Padronização dos extratos polares das partes aéreas de Actinocephalus divaricatus (Körn.) Sano (Eriocaulaceae)
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Data
2016-08-05
Autores
Silva, Ana Caroline Zanatta [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Este trabalho descreve a caracterização química dos extratos metanólicos de capítulos, escapos e folhas de Actinocephalus divaricatus, descrito pela primeira vez na literatura. Primeiramente, as amostras foram analisadas por cromatografia líquida de alta eficiência (HPLC) acoplada com detector de arranjo de fotodiodos (PAD) e por espectrometria de massas em tandem acoplado a um orbitrap com interface de ionização por electrospray, usando um sistema LC para a separação cromatográfica dos metabólitos (HPLC-ESI-HRMS). Esta estratégia permitiu explorar e diferenciar os metabólitos secundários presentes, sendo identificados 31 compostos incluindo 12 flavonoides, 5 naftopiranonas e 14 saponinas. Esta análise diferencial das três partes da planta contribuirá com a discussão do gênero Actinocephalus, uma vez que os estudos na literatura são relatados apenas para Paepalanthus sect. Actinocephalus. A separação dos metabólitos presentes nos extratos metanólicos dos escapos e folhas foi realizada empregando técnicas cromatográficas, como Sephadex, MPLC, HPLC-PAD e HPLC-IR, em que foi possível obter treze compostos: três ácidos fenólicos, sete flavonoides, duas naftopiranonas e uma saponina. As estruturas de todos os compostos foram determinadas por análise dos dados obtidos por RMN mono e bidimensionais. A padronização dos extratos metanólicos foi realizada por meio da quantificação dos flavonoides e naftopiranonas por HPLC-PAD. A metodologia foi validada avaliando-se os parâmetros de seletividade, linearidade, limites de detecção e quantificação, exatidão e precisão. A seletividade foi confirmada por meio dos tempos de retenção e dos espectros de absorção no UV dos padrões de flavonoide e naftopiranona. Os métodos validados apresentaram alta seletividade e permitiram identificar derivados de quercetina e paepalantina glicosilados nos extratos, com limites de detecção (2,56 μg.mL-1) e quantificação (7,76 - 7,77 μg.mL-1) satisfatórios para as condições analisadas, e valores de precisão (0,0028 a 3,90%) e exatidão (83 a 119%) dentro dos limites recomendados pela ANVISA. Os valores encontrados para derivados de quercetina glicosilados foram de 9,64, 39,96, 123,81 mg.g-1 de extrato para capítulos, escapos e folhas, respectivamente. Derivados de paepalantina glicosilados foram quantificados apenas nos capítulos e folhas e apresentaram 39,51 e 5,99 mg.g-1 de extrato de capítulos e folhas, respectivamente. O teor de fenóis encontrados foi de 126,41 246,58 mg.g-1 e 512,24 mg.g-1 de extrato para os capítulos, escapos e folhas, respectivamente. A atividade antiradicalar utilizando DPPH indicou IC50 superior às concentrações de extratos utilizadas (250 µg.mL-1). Paralelamente foi avaliada a atividade antimicrobiana utilizando cepas de bactéria (Gram-positiva e Gram-negativa) e levedura, sendo que os extratos metanólicos de capítulos, escapos e folhas apresentaram fraca sensibilidade frente aos microrganismos testados. Os valores encontrados para a citotoxicidade dos extratos utilizando o método do MTT foram baixos, sendo o melhor resultado encontrado para o extrato metanólico dos capítulos (IC50 = 66,98 µg.mL-1) contra MCF7 (cancro da mama humana). Assim, o estudo químico realizado com a espécie A. divaricatus corrobora com dados da literatura que permitem afirmar que Actinocephalus é um novo gênero, diferenciado de Paepalanthus com a presença de saponinas nos escapos e folhas.
This work describes the chemical characterization of the methanolic extracts of the capitulae, scapes and leaves from Actinocephalus divaricatus, described the first time in the literature. First, the samples were analyzed by high-performance liquid chromatography (HLPC) coupled with a photodiode array detector (PAD) and mass spectrometry tandem coupled to an Orbitrap with electrospray ionization interface using an LC system for separating chromatographic metabolites (HPLC-ESI-HRMS). This strategy has allowed us to explore a differentiate the secondary metabolites present, identified as 31 compounds comprising 12 flavonoids, 5 naphthopyranones and 14 saponins. This differential analysis of the three parts of the plant contributes to the discussion of the Actinocephalus genus, since the studies reported in the literature are only of the Paepalanthus sect. Actinocephalus. The separation of the metabolites present in the methanolic extracts of the scapes and leaves was performed using chromatographic techniques, such as Sephadex, MPLC, HPLC-PAD and HPLC-IR. It was possible to obtain thirteen compounds: three phenolic acids, seven flavonoids, two naphthopyranones and one saponin. The structures of all the compounds were determined by data analysis by mono and bidimensional NMR. The standardization of the methanolic extracts was performed by the quantification of flavonoids and naphthopyranones by HPLC-PAD. The methodology was validated by evaluating the parameters of selectivity, linearity, detection and quantitation limits, accuracy and precision. The selectivity was confirmed by the retention time and UV absorption spectra of flavonoid and standard of naphthopyranone. Validated methods showed high selectivity and identified quercetin glycosides and paepalantine derivatives in extracts, with detection limits (2.56 μg.mL-1) and quantification (7.76 to 7.77 μg.mL-1) satisfactory to the analyzed conditions, and precision values (0.0028 to 3.90%) and accuracy (83-119%) within the limits recommended by ANVISA. The values found for the quercetin glycosides derivatives were 9.64, 39.96, 123.81 mg.g-1 of extract for the capitula scapes and leaves respectively. Paepalantine glycosylated derivatives were quantified only in the capitula and leaves and showed 39.51 and 5.99 mg.g-1 of extract for the capitula and leaves respectively. The phenols contents found were 126.41, 246.58 mg.g-1 and 512.24 mg.g-1 extract for the capitula scapes and leaves respectively. The antiradical activity using DPPH showed IC50 higher than the concentrations of the used extracts (250 μg.mL-1). At the same time, the antimicrobial activity was evaluated by using bacteria and yeast strains, and the methanolic extracts of the capitula, scapes and leaves showed poor sensitivity against the tested microorganisms. The values for the cytotoxicity of the extracts using the MTT assay were low, with the best results found in the methanolic extract of the capitula (IC50 = 66.98 μg.mL-1) against MCF7 (human breast cancer). Thus, the chemical study of the species A. divaricatus corroborates with the literature data has allowed us to state that Actinocephalus is a new genus, different from Paepalanthus with the presence of saponins in the scapes and leaves.
This work describes the chemical characterization of the methanolic extracts of the capitulae, scapes and leaves from Actinocephalus divaricatus, described the first time in the literature. First, the samples were analyzed by high-performance liquid chromatography (HLPC) coupled with a photodiode array detector (PAD) and mass spectrometry tandem coupled to an Orbitrap with electrospray ionization interface using an LC system for separating chromatographic metabolites (HPLC-ESI-HRMS). This strategy has allowed us to explore a differentiate the secondary metabolites present, identified as 31 compounds comprising 12 flavonoids, 5 naphthopyranones and 14 saponins. This differential analysis of the three parts of the plant contributes to the discussion of the Actinocephalus genus, since the studies reported in the literature are only of the Paepalanthus sect. Actinocephalus. The separation of the metabolites present in the methanolic extracts of the scapes and leaves was performed using chromatographic techniques, such as Sephadex, MPLC, HPLC-PAD and HPLC-IR. It was possible to obtain thirteen compounds: three phenolic acids, seven flavonoids, two naphthopyranones and one saponin. The structures of all the compounds were determined by data analysis by mono and bidimensional NMR. The standardization of the methanolic extracts was performed by the quantification of flavonoids and naphthopyranones by HPLC-PAD. The methodology was validated by evaluating the parameters of selectivity, linearity, detection and quantitation limits, accuracy and precision. The selectivity was confirmed by the retention time and UV absorption spectra of flavonoid and standard of naphthopyranone. Validated methods showed high selectivity and identified quercetin glycosides and paepalantine derivatives in extracts, with detection limits (2.56 μg.mL-1) and quantification (7.76 to 7.77 μg.mL-1) satisfactory to the analyzed conditions, and precision values (0.0028 to 3.90%) and accuracy (83-119%) within the limits recommended by ANVISA. The values found for the quercetin glycosides derivatives were 9.64, 39.96, 123.81 mg.g-1 of extract for the capitula scapes and leaves respectively. Paepalantine glycosylated derivatives were quantified only in the capitula and leaves and showed 39.51 and 5.99 mg.g-1 of extract for the capitula and leaves respectively. The phenols contents found were 126.41, 246.58 mg.g-1 and 512.24 mg.g-1 extract for the capitula scapes and leaves respectively. The antiradical activity using DPPH showed IC50 higher than the concentrations of the used extracts (250 μg.mL-1). At the same time, the antimicrobial activity was evaluated by using bacteria and yeast strains, and the methanolic extracts of the capitula, scapes and leaves showed poor sensitivity against the tested microorganisms. The values for the cytotoxicity of the extracts using the MTT assay were low, with the best results found in the methanolic extract of the capitula (IC50 = 66.98 μg.mL-1) against MCF7 (human breast cancer). Thus, the chemical study of the species A. divaricatus corroborates with the literature data has allowed us to state that Actinocephalus is a new genus, different from Paepalanthus with the presence of saponins in the scapes and leaves.
Descrição
Palavras-chave
Metabólitos, Saponinas, Flavonoides, Espectrometria de massa, Citotoxicidade, Metabolites, Saponins, Flavonoids, Mass spectrometry, Cytotoxicity