RESUMO
INTRODUCTION: The genus Clusia L. is mostly recognised for the production of prenylated benzophenones and tocotrienol derivatives. OBJECTIVES: The objective of this study was to map metabolome variation within Clusia minor organs at different developmental stages. MATERIAL AND METHODS: In total 15 organs/stages (leaf, flower, fruit, and seed) were analysed by UPLC-MS and 1H- and heteronuclear multiple-bond correlation (HMBC)-NMR-based metabolomics. RESULTS: This work led to the assignment of 46 metabolites, belonging to organic acids(1), sugars(2) phenolic acids(1), flavonoids(3) prenylated xanthones(1) benzophenones(4) and tocotrienols(2). Multivariate data analyses explained the variability and classification of samples, highlighting chemical markers that discriminate each organ/stage. Leaves were found to be rich in 5-hydroxy-8-methyltocotrienol (8.5 µg/mg f.w.), while flowers were abundant in the polyprenylated benzophenone nemorosone with maximum level detected in the fully mature flower bud (43 µg/mg f.w.). Nemorosone and 5-hydroxy tocotrienoloic acid were isolated from FL6 for full structural characterisation. This is the first report of the NMR assignments of 5-hydroxy tocotrienoloic acid, and its maximum level was detected in the mature fruit at 50 µg/mg f.w. Seeds as typical storage organ were rich in sugars and omega-6 fatty acids. CONCLUSION: To the best of our knowledge, this is the first report on a comparative 1D-/2D-NMR approach to assess compositional differences in ontogeny studies compared with LC-MS exemplified by Clusia organs. Results derived from this study provide better understanding of the stages at which maximal production of natural compounds occur and elucidate in which developmental stages the enzymes responsible for the production of such metabolites are preferentially expressed.
Assuntos
Clusia , Clusia/química , Frutas/química , Cromatografia Líquida , Espectrometria de Massas em Tandem , Benzofenonas/análise , Benzofenonas/química , Benzofenonas/metabolismo , Flores/química , Folhas de Planta/química , Metabolômica/métodos , Sementes/química , Açúcares/análiseRESUMO
Leishmaniasis is a widespread tropical infection caused by different species of Leishmania protozoa. Many of the available drugs against the disease are toxic and in certain cases parasite drug resistance is developed. The discovery of drugs for the treatment of leishmaniasis is a pressing concern. In the present work, we describe in vitro studies of the phenolic compound methyl gallate (MG) against Leishmania (Leishmania) amazonensis and its possible mechanisms of action. The in vitro activity of MG was assayed against L. amazonensis (promastigotes, axenic amastigotes, and intramacrophagic amastigotes). Cytotoxicity tests were performed with J774A.1 macrophages and THP-1 cell derived macrophages. To evaluate mechanisms of action, we analyzed cellular TNF-α, IL-12, IFN-γ, IL-10, IL-6, NO, ROS levels, arginase activity, and structural mechanisms (phagocytic and lysosomal activities) involving macrophage activation. Meglumine antimoniate and amphotericin B were used as reference drugs. It was observed that MG effectively inhibited the growth of both promastigote (IC50 5.71 µM) and amastigote-like forms (EC50 5.39 µM), with much higher selectivity indexes than the reference drugs, being more benign towards J774A.1 macrophages than meglumine antimoniate and amphotericin B, at 1631- and 70.92-fold respectively, with respect to the promastigote form. Additionally, MG proved to be even more active against intracellular amastigotes of the parasite (EC50 4.24 µM). Our results showed that antileishmania activity was associated with increased TNF-α, IL-12, NO and ROS levels, as well as decreased IL-6 and decreased arginase activity. In addition, MG induced increased phagocytic capability, and lysosomal volume in macrophages; structural parameters of microbicidal activity. Taken together, our results suggest that MG may be a promising candidate for new drug development against leishmaniasis.