IJCS | Volume 32, Nº2, May/June 2019

210 Brozzo et al. Curcuma longa abolishes contractions in the aortic artery Int J Cardiovasc Sci. 2019;32(3)207-216 Original Article contractions caused by PHE, contractions induced by increased K + concentrations in the bath – extracellular medium ([K + ]e), were produced; these operations were performed with 20 mM KCl or 80 mM KCl solutions, in separate individual samples; at their plateaus, a cumulative administration of increasing concentrations of AECL was performed. Statistical analysis All points plotted in the graphs are expressed as mean ± standard deviation (SD) and represent the individual experiments (control and treatments) (n sample = 6, by convenience) for each one of them, performed individually. The nature of biological responsiveness and the biochemical processes triggered by the injection of exogenous substances and the reactivity due to the induction of endogenous releases, as well as considering the very nature of the crude extracts (consisting of many active principles), naturally justify the fact that the EC 50 values ​resulting from the analyses on individual concentration-response curves were carried out through non-linear regression. The D’Agostino normality test was performed. As the data were normal, the two-way analysis of variance (ANOVA) was applied. Statistical differences between the means of the results were determined by the paired Student’s t test and when appropriate (for multiple comparisons). The authors did not observe limiting factors in this study. The analyses and representations were carried out with the software GraphPad Prism6.0 (GraphPad Software Inc., San Diego, CA, USA), with significance being set at p < 0.05. Results Toxicity None of the animals died or showed deleterious behavioral changes capable of compromising the limits of normality during the toxicity studies (data not shown). The group receiving the 2000 mg/kg dose, even up to the fifteenth day of observation, showed no alterations as a result of the treatment. At the dose of 5000 mg/kg there was mild sedation in the first hour after the extract administration in only one animal in the group. At the subsequent hours and days, none of the animals showed any behavioral change or died. Therefore, AECL did not show any alterations of clinical importance in the animals submitted to the treatments. Influence of the vascular endothelium on AECL activity in isolated aortic artery annuli of normotensive rats pre-contracted with PHE In E + annuli, the AECL abolished contractions induced by PHE, with an EC 50 = 4.32 ± 0.05 μg/mL. Significant vasorelaxation (p < 0.01) occurred in a concentration- dependent manner. In the E- annuli, it also reached 100%, but the AECL response was significantly (p < 0.01) modified by the removal of the vascular endotheliumand EC 50 increased to 134.20 ± 1.80 μg/mL, indicating that the effect of AECL strongly depends on factors derived from the vascular endothelium (Figure 1). Involvement of NO in the AECL relaxant effect on isolated aortic artery annuli of normotensive rats pre-contracted with PHE After inhibition of the NO synthesis enzyme by L-NAME, the AECL vasorelaxant effect on intact aorta annuli was significantly (p < 0.01) inhibited, an event evidenced by an increase in the EC 50 , from 4.32 ± 0.05 μg/mL to 126.50 ± 2.35 μg/mL (Figure 2), indicating clear involvement of the NO production pathway in the AECL effect. Involvement of the prostacyclin production pathway in the AECL relaxant effect on isolated aortic artery annuli of normotensive rats pre-contractedwith PHE After blocking prostaglandin synthesis with indomethacin, the vasorelaxant response induced by AECL was significantly (p < 0.01) inhibited after indomethacin incubation, a fact disclosed by the EC 50 elevation, from 4.32 ± 0.05 μg/mL to 124.6 ± 0.05 μg/mL), suggesting that the AECL effect is also related to the prostacyclin production pathway (Figure 3). Involvement of themuscarinic pathway in the AECL relaxant effect on isolated aortic artery annuli of normotensive rats pre-contracted with PHE In this approach, the vasorelaxant response induced by AECLwas themost significantly attenuated, as shown by the prominent (p < 0.01) increase in EC 50 , from 4.32 ± 0.05 μg/mL to 437.10 ± 0.2 μg/mL (Figure 4). These data suggest that, of the investigated pathways, muscarinic receptors seem to be the most significantly implicated in the vasorelaxant effect induced by the AECL on the isolated thoracic aorta of rats.