Prospection of the anti-inflamatory activity of Eugenia leitonii D. Legrand, a Brazilian native fruit

Orientador: Pedro Luiz RosalenDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Na busca por substâncias alternativas com atividade biológica, as espécies frutíferas nativas do Brasil demonstram potencial funcional, devido principalmente à presença de compostos fenólicos como flavonoides e antocianinas que apresentam em sua composição. Estudos realizados pelo nosso grupo de pesquisa relataram partes (semente, folha e polpa) das espécies de Eugenia (E. brasiliensis, E. uniflora, E. leitonii e E. myrcianthes) exibiram atividades antioxidante e anti-inflamatória. Em adição, a semente da espécie E. leitonii exibiu o melhor efeito na inibição da migração de neutrófilos na cavidade peritoneal em camundongos. Desta forma, dando continuidade à investigação de frutas nativas brasileiras pouco exploradas, os objetivos deste estudo foram avaliar a atividade anti-inflamatória, mecanismo(s) de ação e o perfil fitoquímico do extrato de sementes de Eugenia leitonii. O extrato hidroetanólico (80:20, v/v) da semente de E. leitonii (EL) foi avaliado por meio dos testes de viabilidade celular, determinação de mediadores inflamatórios (TNF- ?, IL-1? and CXCL2/MIP-2) e ativação de NF-?B em células RAW 264.7. O extrato de EL também foi avaliado in vivo, quanto aos níveis de mediadores inflamatórios (TNF- ?, IL-1? and CXCL2/MIP-2) no lavado peritoneal; ensaio de migração de neutrófilos induzido por carragenina; avaliação da expressão de moléculas de adesão (ICAM-1) e rolamento (P-selectina) no endotélio vascular e edema de pata induzido por carragenina. Por fim, foi quantificado o conteúdo total de compostos fenólicos e identificados por LC-MS/MS. Os resultados mostraram que o extrato de EL não alterou a viabilidade celular significativamente (P > 0,05) nas concentrações de 0,02 a 200 µg mL-1; em 200 µg mL-1 reduziram os níveis de TNF-? e CXCL2/MIP-2 e a ativação de NF-?B em células RAW 264.7 (P 0.05) cell viability at the concentrations ranging from 0.02 to 200 mg µl-1; at 200 mg µl-1 reduced the levels of TNF-? and CXCL2/MIP-2, and NF-kB activation in RAW 264.7 cells (P < 0.05). Mice pre-treated orally with EL extract (300 mg kg-1 ) showed significantly decreased levels of TNF-? and CXCL2/MIP-2, also there was a decrease in ICAM-1 and P¿selectin expression as compared with the control group (P < 0.05). The mice pretreated at doses 30, 100 e 300 mg kg-1 showed decrease of the influx of neutrophils into the peritoneal cavity of the mice as compared with the negative control group carrageenan (P < 0.05) and finally, the mice pretreated orally with EL extract at 300 mg kg-1 demonstrated maximum reduction of edema at 3 h (64 %) when compared with carrageenan (P < 0.05). The EL extract showed total phenolic contents yielded 158.74 ± 3.5 mg GAE/g and flavonoids and anthocianins were identified by LC-MS/MS. Thus, this genuine Brazilian species founded in Atlantic Forest, an ecosystem threatened, showed to be a promising source of bioactive compounds with anti-inflammatory potential, acting on pathways of inflammatory mediators involving rolling and adhesion molecules and showed anti-edematogenic effect in the paw edema. The EL extract exhibited content of phenolic compounds (flavonoids and anthocyanins) higher than that of traditional fruits, which may be related with the anti-inflamatory effects observed allowing it use to enriching foodsMestradoFarmacologia, Anestesiologia e TerapeuticaMestra em Odontologia2013/26251-013426120143FAPESPCNP

Fitoquímica, toxicidade e avaliação das atividades anti-inflamatória e antioxidante de "Eugenia neonitida" Sobral (pitangatuba), uma fruta nativa do Brasil

Orientador: Pedro Luiz RosalenTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: As frutas nativas brasileiras (FNB) podem ser classificadas como "superfrutas", devido à sua rica composição polifenólica que pode modular o processo inflamatório, bem como reduzir a produção de espécies reativas de oxigênio e nitrogênio (ERO/ERN). Este estudo determinou a composição polifenólica, avaliou o mecanismo de ação anti-inflamatório, a atividade antioxidante e perfil de toxicidade sistêmica do extrato, fração (F3) e subfração (S8) de Eugenia neonitida (Ene). Os compostos presentes em Ene, F3 e S8 foram quantificados e identificados por LC-ESI-QTOF-MS. Para os estudos de atividade anti-inflamatória in vitro, utilizou-se cultura de macrófagos RAW 264.7 transfectados (NF-kB-pLUC gene) e quantificação de TNF-'alfa' e CXCL2/MIP-2, por meio de ELISA. Para a avaliação do mecanismo de ação anti-inflamatório in vivo, foram conduzidos os ensaios de migração de neutrófilos induzida por carragenina, quantificação de TNF-'alfa' e CXCL2/MIP-2, microscopia intravital e expressão de molécula de adesão por Western Blotting. Para a atividade antioxidante, foram utilizados os ensaios contra ERO/ERN (ROO, O2 -, HOCl e NO ). Por fim, o perfil de toxicidade sistêmica foi testado em larvas de Galleria mellonella. A análise química revelou a presença de ácido hidroxibenzoico, flavanoides e elagitaninos para Ene e F3 e derivados de quercetrina, ácido vanílico e ácido cumárico para S8. Ene reduziu, in vitro, a ativação de NF-kB e níveis de TNF-'alfa', entretanto, não reduziu CXCL2/MIP-2; in vivo, diminuiu a migração de neutrófilos bem como os níveis de TNF-a e CXCL2/MIP-2. Com relação à fração, a F3 reduziu a ativação de NF-kB, os níveis de TNF-'alfa' e CXCL2/MIP-2 (in vitro e in vivo) e migração de neutrófilos. Finalmente, S8 reduziu ativação de NF-kB, níveis de TNF-'alfa' e CXCL2/MIP-2 (in vitro e in vivo) e migração neutrofílica, comprovada por microscopia intravital e redução da expressão de ICAM-1 (molécula de adesão) em endotélio. Em geral, Ene, F3 e S8 exibiram atividades contra ERO/ERN e nenhuma das amostras induziu a toxicidade sistêmica em G. mellonella. Assim, Ene, F3 e S8 não apresentaram toxicidade e atuaram como anti-inflamatório e antioxidante, devido à sua composição fenólica. A E. neonitida é uma promissora fonte de compostos bioativos pouco explorada, que pode favorecer setores como o agronegócio, indústrias farmacêutica e alimentícia proporcionando o desenvolvimento de novos produtos ou insumos para a saúde humanaAbstract: Brazilian native fruits (FNB) can be classified as "superfruits" due to their rich polyphenolic composition that can modulate the inflammatory process as well as reduce the production of reactive oxygen and nitrogen species (ROS/RNS). This study determined the polyphenolic composition, evaluated the mechanism of anti-inflammatory action, antioxidant activity and systemic toxicity profile of the extract, fraction (F3) and subfraction (S8) of Eugenia neonitida (Ene). The compounds present in Ene, F3, and S8 were quantified and identified by LC-ESI-QTOF-MS. For the studies of anti-inflammatory activity in vitro, RAW 264.7 macrophages (NF-kB-pLUC gene) transfected were used stimulated with LPS and quantification of inflammatory cytokines by ELISA. For the evaluation of the anti-inflammatory mechanism of action in vivo, neutrophil migration tests were performed in the peritoneal cavity of mice challenged with carrageenan, quantification of inflammatory cytokines in vivo using ELISA, intravital microscopy (rolling and leukocyte adhesion) and expression of adhesion molecule (ICAM-1) by Western Blotting. For antioxidant activity, tests against ROS/RNS (ROO, O2 -, HOCl and NO ) were used. Finally, the systemic toxicity profile was tested in Galleria mellonella larvae. Chemical analysis revealed the presence of hydroxybenzoic acid, flavanoids and ellagitannins for Ene and F3 and derivatives of quercetrin, vanillic acid and cumaric acid for S8. Ene reduced the activation of NF-kB and TNF-'alfa' levels in vitro, however, it did not reduce CXCL2/MIP-2; Ene treated in vivo decreased neutrophil migration as well as TNF-'alfa' and CXCL2/MIP-2 levels. Regarding the fraction, F3 reduced the activation of NF-kB, the levels of TNF-'alfa' and CXCL2 / MIP-2 (in vitro and in vivo) and migration of neutrophils. Finally, S8 reduced activation of NF-kB, levels of TNF-'alfa' and CXCL2 / MIP-2 (in vitro and in vivo) and neutrophil migration confirmed by intravital microscopy and reduced expression of ICAM-1 (adhesion molecule) in endothelium. In general, Ene, F3 and S8 exhibited activities against ROS/RNS and none of the samples induced systemic toxicity in G. mellonella. Thus, Ene, F3 and S8 showed no toxicity and exibited anti-inflammatory and antioxidant activities due to their phenolic composition. E. neonitida is a promising source of bioactive compounds little explored, which can favor sectors such as food industries, pharmaceutical and agribusiness, providing the development of new products for human healthDoutoradoFarmacologia, Anestesiologia e TerapeuticaDoutora em Odontologia2016/02926-6FAPESPCAPE

Brazilian Organic Honey from Atlantic Rainforest Decreases Inflammatory Process in Mice

Honey is an ancient food in the human diet, and the chemical composition of some types of honey has been associated with several beneficial biological effects. Among them, honey has been highlighted to improve health and control inflammatory processes. However, there is no study elucidating the mechanism of action of honey produced organically. Here, we separated organic honey (OH) samples from the Brazilian Atlantic Rainforest into eight different profiles (OH-1 to OH-8) and evaluated, in vitro and in vivo, their anti-inflammatory potential. To determine cell viability, RAW 264.7 macrophages were treated with several concentrations of OH-1 up to OH-8, and anti-inflammatory activity was assessed through NF-&kappa;B activation and TNF-&alpha; levels. All types of the studied honey up to a concentration of 4% (w/v) did not interfere with macrophage viability and decreased NF-kB activation and TNF-&alpha; levels in macrophage culture in vitro. OH-7 was selected as the most promising anti-inflammatory and used in subsequent assays. Mice pretreated orally with OH-7 showed a decrease in neutrophil migration and TNF-&alpha; level. Thus, these types of Brazilian organic honey show promising anti-inflammatory potential, particularly the OH-7 variety. Brazilian organic honey may lead to the development of new products and/or be incorporated into food for use in veterinary medicine and human health as well

Antioxidant and Anti-Inflammatory Activities of Unexplored Brazilian Native Fruits

<div><p>Brazilian native fruits are unmatched in their variety, but a poorly explored resource for the development of food and pharmaceutical products. The aim of this study was to evaluate the phenolic composition as well as the antioxidant and anti-inflammatory activities of the extracts of leaves, seeds, and pulp of four Brazilian native fruits (<i>Eugenia leitonii</i>, <i>Eugenia involucrata</i>, <i>Eugenia brasiliensis</i>, and <i>Eugenia myrcianthes</i>). GC—MS analyses of the ethanolic extracts showed the presence of epicatechin and gallic acid as the major compounds in these fruits. Antioxidant activity was measured using synthetic DPPH free-radical scavenging, β-carotene bleaching assay, and reactive oxygen species (ROO·, O₂·⁻, and HOCl). The fruit extracts also exhibited antioxidant effect against biologically relevant radicals such as peroxyl, superoxide, and hypochlorous acid. In general, the pulps were the fruit fractions that exhibited the lowest antioxidant activities, whereas the leaves showed the highest ones. The anti-inflammatory activity was assessed in an <i>in vivo</i> model using the carrageenan-induced neutrophil migration assay, which evaluates the inflammatory response in the acute phase. The pulp, seeds, and leaves of these fruits reduced the neutrophil influx by 40% to 64%. Based on these results, we suggest that the anti-inflammatory activity of these native fruits is related to the modulation of neutrophil migration, through the inhibition of cytokines, chemokines, and adhesion molecules, as well as to the antioxidant action of their ethanolic extracts in scavenging the free-radicals released by neutrophils. Therefore, these native fruits can be useful to produce food additives and functional foods.</p></div

Unexplored endemic fruit species from Brazil: antibiofilm properties, insights into mode of action, and systemic toxicity of four Eugenia spp

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOBrazilian endemic fruit species have aroused attention due to their highly valuable, yet unexplored, agroindustrial, food and therapeutic potential. Herein, we describe the antifungal activity of four Eugenia spp. against Candida albicans biofilms, and further demonstrate insights into their potential mode(s) of action and toxicity in vitro and in vivo. Extracts from different parts (seeds, pulps, leaves) of E. leitonii (EL), E. brasiliensis (EB), E. myrcianthes (EM) and E. involucrata (EI) were obtained (S23 degrees 23',W45 degrees 39') and chemically characterized by GC/MS. The active extracts were tested against C albicans biofilm viability and architecture, as well as mode of action, and toxicology using RAW 264.7 macrophages and Galleria mellonella larvae. The MIC values ranged from 15.62 to > 2000 mu g/mL. The most active extracts were EL (seed, 15.62 mu g/mL) and EB (leaf and seeds, 31.25 and 15.62 mu g/mL, respectively). Treatment with these extracts at 10xMIC reduced biofilm viability by 54-55% (P 0.05) and G. mellonella larvae, with mean in vivo LD50 of 1500 mg/kg (EL, seeds); 2500 mg/kg (EB, seeds); and 1250 mg/kg (EB, leaf). The phenolic compounds epicatechin and gallic acid were the major constituents in the extracts. Our findings may open avenues for the application of these yet unexplored native fruits in the food and pharmaceutical industry. (C) 2017 Elsevier Ltd. All rights reserved.Brazilian endemic fruit species have aroused attention due to their highly valuable, yet unexplored, agroindustrial, food and therapeutic potential. Herein, we describe the antifungal activity of four Eugenia spp. against Candida albicans biofilms, and further demonstrate insights into their potential mode(s) of action and toxicity in vitro and in vivo. Extracts from different parts (seeds, pulps, leaves) of E. leitonii (EL), E. brasiliensis (EB), E. myrcianthes (EM) and E. involucrata (EI) were obtained (S23 degrees 23',W45 degrees 39') and chemically characterized by GC/MS. The active extracts were tested against C albicans biofilm viability and architecture, as well as mode of action, and toxicology using RAW 264.7 macrophages and Galleria mellonella larvae. The MIC values ranged from 15.62 to > 2000 mu g/mL. The most active extracts were EL (seed, 15.62 mu g/mL) and EB (leaf and seeds, 31.25 and 15.62 mu g/mL, respectively). Treatment with these extracts at 10xMIC reduced biofilm viability by 54-55% (P 0.05) and G. mellonella larvae, with mean in vivo LD50 of 1500 mg/kg (EL, seeds); 2500 mg/kg (EB, seeds); and 1250 mg/kg (EB, leaf). The phenolic compounds epicatechin and gallic acid were the major constituents in the extracts. Our findings may open avenues for the application of these yet unexplored native fruits in the food and pharmaceutical industry105280287CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO474335/201352011/15984-0; 2011/25080-7; 2013/26251-

Effect of the oral administration of ethanolic extracts of leaves, seeds, and pulp of four Brazilian native fruit species on the inhibition of neutrophil migration to the peritoneal cavity of mice treated with 500 mg/kg.

<p>(A) <i>Eugenia brasiliensis</i>. (B) <i>Eugenia involucrata</i>. (C) <i>Eugenia leitonii</i>. (D) <i>Eugenia myrcianthes</i>. C: control treated with vehicle; –: carrageenan (500 μg/cavity); Dexa: dexamethasone (2 mg/kg). Data are expressed as mean ± standard deviation of the mean; n = 6. Symbols indicate statistical differences (<i>p</i> < 0.05, Tukey’s test): # <i>p</i> < 0.05 compared with the control group; * <i>p</i> < 0.05 compared with the carrageenan group. The symbol % indicates decrease in the number of neutrophils in the peritoneal cavity.</p

Antioxidant activity of the ethanolic extracts of leaves, seeds, and pulp of four Brazilian native fruit species measured using synthetic DPPH free-radical scavenging, β-carotene bleaching, and reactive oxygen species (ROO·, O₂·⁻, and HOCl) assays.

<p>Antioxidant activity of the ethanolic extracts of leaves, seeds, and pulp of four Brazilian native fruit species measured using synthetic DPPH free-radical scavenging, β-carotene bleaching, and reactive oxygen species (ROO·, O₂·⁻, and HOCl) assays.</p

Anti-inflammatory activity and polyphenolic profile of the hydroalcoholic seed extract of Eugenia leitonii, an unexplored brazilian native fruit

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThis study determined the anti-inflammatory activity, mechanisms of action, and polyphenolic profile of Eugenia leitonii seed extract. The total phenolic content of the hydroethanolic extract from E. leitonii seeds (ESEL) was measured, and its phenolic compounds were identified by LC-MS/MS. The ESEL was submitted to in vitro (RAW264.7) and in vivo assays on neutrophil migration; expression of P-selectin and ICAM-1; release of TNF-alpha, IL-1 beta, and CXCL2/ MIP-2; NF-kappa B activation; and carrageenan-induced paw oedema. ESEL yielded total phenolic content of 158.74 +/- 3.5 mg GAE/g, with the presence of flavonols, anthocyanins and ellagitannins. ESEL significantly reduced neutrophil migration; decreased the expression of P-selectin and ICAM-1; decreased the release of TNF-alpha and CXCL2/MIP-2, as well as NF-kappa B activation and paw oedema in mice. Thus, this extract showed to be a promising source of bioactive compounds with anti-inflammatory potential by inhibiting neutrophil migration through a decrease in the release of TNF-alpha and CXCL2/MIP-2 (NF-kappa B pathway) in macrophages. (C) 2016 Elsevier Ltd. All rights reserved.This study determined the anti-inflammatory activity, mechanisms of action, and polyphenolic profile of Eugenia leitonii seed extract. The total phenolic content of the hydroethanolic extract from E. leitonii seeds (ESEL) was measured, and its phenolic compounds were identified by LC-MS/MS. The ESEL was submitted to in vitro (RAW264.7) and in vivo assays on neutrophil migration; expression of P-selectin and ICAM-1; release of TNF-alpha, IL-1 beta, and CXCL2/ MIP-2; NF-kappa B activation; and carrageenan-induced paw oedema. ESEL yielded total phenolic content of 158.74 +/- 3.5 mg GAE/g, with the presence of flavonols, anthocyanins and ellagitannins. ESEL significantly reduced neutrophil migration; decreased the expression of P-selectin and ICAM-1; decreased the release of TNF-alpha and CXCL2/MIP-2, as well as NF-kappa B activation and paw oedema in mice. Thus, this extract showed to be a promising source of bioactive compounds with anti-inflammatory potential by inhibiting neutrophil migration through a decrease in the release of TNF-alpha and CXCL2/MIP-2 (NF-kappa B pathway) in macrophages26249257FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2013/26251-0; 2013/13190-

Content of total phenolic compounds in the ethanolic extracts of leaves, seeds, and pulp of four Brazilian native fruit species.

<p>Content of total phenolic compounds in the ethanolic extracts of leaves, seeds, and pulp of four Brazilian native fruit species.</p