Estudo químico do extrato etanólico das folhas de Murraya paniculata (L.) Jack e avaliação da ação anti-inflamatória
Carregando...
Data
2018-03-28
Autores
Martín, Celia Magaly Casado
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Murraya paniculata L. Jack (Rutaceae) é uma árvore amplamente utilizada nas Américas como ornamental. Porém, esta espécie vegetal nativa da Índia é considerada medicinal na Ásia e reconhecida pelas propriedades adstringentes, estimulantes, analgésicas e anti-inflamatórias. Diversos estudos revelam a presença de alcaloides, flavonoides, cumarinas e componentes do óleo essencial, fundamentalmente, e as propriedades antibacterianas, antifúngicas, anti-inflamatórias e analgésicas foram observadas para extratos de M. paniculata. Fitoterápicos derivados da planta estão disponíveis no Sistema Nacional de Saúde cubano, como anti-inflamatórios, porém é limitado o conhecimento dos metabólitos secundários majoritários da espécie coletada em Cuba e sua relação com a atividade farmacológica. Neste contexto, foi desenvolvido este trabalho que teve como objetivo realizar o estudo químico do extrato etanólico de folhas de M. paniculata, a fim de contribuir com sua padronização e com o estabelecimento de especificações de qualidade com base na composição química e atividade anti-inflamatória. O fracionamento do extrato etanólico foi iniciado por extração em fase sólida fornecendo 4 frações. A fração EFSMp4 foi fracionada utilizando-se cromatografia em coluna. A purificação final das substâncias majoritárias obtidas a partir de EFSMp4 foi realizada por cromatografia líquida de alta eficiência preparativa de fase reversa. As frações, subfrações e substâncias purificadas foram analisadas por CCD, CLAE-DAD e CLAE-ESI-MS. As substâncias purificadas foram identificadas através dos dados obtidos nas análises espectrométricas (RMN de 1H e 13C, IV, UV e EM). O extrato e as frações obtidas por extração em fase sólida foram submetidos aos ensaios de edema de pata e de pleurisia induzidos por carragenina, em ratos de linhagem Wistar e a avaliação da atividade antioxidante in vitro foi realizada por vários métodos: ensaio de capacidade de captura do cátion radicalar ABTS+• (derivado do ácido 2,2'-azino-bis-(3-etilbenzotiazolina-6-sulfônico); ensaio de captura do ânion radical superóxido (O2) e ensaio de captura do ácido hipocloroso (HClO) ou da Taurina-Cloramina. No caso do extrato e da fração EFSMp4 foram realizados ensaios de mutagenicidade. Adicionalmente a citotoxicidade da fração EFSMp4 e das substâncias purificadas foi avaliada através do ensaio com MTT em macrófagos (células RAW 264.7) nos quais também foi feita a quantificação de nitrito. A partir de neutrófilos (recrutados do peritônio de ratos Wistar sadios), foi avaliada ex vivo a função da enzima mieloperoxidase e a produção total de EROs. O extrato etanólico (300mg/Kg) e a fração EFSMp4 (150mg/Kg) apresentaram o melhor efeito anti-inflamatório pelo modelo de edema de pata, mostrando também o menor efeito ulcerogênico na mucosa gástrica dos animais tratados. Os resultados do experimento de pleurisia não foram conclusivos pois o modelo não permitiu obter resultados associados à atividade anti-inflamatória, porém mostrou evidências de um provável efeito hemorrágico de EFSMp4. Quanto à atividade antioxidante, nos experimentos nos quais conseguiu-se observar a resposta das amostras em estudo, o extrato etanólico e as frações EFSMp3 e EFSMp4 alcançaram os menores valores CE50. Em relação à mutagenicidade foi observado que o extrato, bem como EFSMp4, atuaram induzindo potencial mutagênico nos experimentos sem e com ativação metabólica. Dois metabólitos majoritários de EFSMp4 foram isolados e identificados como as cumarinas hidrato de meranzina e murpanidina, já relatadas em folhas da espécie. O hidrato de meranzina foi quantificado, representando um 8,4% (m/m) do EEMp. Os metabolitos não apresentaram citotoxicidade em macrófagos nas concentrações de 2,5-40,0 g/mL e as porcentagens de inibição de NO na concentração de 40,0 g/mL foram EFSMp4 (54%), murpanidina (30,1%) e hidrato de meranzina (8,0%). A identificação de outra cumarina da mesma classe das já purificadas foi proposta a partir análise por CLAE-ESI-MS (murralongina). Os resultados sugerem que as cumarinas constituem um grupo de metabólitos secundários majoritários no extrato etanólico, bem como sua relação com a ação anti-inflamatória do extrato.
Murraya paniculata L. Jack (Rutaceae) is a widely used tree in America as ornamental. However, this Indian native plant is considered medicinal in Asia and recognized by its astringent, stimulating, analgesic and anti-inflammatory properties. Several studies have revealed the presence of alkaloids, flavonoids, coumarins and essential oil components, fundamentally, and the antibacterial, antifungal, anti-inflammatory and analgesic properties were observed for M. paniculata extracts. Phytotherapics derived from the plant are available in Cuban National Health System, as anti-inflammatory drugs, but knowledge of the major secondary metabolites of M. paniculata collected in Cuba and its relation to pharmacological activity is limited. In this context, the aim of this research was to study the ethanolic extract of M. paniculata leaves, in order to contribute to its standardization and to the establishment of quality specifications based on chemical composition and anti-inflammatory activity. Fractionation of the ethanolic extract was initiated by solid phase extraction yielding 4 fractions, the fraction EFSMp4 being fractionated using column chromatography. The final purification of the major components obtained from EFSMp4 was performed by preparative reverse phase high performance liquid chromatography. The fractions, subfractions and purified substances, were analyzed by CCD, CLAE-DAD and HPLC-ESI-MS. The purified substances were identified by the data obtained in the spectrometric analyzes (1H and 13C NMR, IR, UV and MS). The extract and fractions obtained by solid phase extraction were submitted to carrageenan-induced paw edema and pleurisy tests in Wistar rats and in vitro antioxidant activity evaluation was performed by several methods: radical cation ABTS+• (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonic acid) capture capacity test; superoxide radical anion capture test (O2-•) and hypochlorous acid (HClO) or Taurine-chloramine capture test. In the case of the extract and the EFSMp4 fraction, mutagenicity tests were performed. Additionally, the cytotoxicity of the EFSMp4 fraction and the purified substances was evaluated by the MTT assay on macrophages (RAW 264.7 cells) in which nitrite quantification was also performed. From neutrophils (recruited from the peritoneum of healthy Wistar rats), myeloperoxidase enzyme function and total ROS production were evaluated ex vivo. Ethanolic extract (300mg/kg) and EFSMp4 fraction (150mg/kg) presented the best anti-inflammatory effect by the paw edema model, also showing the lowest ulcerogenic effect on the gastric mucosa of treated animals. The results of the pleurisy experiment were not conclusive because the model did not showed results associated with the anti-inflammatory activity but showed evidence of a probable EFSMp4 hemorrhagic effect. As for the antioxidant activity, in the experiments in which it was possible to observe the response of the samples under study, the ethanol extract, EFSMp3 and EFSMp4 reached the lowest IC50 values. Regarding the mutagenicity, it was observed that the extract, as well as EFSMp4, acted to induce mutagenic potential in the experiments without and with metabolic activation. Two major metabolites of EFSMp4 were isolated and identified as the coumarins meranzine hydrate and murpanidine, already reported in leaves of the species. Meranzine hydrate was quantified representing a 8.4% (m/m) of EEMp. Macrophage cytotoxicity in the concentration range evaluated (2.5-40.0 g/mL) was not observed and NO inhibition percent at 40.0 μg/ml were EFSMp4 (54.0%), murpanidine (30.1%) and meranzine hydrate (8.0%). The identification of another coumarin of the same class of the two already purified was proposed from analysis by HPLC-ESI-MS (murralongin). The results suggest that coumarins are a group of major secondary metabolites in the ethanolic extract, as well as their relationship with the anti-inflammatory action of the extract.
Murraya paniculata L. Jack (Rutaceae) is a widely used tree in America as ornamental. However, this Indian native plant is considered medicinal in Asia and recognized by its astringent, stimulating, analgesic and anti-inflammatory properties. Several studies have revealed the presence of alkaloids, flavonoids, coumarins and essential oil components, fundamentally, and the antibacterial, antifungal, anti-inflammatory and analgesic properties were observed for M. paniculata extracts. Phytotherapics derived from the plant are available in Cuban National Health System, as anti-inflammatory drugs, but knowledge of the major secondary metabolites of M. paniculata collected in Cuba and its relation to pharmacological activity is limited. In this context, the aim of this research was to study the ethanolic extract of M. paniculata leaves, in order to contribute to its standardization and to the establishment of quality specifications based on chemical composition and anti-inflammatory activity. Fractionation of the ethanolic extract was initiated by solid phase extraction yielding 4 fractions, the fraction EFSMp4 being fractionated using column chromatography. The final purification of the major components obtained from EFSMp4 was performed by preparative reverse phase high performance liquid chromatography. The fractions, subfractions and purified substances, were analyzed by CCD, CLAE-DAD and HPLC-ESI-MS. The purified substances were identified by the data obtained in the spectrometric analyzes (1H and 13C NMR, IR, UV and MS). The extract and fractions obtained by solid phase extraction were submitted to carrageenan-induced paw edema and pleurisy tests in Wistar rats and in vitro antioxidant activity evaluation was performed by several methods: radical cation ABTS+• (2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonic acid) capture capacity test; superoxide radical anion capture test (O2-•) and hypochlorous acid (HClO) or Taurine-chloramine capture test. In the case of the extract and the EFSMp4 fraction, mutagenicity tests were performed. Additionally, the cytotoxicity of the EFSMp4 fraction and the purified substances was evaluated by the MTT assay on macrophages (RAW 264.7 cells) in which nitrite quantification was also performed. From neutrophils (recruited from the peritoneum of healthy Wistar rats), myeloperoxidase enzyme function and total ROS production were evaluated ex vivo. Ethanolic extract (300mg/kg) and EFSMp4 fraction (150mg/kg) presented the best anti-inflammatory effect by the paw edema model, also showing the lowest ulcerogenic effect on the gastric mucosa of treated animals. The results of the pleurisy experiment were not conclusive because the model did not showed results associated with the anti-inflammatory activity but showed evidence of a probable EFSMp4 hemorrhagic effect. As for the antioxidant activity, in the experiments in which it was possible to observe the response of the samples under study, the ethanol extract, EFSMp3 and EFSMp4 reached the lowest IC50 values. Regarding the mutagenicity, it was observed that the extract, as well as EFSMp4, acted to induce mutagenic potential in the experiments without and with metabolic activation. Two major metabolites of EFSMp4 were isolated and identified as the coumarins meranzine hydrate and murpanidine, already reported in leaves of the species. Meranzine hydrate was quantified representing a 8.4% (m/m) of EEMp. Macrophage cytotoxicity in the concentration range evaluated (2.5-40.0 g/mL) was not observed and NO inhibition percent at 40.0 μg/ml were EFSMp4 (54.0%), murpanidine (30.1%) and meranzine hydrate (8.0%). The identification of another coumarin of the same class of the two already purified was proposed from analysis by HPLC-ESI-MS (murralongin). The results suggest that coumarins are a group of major secondary metabolites in the ethanolic extract, as well as their relationship with the anti-inflammatory action of the extract.
Descrição
Palavras-chave
Murraya paniculata, Fracionamento biomonitorado, Atividade anti-inflamatória, Atividade antioxidante, Cumarina, Biomonitored fractionation, Anti-inflammatory activity, Antioxidant activity, Coumarin