i
LEILANE HESPPORTE IWAMOTO
AVALIAÇÃO DA ATIVIDADE FARMACOLÓGICA DE EXTRATO
BRUTO DICLOROMETÂNICO DAS FOLHAS DE PIPER
UMBELLATUM MICROENCAPSULADO E LIVRE PADRONIZADO
EM 4-NEROLIDILCATECOL
PHARMACOLOGICAL ACTIVITY EVALUATION OF
MICROENCAPSULATED AND FREE CRUDE DICHLOROMETHANE
EXTRACT FROM LEAVES OF PIPER UMBELLATUM
STANDARDIZED IN 4-NEROLIDYLCATECHOL
Piracicaba 2014
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Universidade Estadual de Campinas Faculdade de Odontologia de Piracicaba
LEILANE HESPPORTE IWAMOTO
AVALIAÇÃO DA ATIVIDADE FARMACOLÓGICA DE EXTRATO BRUTO DICLOROMETÂNICO DAS FOLHAS DE PIPER UMBELLATUM
MICROENCAPSULADO E LIVRE PADRONIZADO EM 4-NEROLIDILCATECOL
PHARMACOLOGICAL ACTIVITY EVALUATION OF MICROENCAPSULATED AND FREE CRUDE DICHLOROMETHANE EXTRACT FROM LEAVES OF
PIPER UMBELLATUM STANDARDIZED IN 4-NEROLIDYLCATECHOL
Dissertação apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Mestra em Odontologia, na Área de Farmacologia, Anestesiologia e Terapêutica
Dissertation presents to the Piracicaba Dental School of University of Campinas in partial fulfillment of the requiriments for the degree of Master in Dentistry in Farmacology, Anesthesiology and Terapeutics area.
Orientador(a): Prof. Dr. Rodney Alexandre Ferreira Rodrigues Coorientador: Prof(a). Dr(a). Mary Ann Foglio
Coorientador: Prof. Dr. João Ernesto de Carvalho
Este exemplar corresponde à versão final da dissertação defendida por Leilane HespporteIwamoto e orientada pelo Prof. Dr. RodneyAlexandre Ferreira Rodrigues
____________________
Assinatura do orientador
Piracicaba 2014
vii RESUMO
A espécie Piper umbellatum L., sinonímia Pothomorphe umbellata (L.) Miquel, é conhecida popularmente como pariparoba, caapeba e malvarisco. Tendo em vista as diferentes atividades biológicas comprovadas para a espécie Piper umbellatum e a técnica de microencapsulação por Spray Dryer, o objetivo desse trabalho foi avaliar a atividade farmacológica de extrato bruto padronizado (EBP) de P.
umbellatum em modelos de câncer, inflamação, úlcera e verificar se as
micropartículas de EBP apresentam atividade antiproliferativa in vitro. O extrato bruto diclorometânico de P. umbellatum possui 23,9% de 4-nerolidilcatecol aproximadamente. Foi avaliado a sua atividade anticâncer em modelo in vivo de tumor sólido de Ehrlich, atividade anti-inflamatória no edema da pata induzido por carragenina e peritonite. EBP foi capaz de reduzir o crescimento do tumor, quando administrado diariamente por via oral, sem sinais de toxicidade. Além disso, diminuiu o edema de pata e a migração de leucócitos no modelo de peritonite dessa forma, acredita-se existir uma relação entre a atividade anticâncer e anti-inflamatória. Também foi comprovada sua a atividade antiulcerogênica e gastroprotetora, relatada pelo uso popular no tratamento de úlceras gástricas. Os mecanismo de ação gastroprotetor envolvido na manutenção dos grupos sulfidrílicos, aumento de glutationa e de muco relacionam sua atividade antiulcerogênica à atividade antioxidante já descrita na literatura. O processo de microencapsulação por Spray Dryer do extrato bruto diclorometânico de P.
umbellatum com o amido modificado Purity Gum 1773® permitiu alterar a
propriedade graxa inerente a este extrato, insolúvel em água e foi capaz de manter sua atividade antiproliferativa in vitro. Dessa forma, a técnica de Spray Dryer pode ser um caminho para desenvolver um extrato seco de P. umbellatum. Portanto, podemos concluir que P. umbellatum pode ser fonte promissora para o desenvolvimento de novos agentes terapêuticos, tanto para o tratamento de úlceras gástricas quanto para inflamação e câncer.
ix ABSTRACT
The species Piper umbellatum, is popularly known as “pariparoba”, “caapeba” and “malvarisco”. Despite the different biological activities certified for the species P.
umbellatum and the technique of microencapsulation by Spray Dryer, the aim of this
study was to evaluate the pharmacological activity of dichloromethane crude extract (DCE) from Piper umbellatum leaves in models of cancer, inflammation and gastric ulcer and also evaluate the antiproliferative activity of microparticles containing standardized crude extract (SDE) in a panel of human tumor cell lines. The SDE from P. umbellatum containing 23,9% of 4-nerolidylcatechol approximately and its in
vivo anticancer activity in the Ehrlich solid tumor model as well as its
anti-inflammatory activity on carrageenan induced paw edema and peritonitis. The SDE presented in vitro and in vivo antiproliferative activity, being capable to reduce tumor growth when administered daily by oral route, without signals of toxicity. It was also reduced paw edema induced and leukocyte migration on carrageenan induced peritonitis model. Thus establishing a relationship between the anticancer and anti-inflammatory activities.It was also confirmed the popular use of P. umbellatum in the treatment of gastric ulcers, as SDE presented gastroprotective action dependent of the sulfhydryl groups, increase glutatione and mucus pathway and possibly for its antioxidant potential. The microencapsulation process by Spray Dryer of SDE of P.
umbellatum with modified starch (Purity Gum 1773®) allowed improvement of the
extract solubility, without interfere on its in vitro antiproliferative activity. Thus, the technique of Spray Dryer may be a good alternative to the development of a dry extract of P. umbellatum. Therefore, we conclude that P. umbellatum is a promising source for the development of new therapeutic agents for the treatment gastric ulcers, inflammation and cancer.
xi SUMÁRIO
DEDICATÓRIA xiii
AGRADECIMENTOS xv
LISTA DE FIGURAS xvii
LISTA DE TABELAS xix
INTRODUÇÃO 1
CAPÍTULO 1: Anticancer and anti-inflammatory activities of a Piper
Umbellatum leaves standardized dichloromethane extract.
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CAPÍTULO 2: Atividade antiulcerogênica de extrato bruto
diclorometânico de Piper umbellattum e estudo de mecanismo de ação envolvido.
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CAPÍTULO 3: Microencapsulação do extrato de pariparoba através de spray drying, empregando-se um amido modificado: atividade
antiproliferativa in vitro e características das partículas
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DISCUSSÃO 77
CONCLUSÃO 81
REFERÊNCIAS 83
ANEXO 1: Comitê de ética de uso animal (CEUA/UNICAMP 2868-1) 87 ANEXO 2: Comitê de ética de uso animal (CEUA/UNICAMP 3182-1) 88 ANEXO 3: Comitê de ética de uso animal (CEUA/UNICAMP 3052-1) 89 ANEXO 4: Comitê de ética de uso animal (CEUA/UNICAMP 3301-1) 90 ANEXO 5: Autorização de acesso e de remessa de componente do
patrimônio genético (CGEN)
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ANEXO 6: Confirmação de submissão de artigo na revista científica Phytomedicine
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Dedicatória
Esse trabalho é dedicado à minha família, meu porto seguro, meu tudo. E também ao meu noivo pela força e compreensão. Amo vocês!!!
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AGRADECIMENTOS
A Deus, por me guiar e estar presente em todos os momentos;
Ao meu orientador, Prof Dr. Rodney Alexandre Ferreira Rodrigues pela confiança, amizade e compreensão;
Aos meus co-orientadores João Ernesto de Carvalho e Mary Ann Foglio pelo carinho e atenção em todos os momentos;
À Faculdade de Odontologia de Piracicaba e ao programa de Farmacologia Anestesiologia e Terapêutica;
À minha família, por permitir buscar meus sonhos e me apoiar sempre.
Ao meu noivo Lindo pelo amor, apoio nos momentos mais difíceis e compreensão; Às minha amigas Gica e Marias (Leila e Gabis). Não importa a distância, sempre
estaremos juntinhas.
Aos meus queridos amigos e colegas do laboratório da Divisão de química de produtos naturais: Ilzinha, Núbia, Natália, Rô. E também da Farmacologia: Sica,
Paulete, Mi, Paula, Karissima, Thais, Aninha, Fabi, Rafa, Dri, Laricota, Gi L., Ana P.
Em especial, gostaria de agradecer à Debora Barbosa Vendramini Costa por estar presente em todos os momentos, mesmo estando nos EUA.
Muito obrigada!!!!
À família CPQBA, que me ensinou a trabalhar em equipe e onde quer que esteja, sempre estará no meu coração....
Amo muito tudo isso!!!!
Ao CNPQ, pela concessão da minha bolsa de mestrado; À CAPES e FAPESP
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xvii
LISTA DE FIGURAS
Figura 1- Foto de folhas de Piper umbellatum no campo experimental do CPQBA/UNICAMP.
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Figura 2 - Estruturas químicas de compostos isolados das folhas de
Piper umbellatum.
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Capítulo 1:
Figure S1: Analytical curve of 4-nerolidylcatechol. 18 Figure S2: HPLC/DAD chromatogram of dichloromethane extract from
P. umbellatum leaves.
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Figure 1: Relative tumor weight of Ehrlich solid tumor after oral daily 12 day-treatment with Vehicle and P. umbellatum SDE.
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Figure 2: Anti-inflammatory effect of P.umbellatum SDE versus time after inflammatory stimulus on carrageenan-induced paw edema.
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Figure 3: Effect of P.umbellatum SDE on carrageenan induced peritonitis, expressed as leukocyte count (cells/mL).
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Capítulo 2:
Figura 1: Efeito da administração oral única de EBP em modelo de úlcera induzida por etanol com tratamento prévio de NEM em ratos
Wistar.
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Figura 2: Efeito da administração oral única de EBP em modelo de úlcera induzida por etanol com tratamento prévio de L-Name em ratos
Wistar.
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Figura 3: Efeito da administração oral única de EBP em modelo de úlcera induzida por etanol com tratamento prévio de Indometacina em ratos Wistar.
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Figura 4: Efeito da administração oral de EBP (18 mg/kg) na produção do muco gastroprotetor em modelo experimental de úlcera induzida por etanol em ratos Wistar.
xviii Capítulo 3:
Figura 1: As amostras de micropartículas de Piper umbellatum e o controle processados por Spray drying (A e B= M10, M20 e C= controle).
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Figura 2: Morfologia externa de micropartículas de EBP e controle pela técnica de Spray drying por microscopia eletrônica de varredura (MEV/FEG) em um aumento de 2000 e 5000 X. (1 e 2- M10 e 3 e 4-M20 e 5-6 Controle).
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Figura 3: Gráficos demonstrativos da atividade antiproliferativa in vitro das amostras 1- Doxo (controle positivo), 2- Controle (controle negativo), 3- EBP, 4- M10, 5- M20 em cultura de células tumorais humanas relacionando porcentagem de crescimento versus concentração (µg/ml). Ensaio da Sulforrodamina B após 48h de exposição).
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LISTA DE TABELAS
Capítulo 1:
Table 1: Concentration (µg/mL) of Piper umbellatum SDE and Doxorubicin (DOXO) required for total growth inhibition of cell lines (TGI valuesa).
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Table 2: Haemogram parameters and organs weight (mean ± SEM) from animals treated (p.o) with Vehicle and P. umbellatum SDE during 21 days.
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Table S1: Inhibitory effect of P.umbellatum SDE versus time after inflammatory stimulus on carrageenan-induced paw edema.
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Capítulo 2:
Tabela 1: Parâmetros avaliados nas lesões ulcerativas no estômago. 39 Tabela 2: Efeito da administração oral única de EBP em modelo de úlcera gástrica induzida por etanol em ratos Wistar.
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Tabela 3: Efeito da administração oral única de EBP em modelo de úlcera gástrica induzida por AINEs em ratos Wistar.
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Tabela 4: Efeito da administração intraduodenal de EBP em modelo de ligadura do piloro em ratos.
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Tabela 5: Efeito da administração oral de EBP (18mg/kg) na determinação de níveis de glutationa em modelo experimental de úlcera induzida por etanol em ratos Wistar
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Capítulo 3:
Tabela 1: Formulação de emulsão para obter microcápsula de EBP de
Piper umbellatum secas em mini - Spray Dryer.
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Tabela 2: Distribuição granulométrica das amostras de EBP de Piper
umbelatum microencapsuladas (M10, M20) e seu respectivo controle
expresso µm
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Tabela 3: Valores de TGI em µg/mL, de Doxo (controle positivo), EBP, M10, M20 e PLA (controle negativo), concentrações necessárias para inibir totalmente o crescimento celular.
1 INTRODUÇÃO
Atualmente, a descoberta de novos fármacos é um grande desafio para a indústria farmacêutica. Os investimentos em pesquisa e desenvolvimento são grandes, porém contrasta com poucos medicamentos que chegam ao mercado (Ferreira et al., 2011; Munos, 2009). No Brasil, ainda é muito pouco explorada a diversidade dos biomas como fonte de novas substâncias de interesse farmacêutico, contudo, as pesquisas para descoberta de protótipos de fármacos e também de fitoterápicos, desencadeiam além do avanço da pesquisa, o desenvolvimento tecnológico do país (Barreiro e Bolzani, 2009).
O uso de plantas na medicina popular é considerado como uma prática frequente e antiga, utilizada pelo homem no tratamento de vários tipos de enfermidades. Esse uso está ligado à sabedoria popular, uma vez que isso acontece dentro de um contexto histórico (Oliveira e Araújo, 2007). Dessa forma, as plantas medicinais possuem um enorme potencial terapêutico. Estudos recentes, estimam que cerca de 75% de todos os fármacos utilizados são derivados diretamente ou indiretamente de produtos naturais (Newman e Cragg, 2013).
A espécie Piper umbellatum L., sinonímia Pothomorphe umbellata (L.)
Miquel (Figura 1), nome popular pariparoba, caapeba e malvarisco é utilizada
popularmente como analgésica, antimalárica, sedativa, diurética (Hammer e Johns, 1993) e antiulcerogênica (Balbach, 1971).
Na literatura científica, o extrato etanólico das folhas de P. umbellatum possui ação anti-inflamatória e analgésica (Perazzo et al., 2005), antifúngica (Rodrigues et al., 2012) e antioxidante (Lopes et al. 2013). Além disso, esse mesmo extrato etanólico de raízes é fotoprotetor (Ropke et al., 2003, 2005, 2006), antioxidante (Tabopda et al., 2008) e antituomoral (Brohem et al., 2009). Já o extrato metánólico das folhas é antioxidante (Agbor et al., 2007), o extrato diclorometânico é considerado antitumoral (Sacoman et al., 2008) e o extrato aquoso anticonvulsivante (Okunrobo et al., 2013). Existe cerca de 94 usos medicinais tradicionais para P. umbellatum (Roersch, 2010).
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P. umbellatum é um arbusto ereto de até 2,5 metros de altura, ramificado
com hastes articulares, possui influrescência em formato de espigas de 4 a 8 cm de comprimento. As folhas são largas (15 - 23 cm), pecioladas e codiformes (em formato de coração) (Gilbert e Favoreto, 2010). Segue foto de P. umbellatum no campo experimental do CPQBA/UNICAMP.
Figura 1: Foto de Piper umbellatum no campo experimental do CPQBA/UNICAMP. Legenda: 1-Arbusto, 2- espigas e 3- folhas.
Além dos compostos fitoquímicos isolados, alguns estudos de toxicidade aguda, sub-crônica e de mutagenicidade através do teste de micronúcleo não revelaram toxicidade (Perazzo et al., 2005; Barros et al., 2005; Bouzada et al., 2009; Valadares et al., 2007).
Por meio dos estudos fitoquímicos, foram identificados nos extratos de
P. umbellatum o terpeno glicosilado (roseosídeo presente também na Vinca rosea),
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(fenilpropanoide e principal metabólito secundário dessa espécie) (Kijoa et al., 1980; Isobe et al., 2002; Tabopda et al., 2008; Baldoqui et al., 2009) (Figura 2).
Figura 2: Estruturas químicas de compostos isolados das folhas de Piper umbellatum. (Fonte: Adaptado de Tabopda et al., 2008 e Baldoqui et al., 2009). Legenda: 1- piperumbellactama A, 2- piperumbellactama B, 3 - piperumbellactama C, 4 – piperumbellactama D, 5 – N-hidroxi-aristolactama II , 6- N-p-cumaroil-tiramina ama, 7- 4-Nerolidilcatecol, 8 -rel-(6S, 9S)-roseosídeo (terpeno glicosilado), 9- C-glicosilflavona-O-glicosídeo, vitexina 2"-O-β-D-glucopiranosídeo, 10- C-glicosilflavonas, apigenina-8-C-β-D-glucopiranosídeo, 11- orientina 8-C-β-D-apigenina-8-C-β-D-glucopiranosídeo, 12- sesamina, 13- 5-hidroxi-7,3',4'-trimetoxi-flavona, 14- Velutina, 15- diidrocubebina.
CÂNCER E INFLAMAÇÃO
O câncer consiste em alterações celulares caracterizadas pela falta de controle sobre a proliferação, diferenciação e morte celular (Mesquita et al., 2009). Essas alterações podem originar um conjunto de mais de 100 doenças nas quais as células apresentam um crescimento desordenado e podem espalhar-se para
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outras regiões do corpo (INCA, 2014). No Brasil, as estimativas realizadas para o ano de 2014 e válidas para 2015 apresentam 576.000 novos casos de câncer sendo os mais incidentes: pele do tipo não melanoma, próstata e mama feminina (INCA, 2014).
O surgimento do câncer (carcinogênese) se caracteriza pela progressiva aquisição de um fenótipo neoplásico pelas células normais (Hanahan e Weinberg, 2011). Cada modificação genética adquirida proporciona, às células tumorais, um tipo de vantagem, constituindo características como a proliferação auto-sustentada, evasão dos sinais de supressão de crescimento, ativação de invasão e metástase, imortalidade replicativa, indução de angiogênese e resistência aos sinais de morte celular (Hanahan e Weinberg, 2000).
Em 1863, Rudolf Virchow foi o primeiro pesquisador a relatar a presença de um infiltrado leucocitário no tecido neoplásico, que foi interpretado como prova da origem de um tumor, a partir de regiões inflamadas (Balkwill e Mantovani, 2001). A inflamação é uma resposta fisiológica à infecção ou lesão tecidual, caracterizada por vasodilatação com aumento da permeabilidade vascular e recrutamento de células inflamatórias como neutrófilos, monócitos, macrófagos e linfócitos. (Medzzhitov, 2010).
Há uma ligação entre câncer e inflamação, que pode ser descrita em duas vias: uma extrínseca, onde as condições inflamatórias ajudam no desenvolvimento do câncer e uma intrínseca, cujas células tumorais promovem o início do processo inflamatório, constituindo um microambiente favorável ao desenvolvimento tumoral (Colotta et al., 2009). Schetter e seus colaboradores (2010) demonstraram que 25% dos tipos de câncer estão relacionados a infecções crônicas e outros tipos de inflamação.
Atualmente, maioria dos agentes antineoplásicos utilizados possuem ação antiproliferativa, atuando sobre o processo de divisão celular, porém, afetam também as células normais de proliferação rápida, promovendo efeitos tóxicos indesejáveis. Nesse sentido, a busca por novos medicamentos que tenham como
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alvos componentes do microambiente tumoral e que tenham menos efeitos colaterais torna-se um desafio.
Estudos prévios realizados por nosso grupo avaliaram a ação in vitro e in
vivo do extrato bruto diclorometânico (EBP) obtido das folhas de P. umbellatum,
mostrando que o tratamento intraperitoneal com EBP (200 mg/kg) aumentou o tempo de sobrevida de animais portadores de tumor ascítico de Ehrlich (Sacoman
et al., 2008).
Tendo em vista a necessidade por novas terapias, especialmente focando o ambiente tumoral e o potencial de Piper umbellatum como um agente anticâncer, um dos objetivos desse estudo foi avaliar a ação do extrato bruto diclorometânico, agora padronizado em 23,92% de 4-NC em modelo de tumor sólido de Ehrlich, bem como avaliar sua toxicidade após tratamento de 21 dias. Além disso, avaliou-se também sua ação anti-inflamatória, buscando evidências da relação entre a ação antitumoral e anti-inflamatória.
ÚLCERA
As úlceras pépticas são caracterizadas por danos na mucosa gástrica ou duodenal e afetam cerca de 4 milhões de pessoas por ano em todo o mundo (Thorsen et al., 2013). Segundo a Federação Brasileira de Gastroenterologia (2014) não há uma estatística em relação ao número de potenciais portadores de úlcera péptica no país.
A fisiopatologia da úlcera consiste em um desequilíbrio de fatores protetores como prostaglandina, muco, bicarbonato, fluxo sanguíneo adequado e óxido nítrico e fatores lesivos como pepsina, ácido clorídrico e radicais livres (Malfertheiner et al., 2009). Também pode ser causada em processo infeccioso por
Helicobacter pylori, agentes antiplaquetários tais como o ácido acetilsalicílico,
fármacos anti-inflamatórios não-esteróides (AINES), bisfosfonatos orais, cloreto de potássio, medicações imunossupressoras, consumo de álcool e tabagismo (Al Batran et al., 2013).
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O muco e o bicarbonato constituem a primeira linha de defesa da mucosa gástrica, sendo secretados pelas células superficiais do epitélio gástrico. O muco é viscoso, como um gel transparente, é constituído de 95% de água e 5% de glicoproteínas (mucinas) cuja produção é estimulada pela gastrina, secretina, agentes colinérgicos e prostaglandinas (Reppeto e Lleusy, 2002). Já o bicarbonato após ser liberado é retido pelo muco, mantendo o pH neutro na superfície das células epiteliais (Banić et al., 2011).
Um importante tratamento para a úlcera gástrica é a administração de antagonistas de receptores H2, tais como cimetidina, ranitidina, famotidina e nizatidina, que competem com a histamina pela ação sobre o receptor. A administração de inibidores da bomba de prótons (omeprazol, lanzoprazol, rabeprazol, pantoprazol e esomeprazol) também constitui uma importante linha terapêutica (Goodman e Gilman, 2010). As substâncias antiácidas como bicarbonato de sódio, magnésio, alumínio ou anti-ácidos neutralizam o ácido gástrico e são usados como adjuvantes no tratamento da úlcera gástrica (Rang e Dale, 2007).
No tratamento da úlcera induzida por Helicobacter pylori, administra-se antibióticos associados aos antagonistas ou inibidores da bomba de prótons, conhecido como esquema tríplice com omeprazol 20 mg, lanzoprazol 30 mg, pantoprazol 40 mg, rabeprazol 20 mg ou esomeprazol 40 mg + claritromicina 500 mg + amoxicilina 1g. Ainda tratamentos baseados na inclusão de azitromicina possibilitaram redução dos efeitos colaterais (Mincis et al., 2011).
Por outro lado, o uso crônico dos inibidores de bomba de prótons inibe irreversivelmente a enzima H+/K+-ATPases, como consequência há aumento da secreção gástrica compensatória e hipergastrinemia. Os efeitos adversos são náuseas, dor abdominal, prisão de ventre, flatulência e diarréia (Goodman e Gilman, 2010). Segundo Braga e colaboradores (2011), o uso a longo prazo de omeprazol em humanos pode estar relacionado com a proliferação de células e tumores carcinoides.
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Apesar da eficiência das terapias em uso, muitas apresentam efeitos colaterais, o que estimula a busca por novos agentes antiulcerogênicos. Nesta busca, os produtos naturais se destacam pois muitas plantas são usadas na medicina popular por suas propriedades antiulcerogênicas. Dentre as espécies relatadas popularmente como antiulcerogênica encontra-se a Piper umbellatum. A fim de comprovar seu uso popular, um dos objetivos desse trabalho foi avaliar a ação gastroprotetora do extrato diclorometânico das folhas de P. umbellatum.
Novas tecnologias e inovações para garantir o efeito terapêutico das plantas medicinais vêm sendo empregados na fabricação de medicamentos fitoterápicos (Feltrin e Chorilli, 2010).
MICROENCAPSULAÇÃO
A microencapsulação é uma tecnologia capaz de encapsular materiais sólidos, líquidos ou gasosos em pequenas cápsulas permitindo a liberação controlada sob condições específicas (Fang e Bhandari, 2010).
A primeira aplicação deste processo foi em 1954 com a produção de um papel de cópia sem carbono. Nessa mesma década, ocorreram as primeiras pesquisas na área farmacêutica, com o uso das microcápsulas visando aumentar a estabilidade tanto física, quanto química de uma droga ou modificar sua liberação em alvos específicos de ação (Ré, 2000)
A microencapsulação pode originar partículas com diferentes morfologias que podem ser definidas como microesferas e microcápsulas. Nas microesferas, o recheio (princípio ativo) está uniformemente distribuído na matriz do material encapsulante, já nas microcápsulas existe um núcleo único envolvido por uma cápsula do material encapsulante (Munin e Edwards-Lévy, 2011). Além disso, o tamanho da micropartícula deve ser entre 1 e 1000 µm (Pereira, 2006).
A obtenção das micropartículas pode ser método físico: atomização (spray drying), leito fluidizado, extrusão centrífuga com múltiplos orifícios, co- cristalização e liofilização (freeze drying); métodos químicos: inclusão molecular e
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polimerização interfacial; métodos físico-químicos: coacervação, separação por fase orgânica, pulverização em agente formador de reticulação química e envolvimento lipossômico (Munin e Edwards-Lévy, 2011)
A técnica de Spray Drying consiste na formação de micropartículas por meio da atomização de uma solução constituída de princípio ativo e material encapsulante. A formação das micropartículas ocorre por contato destas com um gás (ar) aquecido promovendo a evaporação do solvente. As micropartículas passam pelo ciclone e caem no frasco coletor (Fang e Bhandari, 2010).
Existem muitos materiais de paredes usados como encapsulantes para compor a microcápsula, como amido, ciclodextrinas, dióxido de silício coloidal, fosfato tricálcico, gelatina, goma arábica, lactose, maltodextrina, celuloses (carboximetilcelulose, acetilcelulose, metilcelulose, etilcelulose e nitrocelulose); lipídios (parafina, cera, ácido esteárico, triesterina, monoglicerídeo, óleos, gordura hidrogenada e diglicerídeos); proteínas (glúten, caseína, isolado protéico de soro de leite, gelatina e albumina).
O amido é um tipo de polímero degradável renovável, oriundo de várias fontes tais como o milho, a mandioca, dentre outras (Zhang et al., 2010). Apresenta-se de forma modificada, obtida por processos físicos, químicos, enzimáticos ou por meios genéticos, para melhorar suas propriedades funcionais específicas não encontrada nos amidos nativos (Zavareze et al., 2010). Essas modificações químicas e físicas podem ser por intercruzamento, a substituição, a oxidação e a pré-gelatinização (Kaur et al., 2012), tornando-se uma alternativa para melhorar suas limitações, aumentando sua viabilidade na indústria (Spier et al., 2010).
Dessa forma, considerando as diferentes atividades biológicas comprovadas para a espécie Piper umbellatum e a técnica de microencapsulação por Spray drying, o objetivo desse trabalho foi verificar a atividade farmacológica de extrato bruto padronizado (EBP) de Piper umbellatum para a obtenção de um fitoterápico. Assim, avaliou-se sua ação em modelos de câncer, inflamação e úlcera gástrica e verificou se a microencapsulação foi capaz de melhorar as características do extrato e manter sua atividade antiproliferativa in vitro.
9
CAPÍTULO 1: Anticancer and anti-inflammatory activities of a Piper
umbellatum leaves standardized dichloromethane extract
L.H. Iwamotoa,b,#, D.B. Vendramini-Costab,c,#,*, P.A. Monteirob, A.L.T.G. Ruiza,b, I.M.O. Sousab, M.A. Foglioa,b,d, J.E. de Carvalhoa,b,d, R.A.F. Rodriguesa,b
Affiliation
a Department of Pharmacology, Anesthesiology and Therapeutics, Faculty of Dentistry, University of Campinas. Av. Limeira, 901, Piracicaba-SP, 13414-903, Brazil.
b Chemical, Biological and Agricultural Pluridisciplinary Research Center, CPQBA, University of Campinas. Rua Alexandre Cazelatto, 999, Vila Betel, Paulínia – SP, 13148-218, Brazil.
c Department of Organic Chemistry, Institute of Chemistry, University of Campinas. Rua Josué de Castro s/n, Cidade Universitária Zeferino Vaz, Barão Geraldo, Campinas –SP, 13081-970, Brazil.
d Faculty of Pharmacy, University of Campinas, Cidade Universitária Zeferino Vaz, Campinas – SP, 13081-970, Brazil.
# equally contribution
* Correspondence to: Débora Barbosa Vendramini Costa. 8048 Oxford Avenue, B24. Philadelphia, PA, 19111, United States of America. Phone +1 (267) 9922027. Email: vendramini.debora@gmail.com
Abstract
Despite anticancer drug discovery advances, the worldwide cancer incidence is remarkable. Thus, there is continuous necessity on new therapies development considering also the tumor microenvironment that offers multiple targets for cancer therapy, including inflammation. Nature has been a source of medicinal substances for millennia. Nowadays, almost 75% of the anticancer agents used in chemotherapy
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are derived from natural products. Continuing our research on Piper umbellatum leaves anticancer activity, we evaluated a P. umbellatum leaves standardized dichloromethane extract (SDE), containing 23.92 ± 0.46% of 4-nerolidylcatechol, with anticancer (in vitro antiproliferative activity and Ehrlich solid tumor model) and anti-inflammatory (carrageenan-induced paw edema and peritonitis models) activities. SDE showed in vitro and in vivo antiproliferative activity, being capable to reduce Ehrlich tumor growth when administered daily by oral route, without signals of toxicity. SDE also reduced carrageenan-induced paw edema and leukocyte migration on carrageenan-induced peritonitis model. These results corroborated with our previous report suggesting that Piper umbellatum leaves anticancer activity could evolve antiproliferative and anti-inflammatory effects.
Key words: Piperaceae, Piper umbellatum, cancer, inflammation, mice, 4-nerolidylcatechol.
Abbreviations: 4-NC (4-nerolidylcatechol), SDE (standardized dichloromethane extract), DCE (dichloromethane crude extract), DMSO (dimethyl sulfoxide), DOXO (doxorubicin), total growth inhibition (TGI), phosphate buffered saline (PBS), one-way ANOVA (analysis of variance) sulforhodamine B (SRB), maximum tolerated dose (MTD), cyclooxygenase (COX), lipoxygenase (LOX), phospholipase A2 (PLA2).
1 Introduction
Nature has been a source of medicinal products for millennia, going along with the history of humanity. Due to the improvement on methods for isolation, identification and synthesis during the last century, many drugs have surged from natural sources. In chemotherapy field, around 75% of the anticancer agents used nowadays are derived from natural products of different origins, including plants, microorganisms and marine organisms (Newman and Cragg, 2012). One important example is the Piper genus (Piperaceae family), which comprises approximately 2000 species, distributed mainly in tropical areas and widely valued for their medicinal properties (Wang et al., 2014). Piper umbellatum L. is a shrub, popularly known in Brazil as “pariparoba”, “caapeba” and “malvarisco” (Roersch, 2010).
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This specie was included in the Brazilian Pharmacopoeia first edition (1926) and 94 traditional medicinal uses for P. umbellatum are registered (Roersch, 2010). Indeed, there are several pharmacological activities described, such as antioxidant (Lopes et al., 2013), anti-inflammatory and analgesic (Perazzo et al., 2005), antibacterial (Isobe et al., 2002), antifungal (Rodrigues et al., 2012), antitumor (Sacoman et al., 2008), protection against photoaging (Silva et al., 2009) and anticonvulsants (Okunrobo et al., 2013). Moreover, phytochemical studies of P.
umbellatum leaves extracts have demonstrated the presence of terpenes, alkaloids,
flavonoids, sterols, being the catechol 4-nerolidylcatechol (4-NC) the mainly compound (Isobe et al., 2002; Baldoqui et al., 2009; Roersch, 2010).
Previous studies performed by our group evaluated the in vitro and in vivo anticancer activities of P. umbellatum leaves dichloromethane crude extract (DCE) and its fractions, showing that intraperitoneal (i.p.) treatment with DCE (200 mg/kg) increased life span of Ehrlich ascitic tumor-bearing animals (Sacoman et al., 2008). Another study conducted by our group demonstrated that Piper regnellii DCE and its fractions were capable of inhibiting Ehrlich solid tumor development in mice model (Longato et al., 2011).
The emergence of a cancer (carcinogenesis) is a complex and multi-step process during which normal cells progressively acquire a neoplastic phenotype. Each genetic modification confers to tumor cells a kind of advantage, constituting the hallmarks of cancer, such as self-sustained proliferation, evasion of suppressing growth signals, resistance to cell death, limitless replication, inducing angiogenesis, and activating invasion and metastasis (Hanahan and Weinberg, 2011). Besides cancer hallmarks, tumor microenvironment also influence cancer development, and one prominent microenvironment stimuli in carcinogenesis is inflammation (Grivennikov et al., 2010).
Despite the advances in the field of anticancer drug discovery, the statistics are noteworthy; in 2012, 14.1 million new cases of cancer were diagnosed worldwide, with 8.2 million of deaths (Ferlay et al., 2013). Thus, there is still a necessity for the development of new therapies and in this search the tumor
12
microenvironment offers multiple targets for cancer therapy, including inflammation (Li et al., 2007).
Bearing in mind the need for new therapies, specially focusing in the tumor microenvironment and the potential of Piper umbellatum as an anticancer agent, in this study we evaluated the in vitro and in vivo antiproliferative activities of a standardized dichloromethane crude extract (SDE) from P. umbellatum leaves, containing 23.92% of 4-NC. We also evaluated its anti-inflammatory activity, looking for evidences of the relationship between the SDE anticancer and anti-inflammatory activities.
2 Materials and Methods
2.1 Phytochemistry
2.1.1 Plant material. Piper umbellatum (L.) Miq. leaves were collected in February 2013 at experimental field of the Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA – UNICAMP, Paulínia-SP, Brazil). A voucher specimen was deposited at the Herbarium of Institute of Biology, University of Campinas (UEC nº181.451). As P. umbellatum is a Brazilian native genetic material, the Genetic Patrimony Management Board (CGEN/MMA) has approved the present study, through Access and Shipment Component of Genetic Heritage for scientific research purpose (no. 010646/2012-4).
2.1.2 Dichloromethane crude extract production. Milled fresh leaves (1kg) were extract by maceration with dichloromethane (Dinamica®) (1:5 leaves:solvent, 3 x 90 min) at room temperature. After filtration, the filtrates were pooled, evaporated (40°C, BUCHI model RE 215) and lyophilized (Virtis, model 8L) until dryness, affording DCE (2% yield).
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2.1.3 Isolation of 4-Nerolidycatechol. DCE was previously cleaned-up for pigments and other lipophilic compounds (1g) through liquid partition with hexane: acetonitrile (1:1) (3 x 100mL). The acetonitrile phase (680mg) was then applied on a solid-phase extraction (SPE) cartridges C18-E (55 µM, 70 A, 5 g/20 mL) Phenomenex® preliminarily conditioned with 10 mL methanol and 10mL water, at 5 mL/min flow rate. SPE cartridge was eluted with 2 × 10 mL water:methanol (95:5; 50:50; 85;15 and 0:100, named as FA, FB, FC and FD, respectively), at 3.5 mL/min flow rate. Fraction FC (190mg) was analysed by RMN1H and 13C.
2.1.4 Chromatographic analysis. HPLC analysis followed a previously described protocol (Rezende and Barros, 2004). It was performed with a Shimadzu series HPLC system equipped with on-line degasser (DUG-2A), quaternary pump (LC-10AT), autosampler (SIL 20A HT), column heater (CTO 10AS Vp), and photodiode array detector (SPD-M10Vp), using a C18 column (4.6 mm X 250 mm, 5 µm particle size, Gemini, Phenomenex, Mac-clesfield, UK). Instrument control and data analysis was carried out using software Class VP 6.13 edition. The isocratic mobile phase was methanol-acetonitrile-water (62:20:18). Flow was set at 1.0 mL/min, injection volume 20µL and ultraviolet detection was at 282nm.
2.1.5 Quantification of 4-Nerolidycatechol. 4-NC was quantified in SDE by analytical curve. Stock solutions (2396 µg/mL) were prepared in methanol and successively diluted in the range of 48 to 957 μg /mL, two replicates each, in methanol. All samples were analyzed by HPLC as described in Chromatographic Analysis. A graphic correlating area under the curve (AUC) with the respective concentration was plotted and analyzed by linear regression using MS Excel software (supplementary information section).
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2.2 In vitro antiproliferative assay
2.2.1 Cell lines. Human tumor cell lines [UACC-62 (melanoma), U251 (glioma), MCF-7 (breast), NCI-H460 (lung, non-small cells), HT-29 (colon), PC-3 (prostate), 786-0 (kidney), NCI-ADR/RES (ovarian expressing multiple drugs resistance phenotype) and OVCAR-3 (ovary)] were kindly provided by National Cancer Institute (Frederick, MA, USA). Non-tumor cell line HaCat (human keratinocytes) was donate by Prof. Dr. Ricardo Della Coletta, FOP/UNICAMP.
2.2.2 Cell culture. Stock cultures were grown in medium RPMI 1640 (GIBCO) supplemented with 5% fetal bovine serum (FBS, GIBCO) and 10 U/mL penicillin, 10 µg/mL streptomycin at 37 ºC with 5% CO2.
2.2.3 Antiproliferative assay. Cells in 96-well plates (100 µL cells/well) were exposed to SDE (0.25, 2.5, 25 and 250 µg/mL in DMSO/RPMI) at 37 ºC, 5% of CO2 in air for 48 h. Doxorubicin (DOXO) was used as standard (0.025, 0.25, 2.5 and 25 µg/mL). Final DMSO concentration did not affect cell viability (0.25%). Before (T0 plate) and after sample addition (T1 plates), cells were fixed with 50% trichloroacetic acid and cell growth determined by spectrophotometric quantification (540 nm) of cellular protein content using sulforhodamine B (SRB) assay (Monks et al., 1991). The TGI (concentration that produces total growth inhibition) was determined through non-linear regression analysis using the concentration-response curve for each cell line in software ORIGIN 8.0® (OriginLab Corporation) (Shoemaker, 2006).
2.3 In vivo assays
2.3.1 Animals. Experiments were conducted with Balb/C and Swiss female mice (20-30g, 90 days old) from the Multidisciplinary Centre for Biological Investigation on Laboratory Animals Sciences (CEMIB-UNICAMP). Animals were maintained at the Animal facilities of Pharmacology and Toxicology Division, CPQBA, UNICAMP
15
(Paulínia-SP, Brazil), in a room with controlled temperature 25 ± 2ºC for 12 h light/dark cycle, with free access to food and water. Animal care and research protocols were in accordance with the principles and guidelines adopted by the Brazilian College of Animal Experimentation (COBEA). Protocols were approved by the Ethical Committee for Animal Research (CEUA), Institute of Biology, UNICAMP (# 2868-1, 3182-1, 3052-1 and 3183-1). Euthanasia was performed by deeping anaesthesia followed by cervical dislocation.
2.3.2 Drugs. Indocid (indomethacin 50 mg; Merck Sharp & Dohme), Carrageenan (Sigma-Aldrich), Dexametasone (Sigma-Aldrich). SDE was emulsified in Tween 80 (Sinth) 0.3% and dissolved in PBS, pH 7.0. Vehicle was PBS, pH 7.0 + Tween 80 (Sinth) 0.3%.
2.3.3 Acute toxicity. Swiss mice (n = 5) were kept in 12h fasting and then treated orally with SDE 1000 and 2000 mg/kg. Groups were observed during 4 hours and then daily for 15 days, for general toxicity signals evaluation [body weight loss, locomotion, behavior (agitation, lethargy), respiration, salivation, tearing eyes, cyanosis and mortality] (Lapa et al., 2008).
2.3.4 Subcronic toxicity. Balb/C mice (n = 6) were treated orally with vehicle and SDE (100, 200 and 400 mg/kg), daily, for 21 days. Mice were weighted (every three days) and daily observed for possible signals of toxicity (Lapa et al., 2008). At the 21th day, blood was collected from retro orbital plexus of each animal for hemogram analyses (Sysmex ® model Poch-100iV) evaluating total leukocytes (WBC), erythrocytes (RBC) and platelets (Pt) parameters. Animals were euthanized and liver, spleen and kidneys were macroscopically evaluated and weighted.
16 2.3.5 Ehrlich solid tumor assay
2.3.5.1 Cells maintenance and preparation. Ehrlich tumor cells were maintained in the ascitic form in Swiss mice by weekly transplantation of 5x105 cells/animal in PBS (pH 7.0) (Oloris et al., 2002). For the experiments, cells were prepared at the density of 1x106 cells/50 L/animal in PBS (Marchetti et al., 2012) after counting in Neubauer chamber with trypan blue, to exclude non-viable cells and debris.
2.3.5.2 Induction and treatments. Ehrlich cells suspension (1x106 cells/50
L/animal) were inoculated subcutaneously in the flank of Balb/C mice (n = 8). On 5th day, animals with palpable tumors were randomly divided into negative control (vehicle) and experimental (SDE – 100, 200 and 400 mg/kg) groups that were treated every day, orally, for 12 days. On 17th day, animals were euthanized and tumors were removed and weighted. The relative tumor weight was calculated as tumor weight divided by corporal weight. The growth inhibition ratio was calculated according to the formula: [(A-B)/A] x100, where A: mean of group control relative tumor weight, B: mean of group treated relative tumor weight (Marchetti et al., 2012).
2.3.6 Carrageenan-induced paw edema. Experiments were designed according to Posadas et al. (2004) with modifications. Right hind paw basal volume of Balb/C mice (n = 8) was measured using a caliper (Mitutoyo®) according to the elipse oblate formula: V=4
3𝜋𝑎
2𝑏, where a is the paw latero-lateral width and b is the dorsal-ventral width. Then animals were randomly divided into negative control (vehicle), positive control (indomethacin, 10 mg/kg) and experimental (SDE – 100, 200 and 400 mg/kg) groups that were orally treated one hour before inflammation induction by carrageenan solution inoculation (2.5 mg/mL, 40 µL/animal) into the right hind footpad. The right footpad volume was evaluated at 1.5, 3.0, 4.5, 6.0, 24, 48, and 72 h after carrageenan inoculation. Results were expressed as paw edema variations (mL, difference between measured and basal paw volumes) versus time.
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2.3.7 Carrageenan-induced peritonitis. Balb/C mice (n = 8) were randomly divided into negative control (vehicle), positive control (dexamethasone, 2.5 mg/kg) and experimental (SDE, 200 mg/kg) groups that were orally treated one hour before peritonitis induction by carrageenan solution inoculation (500 μg/250μL/animal) into peritoneal cavity. Four hours later, mice were euthanized and the peritoneal cavity was washed with 5 mL of PBS containing heparin 5 IU/mL. Total leukocyte was analysed in peritoneal fluid using a haematology analyser (Sysmex ® model Poch-100iV).
2.4 Statistical analyses. The results are shown as mean ± SEM. The statistical significance of difference between groups was assessed by one-way ANOVA, followed by Newman-Keuls post hoc test using Graph Pad Prism 5.0 software. Values of p ≤ 0.05 were considered significant.
3 Results and Discussion
3.1 Quantification of 4-NC
4-nerolidylcatechol (94% of analytical purity) was identified by experimental data comparison with those reported by Baldoqui et al. (2009). In our study, it was possible to determine by HPLC-DAD quantitative analysis (correlation coefficient R2 = 0.9995 ± 0.0005; detection limit (LOD) = 11.6 µg/mL; quantification limit (LOQ) = 35.1 µg/mL) that P. umbellatum SDE presented 23.92 ± 0.46% of 4-NC yield, considering initial fresh leaves mass.
In spite of Sacoman et al. (2008) described that the most active fraction
in vitro comprised a mixture of steroids instead of the 4-NC fraction, this last
compound was selected as a chemical marker for extract standardization since this compound is readily isolated and easily quantified both by HPLC-UV-DAD or by
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GC/MS. Moreover, considering 4-NC activity as a potent antioxidant, this substance may be involved in the possible anti-inflammatory activity of SDE.
Figure S1: Analytical curve of 4-nerolidylcatechol.
Figure S2: HPLC/DAD chromatogram of dichloromethane extract from P.
19 3.2 In vitro antiproliferative assay
SDE showed a potent antiproliferative activity, leading to total growth inhibition of almost all tumor cell lines (TGI values between 6.8 and 14.9 µg/mL), excepting HT-29 cell line (colon, TGI = 207.3 µg/mL) (Table 1). Moreover, TGI value (144.6 µg/mL) for HaCaT cells (non-tumor cell line) was higher than those observed for most tumor cell lines, thus suggesting a selectivity for tumor cell lines. These promising in vitro antiproliferative results were in accordance with our previous work (Sacoman et al., 2008) and prompted the study in vivo models.
Table 1. Concentration (µg/mL) of Piper umbellatum SDE and Doxorubicin (DOXO) required for total growth inhibition of cell lines (TGI valuesa).
Cell lines Total growth inhibition (µg/ml)
Doxo SDE UACC-62 0.9 6.8 U251 1.6 8.2 MCF-7 0.2 9.3 NCI-ADR/RES 1.9 14.9 786-0 1.1 9.1 CNI-H460 1.9 11.5 PC-3 1.9 8.2 OVCAR-3 1.2 8.3 HT-29 6.2 207.3 HaCat 29.1 144.6
aTGI values were determined by non-linear regression analysis using the ORIGIN 8.0®
(OriginLab Corporation). Experiments were conducted in triplicate and results are representative of three different experiments.
Considering SDE chemical composition, the observed antiproliferative effect could be partially attributed to the presence of 4-NC and sterols β-sitosterol, stigmasterol and campesterol, as Sacoman et al. (2008) and Lopes et al. (2013) had identified these compounds in P. umbellatum dichloromethane extracts.
β-sitosterol induces apoptosis and G2/M arrest in MDA-MB-231 (breast), PC-3 (prostate) and HCT (colon) human tumor cell lines (Awad et al., 2001). Moon et
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al., (2008) in murine fibrosarcoma cells and human leukaemia also reported a
pro-apoptotic activity of β-sitosterol. Moreover, 4-NC also induces alterations in cell cycle profile of SK-Mel-147 (melanoma), promoting a G1 arrest (Brohem et al., 2012). It is interesting to notice that Sacoman et al. (2008) described that the steroids fraction showed a higher in vitro antiproliferative effect compared to the 4-NC fraction, and that Lopes et al. (2013) observed that dichloromethane extract was more potent than the 4-NC and sterol fractions in an in vitro antioxidant activity model, hypothesizing a synergic activity of these compounds.
3.3 In vivo assays
In order of confirm the in vitro P. umbellatum antiproliferative effect, SDE was evaluated in an Ehrlich solid tumor model in mice. Previous studies with P.
umbellatum DCE had described its in vivo activity in the Ehrlich ascitic tumor model
after intraperitoneal treatment (Sacoman et al., 2008). This model allows evaluation of bearing-animals life span; however, presents one limitation: when treatments were conducted by intraperitoneal route, samples were applied at the same place of Ehrlich tumor cells growth. This way, it was difficult to elucidate parameters of sample absorption and distribution. Herein, we described the systemic effects of P.
umbellatum SDE, as treatments were performed by oral route and tumor cells were
implanted in the flank subcutaneous of the animals.
3.3.1 Acute and Subchronic toxicity
Before the in vivo anticancer and anti-inflammatory experiments, an acute toxicity evaluation was conducted in order to determine the maximum tolerated dose (MTD) that could be used in the long-term studies without adverse effects. No evidence of toxicity was observed 4 hours after administration of SDE 1000 mg/kg by oral route, as well as during the following 14 days, when the animals were kept under observation. However, animals treated with 2000 mg/kg died after 4 hours.
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Therefore, MTD was determined as 1000 mg/kg dose for single treatment and to determine doses for repetitive treatments we considered the higher dose as 40% of MTD, as described by Mi et al. (2009), together with two lower doses. This way, in
vivo experiments were carried out with 100, 200 and 400 mg/kg of SDE, oral route.
In our previous study, a LD50 of 533.71 mg/kg was determined for a single treatment with P. umbellatum DCE by intraperitoneal route (Sacoman et al., 2008). Herein, oral LD50 of SDE could be considered in the range of 1000 to 2000 mg/kg. Such loss of toxicity after change the treatment route (intraperitoneal to oral route) may suggest that substances responsible for adverse effects in SDE could show low
bioavailability and/or be quickly metabolized when administrated by oral route. Moreover, when mice were treated every day, during 21 days, with P.
umbellatum SDE 100, 200 and 400 mg/kg, none toxic signals and none
haematological alterations were observed (Table 2). As most chemotherapeutic agents induce collateral effects, the observed results for P. umbellatum SDE were encouraging.
Table 2. Haemogram parameters and organs weight (mean ± SEM) from animals treated (p.o) with Vehicle and P. umbellatum SDE during 21 days.
Organs Vehicle 100 200 400 Liver (g) 0.048 ± 0.001 0.045 ± 0.001 0.046 ± 0.0029 0.051 ± 0.0009 Kidneys (g) 0.012 ± 0.0001 0.012 ± 0.0002 0.013 ± 0.0002 0.012 ± 0.0003 Spleen (g) 0.004 ± 0.0002 0.005 ± 0.0002 0.005 ± 0.0007 0.004 ± 0.0001 Haemogram WBC (106 /µL) 4.4 ± 0.001 3.3 ± 0.3 3.7 ± 0.7 4.7 ± 0.6 Haemoglo bin (g/dL) 0.012 ± 0.0001 14.2 ± 0.2 13.9 ± 0.5 13.9 ± 0.2 Platelet (103/µL) 0.004 ± 0.0002 1278 ± 28.9 1403 ± 76.3 1388 ± 61.9 Vehicle = PBS + tween 80 0.3%, pH 7.0; SDE doses = 100, 200 and 400 mg/kg
22 3.3.2 In vivo Ehrlich solid tumor assay
Solid tumors are structures resembling organs in their complexity and heterogeneity. Inside these tumors there are differences in pH, oxygen pressure, and nutrient flux which often contribute to tumor resistance to chemotherapy due to irregular drugs distribution inside the tumor matrix. Therefore, the development of experimental models to complement in vitro drug screening is necessary due to the limitations inherent to cell cultures to predict the behavior of solid tumors to chemotherapy (Tredan et al., 2007; Smith et al., 2005).
The in vivo anticancer activity of P. umbellatum SDE was evaluated in the Ehrlich solid tumor in mice. Ehrlich tumor is an aggressive and fast growing murine breast adenocarcinoma, which is able to develop both in ascitic or in solid form, depending on whether inoculated (intraperitoneally or subcutaneously, respectively) (Oloris et al., 2002). Ehrlich tumor cells generate a local inflammatory response characterized by the increased vascular permeability, which accounts for edema formation, cell migration and recruitment of the immune response (Stewart, 1959).
After 12 days of P. umbellatum SDE daily treatment, doses of 200 and 400 mg/kg inhibited tumor growth in 38.7 and 52.2%, respectively (p < 0.05), without signals of toxicity, while 100 mg/kg treatment was not effective (Figure 1). These results are in accordance with those previous described by our group (Sacoman et
al., 2008), with the benefit of the toxicity loss by changing route and treatment
frequency. As discussed for P. umbellatum SDE in vitro antiproliferative activity, 4-NC and sterols presents in SDE could be partly responsible for SDE in vivo antitumor activity.
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Figure 1. Relative tumor weight of Ehrlich solid tumor after oral daily 12 day-treatment with Vehicle and P. umbellatum SDE. Relative tumor weight expressed as tumor weight divided by body weight; Groups (n = 8) were treated daily (during 12 days) by oral route with Vehicle (PBS, pH 7.0 + Tween 80 0.3%) and SDE 100, 200 and 400 mg/kg; ANOVA (one-way), Newman-Keuls Multiple Comparison Test, *p<0.05, **p<0.01 = a significant compared to vehicle group.
Solid tumors are among the leading death causes in western countries, with growing incidence every year. Although the prognosis of these patients has been evolved because of early diagnosis and new antitumor therapies, there is still a need for new treatments (de Groot et al., 2007). Therefore, inhibition of tumor development by P. umbellatum SDE associated with low toxicity is an exciting result. Certain types of cancers induce an inflammatory microenvironment formation, which contributes to tumor development (Walczak, 2011). As previously mentioned, Hanahan and Weinberg (2011) included inflammation as a facilitator process, as it provides bioactive molecules such as growth, survival and angiogenic factors, enzymes that modify the extracellular matrix, among others. In some cases, the inflammation is already evident in early stages of tumor progression by promoting tumor development since the action of inflammatory cells can lead to mutagenic agents’ release (Vendramini-Costa and Carvalho, 2012).
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In view of the relationship between cancer and inflammation, we evaluated P. umbellatum SDE anti-inflammatory potential in experimental inflammation models in mice.
3.3.3 In vivo anti-inflammatory assays
The administration of carrageenan 2.5% in mouse hind footpad induced a biphasic inflammatory edema (Henriques et al., 1987). Immediately after carrageenan injection, there is a cascade of mediators release initiates with histamine, serotonin, bradykinin and phospholipase A2 (PLA2). These mediators promote an increase in vascular permeability and signal for arachidonate metabolites (prostaglandins, leukotrienes) and nitric oxide release, until the 6th hour. The second phase initiates after 24 hours, coinciding with a decrease in edema, associated with an increase in leukocytes migration, amplifying the inflammatory response and promoting a second edema within 72h (Posadas et al., 2004).
P. umbellatum SDE treatment significantly inhibited the first phase of
inflammation, in an independent-dose way, as well as indomethacin 10 mg/kg (Figure. 2). SDE was capable to inhibit inflammation up to 4.5 hours, period coincident with prostaglandin release, which could suggest an action on prostaglandins production (Fig. 2). In the second phase, all SDE doses inhibited inflammation at 48 hours while 400 mg/kg dose also inhibited the second inflammatory peak (72 h). This result suggests an effect on neutrophil mobilization, quite similar to the corticosteroids effects that greatly inhibit the cellular phase of inflammation. Table with % of inhibition per group can be found on support information section (Table S1).
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Figure 2. Anti-inflammatory effect of P.umbellatum SDE versus time after inflammatory stimulus on carrageenan-induced paw edema. Paw edema was measured with a caliper; results expressed as paw edema in mm3 (mean ± SEM); treatments: P.umbellatum SDE
(100, 200 and 400 mg/kg), Vehicle (PBS, pH 7.0 + Tween 80 0.3%) or indomethacin (10 mg/kg) one hour before intraplantar carrageenan 2.5% injection, n = 8 animals/group. ANOVA (one-way), Newman-Keuls Multiple Comparison Test; *p<0.05, **p<0.01 and ***p<0.001 in comparison to vehicle group.
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Table S1. Inhibitory effect of P.umbellatum SDE versus time after inflammatory stimulus on carrageenan-induced paw edema.
Treatments (mg/kg)
Time (hours) and % inhibition
1.5 3 4.5 6 24 48 72 96 Vehicle 0.15 ± 0.03 0.25 ± 0.03 0.19 ± 0.03 0.05 ± 0.01 0.04 ± 0.001 0.15 ± 0.01 0.16 ± 0.01 0.10 ± 0.02 Indometacin 10 0.11 ± 0.03 (28.7%) 0.10 ± 0.01*** (60.6%) 0.09 ± 0.02** (54.6%) 0.10 ± 0.02 0.02 ± 0.001 (51.2%) 0.08 ± 0.02 (45.3%) 0.13 ± 0.03 (17.7%) 0.10 ± 0.03 (4.9%) SDE 100 0.10 ± 0.02 (35.6%) 0.08 ± 0.02 *** (69.6%) 0.05 ± 0.02*** (74.5%) 0.03 ± 0.01 (45.6%) 0.01 ± 0.01 (75.3%) 0.11 ± 0.03 (27.3%) 0.11 ± 0.03 (29.8%) 0.07 ± 0.03 (29.9%) SDE 200 0.03 ± 0.01*** (79.7%) 0.04 ± 0.01*** (83.1%) 0.04 ± 0.01*** (77.0%) 0.07 ± 0.02 0.021 ± 0.01 (44.2%) 0.06 ± 0.02* (59.2%) 0.08 ± 0.02 (47.5%) 0.06 ± 0.02 (37.3%) SDE 400 0.02 ± 0.01*** (82.4%) 0.06 ± 0.01*** (76.9%) 0.058 ± 0.01*** (67.8%) 0.045 ± 0.01 (20.9%) 0.20 ± 0.01 (47.7%) 0.05 ± 0.02* (69.9%) 0.05 ± 0.02 (70.9%) 0.03 ± 0.01 (69.8%)
Paw edema was measured with a caliper; results expressed as paw edema in mm3 (mean
± SEM); treatments: P.umbellatum SDE (100, 200 and 400 mg/kg), Vehicle (PBS, pH 7.0 + Tween 80 0.3%) or indomethacin (10 mg/kg) one hour before intraplantar carrageenan 2.5% injection, n = 8 animals/group. ANOVA (one-way), Newman-Keuls Multiple Comparison Test; *p<0.05, **p<0.01 and ***p<0.001 in comparison to vehicle group.
Previous studies performed with P. umbellatum ethanolic extract demonstrated its anti-inflammatory activity, with inhibition of the first phase of inflammation (Perazzo et al., 2005). Another study showed the anti-inflammatory activity of a β-sitosterol rich fraction obtained from Sideris foetens, which was able to inhibit paw edema from 3 to 7 hours after inflammatory stimulus (Navarro et al., 2001). According to these authors, β-sitosterols could be responsible for inhibition of
27
arachidonate metabolites generation and neutrophil migration phase. This way, the anti-inflammatory effect herein described for P. umbellatum SDE, at higher dose, could be partly explained by the presence of sitosterols derivatives. Additionally, Nuñez et al. (2005) observed that P. umbellatum ethanolic crude extract and 4-NC inhibited the PLA2 enzymatic activity, which could also explain the SDE inhibitory effect on the first phase of inflammation (arachidonate metabolites generation).
Cytotoxic agents are able to inhibit the cellular phase of inflammation as demonstrated by Vendramini-Costa et al. (2010). These authors showed that doxorubicin inhibited the second phase of carrageenan-induced inflammation (after 24 hours of inflammation induction). As P. umbellatum SDE was able to inhibit carrageenan-induced inflammation second phase (Fig. 2,) and tumor cell proliferation (Table 1 and Fig. 1), we performed the carrageenan-induced peritonitis model to evaluate SDE activity on leukocyte migration.
Carrageenan when inoculated in the peritoneum exerts a chemotactic effect on inflammatory cells mediated by a synergistic action between prostaglandins, leukotriens and other chemotactic agents producing a sustained increase in postcapillary venule permeability, which leads to cellular infiltration (Damasceno et al., 2014).
In carrageenan-induced peritonitis model, leukocytes migration was inhibited both by dexamethasone (60.5%, 5 mg/kg) and P. umbellatum SDE (52%, 200mg/kg) (Figure. 3). These results corroborated that SDE could inhibit PLA2 activity in a similar way as dexamethasone. PLA2 is involved in arachidonic acid release from membrane phospholipids, which can be metabolized by COX, lipoxygenase (LOX) and cytochrome P450 enzymes. (Vendramini-Costa and Carvalho, 2012).
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Figure 3. Effect of P.umbellatum SDE on carrageenan induced peritonitis, expressed as leukocyte count (cells/mL). Results expressed as mean ± SEM (n = 8 animals/group); treatment: P.umbellatum SDE (200 mg/kg), Vehicle (PBS, pH 7.0 + Tween 80 0.3%) or dexamethasone (5 mg/kg) one hour before intraperitoneal carrageenan (500 µg/250 µL) injection. Peritoneal fluid was collected 4 hours after carrageenan stimulus. ANOVA, Newman-Keuls Multiple Comparison Test, **p<0.01 in comparison to vehicle group.
Based on the results presented here, we conclude that P.umbellatum SDE has promising antitumor and anti-inflammatory activities, which could be partially attributed to 4-nerolidylcatechol and phytosterols. In line with P. umbellatum SDE profile on paw edema and peritonitis model and previously described results on PLA2 inhibition, we hypothesize that SDE interferes on arachidonate metabolites generation, by inhibiting PLA2 or COX-2 activities. Further studies will be performed to clarify the biochemical pathways involved in these activities.
Acknowledgments
The authors thank the Chemical, Biological and Agricultural Pluridisciplinary Research Center (CPQBA/UNICAMP) for the infrastructure. Authors are grateful to financial support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the National Council for Scientific and
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Technological Development (CNPq) for the research fellowship (L.H. Iwamoto, 133897/2012-5).
Conflict of Interest
The authors declare no conflict of interests.
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