Estudo do efeito anti-inflamatório da Calea pinnatifida (R. Br.) Less. no modelo da pleurisia induzida pela carragenina em camundongos

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2016.Introdução: O gênero Calea pertence à família Asteraceae e contém cerca de 125 espécies, distribuídas mundialmente em áreas tropicais e subtropicais, sendo que o maior número dessas espécies é registrado no Brasil. Espécies do gênero Calea são popularmente utilizadas para o tratamento de reumatismo, doenças respiratórias e, problemas digestivos. A Calea pinnatifida (C. pinnatifida), popularmente conhecida como ?aruca? e ?cipó-cruz?, é uma planta perene e subarbustiva encontrada principalmente nos estados do sul e sudeste do Brasil, utilizada na medicina popular para o tratamento de dores estomacais, giardíase e amebíase. Embora já existam trabalhos que demonstrem a atividade antiproliferatica e leishmanicida da C. pinnatifida, ainda não existem estudos farmacológicos descritos que tenham avaliado sua atividade anti-inflamatória. Objetivos: Investigar a atividade anti-inflamatória do extrato bruto, frações e compostos isolados das folhas da C. pinnatifida, utilizando um modelo murino de pleurisia induzida pela carragenina. Metodologia: As folhas da C. pinnatifida foram submetidas a um processo de maceração com etanol 92%, para obtenção do extrato bruto etanólico (EB). O EB foi submetido a uma partição líquido/líquido sob agitação manual de forma exaustiva e sucessiva, utilizando os solventes: hexano, clorofórmio e acetato de etila, respectivamente, para obter as frações hexano (Hex), diclorometano (DCM), e acetato de etila (AcOEt), bem como a fração aquosa remanescente, pelo qual foi obtido a fração metanólica (MeOH), a partir de uma cromatografia em coluna, utilizando da resina Amberlite XAD-4 como fase estacionária e metanol como fase móvel. Os compostos foram isolados a partir da fração acetato por métodos cromatográficos e identificados por meio de dados espectroscópicos de infra-vermelho (IV) e ressonância magnética nuclear de hidrogênio e carbono treze. A pleurisia foi induzida segundo metodologia descrita por SALEH et al., 1996 e os parâmetros inflamatórios como: migração de leucócitos, exsudação, atividade das enzimas mieloperoxidase (MPO) e adenosina-desaminase (ADA), concentrações dos metabólitos do óxido nítrico (NOx) e das citocinas pró-inflamatórias, fator de necrose tumoral alpha (TNF-a), interleucina- 1 beta (IL-1ß) e interleucina- 17A (IL-17A), foram avaliados 4 h após a indução da inflamação. Além disso, também avaliou-se a capacidade dos compostos isolados, ácido 3,5-di-O-E-cafeoil quínico (3,5-diACQ) e ácido 4,5-di-O-E-cafeoil quínico (4,5-diACQ), em inibir a fosforilação da subunidade p65 do NF-?B e p38 MAPK. Para isso, diferentes grupos deanimais foram tratados com EB (25-100 mg/kg), Hex (5-25 mg/kg), DCM (5-25 mg/kg), MeOH (5-25 mg/kg), AcOEt (2,5-25 mg/kg), 3,5-diACQ (1-5 mg/kg) ou 4,5-diACQ (1-5 mg/kg) administrados por via intraperitoneal (i.p.) 0,5 h antes da carragenina (1%) administrada por via intrapleural (i.pl.). Para avaliar a exsudação, os animais foram tratados previamente com solução de Azul de Evans (25 mg/kg) por via intra-venosa (i.v.), 10 min antes da administração de EB, frações e compostos isolados da C. pinnatifida. Ensaios colorimétricos foram utilizados para analisar a atividade das enzimas MPO e ADA, bem como NOx. Testes imunoenzimaticos (ELISA) foram realizados, para determinar as concentrações das citocinas TNF-a, IL-1ß e IL-17A e também para avaliar a fosforilação de p65 (NF-?B) e p38 MAPK. Diferenças estatísticas entre os grupos foram determinados utilizando análise de variância (ANOVA), complementados pelo teste post-hoc de Newman-Keuls, valores de p Abstract : Introduction: Calea genus belongs to the Asteraceae family and contains about 125 species, distributed worldwide in tropical and subtropical areas, with the largest number of these species is registered in Brazil. Calea species of the genus are commonly used to treat rheumatism, respiratory diseases, and digestive problems. The Calea pinnatifida (C. pinnatifida), popularly known as "aruca" and "cipó-cruz", is a perennial and subshrub plant found mainly in the southern and southeastern Brazil states, used in folk medicine to treat stomach aches, giardiasis and amebiasis. Although there are studies that demonstrate the antiproliferative and leishmanicidal activities of C. pinnatifida, there are no pharmacological studies to describe its anti-inflammatory activity. Objectives: To evaluate the anti-inflammatory activity of crude extract, fractions and isolated compounds from C. pinnatifida leaves using a murine model of pleurisy induced by carrageenan. Methodology: C. pinnatifida leaves were subjected to a maceration process with ethanol 92% to obtain a crude hydroalcoholic extract (CE). CE was subjected to a liquid/liquid partition under manual exhaustive and successively stirring using hexane, chloroform and ethyl acetate eluents, respectively, to obtain the hexane fractions (Hex), dichloromethane (DCM) and ethyl acetate (EtOAc) as well as the remaining aqueous fraction from which was obtained the methanolic fraction (MeOH), from the use of XAD-4 resin. The compounds were isolated from the EtOAc fraction by chromatographic methods and identified by spectroscopic data of infrared (IR) and nuclear magnetic resonance of hydrogen and carbon thirteen. Pleurisy was induced according to the methodology described by Saleh et al, 1996 and inflammatory parameters such as: Leukocyte migration, exudation, activity of myeloperoxidase (MPO) and adenosine-deaminase (ADA), concentrations of nitric oxide metabolites (NOx) and pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-1ß) and interleukin-17A (IL-17A) were evaluated 4 hours after inflammation induction. In addition, we also evaluated the ability of the isolated compounds, 3,5-di-O-E-caffeoylquinic acid (3,5-diCQA) and 4,5-di-O-E-caffeoylquinic (4,5-diCQA) acid to inhibit the phosphorylation of the NF-?B p65 subunit and p38 MAPK. For this, different groups of animals were treated with CE (25-100 mg/kg) Hex (5-25 mg/kg), DCM (5-25 mg/kg), MeOH (5-25 mg/kg), EtOAc (2.5-25 mg/kg), 3,5-diCQA (1-5 mg/kg) and 4,5-diCQA (1-5 mg/kg) administered by intraperitoneally route (i.p.) 0.5 h before carrageenan (1%) administered by intrapleural route (i.pl.). To evaluate the exudation, the animals were pretreated with Evans blue solution (25 mg/kg) administred by intravenous (i.v.) route. Colorimetric assays were used to analyze the MPO and ADA activities, as well as NOx and immunoenzymatic assays (ELISA) to determine the TNF-a, IL-1ß and IL-17A concentrations and also to evaluate the phosphorylation of p65 (NF-?B) and p38 MAPK. The significant differences between groups were determined using analysis of variance (ANOVA) followed by Newman?Keuls post hoc test and the values of p < 0.05 were considered significant. Results: CE (50-100 mg/kg), Hex (10-25 mg/kg), MeOH (10-25 mg/kg), EtOAc (5-25 mg/kg), 3,5-diCQA (2,5- 5 mg/kg) and 4,5-diCQA (5 mg/kg) inhibited: leukocytes, neutrophils, and exudation (p < 0.05). CE (50 mg/kg), Hex (10 mg/kg), MeOH (10 mg/kg), EtOAc (5 mg/kg), 3,5-diCQA (2.5 mg/kg) and 4,5-diCQA (5 mg/kg) inhibited the MPO and ADA activities, NOx, IL-1ß and IL-17A concentrations (p < 0.05). At the same dose, only CE, EtOAc, 3,5-diCQA and 4,5-diCQA were able to decrease TNF-a concentrations (p < 0.05). The compounds 3,5-diCQA and 4,5-diACQ also inhibited phosphorylation of p65 and p38 (p < 0.05). Conclusion: This study demonstrated that C. pinnatifida has an important anti-inflammatory activity by inhibiting leukocyte migration, neutrophils and decreased exudation. This effect may be occurred by decrease of the concentration of pro-inflammatory mediators (NOx, TNF-a, IL-1ß and IL-17A) in pleural leakage and these effects appear to be related in part to the ability of the isolated compounds to inhibit important cellular pathways involved in inflammatory process, such NF-?B and p38 MAPK

new pre clinical evidence of anti inflammatory effect and safety of a substituted fluorophenyl imidazole

Abstract Acute Respiratory Distress Syndrome (ARDS) is an inflammatory condition with high mortality rates, and there is still no pharmacological approach with proven effectiveness. In the past few years, several imidazole small molecules have been developed to treat conditions in which inflammation plays a central role. In the present work, we hypothesize that a novel substituted fluorophenyl imidazole synthetized by our research group would present in vivo anti-inflammatory effect in an ARDS murine model induced by LPS. Results shows that the fluorophenyl imidazole has the ability to inhibit leukocyte migration to the bronchoalveolar lavage fluid and lung tissue of animals challenged intranasally with LPS. Furthermore, this inhibition is followed with reduction in myeloperoxidase activity, nitric oxide metabolites generation and cytokines (TNF-α, IL-6, IL-17, IFN-γ and IL-10) secretion. This effect is at least partly related to the capacity of the fluorophenyl imidazole in inhibit p38 MAPK and NF-κB phosphorylation. Finally, fluorophenyl imidazole showed no signs of acute oral toxicity in the toxicological protocol suggested by OECD 423. Taken together, the results shows that fluorophenyl imidazole is a promising prototype for the development of a novel anti-inflammatory drug in which p38 MAPK and NF-κB plays a pivotal role

The anti-inflammatory effect of Ilex paraguariensis A. St. Hil (Mate) in a murine model of pleurisy.

Ilex paraguariensis is a native plant from Southern America, where it is used as a beverage. In traditional medicine, it is used to treat many diseases including inflammation. However, we do not yet know precisely how this effect occurs. We therefore evaluated its anti-inflammatory effect in a murine model of pleurisy. The standardized CE, BF and ARF fractions, Caf, Rut and CGA were able to reduce leukocyte migration, exudate concentration, MPO and ADA activities and NOx levels. Moreover, I. paraguariensis also inhibited the release of Th1/Th17 pro-inflammatory cytokines, while increasing IL-10 production and improving the histological architecture of inflamed lungs. In addition, its major compounds decreased p65 NF-κB phosphorylation. Based on our results, we can conclude that I. paraguariensis exerts its anti-inflammatory action by attenuating the Th1/Th17 polarization in this model. This fact suggests that the use of this plant as a beverage can protect against Th1/Th17 inflammatory diseases

Estudo do efeito anti-inflamatório de derivados da 1,4-dihidropiridina e a importância das determinações de mediadores pró-inflamatórios na fase de triagem de compostos com possível atividade anti-inflamatória in vitro : uma Revisão Sistemática e Meta-análise

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2023.Introdução: O processo inflamatório representa a base fisiopatológica de inúmeras patologias. Trata-se de um mecanismo de defesa do organismo contra agentes infecciosos ou injúria tecidual. Entender os mecanismos subjacentes à inflamação e desenvolver novos agentes terapêuticos que reduzam os danos inflamatórios ainda continuam sendo os pilares de estudos farmacológicos. Derivados dihidropiridinicos apresentam inúmeras atividades biológicas já descritas, podendo ser bons candidatos a novas terapias anti-inflamatórias. Objetivos: Este estudo teve como objetivo investigar a racionalidade da dosagem de diferentes mediadores inflamatórios no modelo de inflamação in vitro utilizando células RAW 264.7, através de uma revisão sistemática (RS) e meta-análise, bem como investigar o efeito anti-inflamatórios de derivados da 1,4-Dihidropiridina (1,4-DHP) em modelos in vitro, utilizando células RAW264.7 induzidas por LPS e in vivo, através do modelo de lesão pulmonar aguda (LPA) em camundongos. Metodologia: Realizamos uma RS com meta-análise com base na declaração PRISMA, selecionando a partir de busca em bases de dados, trabalhos científicos específicos que tivessem realizado a dosagem de mediadores inflamatórios utilizando o modelo de células RAW 264.7 induzidas com LPS. Posteriormente os dados foram extraídos e a meta-análise realizada, com o intuito de verificar qual a eficácia da dosagem do TNF-a, IL-6 e IL-1ß como biomarcadores inflamatórios, comparados ao Óxido Nítrico (NO). Na segunda fase do trabalho, quinze compostos derivados da 1,4-DHP foram testados em células RAW 264.7 para avaliar seus efeitos citotóxicos. Posteriormente, foram avaliados quanto seus efeitos sobre fagocitose de macrófagos e na produção de mediadores pró-inflamatórios e anti-inflamatórios. Por último, verificamos esses efeitos in vivo, utilizando o de LPA em camundongos, dosando mediadores inflamatórios (citocinas, NO e Mieloperoxidase (MPO)) e a migração leucocitária e exsudação. Resultados: Na primeira etapa da revisão, um total de 17 estudos foram incluídos. Células induzidas por LPS produziram altos níveis de NO em comparação com células não induzidas e essa produção não apresentou relação com a densidade celular utilizada. TNF-a, IL-1ß e IL-6 também apresentaram níveis elevados após as células terem sido estimuladas com LPS; no entanto, parte desses efeitos parecem ter relação com a densidade celular. Todos os estudos foram confiáveis com restrições, tendo em vista que foram identificados problemas quanto a qualidade metodológica e qualidade de relatórios. Em seguida, na realização de experimentos, verificou-se que um dos compostos derivados da 1,4-DHP, foi efetivo em inibir a produção de citocinas pró-inflamatórias e do mediador NO em células RAW264.7 induzidas por LPS, além disso, o composto em questão aumentou a fagocitose das células e da produção da citocina IL-10 no sobrenadante das culturas celulares, indicando uma possível polarização de macrófagos para o fenótipo M2. Por fim, no modelo de LPA, o composto selecionado inibiu a migração leucocitária e a exsudação no LBA, além de reduzir a atividade da enzima MPO e a produção de NO e citocinas pró-inflamatórias IL-6 e TNF-a. Conclusão: Dosar os níveis de NO é uma opção suficiente e racional para iniciar os ensaios de triagem anti-inflamatória de compostos utilizando células RAW 264.7 como plataforma experimental. Além disso, o composto selecionado derivado da 1,4-DHP apresentou importante atividade anti-inflamatória, reduzindo a produção de mediadores pró-inflamatórios e aumentando a atividade fagocítica de macrófagos e secreção de IL-10. Esses achados indicam a que a atividade anti-inflamatória do composto deve-se a capacidade de reverter a polarização dos macrófagos para um perfil anti-inflamatório (M2).Abstract: Introduction: Although essential in the innate immune response, the inflammatory process represents the pathophysiological basis of numerous pathologies. It is a defense mechanism of the body against infectious agents or tissue injury. Understanding the mechanisms underlying inflammation and developing new therapeutic agents that reduce inflammatory damage still remain the mainstays of pharmacological studies. Dihydropyridine derivatives have numerous biological activities already described, and may be good candidates for new anti-inflammatory therapies. Objectives: This study aimed to investigate the rationality of the dosage of different inflammatory mediators in the in vitro inflammation model using RAW 264.7 cells, through a systematic review (RS) and meta-analysis, as well as to investigate the anti-inflammatory effect of 1 ,4-Dihydropyridine (1,4-DHP) in in vitro models, using RAW264.7 cells induced by LPS and in vivo, through the model of acute lung injury in mice. Methodology: In the first stage of this work, we carried out an SR with meta-analysis based on the PRISMA statement, selecting from a search in databases, specific scientific works that had performed the measurement of inflammatory mediators (NO, TNF-, IL-6 and IL -1ß) using the LPS-induced RAW 264.7 cell model. Subsequently, the data were extracted and the meta-analysis performed, with the aim of verifying the effectiveness of the dosage of TNF-a, IL-6 and IL-1ß as inflammatory biomarkers, compared to Nitric Oxide (NO). In the second phase of the work, fifteen compounds derived from 1,4-DHP were tested in RAW 264.7 cells to evaluate their cytotoxic effects. Subsequently, their effects on macrophage phagocytosis and on the production of pro-inflammatory (NO, TNF-a, INF-?, IL-12, MCP-1, IL-6) and anti-inflammatory (IL-10). Finally, we verified whether these effects would be repeated in vivo, using the acute lung injury (ALI) model in mice, measuring inflammatory mediators (cytokines, NO and Myeloperoxidase (MPO)) and leukocyte migration and exudation. Results: In the first step of the review, a total of 17 studies were included. As expected, cells induced by LPS produced higher levels of NO compared to non-induced cells and this production was not related to the cell density used. TNF-a, IL-1ß and IL-6 also showed elevated levels after cells were stimulated with LPS; however, part of these effects seem to be related to cell density. All studies were reliable, although with some restrictions, considering that problems were identified regarding methodological quality and quality of reports for specific items, mainly related to compounds and test systems. Then, in carrying out experiments, it was found that one of the compounds derived from 1,4-DHP was effective in inhibiting the production of cytokines IL-12, MCP-1, IFN-? and IL-6 and the mediator NO in RAW264.7 cells induced by LPS, moreover, the compound in question increased the phagocytosis of the cells and caused an increase in the production of the cytokine IL-10 in the supernatant of cell cultures, indicating a possible polarization of macrophages from the M1 phenotype to M2. Finally, in the ALI model, the selected compound was effective in inhibiting leukocyte migration and exudation in BALF, reducing the activity of the MPO enzyme and the production of NO and pro-inflammatory cytokines IL-6 and TNF-a. Conclusion: Measuring NO levels is a sufficient and rational option to start anti-inflammatory compound screening assays using RAW 264.7 cells as an experimental platform. Furthermore, the selected compound derived from 1,4-DHP showed important anti-inflammatory activity, reducing the production of pro-inflammatory mediators and increasing the phagocytic activity of macrophages and IL-10 secretion. These findings indicate that the anti-inflammatory activity of the compound is due to the ability to reverse the polarization of macrophages to an anti-inflammatory profile (M2)

Systemic Administration of Calea pinnatifida Inhibits Inflammation Induced by Carrageenan in a Murine Model of Pulmonary Neutrophilia

Objective. The aim of this study was to investigate the anti-inflammatory effects of the crude extract (CE), derived fraction, and isolated compounds from Calea pinnatifida leaves in a mouse model of pulmonary neutrophilia. Methods. The CE and derived fractions, hexane, ethyl acetate, and methanol, were obtained from C. pinnatifida leaves. The compounds 3,5- and 4,5-di-O-E-caffeoylquinic acids were isolated from the EtOAc fraction using chromatography and were identified using infrared spectroscopic data and nuclear magnetic resonance (1H and 13C NMR). Leukocytes count, protein concentration of the exudate, myeloperoxidase (MPO) and adenosine deaminase (ADA), and nitrate/nitrite (NOx), tumor necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1β), and interleukin-17A (IL-17A) levels were determined in the pleural fluid leakage after 4 h of pleurisy induction. We also analyzed the effects of isolated compounds on the phosphorylation of both p65 and p38 in the lung tissue. Results. The CE, its fractions, and isolated compounds inhibited leukocyte activation, protein concentration of the exudate, and MPO, ADA, NOx, TNF-α, IL-1β, and IL-17A levels. 3,5- and 4,5-di-O-E-caffeoylquinic acids also inhibited phosphorylation of both p65 and p38 (P<0.05). Conclusion. This study demonstrated that C. pinnatifida presents important anti-inflammatory properties by inhibiting activated leukocytes and protein concentration of the exudate. These effects were related to the inhibition of proinflammatory mediators. The dicaffeoylquinic acids may be partially responsible for these anti-inflammatory properties through the inhibition of nuclear transcription factor kappa B and mitogen-activated protein kinase pathways

Qualitative and quantitative analysis data of the major constituents of Ilex paraguariensis leaves by UPLC-PDA and QTOF-MS

Ilex paraguariensis A. St. Hil. is a native plant of South America widely consumed as beverages for its ethno pharmacological properties, such as antioxidant, anti-inflammatory, hypocholesterolemic as well as its benefits on the cardiovascular system. Since these properties are related to its chemical composition, the identification and quantification of the major compounds of I. paraguariensis extracts still remains relevant. The data described in this article supports previous results on the anti-inflammatory effect of I. paraguariensis A. St. Hil (Mate), “The anti-inflammatory effect of I. paraguariensis A. St. Hil (Mate) in a murine model of pleurisy” [1]. The present data article reports on nine major compounds identified in I. paraguariensis extracts and its related fractions by using UPLC-PDA and UPLC-QTOF. Identification of the constituents was based on their retention times, UV absorption spectra and mass spectra data, as well as by comparison with authentic samples. The validated parameters show that the quantification by UPLC-PDA methodology developed is sensitive, precise and accurate. Keywords: Ilex paraguariensis, Mate, Chemical composition, LC-PDA, LC-M