Caracterização por espectrometria de massas e investigação das atividades anti-inflamatória e gastroprotetora do extrato etanólico e diterpenos clerodânicos de folhas de Casearia sylvestris Swartz
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Data
2016-02-26
Autores
Castro, Rogério Cardoso de [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O objetivo geral deste trabalho foi realizar um estudo químico-farmacológico do extrato etanólico de folhas de Casearia sylvestris Swartz (EEtCs), frações obtidas por extração em fase sólida (EFS1: apolar; EFS2: rica em diterpenos clerodânicos; EFS3: polar) e padrões de diterpenos clerodânicos (DC). Os objetivos da parte química foram caracterizar os padrões de DC por espectrometria de massas (full scan e fragmentação) com fonte de ionização por spray de elétrons (ESI-MS e MSn) e identificar DC no EEtCs e na EFS2 por ESI-MS, ESI-MSn e por cromatografia em camada delgada de alta eficiência com detecção por imagem química formada por espectrometria de massas com fonte de dessorção por spray de elétrons (HPTLC-DESI-MS-IMAGING). A parte farmacológica consistiu na investigação da atividade anti-inflamatória e gastroprotetora do EEtCs, frações (EFS1-3) e padrões de DC. A atividade anti-inflamatória foi avaliada por meio de ensaios em ratos, pleurisia induzida pela carragenina e edema de pata induzido por uma fosfolipase A2 de veneno de Bothrops jararacussu (BthTX-II), e os seguintes ensaios in vitro: atividade enzimática das ciclo-oxigenases 1 e 2 (COX-1 e COX-2) e produção de óxido nítrico (NO) e prostaglandina E2 (PGE2) por macrófagos estimulados por lipopolissacarídeo. A atividade gastroprotetora foi avaliada em modelo de lesões gástricas induzidas por indometacina em ratos e ensaio in vitro de atividade anti-Helicobacter pylori. Os resultados levaram às conclusões a seguir. A caracterização dos padrões de DC por espectrometria de massas permitiu estabelecer o padrão de fragmentação dos DC com anel diacetálico do tipo das casearinas, o qual representa uma importante fonte de informação para desreplicação em estudos metabolômicos de extratos vegetais. A análise do EEtCs e da EFS2 por ESI-MS (full scan e fragmentação) e HPTLC-DESI-MS-IMAGING permitiu identificar as casearinas B, C, D, F, J, O e X. Os ensaios farmacológicos confirmaram que o EEtCs apresenta atividade anti-inflamatória e gastroprotetora e que estas dependem essencialmente dos DC, uma vez que a EFS2 e DC isolados, mas não a EFS1 e a EFS3, reproduziram tais efeitos, exceto a atividade anti-H. pylori que foi reproduzida apenas pela EFS2. O efeito anti-inflamatório do EEtCs não depende apenas dos DC, mas também de prováveis metabólitos secundários capazes de inibir a PLA2 e a COX-2, uma vez que o EEtCs inibiu o edema de pata induzido pela BthTX-II, efeito não reproduzido por DC isolados, e a atividade enzimática da COX-2, efeito não reproduzido pela EFS2 ou por DC isolados. A atividade anti-inflamatória dos DC do tipo das casearianas não foi associada à inibição da produção de NO, mas envolveu a redução da produção da PGE2 por macrófagos por mecanismos independentes da inibição da COX-2. É provável que o efeito gastroprotetor do EEtCs envolva tanto mecanismos de proteção contra lesões gástricas induzidas por anti-inflamatórios não-esteroidais quanto a atividade anti-H. pylori. No primeiro caso, a atividade deve estar relacionada com os DC, pois tanto a EFS2 quanto DC isolados reproduziram tal atividade. No segundo caso, apenas a EFS2 apresentou atividade anti-H. pylori, sugerindo a participação de outros metabólitos secundários presentes na EFS2.
The aim of this study was to carry out a chemical-pharmacological study of the ethanol extract from leaves of Casearia sylvestris Swartz (EtECs), fractions obtained by solid phase extraction (SPE1: nonpolar; SPE2: rich in clerodane diterpenes; SPE 3: polar) and standards of clerodane diterpenes (CD). The objectives of the chemical step was to characterize standards of CD by mass spectrometry (full scan and fragmentation) using electrospray ionization (ESI-MS and MSn) and to identify CD in EtECs and SPE2 by ESI-MS, ESI-MSn and high performance thin layer chromatography/desorption electrospray ionization mass spectrometry imaging (HPTLC-DESI-MS-IMAGING). The pharmacological study consisted in investigation of anti-inflammatory and gastroprotective activities of EtECs, fractions (SPE1-3) and CD standards. The anti-inflammatory activity was assessed in rats through pleurisy induced by carrageenan and paw edema induced by phospholipase A2 from Bothrops jararacussu venom (BthTX-II), and the following in vitro assays: enzymatic activity of cyclooxygenases 1 and 2 (COX-1 and COX-2) and macrophage stimulation by lipopolysaccharide to investigate the production of nitric oxide (NO) and prostaglandin E2 (PGE2). The gastroprotective activity was assessed in a model of indomethacin-induced gastric lesions in rats and in vitro assay of anti-Helicobacter pylori activity. The results led to the following conclusions. The characterization of the standards of CD by mass spectrometry demonstrated that it was possible to establish the fragmentation pattern of CD with diacetalic ring like casearins, which is an important source of information for dereplication in metabolomics studies of plant extracts. The analysis of EtECs and SPE2 by ESI-MS (full scan and fragmentation) and HPTLC-DESI-MS-IMAGING identified the casearins B, C, D, F, J, O and X. The pharmacological assays have confirmed that the EtECs exhibits anti-inflammatory and gastroprotective activities and these depend essentially on the CD, since SPE2 and isolated CD, but not SPE1 and SPE3, reproduced these effects, except anti-H. pylori activity that has been reproduced only by the SPE2. The anti-inflammatory effect of EtECs depends not only on CD, but also likely secondary metabolites can inhibit PLA2 and COX-2, since EtECs inhibited the paw edema induced by BthTX-II, which was not reproduced for CD isolation, and the enzymatic activity of COX-2, which was not reproduced by SPE2 and isolated CD. The anti-inflammatory activity of the CD casearins-like were not associated with inhibition of NO production but involved the reduction of PGE2 production by macrophages through independent mechanisms of inhibition of COX-2. It is likely that the gastroprotective effect of EtECs involving both mechanisms of protection against gastric lesions induced by anti-inflammatory nonsteroidal and anti-H. pylori activity. In the first case, it must be related to the CD, since both the SPE2 and isolated CD reproduced this activity. In the second case, only SPE2 showed anti-H. pylori activity, suggesting the involvement of other secondary metabolites present in SPE2.
The aim of this study was to carry out a chemical-pharmacological study of the ethanol extract from leaves of Casearia sylvestris Swartz (EtECs), fractions obtained by solid phase extraction (SPE1: nonpolar; SPE2: rich in clerodane diterpenes; SPE 3: polar) and standards of clerodane diterpenes (CD). The objectives of the chemical step was to characterize standards of CD by mass spectrometry (full scan and fragmentation) using electrospray ionization (ESI-MS and MSn) and to identify CD in EtECs and SPE2 by ESI-MS, ESI-MSn and high performance thin layer chromatography/desorption electrospray ionization mass spectrometry imaging (HPTLC-DESI-MS-IMAGING). The pharmacological study consisted in investigation of anti-inflammatory and gastroprotective activities of EtECs, fractions (SPE1-3) and CD standards. The anti-inflammatory activity was assessed in rats through pleurisy induced by carrageenan and paw edema induced by phospholipase A2 from Bothrops jararacussu venom (BthTX-II), and the following in vitro assays: enzymatic activity of cyclooxygenases 1 and 2 (COX-1 and COX-2) and macrophage stimulation by lipopolysaccharide to investigate the production of nitric oxide (NO) and prostaglandin E2 (PGE2). The gastroprotective activity was assessed in a model of indomethacin-induced gastric lesions in rats and in vitro assay of anti-Helicobacter pylori activity. The results led to the following conclusions. The characterization of the standards of CD by mass spectrometry demonstrated that it was possible to establish the fragmentation pattern of CD with diacetalic ring like casearins, which is an important source of information for dereplication in metabolomics studies of plant extracts. The analysis of EtECs and SPE2 by ESI-MS (full scan and fragmentation) and HPTLC-DESI-MS-IMAGING identified the casearins B, C, D, F, J, O and X. The pharmacological assays have confirmed that the EtECs exhibits anti-inflammatory and gastroprotective activities and these depend essentially on the CD, since SPE2 and isolated CD, but not SPE1 and SPE3, reproduced these effects, except anti-H. pylori activity that has been reproduced only by the SPE2. The anti-inflammatory effect of EtECs depends not only on CD, but also likely secondary metabolites can inhibit PLA2 and COX-2, since EtECs inhibited the paw edema induced by BthTX-II, which was not reproduced for CD isolation, and the enzymatic activity of COX-2, which was not reproduced by SPE2 and isolated CD. The anti-inflammatory activity of the CD casearins-like were not associated with inhibition of NO production but involved the reduction of PGE2 production by macrophages through independent mechanisms of inhibition of COX-2. It is likely that the gastroprotective effect of EtECs involving both mechanisms of protection against gastric lesions induced by anti-inflammatory nonsteroidal and anti-H. pylori activity. In the first case, it must be related to the CD, since both the SPE2 and isolated CD reproduced this activity. In the second case, only SPE2 showed anti-H. pylori activity, suggesting the involvement of other secondary metabolites present in SPE2.
Descrição
Palavras-chave
Casearia sylvestris, Diterpenos clerodânicos, Casearinas, Espectrometria de massas, Atividade anti-inflamatória, Atividade gastroprotetora, Clerodane diterpenes, Casearins, Mass spectrometry, Anti-inflammatory activity, Gastroprotective activity