received same
volume of MNE (5% of MOD, 5% of surfactants and 90 % of water) 118
SUMÁRIO
INTRODUÇÃO 19
OBJETIVOS 21
CAPÍTULO 1. Revisão da Literatura 23
Inseticidas de origem natural 23
Manilkara subsericea (Mart.) Dubard 24
Nanoemulsões 28
Referências 32
CAPÍTULO 2. Materiais e Métodos 39
Referências 51
CAPÍTULO 3. Atividades biológicas e ésteres triterpênicos provenientes de
frutos comestíveis de Manilkara subsericea (Mart.) Dubard (Sapotaceae) 53
Introdução. 53
Referências 54
Artigo 1. Triterpene esters and biological activities from edible fruits of
Manilkara subsericea (Mart.) Dubard, Sapotaceae 55
CAPÍTULO 4. Metabólitos especiais de folhas de Manilkara subsericea
(Mart.) Dubard 76
Introdução 76
Referências 77
Artigo 2. Secondary metabolites from leaves of Manilkara subsericea (Mart.) 78
Dubard
CAPÍTULO 5. Desenvolvimento de uma nanoemulsão inseticida com extrato
de Manilkara subsericea (Sapotaceae) 100
Introdução 100
Referências 101
Artigo 3. Development of an insecticidal nanoemulsion with Manilkara subsericea
(Sapotaceae) extract 102
CONCLUSÕES 132
PERSPECTIVAS FUTURAS 134
- 19 -
INTRODUÇÃO
Plantas são utilizadas no controle de pragas desde tempos remotos e elas foram a
principal fonte de agentes inseticidas até o desenvolvimento de substâncias sintéticas para
essa finalidade. Os efeitos tóxicos dessas substâncias para o meio ambiente e outros
organismos, além da seleção de insetos mais resistentes frente a inseticidas usuais, fez com
que as plantas ocupassem novamente um papel de destaque na busca por novos agentes
pesticidas. Dentre as diversas espécies investigadas, Manilkara subserica apresentou grande
potencial para o controle de Dysdercus peruvianus, praga do cultivo de algodão. Entretanto,
as frações mais ativas e substâncias avaliadas possuem limitada solubilidade em água, sendo
solúveis apenas em solvente orgânicos tóxicos, como o diclorometano e clorofórmio. A
utilização da nanotecnologia apresenta-se como uma alternativa para o desenvolvimento de
um produto viável, eficaz, seguro e capaz de ser disperso em meio aquoso.
Neste contexto, o presente trabalho teve o intuito de apresentar uma nanoemulsão
inseticida para ser utilizada frente a D. peruvianus. Foi utilizada como matéria prima ativa a
fração apolar oriunda de frutos de Manilkara subsericea.. Adicionalmente, foram realizados
estudos fitoquímicos para aumentar a base de informações relacionadas aos metabólitos
secundários produzidos por essa espécie, endêmica da flora brasileira e praticamente
inexplorada.
O presente trabalho será apresentado sob a forma de cinco capítulos, relacionados
respectivamente a:
- 20 -
Revisão da literatura, apresentando os principais pontos abordados nos presentes estudos;
Materiais e métodos empregados nos estudos;
Avaliação da atividade biológica e elucidação estrutural de triterpenos presentes em
frutos de Manilkara subsericea;
Identificação de metabólitos secundários de folhas de Manilkara subsericea, como
triterpenos, flavonóides e constituintes de óleo essencial;
Desenvolvimento de uma nanoemulsão bioinseticida contendo fração apolar proveniente
do extrato de frutos de Manilkara subsericea. Adicionalmente foram feitos testes de
toxicidade, verificando o impacto da formulação em outros organismos.
- 21 -
OBJETIVOS
Objetivo geral
O objetivo deste trabalho foi desenvolver uma nanoemulsão bioinseticida contendo
fração apolar proveniente do extrato de frutos de Manilkara subsericea, além de identificar
substâncias químicas produzidas por essa espécie vegetal.
Objetivos específicos
Elucidar a estrutura química de flavonóides presentes nas folhas de Manilkara
subsericea;
Elucidar a estrutura química de triterpenos presentes nos frutos e folhas de
Manilkara subsericea;
Elucidar a estrutura química de constituintes do óleo essencial extraído de folhas de
Manilkara subsericea;
Desenvolver nanoemulsão contendo fração apolar proveniente do extrato etanólico
de frutos de Manilkara subsericea;
Caracterizar fisicamente as nanoemulsões obtidas;
Avaliar a atividade inseticida da nanoemulsão contendo fração apolar proveniente
do extrato de frutos de Manilkara subsericea;
- 22 -
Verificar a influência da nanoemulsão bioinseticida sobre a acetilcolinesterase de
peixe elétrico;
Verificar a toxicidade da nanoemulsão bioinseticida frente a camundongos (Mus
musculus).
- 23 -
CAPÍTULO 1
Revisão da Literatura
Inseticidas de origem natural
Os insetos se tornam pragas quando eles conflitam com nosso bem-estar ou interferem
nos lucros agrícolas (Gullan & Cranston, 2008). Pesticidas de origem sintética são
comumente utilizados no controle de insetos, entretanto, eles são geralmente nocivos para o
meio ambiente. Neste contexto, há uma crescente preocupação mundial com o uso
indiscriminado dessas substâncias, que está associado à poluição do meio ambiente e
intoxicação de outros organismos (Rao et al., 2003; Varona et al., 2009). Os produtos de
origem natural já foram a principal arma contra pragas agrícolas até aproximadamente 1940,
quando os inseticidas sintéticos assumiram um papel de destaque (Isman et al., 2008).
Entretanto, problemas oriundos do uso indiscriminado desses pesticidas fizeram com que
programas integrados de controle, utilizando produtos de origem natural, voltassem a ser
debatidos (Tunaz, 2004).
Plantas são reconhecidamente capazes de produzir suas próprias substâncias
defensivas, incluindo inseticidas para sua proteção contra o ataque de pragas (Gobbo-Neto &
Lopes, 2007). Esses produtos de origem natural incluem flavonóides, esteróides, alcalóides,
substâncias fenólicas e terpenóides (Viegas Jr, 2003; Castillo-Sánchez et al., 2010). Eles
atuam por uma ampla variedade de mecanismos, incluindo a inibição da acetilcolinesterase
(López & Pascual-Villalobos, 2010) e surgem como fontes potenciais de novos bioinseticidas
- 24 -
biodegradáveis, sendo uma alternativa para o controle de insetos na agricultura (Rattan,
2010).
Dentre os diversos insetos que causam graves prejuízos econômicos, podemos citar o
percevejo Dysdercus peruvianus (Hemiptera), que causa graves danos no cultivo do algodão
(Milano et al., 1999; Stanisçuaski et al., 2005). Estudos prévios realizados com extratos
provenientes de Manilkara subsericea indicaram a capacidade de indução de efeitos severos
sobre essa espécie, incluindo mortalidade, atraso na muda e metamorphose, além de animais
deformados. Possivelmente essa atividade é modulada, ao menos parcialmente, por triterpenos
pentacíclicos, como os acetatos de beta- e alfa-amirina (Figura 1) (Fernandes et al., 2013a).
O
O
O
O
Figura 1. Estruturas químicas do acetato de beta-amirina (esquerda) e acetato de alfa-amirina
(direita).
Manilkara subsericea (Mart.) Dubard
A família Sapotaceae possui aproximadamente 58 gêneros e 1250 espécies (Bartish et
al., 2011). No Brasil, essa família é representada por 11 gêneros e 231 espécies, incluindo 2
gêneros e 104 espécies endêmicas (Carneiro et al. 2014).
O gênero Manilkara Adans é constituído por 30 espécies distribuídas nos Neotrópicos,
20 espécies encontradas na África e 12 espécies encontradas na Ásia e no Pacífico
- 25 -
(Pennington, 2006). Devido à circunscrição dos gêneros, algumas espécies anteriormente
descritas como pertencentes aos gêneros Achas L. e Mimusops L. foram incluídas no gênero
Manilkara (Almeida Jr., 2010). O Brasil possui um total de 18 espécies deste gênero, sendo
15 consideradas endêmicas (Almeida Jr. 2014).
Figura 2. Espécime em frutificação de Manilkara subserica (Mart.) Dubard encontrado no
Parque Nacional da Restinga de Jurubatiba (RJ). Fonte: Próprio autor (Caio Pinho Fernandes).
Manilkara subsericea (Mart.) Dubard (Figura 2) é uma espécie endêmica da Mata
Atlântica brasileira (Almeida Jr., 2014), comumente conhecida como “maçaranduba”,
- 26 -
“maçarandubinha” e “guracica” (Lorenzi, 2009; Santos et al., 2009a). Ela é amplamente
distribuída no Parque Nacional da Restinga de Jurubatiba , sendo utilizada nessa localidade
como material para fabricação de mourões e pelos frutos comestíveis (Santos et al., 2009b). A
prancha dessa espécie foi publicada em 15 de janeiro de 1863 no volume VII (Fascículo 32)
da obra Flora Brasiliensis, produzida por Carl Friedrich Philipp von Martius com a
participação de diversos colaboradores, e considerada uma das obras mais importantes
relacionadas à flora brasileira (Figura 3).
Figura 3. Ilustração botânica de Manilkara subsericea (Mart.) Dubard publicada na Flora
Brasiliensis. Disponível em http://florabrasiliensis.cria.org.br/search?taxon_id=5835, acesso
- 27 -
em janeiro de 2014). A nomenclatura utilizada na prancha segue a classificação da época,
sendo uma das sinonímias atualmente aceitas para a espécie (The Plant List, 2013).
Estudos relacionados a biologia reprodutiva (Gomes et al., 2008), biologia floral
(Gomes et al., 2010) e ecologia (Monteiro et al., 2007; Oliveira et al., 2012) de M. subsericea
têm sido observados, enquanto trabalhos relacionados à atividades biológica de extratos,
frações ou substâncias isoladas de M. subsericea são escassos e possivelmente restritos a
avaliação das atividades antimicrobiana (Fernandes, 2011), inseticida (Fernandes et al.,
2013a) e antiofídica (Oliveira et al., 2014).
Historicamente, a maior parte dos estudos fitoquímicos do gênero Manilkara estão
concentrados na espécie M. zapota (L.) P. Royen (Ma et al., 2003; Ahmed et al., 2001; Fayek
et al., 2013), popularmente conhecida como sapoti. Apesar de observarmos a existência de
poucos trabalhos relacionados a fitoquímica de Manilkara subsericea, é possível verificar a
predominância de triterpenos pentacíclicos, basicamente relacionadas aos esqueletos olean-
12-eno e urs-12-eno, incluindo os acetatos de alfa- e beta-amirina (Fernandes, 2011) (Figura
1). Essas substâncias são encontradas em abundância nos extratos dessa espécie e são
consideravelmente apolares. Devido a esse fato, o desenvolvimento de produtos com essa
matéria-prima torna-se um grande desafio, uma vez que são pouco solúveis em água e
solúveis em solventes orgânicos tóxicos, como clorofórmio e diclorometano. Neste contexto,
a nanotecnologia se configura como uma boa alternativa para resolver tal problema,
aumentando a sua disponibilização em meio aquoso e permitindo a obtenção de produtos
adequados para o uso comercial.
- 28 -
Nanoemulsões
Nanotecnologia pode ser definida como a ciência e engenharia envolvidos no design,
síntese, caracterização e aplicação de materiais e dispositivos cuja organização funcional se
encontra em escala nanométrica. (Silva, 2004). A origem conceitual dessa importante área
pode ser datada de 1959, quando o físico Richard Feynman ministrou uma célebre palestra em
uma reunião da Sociedade Americana de Física. Entretanto, Noro Taniguchi é reconhecido
como a primeira pessoa a utilizar o termo “nanotecnologia”, no ano de 1974 (Irache et al.,
2011). Desde então, avanços notáveis foram realizados com o auxílio dessa tecnologia,
incluindo o desenvolvimento de uma série de formulações com potencial aplicação na área de
alimentos, cosméticos, medicamentos e pesticidas (Assis et al., 2012; Patel & Velikov, 2011;
Duncan, 2011; Brumfiel, 2006; Irache et al., 2011; Wang et al., 2007). Dentre as diversas
apresentações para esses produtos, podemos citar as ciclodextrinas (Gomes et al., 2014;
Falcão et al., 2011), nanopartículas (Wissing & Muller, 2002; Falcão et al., 2011),
nanosuspensões (Pardeike et al., 2011; Wang et al., 2011), nanodispersões (Leong et al.,
2011; Cheong et al., 2008) e nanoemulsões (Donsi et al., 2012; Fernandes et al., 2013b), entre
outras.
Esses nanoprodutos apresentam uma série de vantagens, como maior estabilidade,
características organolépticas favoráveis, maior poder de penetração por membranas, maior
biodisponibilidade, incremento da solubilidade em água de substâncias pouco solúveis e até
mesmo liberação controlada de substâncias (Irache et al., 2011). Neste contexto, uma das
formulações que vem ganhando mais destaque são as nanoemulsões, um tipo de formulação
do grupo das emulsões, tendo em vista a facilidade de sua obtenção por diferentes métodos e
versatilidade.
- 29 -
Emulsões são basicamente dispersões constituídas por um líquido disperso sob a
forma de pequenas gotículas em outro líquido, sendo ambos imiscíveis (Brasil, 2010).
Usualmente esses dois líquidos são água e um óleo. Denomina-se o líquido dispersante como
fase externa ou contínua, enquanto o líquido disperso é frequentemente chamado de fase
interna ou descontínua (Aulton, 2005; Ansel et al., 2007). Outras substâncias podem ser
adicionadas, sendo frequentemente necessária a utilização de agentes tensoativos. Eles
permitem a redução da tensão superficial entre a fase oleosa e a fase aquosa, que é
fundamental para a manutenção de pequenos glóbulos, sendo especialmente importante na
estabilidade de nanoemulsões (Surassmo et al., 2010). Além disso, os tensoativos auxiliam na
formação de um filme ao redor da fase dispersa, reduzindo a chance de ocorrência de um
fenômeno de instabilidade chamado de coalescência, que é fusão entre duas gotículas gerando
uma gotícula maior (Ansel et al., 2007).
As emulsões são frequentemente sub-divididas em três grupos, que são as
macroemulsões, microemulsões e nanoemulsões. As macroemulsões são sistemas
termodinamicamente instáveis e contém gotículas da fase dispersa com diâmetro médio que
varia de 0,5 até 100 μm, sendo susceptíveis à força da gravidade. As microemulsões são
termodinamicamente estáveis, possuindo gotículas com diâmetro médio próximo a 10 nm.
Elas são espontaneamente formadas quando as fases aquosa e oleosa são colocadas em
contato com os tensoativos. Entretanto, esse tipo de formulação requer grandes quantidades
de tensoativos, sendo restritas para algumas aplicações. As nanoemulsões, também chamadas
de emulsões ultrafinas ou miniemulsões, possuem gotículas da fase dispersa com tamanho
diminuto (20-500 nm). Elas são transparentes ou translúcidas, frequentemente apresentando
um reflexo azulado (Figura 4) e possuem estabilidade cinética (Forgiarini et al., 2000).
- 30 -
Apesar das nanoemulsões possuírem tamanho de gotícula diminuto, quando
comparados a macroemulsões, elas frequentemente podem ser obtidas com quantidades
inferiores de tensoativos do que o requerido para as microemulsões. Isso se configura como
uma importante vantagem, visto que a menor concentração de tensoativos reduz custos e
possíveis restrições de uso. Elas são frequentemente caracterizadas como formulações do tipo
óleo em água (O/A), sendo a fase interna (dispersa) oleosa e a fase externa aquosa. Essa
abordagem permite o incremento na solubilidade em água de substâncias lipofílicas e a
diluição das nanoemulsões em água e/ou outras soluções miscíveis neste líquido. Por outro
lado, nanoemulsões do tipo A/O, em que a fase dispersa é aquosa e a fase externa é oleosa
têm sido historicamente menos frequentes (Solans et al., 2005). Essas formulações possuem
uma ampla variedade de aplicações industriais (Izquierdo et al., 2002; Tadros et al., 2004),
como adjuvantes em alimentos, medicamentos e produtos agrícolas, indicando o alto potencial
econômico das nanoemulsões.
Figura 4. Macroemulsão (esquerda) e nanoemulsão (direita). Fonte: Próprio autor (Caio Pinho
Fernandes).
- 31 -
Sistemas nanoemulsionados contendo substâncias naturais com atividade inseticida
têmchamado atenção nos últimos anos. Além de serem possivelmente biodegradáveis e menos
tóxicos para o meio ambiente (Varona et al., 2009), eles permitem uma maior molhabilidade,
espalhamento e penetração, que são vantagens adicionais para o uso de nanoemulsões como
pesticidas agrícolas (Wang et al., 2007). Tendo em vista que muitos pesticidas de origem
natural são pouco solúveis em água, o desenvolvimento de nanoemulsões inseticidas é uma
alternativa viável para o lançamento de novos produtos. Além disso, a mudança no perfil dos
consumidores tem levado a uma busca por “produtos verdes”, indicando o potencial das
nanoemulsões contendo produtos naturais para agroindústria (Lim et al., 2012).
- 32 -
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CAPÍTULO 2
Materiais e Métodos
Material Vegetal
Frutos e folhas de Manilkara subsericea foram coletados respectivamente em 2009 e
2010 no Parque Nacional da Restinga de Jurubatiba. Amostras do material vegetal foram
herborizadas e depositadas no Herbário da Faculdade de Formação de Professores da
Universidade do Estado do Rio de Janeiro (RFFP 13.416 e 15.316).
Solventes
Os solventes utilizados na preparação de reagentes, soluções, extração, partições
apresentaram grau de análise (P.A.) enquanto aqueles utilizados nos métodos cromatográficos
apresentaram grau espectroscópico, sendo obtidos do fabricante VETEC® (RJ, Brasil). Foi
utilizada água destilada para os procedimentos efetuados nestes estudos.
Extratos e partições
Os frutos frescos (1.1 kg) foram triturados em turbilhonador e extraídos pela técnica
de maceração em etanol 96 % (v/v) até completo esgotamento. Após este período o extrato
obtido foi filtrado e concentrado em evaporador rotatório (Fisatom), obtendo-se 170 g de
extrato. Ele foi ressuspendido em 500 mL de uma solução hidroacoólica 90% (v/v) e
particionado com hexano (2 x 600 mL). A fração orgânica foi evaporada até a secura para
obtenção de uma fração em hexano de coloração amarela (FH 14 g) (Fernandes, 2011).
- 40 -
Folhas (0,8 kg) foram submetidas a processo de secagem em estufa com ventilação
forçada (QUIMIS), com temperatura de aproximadamente 40°C pelo período de 2 dias,
moídas em moinho de facas e em seguida submetidos à extração por maceração em etanol 96
% (v/v) até completo esgotamento. Após este período, o extrato obtido foi filtrado e
concentrado em evaporador rotatório (Fisatom) até completa remoção do solvente, obtendo-se
o extrato etanólico de folhas (109,5 g). Em seguida, o extrato bruto de folhas foi submetido à
lavagem com hexano (5 x 2000 mL) e diclorometano (5 x 2000 mL), com o intuito de
remover os constituintes menos polares. A fração insolúvel nesses solventes foi lavada com
acetato de etila (5 x 2000 mL). A fração solúvel em acetato de etila foi filtrada e concentrada
em evaporador rotatório, permitindo a obtenção de 17,3 g de fração em acetato de etila de
folhas.
Óleo essencial
Folhas frescas de Manilkara subsericea (340 g) foram fragmentadas utilizando-se um
turbolizador com água destilada. Em seguida, o material vegetal foi adicionado em balão de 5
L, contendo fragmentos de porcelana porosa para regularização da ebulição. O balão foi
colocado em manta de aquecimento e acoplado a um aparato do tipo Clevenger. Foi
adicionada quantidade suficiente de água pelo topo do condensador até que o material vegetal
em suspensão ocupasse volume aproximado de 3 L, seguida da adição de 2 mL de hexano no
condensador. Após o sistema entrar em ebulição, procedeu-se a extração do óleo essencial por
3 horas. Ao término deste período, o hidrolato foi recolhido e a fase hexânica contendo as
substâncias voláteis foi recolhida, filtrada em sulfato de sódio anidro e armazenada em freezer
até a realização da análise química.
- 41 -
Cromatografia em Camada Fina (CCF)
Para a realização dos ensaios cromatográficos qualitativos, utilizou-se gel de Sílica G
60 de fase normal em cromatofolhas de alumínio ALUGRAM® SIL G/UV254 20 x 20 com
0,20 mm de espessura.
Reveladores (Wagner & Bladt, 1996)
Solução de anisaldeído sulfúrico – solução de 0,5 mL de p-anisaldeído, 98% (Aldrich
Chemical Company, Inc, Milwaukee, USA) em 10 mL de ácido acético glacial, seguida da
adição de 85 mL de metanol e 5 mL de ácido sulfúrico concentrado. A placa deve ser
borrifada com a solução e aquecida até aparecimento das manchas cromatográficas.
NP/PEG - – preparar reagente I constituído por solução metanólica de ácido difenil bórico β-
etilamino éster (1 %, v/v) e reagente II, constituído por solução etanólica de polietilenoglicol-
4000 (5%, v/v). Borrifar a placa com aproximadamente 10 mL da solução I e 8 mL da solução
II.
Cromatografia em coluna
O isolamento das substâncias foi feito por cromatografia em coluna utilizando-se
diferentes fases estacionárias e sistemas de eluição (Figura 1). A fração solúvel em acetato de
etila proveniente do extrato bruto de folhas de M. subsericea foi inicialmente fracionada
utilizando-se a resina Amberlite XAD-2 como fase estacionária (Sigma-Aldrich, St Louis).
Foi efetuado um gradiente de eluição com água, misturas de metanol/água (5:95→9:1),
metanol e acetona. Após análise do perfil cromatográfico por cromatografia em camada fina
(CCF), as frações 28-40 foram reunidas. Em seguida foi realizada uma purificação final por
cromatografia em coluna utilizando-se Sephadex LH-20 como fase estacionária e metanol
- 42 -
como fase móvel, permitindo a obtenção de três agliconas de flavonoides (40.2 mg de
miricetina, 35.7 mg de quercetina e 11.9 mg de kaempferol).
Figura 1. Esquema ilustrativo do isolamento de miricetina, quercetina, kaempferol,
miricetrina, quercitrina e ácido pomólico da fração solúvel em acetato de etila, proveniente do
extrato etanólico de folhas de Manilkara subsericea.
As frações 8-19 foram reunidas com base em seu perfil cromatográfico (CCF) e
submetidas a purificação utilizando-se C-18 (Sigma-Aldrich, St Louis) como fase estacionária
e gradiente de metanol em água (60%→63%, v/v) como fase móvel. Foi feita uma purificação
final utilizando-se Sephadex LH-20 como fase estacionária e metanol como fase móvel,
permitindo a obtenção de dois flavonoides glicosilados (7.4 mg de uma mistura de miricetrina
e quercitrina). As frações 41-52 foram submetidas a purificação utilizando-se silica gel como
fase estacionária e um sistema de eluição isocrático constituído por mistura de hexano :
acetato de etila : metanol (5:5:1), permitindo a obtenção de 9.7 mg do triterpeno ácido
pomólico.
- 43 -
Elucidação estrutural
Cromatografia Gasosa acoplada à Espectrometria de Massas (CG-EM)
O cromatograma e espectros de massas do óleo essencial de M. subsericea foram
obtidos em cromatógrafo gasoso acoplado à espectrômetro de massas (GCMS-
QP5000,SHIMADZU) equipado com detector por impacto de elétrons. 1 µL da solução do
óleo essencial diluído em hexano desta solução foi injetada em coluna RTX-5MS (30m x 0,25
mm x 0,25 µm). As condições de análise foram: hélio como gás de arraste; fluxo com taxa de
1mL/min; injeção de split com taxa de 1:40; temperatura do injetor, 260°C, temperatura do
detector, 290 ºC; temperatura inicial da amostra 60ºC e final 290 ºC, com taxa de variação de
3º/min. As condições da EM foram: voltagem de ionização de 70eV e taxa de varredura 1
scan/s. Os indices de retenção foram calculados em relação a uma mistura de hidrocarbonetos
alifáticos (C7-C40) (Sigma-Aldrich, EUA) analisados sob as mesmas condições (Van den
Dool and Kratz, 1963). A identificação das substâncias foi feita por comparação dos indices
de retenção e padrão de fragmentação com dados da literatura (Adams, 2007). A análise
quantitativa foi realizada através de análise em cromatógrafo gasoso acoplado a detector de
ionização de chama (CG/DIC), sob as mesmas condições da análise por CG/EM).
A análise da fração hexânica proveniente do extrato etanólico de frutos de M.
subsericea foi realizada conforme descrito por Fernandes (2011). A elucidação das estruturas
dos ésteres triterpênicos foi realizada por comparação com dados da biblioteca do aparelho
(NIST) e análise da fragmentação (via Retro-Dials-Alders).
Ressonância Magnética Nuclear (RMN)
Os espectros de RMN 1H e RMN 13C foram obtidos no equipamento Varian VNMRS
com 500 MHz 1H e 125 MHz para 13C. O solvente deuterado para obtenção dos espectros de
- 44 -
RMN foram obtidos da Cambridge Isotope Laboratories (USA). Os deslocamentos químicos
(δ) foram expressos em partes por milhão (ppm). A edição dos espectros foi realizada
utilizando-se o programa MestReNova 6.0.2- 5475 (Mestrelab Research S.L., 2009).
Nanoemulsões
Método de emulsificação
As nanoemulsões foram preparadas utilizando-se o método de inversão de fases
(Aulton, 2005). A fase oleosa e os tensoativos foram adicionados em um béquer e aquecidos a
75 ± 5 ºC, enquanto a fase aquosa constituída de água destilada foi colocada em outro
recipiente e aquecida sob essa mesma temperatura. Quando ambas as fases atingiram a
temperatura desejada, a fase aquosa foi sobre a fase oleosa sob agitação constante (400 rpm)
pelo período de 10 minutos. Em seguida o sistema foi resfriado com agitação constante (400
rpm) por mais 5 minutos. As nanoemulsões contendo fração em hexano obtida do extrato
etanólico de frutos de M. subsericea ou eserina tiveram esses constituintes adicionados ao
bequer contendo fase oleosa, sendo sua quantidade descontada da massa total de água
(Fernandes et al., 2013a).
- 45 -
Caracterização das nanoemulsões
As nanoemulsões foram caracterizadas imediatamente e após 1, 15 e 30 dias. Elas
foram armazenadas em tubos de vidro com tampa e armazenadas sob temperatura ambiente
(25±2 ºC). Adicionalmente foram colocados tubos em estufa (40±5 ºC) para verificação da
estabilidade acelerada. Foram avaliadas características macroscópicas como cor, aspect
visual, separação de fases, cremagem e sedimentação (Fernandes et al., 2013a).
O tamanho das gotículas da fase dispersa e polidispersão foram determinados por
espectroscopia de correlação de fótons, utilizando aparelho ZetaPlus (Brookhaven Inst. Corp.,
USA). Cada emulsão foi diluída utilizando água ultra pura Mili-Q (1:25). Cada análise foi
feita em triplica, sendo os resultados expressos em função da média e desvio padrão.
Desenvolvimento das formulações
Determinação do valor de Equilibrio Hidrófilo-Lipófilo (EHL) requerido da fase oleosa
Foram preparadas diversas emulsões contendo diferentes proporções de tensoativos na
faixa de EHL entre 4,3 (EHL do monooleato de sorbitano) e 15 (EHL do polissorbato 80). O
EHL resultante de cada mistura de tensoativos foi determinado com base na fórmula a seguir:
Onde:
EHLm é o valor de EHL resultante da mistura de dois tensoativos
EHLA é o valor de EHL do tensoativo mais hidrofóbico
EHLB é o valor de EHL do tensoativo mais hidrofílico
- 46 -
A% é o percentual do tensoativo mais hidrofóbico
B% é o percentual do tensoativo mais hidrofílico
A% + B% = 100
As emulsões foram preparadas pelo método de inversão de fases (Aulton, 2005),
contendo 5% de fase oleosa (miristato de octildodecila, MOD®), 5% de tensoativos
(monooleato de sorbitano e polisorbato 80) e 90 % de fase aquosa (água destilada) (Fernandes
et al., 2013a). Com base na estabilidade das emulsões iniciais obtidas (separação de fases e
cremagem), foram preparadas novas emulsões com valor de EHL próximos aquela que
apresentou maior estabilidade, sendo avaliadas macroscopicamente e microscopicamente. O
valor de EHL requerido para o MOD® foi definido como sendo o EHL do tensoativo ou
mistura de tensoativos capaz de formar a emulsão mais estável (Salager, 2000), verificada
através dos ensaios de estabilidade e tamanho de partícula.
Diagrama ternário
Este método se baseia na utilização de um triângulo equilátero, em que cada um dos
vértices corresponde a 100% da composição de cada constituituinte da formulação. A razão
tensoativo/co-tensoativo correspondente ao EHL requerido do óleo empregado. Diversas
emulsões serão preparadas variando-se a proporção entre os constituintes (água,
tensoativo/co-tensoativo/óleo). A análise do tamanho médio das gotículas das emulsões
geradas será realizada com o intuito de se traçar a região de nanoemulsão (Fernandez et al.,
2004).
- 47 -
Avaliação da atividade inseticida
Colônias
As colônias de Dysdercus peruvianus (Hemiptera) foram mantidas em cubas de vidro
fosco (de aproximadamente 19 cm de profundidade por 17 cm de diâmetro) em uma
temperatura média entre 20-25 ºC, umidade relativa de 70-75% e ciclos de 16h (dia) e 8h
(noite). A abertura da cuba deve estar tampada com um pano (filó) para entrada de ar. Os
insetos tinham livre acesso a água e foram alimentados com sementes de algodão (Fernandes
et al., 2013b). Dentro das cubas, foram utilizados dois pedaços de papel filtro: um cortado em
circulo para ser depositado no fundo e outro retangular e sanfonado, depositado verticalmente,
para aumentar a área de movimentação dos insetos. Um bebedouro ficava pendurado por um
arame preso no topo da cuba, a fim de evitar vazamento no fundo da mesma. Placas de petri
eram colocadas no fundo, contendo gazes ou algodão, para que os insetos realizassem as
posturas.
Os testes foram realizados através de tratamento tópico de ninfas no quarto estágio. Os
animais foram separados em grupos de 30 indivíduos e foi realizada aplicação de uma
nanoemulsão contendo fração hexânica proveniente do extrato etanólico de frutos de M.
subsericea (50 μg de extrato / inseto). O controle negativo foi realizado aplicando-se a
nanoemulsão correspondente, sem adição de fração hexânica no dorso dos insetos, submetidos
às mesmas condições do grupo experimental. O grupo não tratado recebeu comida e água,
sem aplicação tópica de nenhuma substâncias. Em seguida foram colocadas em frascos
pequenos de aproximadamente 7 cm de profundidade por 5 cm de diâmetro e iniciou-se a
contagem diária, acompanhando o desenvolvimento dos insetos até atingirem o estágio adulto
(Mello et al., 2007; 2008; Fernandes et al., 2013b)
- 48 -
A significância dos resultados foi analisada utilizando-se programa ANOVA e teste de
Tukey utilizando-se o programa Stats Direct Statistical Software, versão 2.2.7 (Windows 98).
As diferenças entre os grupos tratados, controle e não tratados foram considerados
estatisticamente sem significância para p>0,05. Todos os experimentos foram feitos em
triplicata.
Atividade inibitória sobre a acetilcolinesterase
Para verificar a atividade inibitória sobrea enzima acetilcolinesterase, foi realizado o
ensaio espectrofotométrico baseado no método colorimétrico de Ellman (1961) com algumas
modificações (Rhee et al., 2001), utilizando-se leitor de microplacas de 96 poços. O ensaio
colorimétrico ocorre segundo a reação enzimática de hidrólise do iodeto de acetiltiocolina
(ATCI) catalisada pela enzima AChE, na qual o produto de hidrólise, a tiocolina, reage com o
agente colorimétrico, ácido 5,5'-ditiobis (2-nitrobenzóico) (DTNB), promovendo a formação
de uma substância de coloração amarela, o ácido tionitrobenzóico (TNB) (figura 2).
O volume total em cada poço foi de 200 μL, sendo constituídos por 65 μL de tampão
fosfato salino (PBS), 60 μL de ácido 5,5'-ditiobis (2-nitrobenzóico) (DTNB (1,5 mM), 25 μL
de acetilcolinesterase de peixe elétrico (550 mU/mL) e 25 μL de nanoemulsão. Por fim,
adicionou-se 25 μL de iodeto de acetiltiocolina. Diferentes concentrações das amostras foram
obtidas através de diluições sucessivas em PBS. A hidrólise espontânea do substrato
enzimático (iodeto de acetiltiocolina) foi verificada na ausência de enzima, sendo seu volume
substituído por PBS. As leituras foram feitas no comprimento de onda de 412 nm. Os
resultatos são expressos em função da atividade enzimática remanescente, calculada em
- 49 -
relação ao controle negativo. A análise estatística foi feita através coeficiente de correlação de
Pearson, com 95 % de intervalo de confiança, utilizando-se o programa GraphPad Prism 5.04.
SN
O
O
O
SN
HO
+_+_
+ + 2H+AChE
2
tiocolina acetato
Reação 1
acetiltiocolina
SS
O
OH OH
O
NO2
O2N
OH
O
NO2
(CH3)3NCH
2SS S
O
OH
O2N
(CH3)3NCH
2S+ + +
DTNB amarelo
+
tiocolina
_
Reação 2
Figura 2. Representação da reação de hidrólise do substrato enzimático (acetiltiocolina) pela
enzima acetilcolinesterase. A formação de ácido tionitro benzoico (TNB) indica atividade
enzimática e pode ser observada pela formação de coloração amarela.
Toxicidade aguda
Animais
O estudo envolvendo animais foi aprovado pelo Comitê de Ética em Pesquisa da
Universidade Federal do Amapá sob o número de registro (CEP – UNIFAP – 005AP/2013).
Foram utilizados machos e fêmeas de camundongos suíços albinos (Mus musculus), com 12
semanas de idade, provenientes do Laboratório Central do Estado do Amapá – Macapá
(LACEN/AP). Cada grupo experimental foi constituído de 5 animais. Eles foram mantidos em
caixas sob condições controladas (25°C ± 2°C e períodos de claro/escuro de 12 horas cada).
Os animais tiveram livre acesso a água e comida, exceto nas 24 horas anteriores ao
experimento, quando foram mantidos em jejum.
- 50 -
Protocolo experimental
Os estudos de toxicidade aguda foram realizados utilizando-se camundongos de ambos
os sexos, conforme descrito por Pina et al. (2012), com algumas modificações. Os grupos
tratados receberam uma única dose de nanoemulsão contendo fração hexânica proveniente do
extrato etanólico de frutos de M. subsericea por gavagem, correspondendo a uma dose de
3g/kg de extrato. O controle negativo foi realizado administrado-se mesma quantidade da
nanoemulsão correspondente, sem adição de fração hexânica, em camundongos submetidos às
mesmas condições do grupo experimental.
As observações foram realizadas 30, 60, 120, 240, 360 e 720 minutos após o
tratamento e diariamente por 14 dias. Foram observadas possíveis alterações
comportamentais, como agitação, convulsão, frêmito vocal, irritação, movimentos
estereotipados, resposta ao toque, salivação, tremores, distensão corporal, ptose, sono,
defecação, diarreia, piloereção). O peso, consumo de alimento e água, sinais de intoxicação e
mortalidade foram verificados diariamente. Ao final dos 14 dias de tratamento, os animais
foram sacrificados por deslocamento cervical e foi realizada autópsia para retirada dos órgãos
(coração, pulmões, fígado, rim e baço), para posterior pesagem e observação de possíveis
alterações macroscópicas. A análise estatística foi efetuada através do Test t de Student (95%
de interval de confiança), utilizando o programa GraphPad Prism 5.04. Diferenças foram
consideradas significante para p<0.05.
- 51 -
Referências
Adams R.P., “Identification of essential oil components by gas chromatography/mass
spectrometry” , Allured Publishing, Carol Stream 4th ed. 2007.
AULTON, M.E. Delineamento de Formas Farmacêuticas. 2ª ed. São Paulo: Artmed. 2005.
Ellman G.L., Courtney K.D., Andres V.Jr., Featherstone R.M. (1961). Biochemical
Pharmacology. 7: 88-95.
Fernandes C.P. (2011). Estudo fitoquímico e biológico da espécie vegetal Manilkara
subsericea (Mart.) Dubard. Niterói. 75f.
Fernandes C.P., Mascarenhas M.P., Zibetti F.M., Lima B.G., Oliveira R.P.R.F., Rocha L.,
Falcão D.Q. (2013a). HLB value, an important parameter for the development of essential oil
phytopharmaceuticals. Brazilian Journal of Pharmacognosy. 23: 108-114.
Fernandes C.P., Xavier A., Pacheco J.P.F., Santos M.G., Mexas R., Raticliffe N.A., Gonzalez
M.S., Mello C.B., Rocha L., Feder D. (2013b). Laboratory evaluation of the effects of
Manilkara subsericea (Mart.) Dubard extracts and triterpenes on the development of
Dysdercus peruvianus and Oncopeltus fasciatus. Pest Management Science. 69: 292–301.
Fernandez P, André A., Rieger J., Kuhnle. (2004). Nano-emulsion formation by emulsion
phase inversion. Colloids and Surfaces A: Physicochemical Engineering Aspects. 251: 53-58.
Mello C.B., Uzeda C.D., Bernardino M.V., Mendonça-Lopes C., Kelecom A., Fevereira
P.C.A. (2007). Effects of the essential oil obtained from Pilocarpus spicatus on the
development of Rhodnius prolixus nymphae. Revista Brasileira de Farmacognosia. 17:514–
520.
- 52 -
Mello C.B., Mendonça-Lopes D., Feder D., Uzeda C.D., Carneiro R.M., Rocha M.A. (2008).
Laboratory evaluation of the effects of triflumuron on the development of Rhodnius prolixus
nymph. Memórias do Instituto Oswaldo Cruz. 103: 839–842.
Pina E.M.L., Araújo F.W.C., Souza I.A., Bastos I.V.G.A., Silva T.G., Nascimento S.C.,
Militão G.C.G., Soares L.A.L., Xavier H.S., Melo S.J. (2012). Pharmacological screening and
acute toxicity of bark roots of Guettarda platypoda. Brazilian Journal of Pharmacognosy. 22:
1315-1322.
Rhee I.K., Van De Meent M., Ingkaninan K., Veerporte R. (2001). Journal of
Chromatography A. 915: 217-23.
Salager J.L. (2000). Formulation concepts for the emulsion makers. In: Nielloud F, MartiI-
Mestres G. Pharmaceutical emultions and suspensions: drugs and the pharmaceutical
sciences. New York: Marcel Dekker p. 19-72.
Van den Dool H., Kratz P.D. (1963). A generalization of the retention index system including
linear temperature programmed gas-liquid partition chromatography. Journal of
Chromatography. 2: 463-471.
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CAPÍTULO 3
Atividades biológicas e ésteres triterpênicos provenientes de frutos comestíveis de
Manilkara subsericea (Mart.) Dubard (Sapotaceae)
Introdução
Manilkara subsericea (Mart.) Dubard é uma espécie da família Sapotaceae com ampla
distribuição no Parque Nacional da Restinga de Jurubatiba. Seus frutos são utilizados como
alimento pela população desta localidade, onde é popularmente conhecida como guracica
(Santos et al., 2009). Trata-se de uma espécie endêmica do Brasil e poucas informações estão
disponíveis em relação à constituição química de M. subsericea.
Estudos anteriores realizados no Laboratório de Tecnologia de Produtos Naturais da
Universidade Federal Fluminense (LTPN-UFF) indicaram a presença de triterpenos
pentacíclicos, como os acetatos de alfa- e beta-amirina e os ácidos ursólico e oleanólico nesta
espécie. Adicionalmente, seus extratos foram capazes de inibir o crescimento de uma
linhagem de Staphylococcus aureus (ATCC 25923), incluindo uma fração hexânica de frutos
(Fernandes, 2011).
Neste contexto, o presente capítulo tem como objetivo apresentar a elucidação
estrutural de quatro triterpenos pentacíclicos oriundos de frutos, que não haviam sido
descritos anteriormente para essa espécie.
- 54 -
Referências
Fernandes, C.P. (2011). Estudo fitoquímico e biológico da espécie vegetal Manilkara
subsericea (Mart.) Dubard. Niterói. 75f.
Santos M.G., Fevereiro P.C.A., Reis G.L., Barcelos J.I. (2009). Recursos Vegetais da
Restinga de Carapebus. Revista de Biología Neotropical. 6: 35-54.
- 55 -
Artigo 1
Triterpene esters and biological activities from edible fruits of Manilkara subsericea
(Mart.) Dubard, Sapotaceae
Artigo publicado no periódico “BioMed Research International”
Volume 2013, Article ID 280810, 7 pages
http://dx.doi.org/10.1155/2013/280810
- 56 -
Triterpene esters and biological activities from edible fruits of Manilkara subsericea
(Mart.) Dubard, Sapotaceae
Caio P. Fernandesa,e, Arthur L. Corrêaa, Jonathas F.R. Loboa, Otávio P. Caramela, Fernanda B.
de Almeidaa, Elaine S. Castrob, Kauê F.C.S. Souzab , Patrícia Burthb, Lidia M.F. Amorimb,
Marcelo G. Santosc, José Luiz P. Ferreirad, Deborah Q. Falcãoe, José C. T. Carvalhof &
Leandro Rochaa,e*
aLaboratório de Tecnologia de Produtos Naturais, Faculdade de Farmácia, Universidade
Federal Fluminense, Rua Doutor Mário Viana 523, Santa Rosa, CEP 24241-000, Niterói, RJ,
Brazil
bDepartamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal
Fluminense, Outeiro São João Batista s/no, Centro CEP 24020-141, Niterói, RJ, Brazil
cDepartamento de Ciências, Faculdade de Formação de Professores, Universidade do Estado
do Rio de Janeiro, Dr. Francisco Portela 1470, CEP 24435-000, São Gonçalo, RJ, Brazil
dLaboratório de Farmacognosia, Faculdade de Farmácia, Universidade Federal Fluminense,
Rua Doutor Mário Viana 523, Santa Rosa, CEP 24241-000, Niterói, RJ, Brazil
eDepartamento de Tecnologia Farmacêutica, Faculdade de Farmácia, Universidade Federal
Fluminense, Rua Doutor Mário Viana 523, Santa Rosa, CEP 24241-000, Niterói, RJ, Brazil
fLaboratório de Pesquisa em Fármacos, Colegiado de Ciências Farmacêuticas, Universidade
Federal do Amapá, Campus Universitário - Marco Zero do Equador, Rod. Juscelino
Kubitschek de Oliveira, KM-02 - Bairro Zerão, CEP 68902-280 Macapá, AP, Brazil
- 57 -
*Corresponding author: Tel: 55 21 2629 9578 / E-mail address: [email protected]
Abstract
Manilkara subsericea (Mart.) Dubard (Sapotaceae) is popularly known in Brazil as
“guracica”. Studies with Manilkara spp indicated the presence of triterpenes, saponins and
flavonoids. Several activities have been attributed to Manilkara spp, such as antimicrobial,
antiparasitic and antitumoral, which indicates the great biological potential of this genus. In
all, 87.19% of the hexanic extract from fruits relative composition were evaluated, in which
72.81 % were beta- and alpha- amyrin esters, suggesting that they may be chemical markers
for M. subsericea. Hexadecanoic acid, hexadecanoic acid ethyl ester, (E)-9-octadecenoic acid
ethyl ester and octadecanoic acid ethyl ester were also identified. Ethanolic crude extracts
from leaves, stems and hexanic extract from fruits exhibited antimicrobial activity against
Staphylococcus aureus ATCC25923. These extracts had high IC50 values against Vero cells,
demonstrating weak cytotoxicity. This is the first time, to our knowledge, that beta- and alpha-
amyrin caproates and caprylates are described for Manilkara subsericea.
1. Introduction
Manilkara subsericea (Mart.) Dubard (Sapotaceae) is popularly known in Brazil as
“guracica”, “maçaranduba-pequena”, “maçaranduba-vermelha “maçarandubinha” or “paraju”
(Figure 1). This species is widely spread on the sandbanks of eastern Brazil, from the states of
Espírito Santo to Santa Catarina. M. subsericea has edible fruits, being consumed in natura,
and local population also use its woods for construction [1, 2]. Studies with species from the
genus Manilkara indicated the presence of triterpenes [3], saponins [4] and flavonoids [5].
Several activities have been attributed to Manilkara spp, such as antimicrobial [6, 7],
- 58 -
antiparasitic [8, 9] and antitumoral [10], which indicates the great biological potential of the
genus.
Figure 1: Manilkara subsericea (Mart.) Dubard, Sapotaceae, at Restinga de Jurubatiba
National Park (Rio de Janeiro, Brazil).
On the present study, we evaluated the antibacterial and cytotoxity activity of extracts
from Manilkara subsericea. We also made the phytochemical characterization of the hexanic
extract from edible fruits of M. subsericea.
- 59 -
2. Materials and Methods
2.1. Plant material
Aerial parts with fruits of Manilkara subsericea (Mart.) Dubard (Sapotaceae) were
collected at Restinga de Jurubatiba National Park (Rio de Janeiro, Brazil) in January 2009 and
were identified by the botanist Dr. Marcelo Guerra Santos. A voucher specimen of M.
subsericea was deposited at the herbarium of the Faculdade de Formação de Professores
(Universidade do Estado do Rio de Janeiro, Brazil) under the register number RFFP 13.416.
2.2. Preparation of extracts
Extracts were obtained from fruits, leaves and stems. The M. subsericea freshly fruits
(1.14 kg) were crushed and macerated with ethanol (EtOH) 96 % (v/v) at room temperature
until exhaustion. This ethanolic extract was concentrated in vacuum using a rotary evaporator
to obtain ethanolic crude extract from fruits (170g). This extract was dissolved into 500 mL
EtOH/H2O 90% (v/v) mixture and partitioned with hexane (2 x 600 mL) to obtain, after
evaporation of the hexanic portion, 14.0g of hexanic extract from fruits (FH). The
hydroalcoholic portion from this partition was evaporated in vacuum and ressuspended in 500
mL distilled water. This aqueous suspension was successively partitioned with ethyl acetate
(2 x 600 mL) and butanol (2 x 600 mL), furnishing, after evaporation, 4.5g of ethyl acetate
extract (FEA) and 6.8g of butanol extract (FB) from fruits. Leaves (1.93 kg) and stems (0.96
kg) were individually dried at 40ºC for two days, triturated and macerated with ethanol
(EtOH) 96 % (v/v) at room temperature until exhaustion. Each ethanolic extract was
concentrated in vacuum using a rotary evaporator to obtain 530g of ethanolic crude extract
from leaves (LET) and 169.3 g of ethanolic crude extract from stems (SET).
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2.3. Analysis of FH by Gas-Chromatography-Mass Spectrometry
The hexanic extract from fruits (FH) was analyzed by a GCMS-QP5000
(SHIMADZU) gas chromatograph equipped with a mass spectrometer using electron
ionization, according to these experimental conditions: injector temperature, 270°C; detector
temperature, 290°C; carrier gas, Helium; flow rate 1 mL/min and split injection with split
ratio 1:50. The oven temperature was programmed from 60°C (isothermal for 3 min), with an
increase of 10 °C/min to 290 °C, ending with a 59 min isothermal at 290 °C. One microliter
of the sample, dissolved in CHCl3 (1:100 mg/μL), was injected into a ZB-5MS column (i.d. =
0.25 mm, length 30 m, film thickness = 0.25 mm). Mass spectrometry (MS) conditions were
ionization voltage, 70 eV and scan rate, 1 scan/s. The identification was performed by
comparison of the MS fragmentation pattern of the substances of FH with NIST mass spectra
libraries. Quantitative analysis of the chemical constituents was performed by flame
ionization gas chromatography (GC/FID), under same conditions of GC/MS analysis and
percentages obtained by FID peak-area normalization method.
2.4. Antimicrobial activity
2.4.1. Microbial strain
Staphylococcus aureus ATCC25923 and Escherichia coli ATCC36298 obtained from
the culture collections of the Laboratório de Controle Microbiológico, Faculdade de Farmácia,
Universidade Federal Fluminense, were used for the antibacterial activity experiments.
Overnight cultures were prepared by inoculating approximately 2 mL Tryptic soy broth (TSB;
Difco) with 2-3 colonies of each organism. Bacterial strains were cultured overnight at 37ºC.
Inocula were prepared by diluting overnight cultures in saline to approximately 108 CFU/mL.
- 61 -
2.4.2. Diffusion disk assay
Qualitative antimicrobial tests were carried out by disk diffusion method [11]. Briefly,
a suspension of microorganism (108 UFC/mL) was spread on the solid media plates of
Muller-Hinton agar (Difco). Disks (6 mm in diameter) were impregnated, until saturation,
with the ethanolic crude extracts from leaves and stems, hexanic, ethyl acetate and butanol
extracts from fruits. Then, disks were placed on the inoculated agar. Vancomycin (30 μg) and
ampicillin (30 μg) were used as positive reference standards of the test. Disks impregnated
with solvents used for solubilization of extracts were used as negative control. The inoculated
plates were incubated at 37ºC for 24 h. Antimicrobial activity was evaluated by measuring the
zone of inhibition against the test organisms. Each experiment made in triplicate.
2.4.3. Minimum inhibitory concentration (MIC)
A micro-dilution technique using 96 well micro-plates, as described by Eloff [12] was
used to obtain MIC values of extracts against S. aureus. The method comprised of filling all
the wells of a 96 well micro-plate with 100µl of Muller-Hinton broth (Vetec). Triplicates
(100µl) of the samples (ethanolic crude extracts from leaves and stems, hexanic, ethyl acetate
and butanol extracts from fruits) at starting concentrations of 2 mg/ml in DMSO were
introduced into the first well. Serial doubling dilutions were then performed, rejecting 100 µl
from each well and adding a mixture of test micro-organism (100 µl) having an inoculum size
of approximately 1×106 CFU/ml. The final concentrations per well were 500, 250, 125, 64 e
32 µg/mL. The micro plates were sealed and incubated at 37ºC for 24h. After incubation, 50µl
of a 2.5% solution of the biological indicator TTC (Triphenyl Tetrazolium Chloride) solution
was added, and the plate were incubated again for 2h to visualize growth inhibition. The
- 62 -
lowest concentration of the sample that inhibited the bacterial growth (colourless) after
incubation was taken as the MIC. Vancomycin and DMSO were used as positive and negative
control, respectively.
2.5. Cytotoxic assay
2.5.1. Mammalian Vero cell line culture condition
Vero cell line (ATCC CCL-81) was cultured at 37 °C, 5% CO2 in DMEM medium (GIBCO)
supplemented with 10% heat-inactivated fetal bovine serum (GIBCO) and 0.1 mg/mL
streptomycin (GIBCO), and 100 U/mL penicillin (GIBCO).
2.5.2. Cell viability by LDH assay
To evaluate the toxicity of extracts, Vero cell line was incubated with samples
(ethanolic crude extracts from leaves and stems, hexanic, ethyl acetate and butanol extracts
from fruits) for 24 hours and cell viability measured using LDH assay (Doles). In brief, 5 X
104 cells/well were seeded in a 96-well microplate and incubated for 24 hours to attach. In the
following day, cells were washed with PBS and fresh media DMEM without serum were
replaced containing the plant extract at different concentrations (500 - 31.25 μg/mL). Plates
were incubated for further 24 hours and LDH activity measured by colorimetric assay using
spectrophotometer (micronal-B582) at 510 nm. As control for maximum LDH release, cells
were treated with 0.1% triton-X100 in DMEM medium for 10 min before running the assay.
To determine the normal LDH release, cells were cultured in serum-free medium in presence
of DMSO. Cell viability was determined using absorbance of treated cells at DMSO as a
- 63 -
reference for 100% viability (Absorbance of extract treated cells × 100/ Absorbance of
DMSO treated cells).
Figure 2: Fragmentation pattern for Δ12-oleane/Δ12-ursane series. A: beta-amyrin acetate. B:
alpha-amyrin caproate. C: alpha-amyrin caprylate.
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2.6. Statistical analysis
For antibacterial assay, statistical analysis was performed by ANOVA (One-way
Anova) with 95% confidence interval, using the GraphPad Prism 5.0 software package.
Differences were considered significant when P-values were ≤ 0.05. Vero cell viability (%)
was determined by averaging three repeated experiments and IC50, representing the
concentration at which cell viability was reduced by 50%, was calculated by linear regression
using the GraphPad Prism 5.0 software package.
3. Results and Discussion
Analysis of the chromatogram obtained from the hexanic extract from fruits of M.
subsericea indicated the elution of 20 compounds. Substances with retention time (min) of
16.84, 16.99, 18.71 and 18.94 corresponded, respectively, to hexadecanoic acid (A) (5.41%),
hexadecanoic acid ethyl ester (B) (3.57%), (E)-9-octadecenoic acid ethyl ester (C) (3.95%)
and octadecanoic acid ethyl ester (D) (1.45%). These compounds were identified by
comparison of their MS fragmentation pattern with NIST mass spectra.
On another study, we described the obtainment and identification of a mixture
containing beta-amyrin acetate and alpha-amyrin acetate from edible fruits of this species
[13]. Thus, comparison of the previously fragmentation pattern obtained for these substances
confirmed the major substances, with retention time (min) of 34.63 and 36.15, as beta-amyrin
acetate (E) (10.27%) and alpha-amyrin acetate (F) (42.34%), respectively. The substances
with retention time (min) of 53.31/56.55 and 71.70/76.99 also showed a typical fragmentation
pattern for pairs of triterpenes from the Δ12-oleane/Δ12-ursane series. The characteristic
peaks at m/z 218 (base peak), 203 and 189 due to Retro-Dials–Alder fragmentation [14] were
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observed for these substances. Beta-amyrin type triterpenes presented peak at m/z 203 higher
than peak at m/z 189, while alpha-amyrin type triterpenes showed an equal abundance (Figure
2). According to Oyo-Ita et al. [15], the amyrin caproates have molecular ion peak (M+) at m/z
524, followed by loss of CH3 or the acid moiety to m/z 509 and 408, respectively. Thus, the
substances with retention time (min) of 53.31 and 56.55 could be identified as beta-amyrin
caproate (G) (5.46%) and alpha-amyrin caproate (H) (7.26%). The mass fragment at m/z 408,
due to the loss of 144 (caprylic acid) mass unit from the molecular ion peak (M+) at m/z 552
was in accordance with literature data [16, 17] and suggested substances with retention time
(min) of 71.70 and 76.99 as beta-amyrin caprylate (I) (2.44%) and alpha-amyrin caprylate (J)
(5.04%), respectively.
It is interesting for the chemotaxonomic consideration that several studies carried out
for Manilkara species, such as Mimusops littoralis Kurz [=Manilkara littoralis (Kurz)
Dubard] [18], Mimusops manilkara G.Don [=Manilkara kauki (L.) Dubard] (Misra & Mitra,
1969) and Mimusops hexandra Roxb [=Manilkara hexandra (Roxb.) Dubard] [19] indicated
the presence of triterpene esters. Thus, the chemical substances identified on the hexanic
extract from fruits of M. subsericea (Figure 3) are in accordance with the chemical markers of
the genus Manilkara.
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R1
R3
R2
E R1=CH3 , R2=H , R3= AcO
F R1=H , R2=CH3 , R3= AcO
G R1=CH3 , R2=H , R3= CH3(CH2)4COO
H R1=H , R2=CH3 , R3= CH3(CH2)4COO
I R1=CH3 , R2=H , R3= CH3(CH2)6COO
J R1=H , R2=CH3 , R3= CH3(CH2)6COO
Figure 3: Chemical structures of the amyrin esters: beta-amyrin acetate (E), alpha-amyrin
acetate (F), beta-amyrin caproate (G), alpha-amyrin caproate (H), beta-amyrin caprylate (I)
and alpha-amyrin caprylate (J ) from the hexanic extract from fruits of Manilkara subsericea.
The identified substances corresponded to 87.19% of the total relative composition of
the hexanic extract from fruits of M. subsericea. The individual amounts of each substance are
illustrated in Figure 4. Furthermore, to our knowledge, this is the first time that the beta- and
alpha- amyrin caproates and caprylates are described for the Manilkara subsericea species.
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Figure 4: GC-FID chromatogram of the hexanic extract from fruits of Manilkara subsericea,
Sapotaceae. A: hexadecanoic acid (5.41%), B: hexadecanoic acid ethyl ester (3.57%), C: (E)-
9-octadecenoic acid ethyl ester (3.95%), D: octadecanoic acid ethyl ester (1.45%), E: beta-
amyrin acetate (10.27%), F: alpha-amyrin acetate (42.34%). For analysis conditions see
Materials and Methods, Section 2.3.
Antibacterial assay was performed against Staphylococcus aureus ATCC25923 and
Escherichia coli ATCC36298. There were significant differences (P < 0.05) in the
antibacterial activity of ethanolic crude extract from leaves (7 ± 0.28 mm), ethanolic crude
extract from stems (8 ± 0 mm) and hexanic extract from fruits (6 ± 0 mm), which were
considered active against S. aureus. (Table 1). Ethyl acetate and butanol extracts from fruits
did not inhibit the S. aureus growth. All extracts were considered inactive against E. coli.
(Table1).
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Table 1: Means of the inhibition halos (mm + SD) for Staphylococcus aureus and
Escherichia coli tested with extracts (100 mg/mL) from Manilkara subsericea. LET:
ethanolic crude extract from leaves, SET: ethanolic crude extract from stems, FH: hexanic
extract from fruits, FEA: ethyl acetate extract from fruits, FB: butanol extract from fruits.
Vanc: Vancomycin (30 µg), Ampicillin: (30 µg).
Inhibition halo (mm) + SD
Staphylococcus aureus Escherichia coli
LET 7 + 0.28 b 0 b
SET 8 + 0 c 0 b
FH 6 + 0 d 0 b
FEA 0 e 0 b
FB 0 e 0 b
Vanc 18 + 0.28a Not tested
Amp Not tested 32 + 7 a
Means in the same column with different superscripts are significantly different (P<0.05)
The extracts that exhibited antimicrobial activity during the disk diffusion method
were evaluated for their Minimum inhibitory concentration (MIC). All extracts tested
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inhibited the bacterial growth of the S. aureus strain with MIC of 250 µg/mL. Terpenoids are
active against bacteria but the mechanism of action of terpenes is not fully understood,
although it is speculates to involve membrane disruption by lipophilic compounds [20]. The
isomeric mixture of beta-amyrin and alpha-amyrin is known by its antimicrobial activity [21]
and 72.81% of the relative amount of the hexanic extract from fruits is constituted by esters of
these substances. Beta- and alpha- amyrin acetates are also known by their anti-inflammatory
activity and also inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) in Raji cells
[22]. Furthermore, according to Hichri et al [23], the triterpene beta-amyrin acetate was able
to inhibit the bacterial growth of the Staphylococcus aureus ATCC25923 reference strain at
90 µg/mL. Thus, our results suggest that the antibacterial activity found in the hexanic extract
from fruits may be modulated by the beta- and alpha- amyrin esters identified.
All tested extracts demonstrated weak cytotoxic effects on the mammalian Vero cells.
The Cell viability on treatment with hexanic and ethyl acetate extracts from fruits was 69.66%
and 56.07% in concentration of 250 µg/mL, respectively (Figure 5). Ethanolic crude extract
from leaves (1658 μg/mL; 1164-2525) had highest IC50 value, followed by ethanolic crude
extract from stems (1112 μg/mL; 757-2525), butanol extract from fruits (683.4 μg/mL; 451-
2200), hexanic extract from fruits (482.6 μg/mL; 385-677) and ethyl acetate extract from
fruits (307.6 μg/mL; 276-346).
The triterpene beta-amyrin acetate was reported to have cytotoxicity against A2780
ovarian cancer cell line with IC50 of 12.1 μg/ml [24]. This compound was not considered
active against A549, SK-OV-3, SK-MEL-2, XF498 and HCT15 cancer cell lines [25].
Moreover, some beta- and alpha- amyrin esters were able to induce cell apoptosis in HL-60
leukemia cells [26].
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Figure 5 – Vero cell viability in the presence of extracts for 24 h measured by LDH assay.
LET: ethanolic crude extract from leaves, SET: ethanolic crude extract from stems, FEA:
ethyl acetate extract from fruits, FB: butanol extract from fruits, FH: hexanic extract from
fruits.
4. Conclusions
Although Manilkara subsericea fruits are used as food, to our knowledge, only one
article regarding to its phytochemicals and biological activities was published [13]. The
present study describes the identification of a high percentage of substances from the hexanic
extract from edible fruits of Manilkara subsericea, in which beta- and alpha- amyrin
caproates and caprylates are reported for the first time for this species. Our results suggest that
this hexane extract from fruits and ethanolic crude extract from leaves and stems presented
antimicrobial activity against S. aureus ATCC25923. In addition, these extracts had low
cytotoxicity on Vero cells, in the same concentration which inhibited S. aureus growth.
- 71 -
Several biological studies are carried out for mixtures of beta- and alpha- amyrin type
triterpenes [21, 27, 28], since their separation by conventional chromatographic methods is
quite difficult [29].
Acknowledgments
The authors thank CNPq (Proc. 564745/2010-3), CAPES, FAPERJ and Rede Bionorte 156
(Rede Biodiversidade e Biotecnologia da Amazonia Legal) for their financial support.
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CAPÍTULO 4
Metabólitos secundários de folhas de Manilkara subsericea (Mart.) Dubard
Introdução
O gênero Manilkara Adans encontra-se distribuído nos Neotrópicos, sendo 18
espécies (15 endêmicas) encontradas no Brasil (Almeida Jr., 2013). Devido a circunscrição
dos gêneros, algumas espécies anteriormente descritas como pertencentes aos gêneros Achas
L. e Mimusops L. foram incluídas no gênero Manilkara (Almeida Jr., 2010).
A maioria dos estudos fitoquímicos com espécies do gênero Manilkara têm sido
realizados com M. zapota (L.) P. Royen (Ma et al., 2003; Ahmed et al., 2001; Fayek et al.,
2013), espécie popularmente conhecida como sapoti. Entretanto, dados relacinados a
constituição da maioria das espécies desse gênero ainda são escassos, incluindo M.
subsericea. Estudos prévios com essa espécie indicam umas predominância de triterpenos
pentacíclicos com esqueletos do tipo olean-12-eno e ursan-12-eno (Fernandes et al., 2011;
2013).
Neste contexto, o presente capítulo tem o objetivo de identificar estruturalmente
substâncias não descritas previamente em Manilkara subsericea, incluindo um triterpeno
pentacíclico, cinco flavonoides e constituintes de óleo essencial.
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Referências
Almeida Jr.E.B. (2010). Diversidade de Manilkara Adans. (Sapotaceae) para o Nordeste do
Brasil. PhD Thesis, Universidade Federal Rural de Pernambuco. Pernambuco, Brasil.
Almeida Jr.E.B. Manilkara in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio
de Janeiro. Available at: <http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB14473>.
Accessed in November 2013.
Ahmed R., Ifzal S.M., Usmanghani K. (2001). Studies on Achras zapota L: Part IV chemistry
and pharmacology of wood. Pakistan Journal of Pharmaceutical Sciences.
Fayek N.M., Monem A.R., Mossa M.Y., Meselhy M.R. (2013). New triterpenoid acyl
derivatives and biological study of Manilkara zapota (L.) Van Royen fruits. Pharmacognosy
Research. 5: 55-59.
Fernandes C.P., Corrêa A.L., Cruz R.A.S., Botas G.S., Silva-Filho M.V., Santos M.G., de
Brito M.A., Rocha L. (2011). Anticholinesterasic activity of Manilkara subsericea (Mart.)
Dubard triterpenes. Latin American Journal of Pharmacy. 30: 1631-1634.
Fernandes C.P., Corrêa A.L., Lobo J.F.R., Caramel O.P., de Almeida F.B., Castro E.S., Souza
K.F.C.S., Burth P., Amorim L.M.F., Santos M.G., Ferreira J.L.P., Falcão D.Q., Carvalho
J.C.T., Rocha L. (2013). Triterpene esters and biological activities from edible fruits of
Manilkara subsericea (Mart.) Dubard, Sapotaceae. Bio.Med. Research International. Article
ID 280810, 7 p.
Ma J., Luo X.D., Protiva P., Yang H., Ma C., Basile M.J., Weinstein I.B., Kennelly E.J.
(2003). Bioactive novel polyphenols from the fruit of Manilkara zapota (Sapodilla). Journal
of Natural Products. 66: 983-986.
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Artigo 2
Secondary metabolites from leaves of Manilkara subsericea (Mart.) Dubard
Artigo submetido ao periódico “Brazilian Journal of Pharmacognosy”
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Secondary metabolites from leaves of Manilkara subsericea (Mart.) Dubard
Caio P. Fernandes1,2*, Fernanda B. de Almeida2, Wanderson Romao3,4, Gabriela
Vanini3, Helber B. Costa3, Hildegardo S. França4, Marcelo G. Santos5, José C. T.
Carvalho6, Deborah Q. Falcão7, Leandro Rocha1,8
1Programa de Pós – Graduação em Biotecnologia Vegetal – Centro de Ciências da Saúde-
Bloco K , 2º andar – sala 032 – Universidade Federal do Rio de Janeiro – UFRJ – Av.
Brigadeiro Trompowski s/n – CEP: 21941-590 - Ilha do Fundão – RJ – Brazil
2Laboratório de Nanobiotecnologia Fitofarmacêutica – Colegiado de Farmácia –
Universidade Federal do Amapá – Campus Universitário Marco Zero do Equador – Rodovia
Juscelino Kubitschek de Oliveira – KM – 02 Bairro Zerão – CEP: 68902-280 – Macapá – AP
– Brazil
3Laboratório de Petroleômica e Forense- Departamento de Química- Universidade Federal
do Espírito Santo- CEP: 29075-910, Vitória – ES - Brasil.
4Instituto Federal de Educação - Ciência e Tecnologia do Espírito Santo - CEP: 29106-010 -
Vila Velha – ES - Brazil
5Faculdade de Formação de Professores – UERJ- Rua: Dr. Francisco Portela, 1470 –
Patronato – CEP: 24435-005 – São Gonçalo – Rio de Janeiro – Brazil
6Laboratório de Pesquisa em Fármacos, Colegiado de Farmácia, Universidade Federal do
Amapá, Campus Universitário Marco Zero do Equador, Rod. Juscelino Kubitschek de
Oliveira, KM-02 - Bairro Zerão, CEP 68902-280 Macapá, AP, Brazil
- 80 -
7Laboratório de Tecnologia Farmacêutica – Departamento e Tecnologia Farmacêutica –
Faculdade de Farmácia – Universidade Federal Fluminense – UFF Rua: Mario Viana, 523 –
CEP: 24241-000 – Santa Rosa – Niterói – RJ – Brazil
8Laboratório de Tecnologia de Produtos Naturais – LTPN – Departamento e Tecnologia
Farmacêutica – Faculdade de Farmácia – Universidade Federal Fluminense – UFF Rua:
Mario Viana, 523 – CEP: 24241-000 – Santa Rosa – Niterói – RJ – Brazil
* Corresponding author
Caio Pinho Fernandes
Universidade Federal do Amapá, Campus Universitário Marco Zero do Equador
Rodovia Juscelino Kubitschek de Oliveira – KM – 02 Bairro Zerão – CEP: 68902-280 –
Macapá – AP – Brazil
Tel: +55 (96) 4009 2924 / E-mail address: [email protected]
Abstract
Manilkara subsericea is a species widely spread in the sandbanks of Restinga de Jurubatiba
National Park (Rio de Janeiro, Brazil). To our knowledge, the present study reports the first
phytochemical information about leaves from M. subsericea, being 39 substances found for
the first time on this species, including a polyhydroxy triterpene acid (pomolic acid) and some
flavonoids, such as quercitrin, myricitrin, and their aglycons, quercetin and myricetin.
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Keywords
Essential oil, Flavonoids, Manilkara subsericea, Pomolic Acid, Sapotaceae
1. Introduction
Sapotaceae is a family containing 58 genus and approximately 1250 species with
morphological variation, ranging from shrubs to medium and giant trees (Bartish et al., 2011).
Brazil comprises 11 genera and 231 species, including 1 endemic genus and 104 endemic
species (Carneiro et al. 2013). This family has the following synapomorphies, well-
developed, elongate laticifers with white latex; 2-branched hairs, brownish, T-shaped; berry
fruits, seeds usually with a hard shiny testa and large hilum (Judd et al, 2009).The genus
Manilkara Adans. is constituted by 30 species in the Neotropics, been approximately 20
species found in Africa and 12 species found in Asia and Pacific (Pennington, 2006). Brazil
has 18 species, being 15 endemic to this country (Almeida Jr., 2013). The genus Manilkara is
characterized by calyx of 2 whorls of 3 sepals, presence of staminodes and hilum shape seed
(Almeida Jr., 2010; Pennington, 1990, 2006). Due to this genus circumscription, some species
of the genera Achras L. and Mimusops L. were included in Manilkara (Almeida Jr., 2010).
Manilkara subsericea (Mart.) Dubard is an endemic species from Brazilian Atlantic
Rain Forest (Almeida Jr., 2013), commonly known as “maçaranduba”, “maçarandubinha” and
“guracica”. It is widely distributed at Restinga de Jurubatiba National Park (Rio de Janeiro
state, Brazil), being used in this locality as food and timber (Santos et al., 2009). This species
develop a main role in the ecology of Restinga de Jurubatiba, especially regarding insect-plant
interactions, being an important host plant for some Lepidoptera species (Monteiro et al.,
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2007) and for porcupine (Chaetomys subspinosus) feeding (Oliveira et al., 2012). The aim of
the present study was to perform phytochemical characterization with chemotaxonomic
significance of leaves from Manilkara subsericea.
2. Material and Methods
2.1. Plant material
Leaves of Manilkara subsericea were collected at Restinga de Jurubatiba National
Park, Rio de Janeiro State, Brazil (22°14’46’’S - 41°34’56’’W), October 2010 by Dr. Caio
Pinho Fernandes. Identification was performed by the botanist Dr. Marcelo Guerra Santos and
voucher specimen of M. subsericea were deposited at the herbarium of the Faculdade de
Formação de Professores (Universidade do Estado do Rio de Janeiro, Brazil) under the
register number RFFP 15316. Nomenclatural update was realized in Lista de Espécies da
Flora do Brasil (http://floradobrasil.jbrj.gov.br/jabot/listaBrasil/PrincipalUC/PrincipalUC.do)
and The Plant List: a Working List of All Plant Species (http://www.theplantlist.org/).
2.2. Preparation of extracts
Leaves (0.84 kg) were dried at 40 ºC for 2 days and then crushed and macerated in
ethanol (EtOH) 96% (v/v) at room temperature until exhaustion. After filtration, the ethanolic
extract was concentrated under vacuum using a rotary evaporator in order to obtain 109.5 g of
the ethanolic crude extract from leaves. This dried extract was sequentially washed with n-
hexane (5 x 2000 mL) and dichloromethane (5 x 2000) in order to remove less polar
constituents from the extract. The insoluble fraction was washed with ethyl acetate (5 x 2000
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mL). The ethyl acetate-soluble fraction was filtered and concentrated under vacuum using a
rotary evaporator, affording 17.3 g of the ethyl acetate fraction from leaves.
2.3. Isolation of substances
The ethyl acetate-soluble fraction from leaves was fractionated through column
chromatography using Amberlite XAD-2 resin (Sigma-Aldrich, St Louis). Elution was
performed with water, methanol/water mixtures (5:95→9:1), methanol and acetone. Fractions
28-40 were pooled together according to the TLC profile and purified on Sephadex LH-20
using and methanol as mobile phase, affording1 (40.2 mg), 2 (35.7 mg) and 3 (11.9 mg).
Fractions 8-19 were pooled together according to the TLC profile and chromatographed in C-
18 reversed phase silica gel (Sigma-Aldrich, St Louis) using gradient of mobile phase
constituted by methanol solutions (v/v) in water (60%→63%). Final purification on Sephadex
LH-20 using methanol as mobile phase afforded a fraction (7.4 mg) containing 4 and 5.
Fractions 41-52 was submitted to silica gel chromatography column using an isocratic system
of mobile phase (n-hexane : ethyl acetate : methanol, 5:5:1), affording a fraction (9.7 mg)
constituted by 6.
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O
OH O
OH
R4
R2
R3
R1
1 Myricetin R1 = R2 = R3 = R4 = OH
2 Quercetin R1 = R2 = R4 = OH, R3 = H
3 Kaempferol R1 = R3 = H, R2 = R4 = OH
4 Myricitrin R1 = R2 = R3 = OH, R4 = ORha
5 Quercitrin R1 = R2 = OH, R3 = H, R4 = ORha
OH
COOH
OH
6 Pomolic acid
Figure 1. Structures of flavonoids and triterpene from leaves of Manilkara subsericea.
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2.4. Essential oil extraction
Fresh leaves (340 g) from Manilkara subsericea were ground with distilled water
using an automatic blender (Ética Equipamentos Científicos S.A, Brazil). Hydrodistillation
method was employed for three hours using Clevenger apparatus and plant material was
placed in a 5L flask. At the end of extraction, essential oil was collected, dried over anhydrous
sodium sulphate and stored at 4 °C for further analyses.
2.5. Chemical analysis
1H and 13C NMR spectra of 1, 2, 3 and 6 were recorded at 500 and 125 MHz,
respectively, on a Varian VNMRS 500MHz spectrometer. Deuterated methanol was used for
solubilization of flavonoids, while deuterated dimethyl sulfoxide was used for solubilization
of triterpene fraction. Solvents were obtained from Cambridge Isotope Laboratories (USA)
and TMS peak was used as an internal standard.
Substances 4, 5 and 6 were analyzed by negative-ion electrospray ionization Fourier
transform ion cyclotron resonance mass spectrometry, ESI(−)-FT-ICR MS (Colati et al.,
2013; Dalmaschio et al., 2014; Freitas et al., 2013). Briefly, each sample was diluted to 1.0
mg mL−1 in acetonitrile (containing 0.1% w/v of NH4OH). The resulting solution was directly
infused at a flow rate of 5 μl min−1 into the ESI source. The mass spectrometer (ESI(−)) over a
mass range of m/z 200–2000. The ESI source conditions were as follows: nebulizer gas
pressure of 0.5–1.0 bar, capillary voltage of 2.5–3.5 kV, and transfer capillary temperature of
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250 ºC. All mass spectra were externally calibrated using a NaTFA (m/z from 200 to 1200). A
resolving power (m/Δm50% ≈ 530,000, in which Δm50% is the full peak width at half-maximum
peak height) of m/z 400 and a mass accuracy (mass error) of < 1 ppm provided unambiguous
molecular formula assignments for singly charged molecular ions. Mass spectra were
acquired and processed using the software package Compass Data Analysis (Bruker
Daltonics, Bremen, Germany). ESI(-)-MS/MS experiments were collected after 4–40 eV
collision-induced dissociations (CID) with argon. Selection was performed by quadrupole,
using a unitary m/z window, and collisions were performed in the rf-only hexapole collision
cell, followed by mass analysis of product ions by the ultra-high resolution ICR analyzer.
Structures of flavonoids and triterpene identified are presented in Figure 1.
Essential oil was analyzed by a GCMS-QP2010 (SHIMADZU) gas chromatograph
equipped with a mass spectrometer using electron ionization. The gas chromatographic (GC)
conditions were as follows: Essential oil: injector temperature, 260 °C; detector temperature,
290 °C; carrier gas (Helium), flow rate 1 mL/min and split injection with split ratio 1:40.
Oven temperature was initially 60 °C and then raised to 290 °C at a rate of 3 °C/min. The
sample was diluted with n-hexane (1:100, v/v) and injected at RTX-5 column (i.d. = 0.25 mm,
length 30 m, film thickness = 0.25 µm). The mass spectrometry (MS) conditions were
voltage, 70 eV and scan rate; 1 scan/s. The retention indices (RI) were calculated by
interpolation of retention times of the substances to the retention times of a mixture of
aliphatic hydrocarbons (C7-C40) (Sigma) analyzed in the same conditions (Van den Dool and
Kratz, 1963). The identification of substances was performed by comparison of their retention
indices and mass spectra with those reported in literature (Adams, 2007). MS fragmentation
pattern of compounds was also checked with NIST mass spectra libraries. Quantitative
analysis of the chemical constituents was performed by flame ionization gas chromatography
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(CG/FID), under same conditions of GC/MS analysis and percentages obtained by FID peak
area normalization method.
3. Results and Discussion
1H NMR spectrum of 1 (CD3OD, 500 MHz) showed two doublets at δH 6.18 (J = 1.45
Hz) and δH 6.38 (J = 1.45 Hz), attributable respectively to the protons H-8 and H-6, and a
two-proton singlet observed at δH 7.34, corresponding to the B-ring aromatic protons H-2´
and H-6´ of a flavonol. Signals observed in the 1H and 13C NMR spectrum are in accordance
with literature data for myricetin (Ceruks et al., 2007). Substances 2 and 3 had their 1H and
13C NMR spectra compared with literature data (Koolen et al., 2012; Olennikov et al., 2012)
and therefore were respectively identified as quercetin and kaempferol.
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Figure 2. ESI(-)-FT-ICR mass spectra for (a) FL4 and (b) FL5 samples and CID experiments
for ion of m/z 463 and 447 corresponding to (c) myricitrin and (d) quercitrin.
The remaining flavonoids, 4 and 5, were analyzed by ESI(−)-FT-ICR MS, Figure 2a-
d. For FL4, Figure 2a, ESI(-)-FT-ICR mass spectrum, detected the presence of the O-
glycoside flavonol myricitrin (M = C21H20O12), ions of m/z 463.0882 ([M – H]-), 499.0651
([M + Cl]-) and 927.1844 ([2M – H]-) as deprotonated molecule, chlorine adduct and dimer,
respectively. A DBE (double bound equivalents) of 12 found for ions [M – H]- and [M + Cl]-
agrees with the chemical structure of myricitrin, that presents two aromatic rings (DBE = 8),
one rhamnose (DBE = 1) and a ring containing one double bond, and a ketone group (DBE =
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3). To confirm myricitrin detection, ESI(-)-MS/MS was performed for the ion of m/z 463,
Figure 2c. The CID of [M - H]- of m/z 463 agrees well with its structure and connectivity,
producing fragments with m/z 316, corresponding to neutral loss of C6H11O4. For FL5, Figure
2b, ESI(-)-FT-ICR mass spectrum identified, simultaneously, ion [M – H]- of m/z 463.0882,
and ions [N – H]- and [N + Cl]- of m/z 447.0933 and 483.0703, respectively, where N =
C21H20O11, corresponding to the O-glycoside flavonol quercitrin. CID experiment of ion of
m/z 447, Figure 2d, produces fragments of m/z 301 and 284 corresponding to neutral losses
of C6H10O4 and one rhamnose molecule.
Some flavonoids isolated and identified on the present study have also been found on
this genus. Quercetin was identified on Mimusops manilkara G.Don (Manilkara kauki (L.)
Dubard) and Mimusops littoralis Kurz (Manilkara littoralis (Kurz) Dubard) (Jahan et al,
1996; Misra and Mitra, 1969), while myricetin was identified on Achras zapota L. (Manilkara
zapota (L.) P.Royen) (Subramanian and Nair, 1972). The glycosylated flavonoids quercitrin
and myricitrin were isolated from M. zapota (Ma et al., 2003).
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Figure 3. ESI(-)-FT-ICR mass spectrum of triterpene acids present in the leaves M.
subsericea
Chromatographic fractionation of the ethyl acetate extract from leaves (EAL) also
afforded a white powder. These substances comprise one of the largest classes of special
metabolites and usually are obtained as mixtures (Olea and Roque, 1990). ESI(-)FT-ICR mass
spectrum of the fraction containing substance 6 revealed the presence of precursor ions
[C30H48O4 – H]-, [C30H48O5 – H]- and [C30H48O6 – H]- of m/z 471, 487 and 503, being
attributed to dihydroxy, trihydroxy and tetrahydroxy triterpene acids (Figure 3), probably
derivatives from ursolic and oleanolic acids (DNP version 19.1, 2010). 13C NMR spectra of
this fraction showed typical set of signals for triterpene mixtures. It was observed a typical
signal at δc 181.0, due to the C-28 carboxyl group of triterpene acid. The presence of C-12/C-
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13 olefinic carbons at δc 128.4/140.3, as well as the signals of carbons bonded to hydroxyl
groups at δc 79.8 (C-3) and 73.1 (C-19) suggested that pomolic acid (6) may be the main
constituent of this fraction. Assignments for pomolic acid are in accordance with literature
data and allowed establishment of relative configuration (Mahato and Kundu, 1994). To our
knowledge, this is the first time that this substance is reported as constituent of M. subsericea.
Several triterpenes, such as beta-amyrin acetate, alpha-amyrin acetate, beta-amyrin
caproate, alpha-amyrin caproate, beta-amyrin caprylate, alpha-amyrin caprylate, oleanolic
acid and ursolic acid have been previously isolated from M. subsericea (Fernandes et al. 2011,
2013). Pomolic acid, is structurally related to ursolic acid and was already found in the family
Sapotaceae (Lee et al., 2005). Alpha-amyrin caprylate, alpha-amyrin acetate and β-amyrin-3-
(3'-dimethyl) butyrate were isolated from Manilkara zapota (L.) P.Royen (Ahmed et al.,
2001; Fayek et al., 2013). Alpha-amyrin acetate, beta-amyrin acetate and ursolic acid were
isolated from Mimusops manilkara (Manilkara kauki (L.) Dubard) (Misra et al., 1969).
Alpha- and beta-amyrin and beta-amyrin caproate were isolated from Mimusops littoralis
Kurz (Manilkara littoralis (Kurz) Dubard) (Jahan et al., 1996). Alpha-amyrin acetate and
alpha-amyrin cinnamate were isolated from Manilkara bidentata (A.DC.) A.Chev. (Rhourri-
frih et al., 2012). It is interesting from a chemosystematic overview that substances described
above for Manilkara species are derived from olean-12-ene and urs-12-ene pentaciclic
triterpene type. This data suggests a possible chemotaxonomic significance of pentacyclic
triterpenes, especially from these specific skeletons on the genus.
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Table 1. Relative abundance of essential oil constituents from leaves of Manilkara
subsericea.
Substance RI
Relative
abundance (%)
Substance RI
Relative
abundance (%)
3-hexen-1-ol 850 0.2 Trans-2-decenal 1262 0.2
Hexanol 862 0.2 Beta-damascenone 1388 0.2
2-heptanone 889 0.1 Beta-damascone 1418 0.3
Heptanal 902 0.2 Beta-caryophyllene 1423 0.5
Heptanol 966 0.3 Farnesene 1510 0.3
Octanal 1003 0.2
Caryophyllene
oxide
1588 0.7
(3E)-3-hexenyl
acetate
1006 0.6 Hexadecanoic Acid 1969 8.6
Beta-ocimene 1047 7.3 Eicosene 1994 0.2
Octanol 1069 1.3 Heneicosane 2100 0.3
Linalool oxide 1089 0.5 Phytol 2116 15.6
2-nonanone 1092 0.2 Docosene 2194 0.4
Linalool 1100 27.6 Tricosane 2300 0.6
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Terpineol 1193 0.9 Pentacosane 2500 0.4
Methyl salicilate 1197 1.9 Heptacosane 2700 0.9
Safranal 1202 0.3 Squalene 2832 2.6
Beta-cyclocitral 1223 0.3 Nonacosane 2900 8.9
Geraniol 1256 0.3 Untriacontane 3100 3.4
Total Identified 86.5
RI: Retention Indices calculated by interpolation of retention times of the substances to the
retention times of a mixture of aliphatic hydrocarbons
Gas chromatographic (GC) conditions: injector temperature, 260 °C; detector temperature,
290 °C; carrier gas, Helium; flow rate, 1 mL/min; split ratio, 1:40. Initial temperature, 60 °C;
final temperature, 290 °C; rate of 3 °C/min. RTX-5 column (i.d. = 0.25 mm, length 30 m, film
thickness = 0.25 µm). Mass spectrometry (MS) conditions: voltage, 70 eV; scan rate; 1 scan/s.
Essential oil obtained from fresh leaves of Manilkara subsericea was analysed by GC-
MS in order to determine its chemical composition. In all, 34 substances were identified,
mainly comprising monoterpenes, sesquiterpenes and long chain hydrocarbons, corresponding
to 86.5 % of total relative composition of the oil (Table 1). The monoterpene linalool was the
major substance found, corresponding to 27.6% of the total relative composition of the
essential oil. The relative amount of each substance found is presented in table 1. Some
terpenoids with more isoprene units than monoterpenes and sesquiterpenes were also found
on this essential oil. It was observed an ion peak (M+) at m/z 296 and characteristic fragments
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at m/z 278, due to loss of 18 units of mass (M+ - H2O), m/z 123 and 71 (base peak). This data
is in accordance with literature data for phytol (Siqueira et al., 2003).This substance has wide
distribution among plants, since this substance forms the lipophilic side chain of the
chlorophylls (Dewick, 2009). It was also observed a substance with main fragments at m/z
341, 95, 81 and 69 (base peak), which is in accordance with literature that for squalene
(Bhatia et al., 2013). Detection of this substance may be relevant from a biosynthetic
perception for M. subsericea, since this acyclic substance with 30 atoms of carbon is an
important precursor of triterpenes (Dewick, 2009; Xu et al., 2004).
4. Conclusions
Despite the ecological significance, great abundance, use as food and literature data
concerning its biological activities, Manilkara subsericea remains almost chemically
unexplored. As part of our ongoing studies, the present study allowed the identification of
substances from leaves of this species for the fisrt time, being all substances found identified
for the first time on M. subsericea, with the exception of hexadecanoic acid. Moreover,
flavonoids and a polyhydroxy triterpene acid are reported, contributing for phytochemical
characterization of an almost unexplored species of the Brazilian flora.
Acknowledgment
Authors thank CNPq (nº 306676/2010-9) and FAPERJ for their financial support.
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CAPÍTULO 5
Desenvolvimento de nanoemulsão inseticida contendo extrato de Manilkara subsericea
(Sapotaceae)
Introdução
O uso indiscriminado de pesticidas de origem sintética em lavouras têm sido alvo de
debates, principalmente devido a possibilidade de seleção de populações de insetos com
resistência aos inseticidas e efeitos tóxicos sobre organismos não alvos. Neste contexto, o uso
de substâncias de origem natural têm ganhado força para geração de produtos específicos,
menos tóxicos, menos poluentes e ecologicamente corretos (Rao et al., 2003).
As plantas produzem substâncias de defesa em resposta ao ambiente em que se situam,
incluindo agentes com atividade inseticida, com o intuito de repelir e/ou eliminar herbívoros.
Diversas classes de metabólitos especiais, como alcalóides, substâncias fenólicas e
terpenóides foram descritas como inseticidas (Rattan, 2010). Os triterpenos estão entre os
grupos mais ativos (Viegas Jr, 2003), entretanto, a baixa solubilidade em água e consequente
necessidade de uso de solventes orgânicos potencialmente tóxicos se configura como um
desafio tecnológico para obtenção de produtos viáveis.
A espécie Manilkara subsericea (Mart.) Dubard foi capaz de induzir mortalidade em
Dysdercus peruvianus (Hemiptera), uma praga agrícola que causa graves danos no cultivo do
algodão. Além disso, foi observado que a atividade inseticida da fração hexânica de frutos é,
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ao menos parcialmente, modulada por triterpenos pentacíclios com baixa solubilidade em
água, como os acetatos de alfa- e beta-amirina (Fernandes et al., 2013).
Neste contexto, a utilização de nanoemulsões se configura como uma alternativa
viável para aumentar a disponibilização de substâncias apolares. O tamanho diminuto das
partículas dispersas aumentam a estabilidade da formulação, além de torná-la mais atraente
visualmente e potencialmente aumentar a biodisponibilidade dos ativos. O presente capítulo
tem por objetivo descrever o desenvolvimento de uma nanoemulsão inseticida contendo a
fração em hexano obtida dos frutos de Manilkara subsericea.
Referências
Fernandes C.P., Xavier A., Pacheco J.P.F., Santos M.G., Mexas R., Raticliffe N.A., Gonzalez
M.S., Mello C.B., Rocha L., Feder D. (2013) Laboratory evaluation of the effects of
Manilkara subsericea (Mart.) Dubard extracts and triterpenes on the development of
Dysdercus peruvianus and Oncopeltus fasciatus. Pest Management Science. 69: 292–301.
Rao J.V., Shilpanjali D., Kavitha P., Madhavendra S.S. (2003). Toxic effects of profenofos on
tissue acetylcholinesterase and gill morphology in a euryhaline fish, Oreochromis
mossambicus. Archives of Toxicology. 77: 227-232.
Rattan R.S. (2010). Mechanism of action of insecticidal secondary metabolites of plant origin.
Crop Prot. 29: 913–920.
Viegas Jr.C. (2003). Terpenos com atividade inseticida: uma alternativa para o controle
químico de insetos. Quimica Nova. 26: 390-400.
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Artigo 3.
Development of an insecticidal nanoemulsion with Manilkara subsericea (Sapotaceae)
extract
Artigo publicado no periódico “Journal of Nanobiotechnology”
Volume 2014 12:22
doi:10.1186/1477-3155-12-22
- 103 -
Development of an insecticidal nanoemulsion with Manilkara subsericea (Sapotaceae)
extract
Caio Pinho Fernandes1,2, Fernanda Borges de Almeida1 Amanda Nunes Silveira3, Marcelo
Salabert Gonzalez4, Cicero Brasileiro Mello4, Denise Feder4, Raul Apolinário4, Marcelo
Guerra Santos5, José Carlos Tavares Carvalho6, Luis Armando Cândido Tietbohl7,
Leandro Rocha7 & Deborah Quintanilha Falcão3
* Correspondence
Caio Pinho Fernandes
Universidade Federal do Amapá, Campus Universitário Marco Zero do Equador
Rodovia Juscelino Kubitschek de Oliveira – KM – 02 – Jardim Marco Zero – CEP: 68902-
280 – Macapá – AP – Brazil
Tel: +55 (96) 4009 2924 / E-mail address: [email protected]
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Abstract
Background: Plants have been recognized as a good source of insecticidal agents, since
they are able to produce their own defensives to insect attack. Moreover, there is a
growing concern worldwide to develop pesticides with low impact to environment
and non-target organisms. Hexane-soluble fraction from ethanolic crude extract
from fruits of Manilkara subsericea and its triterpenes were considered active
against a cotton pest (Dysdercus peruvianus). Several natural products with
insecticidal activity have poor water solubility, including triterpenes, and
nanotechnology has emerged as a good alternative to solve this main problem. On
this context, the aim of the present study was to develop an insecticidal
nanoemulsion containing apolar fraction from fruits of Manilkara subsericea.
Results: It was obtained a formulation constituted by 5% of oil (octyldodecyl
myristate), 5% of surfactants (sorbitan monooleate/polysorbate 80), 5% of apolar
fraction from M. subsericea and 85% of water. Analysis of mean droplet diameter
(155.2 ± 3.8 nm) confirmed this formulation as a nanoemulsion. It was able to
induce mortality in D. peruvianus. It was observed no effect against
acetylcholinesterase or mortality in mice induced by the formulation, suggesting the
safety of this nanoemulsion for non-target organisms. Conclusions: The present
study suggests that the obtained O/A nanoemulsion may be useful to enhance water
solubility of poor water soluble natural products with insecticidal activity, including
the hexane-soluble fraction from ethanolic crude extract from fruits of Manilkara
subsericea.
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Keywords: Dysdercus peruvianus, Manilkara subsericea, Nanoemulsions
Background
Chemical pesticides have been used to control pest insects, however, they are usually
toxic to environment. There is a growing concern worldwide regarding indiscriminate use of
these substances, which are associated to environmental pollution and toxicity risk to non-
targeted organisms [1]. Plant species are well recognized by their ability to produce defensive
substances, in order to protect themselves from insect attack [2]. These natural products
appear as potential sources of new biodegradable insecticides with wide range of mechanisms
of action, being an important alternative for insect pest management in agriculture [3]. One of
the most promising and recognized group of substances with insecticidal activity are the
triterpenes [4].
Manilkara subscericea (Mart.) Dubard (Sapotaceae) is an endemic species of
Brazilian Atlantic Forest [5] and widely distributed at Restinga de Jurubatiba National Park
(Rio de Janeiro State, Brazil) [6]. Several non-polar pentacyclic triterpenes have been
described as major constituents of M. subsericea, mainly alpha- and beta-amyrin esters [7,8].
Hexane-soluble fraction from ethanolic crude extract from fruits of M. subsericea and its
major substances (alpha- and beta-amyrin acetate) was able to induce mortality, delayed
development and inhibition of moulting in Dysdercus peruvianus [9], a hemiptera species
which causes serious loss of cotton crops [10]. This apolar fraction and its triterpenes have
poor water solubility and are soluble in toxic organic solvents, such as chloroform and
dichloromethane, being this intrinsic characteristic a technological challenge if development if
a viable product is desired.
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Nanotechnology has emerged as a promising area for development of products in a
wide range of applications, including pesticide agents. Considering that many of the
insecticides known today are organic compounds with poor water solubility, development of
nanoproducts appear to solve this main problem, enhancing water solubility, bioavailability
and resulting in stable formulations without utilization of organic toxic solvents [11].
Nanoemulsions are one of the most important formulations to enhance solubility and
dissolution properties of poorly water soluble substances [12]. They are also referred as
miniemulsions or ultrafine emulsions and have small droplet size (20-200 nm). They are
transparent or translucent, often presenting a bluish reflect and have high kinetic stability [13].
Low energy methods have been used to achieve nanoemulsions, including reverse-phase
composition (RPC) and temperature of inversion phase (TIF) [14]. Formulation screening
stage is crucial if development of a stable nanoformulation is desired, especially if a low
energy method is employed, being determination of required HLB value of an oil [15] and
construction of pseudo-ternary phase diagrams [16] very useful, especially to achieve
nanoemulsions.
On this context, the aim of the present study was to develop an insecticidal
nanoemulsion containing apolar fraction from fruits of Manilkara subsericea and verify its
effects against Dysdercus peruvianus and non-target organisms.
Results and Discussion
Preliminary solubility studies were performed regarding choice of oil phase and
surfactants. Octyldodecyl myristate (MOD®) was the best oil, being able to solubilize equal
amount (1:1, w/w) of hexane-soluble fraction from fruits of M. subsericea (HF). It is
frequently necessary to use blends, such as a pair of hydrophilic and lipophilic non-ionic
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surfactants, to achieve droplets with small diameter [17]. Sorbitan oleate and polysorbate 80
were considered the best pair (Data not shown). These surfactants have been used in low
energy methods, being able to produce nanoemulsions with smaller mean droplet size, when
compared to other surfactants. This could be explained by the ability of this couple to induce
formation of a looser film, which is associated to generation of nanoemulsions [12]. Addition
of water to a surfactant in oil solution was employed in the present study, since it provided
better results, when compared to addition of oil to an aqueous surfactant solution (Data not
shown). This could be attributed to phase transitions and changes in the curvature of the
surfactant from W/O to O/W during emulsification process [18].
In order to predict the best ratio of surfactants to be used, several emulsions were
prepared varying the relative amounts of sorbitan oleate and polysorbate 80. Most of them
presented instable behavior, including critical macroscopical changes, such as creaming and
phase separation. Surfactants can be classified according to their Hydrophile-Lipophile
Balance (HLB), a semi-empirical scale [19] and several HLB values can be obtained using
different amounts of each component of a couple of surfactants [20]. Emulsions with HLB
values of 10 (sorbitan oleate/polysorbate ratio, 1.0/1.1) and 11 (sorbitan oleate/polysorbate
ratio, 1.0/1.7) were considered more stable. A second set of emulsions within this HLB range
was prepared and the obtained formulations presented translucent aspect and bluish reflect,
which is characteristic for nanoemulsions [13]. Mean droplet size analysis indicated that
nanoemulsion with HLB value of 10.75 (sorbitan oleate/polysorbate ratio, 1.0/1.5) presented
the smallest mean diameter (50.6 ± 0.4 nm) and low polydispersity (0.164 ± 0.021). Stable
formulations with low mean droplet size can be obtained when HLB value of the surfactant
couple coincides with required HLB value of the oil [12,20]. Thus, required HLB of oil can be
determined by calculating the HLB value of emulsifier or emulsifier mixture which was able
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to induce formation of the most stable formulation, among a set of emulsions prepared with
different blends of a couple of emulsifiers in a wide range of HLB value [21]. Our results
indicate that 10.75 should be the required HLB value of MOD® used in the present study.
Table 1. Composition, mean droplet size and polydispersity of each formulation prepared
during construction of pseudo-ternary phase diagram for delimitation of nanoemulsion region.
% of Oil
% of
Surfactants
% of Water
Mean Diameter
(nm)
Polydispersity
1a 5 5 90 50.6 ± 0.4 0.164 ± 0.021
2 2.5 5 92.5 234.2 ± 12.5 0.025 ± 0.012
3 2.5 7.5 90 421.5 ± 50.4 0.005 ± 0.000
4a 5 7.5 87.5 196.4 ± 12.5 0.178 ± 0.044
5a 7.5 5 87.5 145.7 ± 8.6 0.132 ± 0.038
6 7.5 2.5 90 256.9 ± 8.6 0.016 ± 0.011
7a 5 2.5 92.5 151.7 ± 6.0 0.155 ± 0.018
8a 10 5 85 139.4 ± 9.6 0.078 ± 0.049
9a 10 7.5 82.5 133.5 ± 0.5 0.247 ± 0.011
10a 7.5 7.5 85 139.5 ± 4.7 0.073 ± 0.039
11a 10 10 80 86.8 ± 0.9 0.294 ± 0.006
12a 7.5 10 82.5 48.7 ± 0.2 0.313 ± 0.002
13 5 10 85 234.4 ± 2.3 0.270 ± 0.016
14 5 12.5 82.5 298.5 ± 31.8 0.005 ± 0.000
15a 7.5 12.5 80 85.4 ± 1.2 0.350 ± 0.005
16a 10 12.5 77.5 51.7 ± 0.2 0.331 ± 0.005
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17a 7.5 15 77.5 162.4 ± 1.3 0.335 ± 0.011
18a 7.5 17.5 75 159.2 ± 2.3 0.334 ± 0.007
19a 10 15 75 68.7 ± 0.8 0.360 ± 0.004
20a 10 17.5 72.5 87.9 ± 1.5 0.365 ± 0.003
21a 12.5 15 72.5 170.6 ± 6.2 0.144 ± 0.027
22a 12.5 12.5 75 66.6 ± 1.1 0.304 ± 0.005
23a 12.5 10 77.5 120.1 ± 1.0 0.225 ± 0.003
24a 15.0 12.5 72.5 95.3 ± 0.6 0.255 ± 0.006
25a 15 15 70 45.9 ± 0.4 0.271 ± 0.005
26a 12.5 7.5 80 75.4 ± 2.3 0.340 ± 0.005
27a 17.5 15.0 67.5 161.6 ± 1.9 0.266 ± 0.007
28a 12.5 17.5 70 97.2 ± 1.0 0.256 ± 0.002
Oil – MOD®
Surfactants – sorbitan monooleate/polysorbate 80 at HLB of 10.75
a Formulations in the nanoemulsion region
It is observed that not every combination of components produces nanoemulsions over
the whole range of possible compositions [22]. Thus, a total of 28 emulsions were prepared
using different percentages of water, MOD® and surfactants (sorbitan monoleate/polysorbate
80, HLB 10.75) and mean droplet size of each formulation was analyzed. Mean droplet size
ranged from 45.9 ± 0.4 nm (oil 15%, surfactants 15%, water 70%) to 421.5 ± 50.4 nm (oil
2.5%, surfactants 7.5%, water 90%) (Table 1). Composition of each emulsion obtained can be
expressed as a pseudo-ternary phase diagram, which is represented by equilateral triangle in
which four or more constituents are investigated [22] and is very useful to determine relation
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between phase behavior of a mixture and its composition [23]. In all, 23 emulsion presented
mean droplet size bellow 200 nm, ranging 45.9 ± 0.4 nm to 196.4 ± 12.5 and were used to
delineate the nanoemulsion region (Figure 1). Small mean droplet sizes, such as 48.7 ± 0.2 nm
(7.5% of oil, 10% of surfactants, 82.5% of water), 51.7 ± 0.2 nm (10% of oil, 12.5% of
surfactants, 77.5% of water) and 45.9 ± 0.4 nm (15% of oil, 15% of surfactants, 70% of
water) were obtained (Table 1). This data may be important for further studies or development
of nanoformulations using MOD®, sorbitan oleate, polysorbate 80 and water.
Figure 1. Pseudo-ternary phase diagram constructed with water, MOD® and surfactants
(sorbitan monoleate/polysorbate 80, HLB =10.75) at different compositions. Nanoemulsion
region is delimited in blue.
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Special attention was given to formulation comprised by 5% of MOD, 5% of
surfactants and 90 % of water, which also presented stable behavior, small mean droplet size
(50.6 ± 0.4 nm) and low polidispersity (0.164 ± 0.021). Low surfactant percentage could be
considered an advantage, since further preparation of this formulation would reduce toxicity
and costs with raw materials, when compared to other nanoemulsions with higher
concentrations of surfactants. Thus, this formulation was chosen to prepare a nanoemulsion
with hexane-soluble fraction from fruits of Manilkara subsericea dispersed through internal
phase (HFNE).
Concentration of extract corresponded to equal percentage of MOD®, based on its
intrinsic solubility. This amount was discounted from water percentage, being HFNE
constituted by 5% of MOD, 5 % of surfactants, 5 % of hexane-soluble fraction from fruits of
M. subsericea and 85% of water. A blank nanoemulsion without hexane-soluble fraction of
M. subsericea extract (HF) was prepared for negative control. Both nanoemulsions presented
a characteristic bluish reflect, associated to Tyndall effect [13] (Figure 2). It was observed an
increase in the nanoemulsion mean droplet size when HF was dispersed through oil phase
(Figure 3). This fact could be explained due to deposition of substances, which may reduce
the flexibility of the surfactant film and result in more compact films instead of looser films
and smaller mean droplets [12]. Previous gas chromatography analysis of HF indicated a high
relative percentage of pentacyclic triterpenes, including beta-amyrin acetate (10.27%), alpha-
amyrin acetate (42.34%), beta-amyrin caproate (5.46%), alpha-amyrin caproate (7.26%), beta-
amyrin caprylate (2.44%) and alpha-amyrin caprylate (5.04%) [8]. These substances may be
contributing to the result described above.
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HFNE and blank nanoemulsion presented zeta potential values of – 47.4 ± 3.2 and –
59.6 ± 4.1, respectively. Zeta potential is a special parameter that should be analyzed, in order
to determine stability of nanoemulsions and is associated to surface potential of the droplets
[24]. Maximum stability is observed when zeta potential value is above ± 30 mV [25]. The
high stability of formulations with great zeta potential values is associated to repulsive forces
that exceed attracting Van der Waals forces, resulting in dispersed particles and a
deflocculated system [23]. Macroscopical analysis of the nanoemulsion with HF and blank
nanoemulsion indicated that these formulations maintained their original fine appearance and
bluish reflection. It was observed no phase separation, creaming and sedimentation under
room temperature (25±2 ºC) and accelerated stability evaluation. Long term physical stability
of a nanoemulsion related to its small droplets, making this type of formulation being also
referred as “approaching thermodynamic stability” [26,13].
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Figure 2. Nanoemulsions obtained by low energy method. HFNE shown in left side and
blank nanoemulsion shown in right side of the picture.
Figure 3. Particle size distribution of (a) negative control (57.0 ± 0.3 nm) and (b)
nanoemulsion with hexane-soluble fraction from fruits of M. subsericea (155.2 ± 3.8 nm).
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Polidispersity was 0.270 ± 0.006 for blank nanoemulsion and 0.150 ± 0.050 for nanoemulsion
with hexane-soluble fraction from fruits of M. subsericea.
Insecticidal assay was performed in order to verify if HFNE is able to induce mortality
in D. peruvianus. During the whole experimental period, it was observed that HFNE (treated
group) did not interfere in body weight, when compared to untreated group, indicating the
absence of antifeedant effect. This effect was also not detected in negative control group. It
was not observed overaged, extranumerary nymphs or insects with body deformations. Figure
4 indicates that mortality in the untreated group ranged from 3.3 ± 1.15 %, between 5° and
14° days of observation, to 10 ± 1.53 %, between 15° and 30° of observation. Negative
control group (treated with blank nanoemulsion) presented higher levels of mortality
throughout the experimental period, reaching (6.6± 1.15%) (p< 0.001) after 4 days, 13.3±
2.52% (p< 0.001) after 14 days and 21.10± 3.06% (p< 0.001) after 30 days of treatment.
Treatment of insects with HFNE exhibited significantly higher levels of mortality. It was
observed that mortality began on the first day after treatment (12.23 ± 0.58 %) (p< 0.001),
reached 22.23± 1.73% (p< 0.001) after 14 days and 44.43± 6.66% (p< 0.0001) after the end of
the experiment. Significant differences between the group of insects treated with hexanic
nanoemulsion containing extract of M. subsericea and the control group were detected in
almost all days of observation until the end of the experiment (ANOVA, p<0.005). However,
it is worth to note that there was no statistical difference between HFNE-treated and blank
nanoemulsion-treated insects among days 21-23 after treatment. Perhaps, differences in the
speed of absorption between HFNE and blank nanoemulsion by insect metabolic systems may
explain this not expectable result. Physiological mechanisms of metabolization of these
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compounds by invertebrates remains unknown [9] and is now under investigation by our
research group.
Changes in the time period in which occur the processes of molt and metamorphosis
were observed in group treated with HFNE and negative group (Data not shown). Moreover,
an associated high mortality rate were displayed continuously and gradually increasing
throughout insects lifecycle regardless whether the insects were in the nymphal or adult stage.
This observation point out to a physiological connection between the neuroendocrine control
of the insect development and the reduced longevity obtained after treatments. These results
suggest that HFNE may able to release insecticidal components from HF, while formulation
used as blank nanoemulsion may be used to disperse other insecticidal agents.
Figure 4. Analysis of mortality after topical treatment of Dysdercus peruvianus with
nanoemulsion containing hexane-soluble fraction from fruits of Manilkara subsericea
(HFNE) (filled column). Negative control group was topically applied with blank
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nanoemulsion (crosshatched columns). Untreated group is represented by open columns. Each
group represents mean of three experiments.
In order to evaluate if the formulation interfere with acetylcholinesterase, HFNE was
tested using a colorimetric assay. Positive control was performed by preparing a
nanoemulsion with eserine, a recognized lipophilic anticholinesterase agent dispersed through
oil phase (MOD®). Mean droplet analysis confirmed this formulation, constituted by 5% of
MOD®, 5% of surfactants (HLB of 10.75), 0.05% of eserine and 89.95% of water, as a
nanoemulsion (67.3 ± 0.3 nm). IC50 of this substance could not be determined, since lowest
eserine concentration (0.6 ppm) was able to inhibit 90% of enzyme activity (Figure 5).
Results suggest that eserine may be able to displace from disperse phase of the nanoemulsion
to the aqueous external phase and induced a dose-dependent inhibition of
acetylcholinesterase, since it should be in external phase to bind to the enzyme. Pesticides are
used as an important tool to protect crops worldwide, however, residues of these substances
can be found in many environments, including rivers, estuaries and oceans [27,28]. Most
insecticides provide harmful impacts on non-target species, especially aquatic organisms,
such as fishes. These animals are especially susceptive to acetylcholinesterase inhibitors,
probably due to lacking of detoxification systems and sharing same neurological and
respiratory mechanisms [27,29]. Acetylcholinesterase inhibitory assay indicated that no
significant inhibition of acetylcholinesterase modulated by HFNE (Figure 5). Considering that
acetylcholinesterase used in this assay is from fish origin, our results suggest that HFNE may
not induce harmful effects over aquatic non-target animals, indicating the potential of this
nanoemulsion as an insecticidal agent. Nanoemulsion without M. subsericea extract also did
not interfere with the enzyme (Data not shown).
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Acute toxicity evaluation was performed in order to verify effects of HFNE in mice. It
was not observed any behavioral change during all tested period and mortality in all groups.
Analysis of body weight also indicated absence of significant difference between HFNE and
negative control group (Table 2). It was also not observed significant difference in food and
water consumption, macroscopical aspects and weight of organs between groups treated with
HFNE and negative control group (Data not shown). Treatment with HFNE was performed at
a single high dose corresponding to 3g/kg of extract per animal. Since no death or toxic
signals were observed, LD50 could not be estimated and HFNE, suggesting that HFNE may
be considered non-toxic [30].
Figure 5. Linear regression between AchE activity (mU) x natural logarithm of (a) effective
concentration of eserine (p<0.05) and (b) effective concentration of hexane-soluble fraction
from fruits of Manilkara subsericea (p>0.05).
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Table 2. Weight variation in adult female and male Swiss albino mice (Mus musculus) treated
with HFNE (5% of MOD®, 5 % of surfactants (HLB of 10.75), 5% of hexane-soluble fraction
from fruits of M. subsericea and 85% of water) by oral route, corresponding to 3g/kg of
extract. Control groups received same volume of blank nanoemulsion (5% of MOD, 5% of
surfactants and 90 % of water)
Body weight (Male) Body weight (Female)
Initial (g) Final (g) Initial (g) Final (g)
HFNE 49.06 ± 0.43 50.39 ± 1.37 52.72 ± 1.62 50.64 ± 0.63
Control 50.65 ± 1.50 52.05 ± 2.71 50.64 ± 0.63 51.76 ± 1.59
p>0.05
Conclusions
Previous study performed by our research group indicated that hexane-soluble fraction
from ethanolic crude extract from fruits of Manilkara subsericea presented insecticidal
activity against Dysdercus peruvianus. This activity may be partially attributed to beta-and
alpha amyrin acetates, which may be used as chemical markers for quality control of products
with M. subsericea extracts. However, these substances, as well as the active fraction are
poorly water soluble. As part of our ongoing studies with this species, we decided to develop
an insecticidal nanoemulsion. This formulation was able to induce mortality in insects and our
results suggest that it may be safe for non-target organisms and environment. The present
study suggests the obtained O/A nanoemulsion may be useful to enhance water solubility of
poor water soluble natural products with insecticidal activity, including the hexane-soluble
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fraction from ethanolic crude extract from fruits of Manilkara subsericea. The absence of
organic toxic solvents and stability makes this nanoemulsion a potential insecticidal product.
Materials and Methods
Chemicals
Sorbitan oleate (HLB: 4.3) and Polysorbate 80 (HLB: 15) were purchased from La
Belle Ativos Ltda (Paraná, Brazil). Octyldodecyl myristate (MOD®) was purchase from
Brasquim Ltda (São Paulo, Brazil). Acetylthiocholine iodide (ATCI), 5,5-dithiobis-2-
nitrobenzoic acid (DTNB), physostigmine (eserine) and acetylcholinesterase from electric eel
(type VI-S, C3389-2UK, lyophilized powder) were purchased from Sigma (Sigma-Aldrich
Corporation, St Louis, MO). Hexane-soluble fraction from fruits of M. subsericea was
previously obtained [9] and stored at 4 °C for further utilization.
Emulsification method
Emulsions were prepared by temperature of inversion phase method [31]. The required
amounts of both emulsifiers were dissolved in the oil phase and heated at 75 ± 5 º C, while the
aqueous phase was separately heated at same temperature. When both phases reached the
same temperature, aqueous phase was gently added and mixed with the oil phase, using a
mechanic agitator model Fisatom 713D at 400 rpm for 10 min and additional 5 min of
agitation under cooling. Aditional constituents was weight an placed together with oil and
surfactants mixture, being its mass discounted from water mass.
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Required HLB Determination
Each emulsion was prepared at a final mass of 25 g, containing 90 % (w/w) of distilled
water, 5% (w/w) of MOD® and 5% of a mixture of emulsifiers [32]. Series of emulsions were
prepared using sorbitan oleate (HLB = 4.3) and polysorbate 80 (HLB = 15), allowing a wide
range of HLB values from 4.3 (5% w/w of sorbitan oleate) to 15 (5% w/w of polysorbate 80)
by blending together the emulsifiers in different ratios.
Pseudo-Ternary Phase Diagram
Nanoemulsion region was determined using pseudo-ternary phase diagram. Each
corner corresponded to 100% of water, surfactants and MOD®. Surfactants blend was kept
constant and corresponded to ratio which results on required HLB value of oil phase.
Composition (w/w) which allowed required HLB value determination was used as starting
point (90 % of distilled water, 5% of oil and 5% of surfactants blend) and mean droplet size of
each prepared composition was performed in order to determine nanoemulsion region.
Macroscopical Analysis
Stability of all emulsions was evaluated immediately and after 1, 15 and 30 days of
manipulation by macroscopic analysis, such as color, visual aspect, phase separation,
creaming and sedimentation. During this period all emulsions were maintained under room
temperature (25±2 ºC) in screw-capped glass test tubes [32]. Acelerated stability evaluation
was performed keeping emulsion under controlled temperature (40 ± 5 ºC)
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Droplet size and zeta potential analysis
The droplet size, polydispersity and zeta potential were determined by photon
correlation spectroscopy using a ZetaPlus (Brookhaven Inst. Corp., USA). Each emulsion was
diluted using ultra-pure Milli-Q water (1:25). Measurements were performed in quintuplicate
and average droplet size was expressed as the mean diameter.
Insect Bioassay
Dysdercus peruvianus were obtained from the colony maintained in the Laboratory of
Insect Biology of the Universidade Federal Fluminense (GBG-UFF), being kept at 24-25 0C,
relative humidity of 70-75% and a 16:8 h light:dark cycle [9].
Fourth-instar insects were randomly chosen and separated in two treated groups, being
one group topically applied with a nanoemulsion containing hexane-soluble fraction from
fruits of M. subsericea (HFNE) (5% of MOD®, 5 % of surfactants (HLB of 10.75), 5% of HF
and 85% of water), corresponding to 50 μg of extract per insect, while negative control group
was treated with blank nanoemulsion (5% of MOD®, 5% of surfactants and 90 % of water).
Untreated insects received no treatment, being only fed. Biological evaluation was performed
in order to determine mortality levels during the entire time required for development from
the fourth instar to the adult stage [9,33,34]. All experiments were repeated at least three
times with samples from 30 insects (n = 30 in each triplicate). Significance of the results was
analysed using ANOVA and Tukey’s test21 according to Stats Direct Statistical Software,
v.2.2.7 for Windows 98. Differences between treated group and control.
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Anticholinesterase assay
Anticholinesterase activity was performed according to method described by Ellman et
al. (1961) [35] with some modifications [36], using a 96-well microplate. A total volume of
200 μL of test media was composed by 65 μL of Phosphate buffered saline (PBS), 60 μL of
5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) 1,5 mM, 25 μL of electric eel
acetylcholinesterase (Sigma) (AchE) 550 mU/mL, 25 μL of nanoemulsion and 25 μL of
acetylthiocholine iodide (ASCh). Different concentrations of HF and eserine (positive control)
were obtained by dilution of each nanoemulsion with PBS. Negative control was performed
using a blank nanoemulsion, without inhibitor or extract. The spontaneous hydrolysis of
substrate was calculated replacing the enzyme solution by PBS. Absorbance was measured at
412 nm. The statistical analysis of the anticholinesterase assay was performed on GraphPad
Prism 5.04 program using Pearson´s correlation coefficient with 95% confidence interval.
Acute toxicity
Animals
This study was approved by the Ethics Committee of the Universidade Federal do
Amapá (CEP – UNIFAP – 005AP/2013). All procedures were performed according to the
International Committee for animal care in accordance with established national regulations
for animal experimentation. The experiments were performed using adult female and male
Swiss albino mice (Mus musculus), 12 weeks age, provided by the Central Laboratory of the
State of Amapá – Macapá (LACEN/AP). Each experimental group was composed of 5
animals. They were kept in polyethylene cages on a temperature-controlled rack (25°C ± 2°C)
under a 12-hour light-dark cycle. They had free access to food and water, except for the 24
hours before the experiments, when they had access only to water.
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Experimental protocol
Acute toxicity studies were performed using both sexes of mice according to Pina et
al. (2012) [30], with some modifications. Treated groups received a single dose of HFNE (5%
of MOD®, 5 % of surfactants (HLB of 10.75), 5% of hexane-soluble fraction from fruits of M.
subsericea and 85% of water) by oral route, corresponding to 3g/kg of extract. Negative
control groups received a blank nanoemulsion (5% of MOD®, 5% of surfactants and 90 % of
water).
Observations were performed at 30, 60, 120, 240, 360 and 720 min after the oral
treatment and daily for fourteen days. Behavioral changes (agitation, convulsions, vocal
fremitus, irritation, stereotyped movements, touch response, salivation, tremors, writhing,
body distension, ptose, sleepiness, defecation, diarrhea, piloerection), weight, food and water
intake, clinical signs of toxicity and mortality were recorded daily. At the end of fourteen
days, they were sacrificed by cervical dislocation and taken to autopsy for macroscopic
observation of the organs (heart, lung, liver, kidney and spleen). Statistical analysis was
performed by Student t test with 95% confidence intererval, using GraphPad Prism 5.04.
Differences between organs, body weight and food and water intake were considered
significant when p<0.05.
Competing interests
All authors declare no conflict of interests.
Authors’ contributions
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CPF contributed in collecting plant sample, running the laboratory work, analysis of the data
and drafted the paper. FBA and ANS contributed in preparation of extracts, HLB
determination and nanoemulsions preparation. MSG, CBM and DF contributed in insect
bioassay. MGS contributed in plant identification and herbarium confection. LACT
contributed in AChE bioassay. JCTC contributed to critical reading of the manuscript and
acute toxicity assay. LR and DQF designed the study, supervised the laboratory work and
contributed to critical reading of the manuscript. All the authors have read the final
manuscript and approved the submission.
Authors’ information
Caio Pinho Fernandes is a professor at Universidade Federal do Amapá and has been
working with natural products, including phytochemistry, nanotechnology and biological
activities of these compounds.
Fernanda Borges de Almeida is an undergraduate student at Universidade Federal do
Amapá and participated in this project as part of her scientific initiaion program.
Amanda Nunes Silveira is an undergraduate student at Universidade Federal Fluminense and
participated in this Project as part of her scientific initiaion program.
Marcelo Salabert Gonzalez is professor at Universidade Federal Fluminense and has been
working with complementary strategies to control insects with secondary metabolites from
plant species.
Cicero Brasileiro Mello is professor at Universidade Federal Fluminense and has been
working with complementary strategies to control insects with secondary metabolites from
plant species.
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Denise Feder is professor at Universidade Federal Fluminense and has been working with
complementary strategies to control insects with secondary metabolites from plant species.
Raul Apolinário is undergraduate student at Universidade Federal Fluminense and
participated in this project as part of her scientific initiation program and did al experiments
with insects.
Marcelo Guerra Santos is professor at Universidade Estadual do Rio de Janeiro. He is a
botanist and has been working with species from sandbanks of Parque Nacional da Restinga
de Jurubatiba (RJ) Brazil.
José Carlos Tavares Carvalho is professor and President of the Universidade Federal do
Amapá (Brazil) and has been working with natural products pharmacology.
Luis Armando Cândido Tietbohl is a Master´s student at Universidade Federal Fluminense
and has been working with acetylcholinesterase inhibition.
Leandro Rocha is professor at Universidade Federal Fluminense and has been working with
natural products and its biological activities.
Deborah Quintanilha Falcão is professor at Universidade Federal Fluminense and has been
working with nanotechnology of natural products.
Acknowledgement
Authors would like to thank CNPQ (nº 306676/2010-9) and FAPERJ for the finantial support
and “Centro Brasileiro de Pesquisas Físicas” for the use of Zeta Potential Analyzer.
Author Details
1Programa de Pós – Graduação em Biotecnologia Vegetal – Centro de Ciências da Saúde-
Bloco K , 2º andar – sala 032 – Universidade Federal do Rio de Janeiro – UFRJ – Av.
Brigadeiro Trompowski s/n – CEP: 21941-590 - Ilha do Fundão – RJ – Brazil
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2Laboratório de Farmacotécnica - Colegiado de Ciências Farmacêuticas – Universidade
Federal do Amapá –– Rodovia Juscelino Kubitschek – KM – 02-Jardim Marco Zero –
CEP: 68903-419 – Macapá – AP – Brazil
3Laboratório de Tecnologia Farmacêutica I - Faculdade de Farmácia – Universidade
Federal Fluminense - Rua: Mario Viana, 523 – CEP: 24241-000 – Santa Rosa – Niterói –
RJ – Brazil
4Laboratório de Biologia de Insetos – LABI, Departamento de Biologia Geral (GBG),
Universidade Federal Fluminense, Morro do Valonguinho S/No, CEP 24001-970, Niterói,
RJ, Brazil
5Faculdade de Formação de Professores – UERJ- Rua: Dr. Francisco Portela, 1470 –
Patronato – CEP: 24435-005 – São Gonçalo – Rio de Janeiro - Brazil
6Laboratório de Pesquisa em Fármacos – Colegiado de Ciências Farmacêuticas –
Universidade Federal do Amapá – Rodovia Juscelino Kubitschek – KM – 02 – Jardim
Marco Zero – CEP: 68903-419 – Macapá – AP – Brazil
7Laboratório de Tecnologia de Produtos Naturais – LTPN – Departamento e Tecnologia
Farmacêutica – Faculdade de Farmácia – Universidade Federal Fluminense – UFF Rua:
Mario Viana, 523 – CEP: 24241-000 – Santa Rosa – Niterói – RJ – Brazil
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CONCLUSÕES
O presente trabalho permitiu concluir que:
As rotas biosintéticas na espécie Manilkara subsericea favorecem a formação de
triterpenos pentacíclicos do tipo olean-12-eno e ursan-12-eno, permitindo sugerir estas classe
como marcadores químicos;
A presença de esqualeno, um precursor acíclico de triterpenos no óleo essencial obtido
reforça a importância da rota de formação de triterpenos nessa espécie;
Os flavonóides obtidos pertencem a classe dos flavonóis, o que está de acordo com
dados da literatura sobre o gênero;
O valor requerido do equilíbrio hidrófilo-lipófilo do miristato de octildodecila é 10,75.
Este dado é importante para obtenção de nanoemulsões contendo este óleo, permitindo a
redução de etapas no desenvolvimento de futuros produtos com essa matéria prima;
A nanoemulsão contendo fração apolar de frutos de Manilkara subsericea é capaz de
induzir mortalidade em Dysdercus peruvianus;
A nanoemulsão contendo fração apolar de frutos de Manilkara subsericea não é capaz
de inibir a acetilcolinesterase de peixe elétrico, sendo que a atividade sobre essa enzima é
umas das principais causas de efeitos tóxicos sobre organismos aquáticos. Portanto, é possível
que esse produto seja seguro para peixes;
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A nanoemulsão contendo fração apolar de frutos de Manilkara subsericea não exerceu
efeito letal sobre camundongos, sugerindo uma possível segurança para mamíferos;
Nanoemulsões contendo produtos de origem natural são uma alternativa viável e
promissora no desenvolvimento de produtos inseticidas.
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PERSPECTIVAS FUTURAS
O presente trabalho abre uma série de perspectivas, que irão contribuir para bases científicas
sólidas, dentre elas, podemos citar:
Agregar valor econômico a plantas das restingas, que são espécies fascinantes capazes
de produzir substâncias que podem servir de protótipo ou matéria-prima para diversos
produtos, como inseticidas;
Criação de cultivares de Manilkara subsericea para aumentar a biomassa e atender as
possíveis demandas futuras;
Modificações genéticas em Manilkara subsericea para favorecer as rotas biosintéticas
produtoras de determinadas substâncias (p.ex. triterpenos), aumentando o rendimento em
termos de substâncias ativas;
Preparação de novas nanoemulsões contendo substâncias inseticidas a partir da
formulação desenvolvida nesse trabalho.