Desenvolvimento de método para determinação simultânea de Prostaglandina E2 e Leucotrieno B4 em plasma humano e avaliação de atividade anti-inamatória de espécies da família Lauraceae.

Abstract

It is believed that substances capable of simultaneously inhibiting the cyclooxygenase (COX) and lipoxygenase (LOX) enzymes have superior anti-inflammatory activity and fewer adverse effects. Therefore, for the search for substances with this mechanism of action, accessible methods are needed to evaluate the inhibition of these enzymes. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) are important mediators of the inflammatory cascade, produced by the COX and LOX enzymes, respectively. There are methods for quantifying PGE2 in human plasma; however, for the quantification of LTB4, the available assays have limitations. The Lauraceae family presents a series of species with promising anti-inflammatory activity reported in the literature, but few have been investigated for the mechanism of action of dual inhibition of the COX and LOX pathways. In this sense, an assay capable of simultaneously evaluating the inhibition of the COX and LOX pathways, through the quantification of PGE2 and LTB4 in human plasma, was developed to enable the evaluation of the anti-inflammatory activity of plant extracts from the Lauraceae family. In this assay, inflammation was induced in human blood, treated with the samples to be tested, and reference drugs as control groups. The extraction of the analytes was carried out by protein precipitation and solid-phase extraction. The analytes (PGE2 and LTB4) were quantified by chromatography coupled to mass spectrometry. The acquired data were treated by analysis of variance, followed by the Dunnett and Mann-Whitney tests. Subsequently, extracts from four species of the Lauraceae family – Ocotea odorifera, Ocotea diopyriofolia, Persea major, and Nectandra membranacea – were investigated for their anti-inflammatory activity. As a result, it was observed that the developed methodology met the criteria of reliability, selectivity, and detection limit of the assay, and it was determined, unprecedentedly, the dual inhibition activity of the COX and LOX pathways for all evaluated species. Therefore, in this work, it was possible to develop and validate a new methodology capable of enabling the discovery of antiinflammatory samples with a mechanism of action differential to those of currently available medication.