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Optimization of screening methods for the evaluation of the antileishmanial potential of halophytes and macroalgae from the Iberian coast
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Orientador(es)
Resumo(s)
Leishmaniases are parasitic diseases caused by Leishmania parasites, which are transmitted to mammals by the bite of sand flies. In the Mediterranean basin, L. infantum is the etiological agent of human visceral leishmaniasis, the most severe form of the disease. The obvious limitations of current available drugs still provide an undoubtedly motivation for research of novel compounds with antileishmanial potential. In the last decades, marine organisms or species living in a terrestrial habitat strongly influenced by the sea have been shown to be potential sources of rich bioactive compounds. Thus, the search for marine natural products with antileishmanial activity has gathered interest and, as a result, some promising extracts and compounds have been described. For assessment of the in vitro activity of natural extracts mainly axenic promastigotes and amastigotes have been used. Although the amastigote-macrophage system remains the ideal model, its use has been neglected mainly due to the lack of sensitive and rapid in vitro screening methods, which rely on the inherent difficulties of the intracellular stage. In this context, the main objective of this work is to contribute for the study of the antileishmanial potential of macroalgae and halophytes, through the optimization of screening methods focusing on the intracellular amastigote form. The direct counting method was optimized and used for the assessment of extracts activity against intracellular amastigotes. Concerning the parasite rescue assay, lysis conditions were established. However, aberrant results were obtained when the effect of amphotericin B and that of different infection ratios was assessed, in comparison to the direct counting method results. As the complete optimization of the parasite rescue assay was not possible and reminding the shortcomings of the direct counting method, halophyte and macroalgae extracts were tested against L. infantum promastigotes. Halophytic extracts were in general inactive against L. infantum promastigotes. Spergularia rubra and Inula crithmoides dichloromethane extracts were the most active, but were also extremely cytotoxic towards THP-1 macrophages. Concerning macroalgae, the hexane and dichloromethane extracts of Cystoseira tamariscifolia and the hexane extract of C. usneoides were highly active towards the promastigotes with IC50 values below 35 μg mL-1, yet they had also poor selectivity. C. baccata hexane extract was the most selective algal extract (SI > 1.32). Additionally, C. baccata and C. barbata hexane:dichloromethane extracts, obtained by hot soxhlet extraction, displayed interesting antileishmanial activities (IC50, 50.2 ± 2.6 and 15.1 ± 2.9, respectively) towards the intracellular stage of L. infantum combined with promising SI values (>2.5 and >8.3, respectively), reinforcing the main argument of this work that intracellular amastigotes are the ideal in vitro model to be used in drug screening purposes, which may yield a larger number of positive hits if implemented in future screening efforts.
Descrição
Palavras-chave
Parasitologia médica Leishmaniose
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical