Bioprospecção em espécies vegetais da família Fabaceae, da mata atlântica mineira, visando a obtenção de substâncias com potencial anti- inflamatório

Abstract

Crude leaf extracts from 47 different Fabaceae species were explored for anti-inflammatory activity in different mechanisms of action in the search for compounds with anti- inflammatory potential. Initially, the extracts were tested for antioxidant activity in vitro, and nine species showed great potential, comparable to ascorbic acid. Then, an ex vivo bioassay with human whole blood was developed for the production of IL-10, under inflammatory conditions. It was possible to evaluate the potential of Fabaceae species samples to inhibit the production of this cytokine, and 22 of these species showed inhibition potential comparable to dexamethasone, with EC50 averages below 50 μg/mL. Using the same methodology ex vivo in human whole blood, another assay was developed to assess the anti-inflammatory activity by monitoring the production of PGE2 by detection in LC-MS/MS. The validated method proved to be effective for screening the anti-inflammatory activity of a large number of samples, and five of the total species studied in this work had the potential to inhibit the production of PGE2. Among the 47 samples tested, the species P. pluviosa was active in these three different mechanisms of action tested against inflammation, corroborating the great anti- inflammatory potential of this species with reports from the literature. Among the five species active in the ex vivo blood test for inhibiting the production of PGE2, four of them also exhibited anti-inflammatory potential in inhibiting ear edema in vivo, confirming the efficacy of the ex vivo assay developed in the blood for screening activity anti-inflammatory. In addition, among these four species, two of them (A. polyphylla and P. pluviosa) still presented potential to inhibit neutrophil recruitment, evaluated by the MPO assay in the ear fragments, demonstrating the great anti-inflammatory potential of these species, being A. polyphylla unprecedented in terms of this pharmacological property so far. The composition study of the active samples against inflammation was carried out by two approaches: the first consisted in peak annotation in a bioguided way, using the modern in silico fragmentation tool MetFrag, of the major compounds present in extract samples that inhibited the production of PGE2 in the blood and qualitative comparison of these compounds in these extracts, to seek to putatively identify the compounds present there and possibly responsible for the inhibition of the production of PGE2 in the blood. The other approach used metabolomic studies in which the main simultaneous biomarkers in the different mechanisms of action (in this case the antioxidant activity and inhibition of the production of IL-10 and PGE2) were determined and later annotated, also with the aid of MetFrag, as a more comprehensive and objective putative identification of the most important compounds against inflammation among the tested Fabaceae samples. Different compounds were annotated in the two approaches, as glycosylated flavonoids or aglycone, triterpenes, saponins, sphingolipids, alkaloids, ellagitannins, and ellagic acid derivatives, and still less common ones such as chlorinated, sulfurized, or nitrogenous flavonoids. These results will allow guiding the isolation to confirm the structure and anti-inflammatory activity of these compounds, in future works. Finally, a method for comparing the levels of PGE2 and LTB4 in the ear fragments from in vivo test was developed, allowing to compare the groups of samples from the negative and positive controls (indomethacin and dexamethasone), as well as the P. pluviosa species. The method proved to be effective as a limit test to differentiate the levels of PGE2 and LTB4 between the groups of tested samples in the ear edema test, allowing us to infer that P. pluviosa may act inhibiting possibly in the COX and LOX pathways simultaneously, or even the PLA2, like steroidal anti- inflammatory drugs, corroborating with other works in the literature about this species. Finally, this work enabled the development of different methodologies for the evaluation of anti-inflammatory activity ex vivo, such as screening methods before evaluation in in vivo tests, and the quantitation methods of PGE2 in blood or PGE2 and LTB4 in the ear fragments may be used in future works, with natural products or synthetic compounds, to evaluate the possible inhibition of the COX and LOX pathways against inflammation.