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ABSTRACT Caryocar brasiliense Cambess., Caryocaraceae (pequi) is a typical Brazilian Cerrado tree. A previous study showed that the butanolic fraction of pequi leaves promotes endothelium-dependent relaxation mediated by nitric oxide and that it causes reversible hypotension in rats. In the present study, we investigated the cell signaling pathways associated with the butanolic fraction-induced nitric oxide release, and we characterized the chemical composition of its fraction. Vascular reactivity tests, a western blotting analysis, and a chemiluminescence assay were used to investigate the signaling pathways involved in the vasorelaxant effect of the butanolic fraction. Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry was used to characterize the butanolic fraction chemical composition. Vasorelaxation was mediated through the activation of the calmodulin and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways, leading to subsequent endothelial nitric oxide synthase phosphorylation and nitric oxide production, as evidenced by western blotting and chemiluminescence assays, respectively. The chemical characterization of the butanolic fraction revealed the presence of 72 oxygenated compounds, whose molecular formulae are compatible with phenolic compounds, suggesting a potential contribution of these compounds for the butanolic fraction vasorelaxant effect. These findings show that the calmodulin and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways are involved in the butanolic fraction-induced endothelial nitric oxide synthase activation and are promoted by polyphenol compounds present in the C. brasiliense leaves.
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Objective To describe and to characterize the relaxing effect of an extract of the bark of Combretum leprosum on isolated arterial rings from different animals.Methods Rings (3 to 4mm) from rabbit, rat, or porcine arteries rings were suspended in an organ bath (Krebs, 37°C, 95%O2/5%CO2) to record isometric contractions. After the stabilization period (2 to 3 hours) contractions were induced by the addition of phenylephrine (0.1 to 0.3µM) or U46619 (10 to 100nM), and Combretum leprosum extract was added on the plateau of the contractions. Experiments were performed to determine the potency, duration, reversibility, and to get insights on the potential mechanism involved in extract-induced relaxations.Results In all rings tested, Combretumleprosum extract (1.5μg/mL) was able to cause relaxations, which were strictly endothelium-dependent. In rabbit or rat thoracic aorta rings, the relaxations were reversed by vitamin B12a or L-NG-nitroarginine. In porcine right coronary arteries and rabbit abdominal aorta, extract caused both L-NG-nitroarginine-sensitive and L-NG-nitroarginine-resistant relaxations. In rabbit thoracic aorta, the extract was relatively potent (EC50=0.20µg/mL) and caused relaxations; intriguingly the endothelium continued to produce relaxing factors for a long period after removing the extract. The magnitude of extract-induced relaxations was significantly reduced in the absence of extracellular Ca2+; in addition, the TRPs channels blocker ruthenium red (10µM) was able to revert extract-induced relaxations. Phytochemical analyses indicated that the extract was rich in polyphenol-like reacting substances.ConclusionsCombretum leprosum extract contains bioactive compounds capable of promoting Ca2+-dependent stimulation of endothelial cells which results in a prolonged production of relaxing factors.
Objetivo Descrever e caracterizar os relaxamentos induzidos por um extrato das cascas de Combretum leprosum em anéis de artérias de diferentes espécies de animais.Métodos Anéis (3 a 4mm) de artérias de coelho, rato e porco foram montados em cubas para órgão isolado (Krebs, 37°C, 95%O2/5%CO2) para registro das contrações isométricas. Após um período de estabilização (2 a 3 horas), as contrações foram induzidas com fenilefrina (0,1 a 0,3µM) ou U46619 (10 a 100nM); no platô dessas contrações, adicionamos o extrato Combretum leprosum. Diferentes protocolos foram realizados para determinar potência, duração, reversibilidade e mecanismo dos relaxamentos induzidos pelo extrato.Resultados Em todas as preparações testadas, o extrato de Combretum leprosum (1,5µg/mL) provocou relaxamentos dependentes de endotélio. Em aorta torácica de coelho ou rato, os relaxamentos foram revertidos pela vitamina B12a ou L-NG-nitro-arginina. Em anéis de aorta abdominal de coelho e de artérias coronárias de porco, o extrato causou relaxamentos sensíveis e resistentes à L-NG-nitro-arginina. Em aorta torácica de coelho, o extrato foi relativamente muito potente (EC50=0,20μg/mL) e quando causou relaxamentos; intrigantemente o endotélio continuou a produzir fatores relaxantes por um longo período após remoção do extrato. A magnitude dos relaxamentos induzidos pelo extrato foi significativamente reduzida em ausência Ca2+ extracelular; ademais, o vermelho de rutênio (10μM), um bloqueador de canais TRPs, foi capaz de reverter os relaxamentos induzidos pelo extrato. Análises preliminares indicaram que o extrato continha compostos com reatividade química semelhante à polifenóis.Conclusão O extrato de Combretum leprosum contem compostos bioativos capazes de promover estimulação dependente de Ca2+ das células endoteliais a qual resulta numa produção prolongada de fatores relaxantes.