IJCS | Volume 32, Nº2, May/June 2019

209 Brozzo et al. Curcuma longa abolishes contractions in the aortic artery Int J Cardiovasc Sci. 2019;32(3)207-216 Original Article two groups (n = 6), being sufficient to perform in vitro experiments for each concentration of the same group with the same nature, defined as group 1 (annulus with present endothelium) and 2 (annulus without endothelium). Subsequently, the subgroups were divided into five groups (n = 6) for the assessment of the verified action mechanism, namely: group 1.1 (L-NAME), group 1.2 (Indomethacin), group 1.3 (Atropine), group 2.1 (KCl 20) and group 2.2 (KCl 80). Preparation and setting-up of the vascular annulus Male rats of the Rattus norvegicus species, of the Wistar lineage, weighing 200-300 g, were euthanized by cervical dislocation and then a ventral incision was performed with opening of the rib cage by performing an excision in the sternum and part of the ribs, for visualization and removal of the intrathoracic organs. The thoracic aorta was removed and transferred to a Petri dish containing Krebs nutrient solution aerated by carbogenic mixture (95% CO 2 + 5% O 2 ) and cleansed by removing the adipose tissue and connective tissue adhered to the artery segment. The artery segment was fractionated into 4 mm-rings and set up, fixed between 2 small wire triangles. One side of one of the triangles was introduced into the annulus lumen, with the vertex opposite to the annulus, being fixed to the base of a thin metallic rod attached to the system, and the opposite vertex of the other triangle, by a cotton thread, attached to a isometric force transducer (AdInstruments). The aorta preparations for all experiments were preliminarily stabilized for 1 hour in Krebs nutrient solution, with replacement of the solution every 15 minutes to eliminate and minimize metabolite effect; constant adjustment of the passive voltage applied to the annulus was also performed to 1 g (baseline initial tension), maintained at 37°C and continuously aerated by carbogen (95% O 2 + 5% CO 2 ). The data on voltage variations, as a function of tissue reactivity to drug administration, were obtained after the conversion of the electrical signals captured by the transducer into digital signals and transmitted by an amplifier-recorder (AdInstruments). The variation records of the degree of contraction of the annulus smooth musculature were sent to a microcomputer, where they were processed and stored in the Protowin organ baths software (Pan Lab) for later analytical and statistical treatment. All protocols were carried out according to the norms issued by the National Council for the Control of Animal Experimentation (CONCEA) and approved by the Ethics Committee on Animal Use ( Comitê de Ética no Uso de Animais – CEUA) of UFAC ( Universidade Federal do Acre ) under number 23107.018498 / 2016- 40. Evaluation of the AECL activity in aortic annuli with and without endothelium contracted with phenylephrine Immediately after the preparations’ stabilization period, the presence (E + ) or absence (E-) of functional endothelium in the annuli was verified by adding ACh on the plateau of the sustained tonic phase of the PHE- induced contraction, establishing as (E + ) annuli those of which relaxation percentages induced by ACh were ≥ 70% and as (E-), those with < 5% of relaxation. The tissues prepared in the recipients were again contracted by PHE and, after five to seven minutes of sustained tonic phase contraction, increasing concentrations of AECL (1, 3, 10, 30, 100, 300 and 1000 μg/mL) were cumulatively added to the preparations containing (E + ) or (E-) annuli. The relaxation percentage was determined by comparing the PHE contraction values​ before and after the cumulative addition of the extract and also by calculating the EC50 values. Evaluation of the AECL effect after inhibition of the enzymes of thenitric oxide (NO) productionpathway, the prostacyclin (PGE2 and PGI2) production pathway and the endothelial muscarinic blockade In annulus-containing preparations (E + ), the cumulative administration of AECL at the PHE-induced contraction plateau was performed before and after the L-NAME (100 μM), and incubated for 30 minutes, for the evaluation of nitric oxide (NO) involvement in the AECL effect. To assess the involvement of prostacyclines, indomethacin (10 μM), instead of L-NAME, was also incubated for 30 minutes. To verify the probable participation of muscarinic receptors, following the same experimental protocol, atropine incubation (1 μM) was previously performed for 15 minutes, instead of the two enzyme inhibitors. Evaluation of AECL activity in contracted aortic rings with depolarizing KCl solution To evaluate the involvement of ion channels, experiments were performed using (E-) annuli. In these approaches, after stabilization of the preparations and other mandatory preliminary procedures, instead of