IJCS | Volume 32, Nº2, May/June 2019

208 Brozzo et al. Curcuma longa abolishes contractions in the aortic artery Int J Cardiovasc Sci. 2019;32(3)207-216 Original Article leaves were used to wrap fish and to give aroma and flavor to the food preparation. 9 More recent studies report cardioprotective, 10 anti-inflammatory, 8,11 antimicrobial, 12 antiproliferative, 12 antioxidant, 13 antifungal 14 and vasorelaxant 15 properties. There is still little available information on its mechanism of action and effects on the cardiovascular system. The present study aims to investigate AECL action on vascular contractions induced in the isolated aortic artery of normotensive rats. Preclinical scientific studies of this nature may not only serve as the basis to obtain alternative phytomedicinal sources of attenuation of cardiovascular system-related diseases among the traditional needy populations, but they can, primarily, indicate to the scientific community and to the pharmaceutical industry new sources of raw materials to obtain medicinal principles available in nature, which already have discreet and crucial scientific information. Methods Preparation of the Curcuma longa L extract The plant was collected in a private property located in Vila Campinas, Ramal das chácaras, in the municipality of Plácido de Castro, in the state of Acre, northern Brazil. A voucher specimen was deposited at the herbarium of Universidade Federal do Acre (UFAC), under number 20002. The healthy-looking leaves were selected, washed under running water and placed in a greenhouse at 40°C for 48 hours, where dehydration occurred, and then they were ground in a knife-type mill at the Food Technology Unit (UTAL) of Universidade Federal do Acre. To obtain the aqueous extract of Curcuma Longa (AECL), 4 liters of distilled water with 370 grams of the crushed plant material were placed in a heated balloon flask for 10 minutes at 100°C, followed by filtration. The filtrate was lyophilized, stored in an amber glass vial and kept under refrigeration at 4°C. Drugs Phenylephrine hydrochloride (PHE), atropine sulfate, acetylcholine chloride (ACh), NG-nitro-L-arginine- methyl ester (L-NAME) and Indomethacinwere used, all fromSIGMA-ALDRICH. Stock solutions of indomethacin were dissolved together with 5 N sodium bicarbonate (NaHCO 3 ) in distilled water. The other drugs, including AECL and solutions, were diluted in distilled water. The perfusion medium used was Krebs-Henseleit nutrient solution, containing (in mM): NaCl (118.4); KCl (4.75); KH 2 PO 4 (1.18); NaHCO 3 (25); MgSO 4 (1.18); CaCl 2 (1.9) and glucose (5). The nutrient solution had the pH adjusted with 1M HCl or 1M NaOH to 7.3 and 7.4. The AECL concentrations that were cumulatively used in all experiments were, invariably, 1, 3, 10, 30, 100, 300 and 1000 μg/mL. PHE and ACh concentrations were 0.1 µM in all experimental protocols. Toxicity Assay To assess possible toxic effects of the AECL, a Fixed Dose Procedure (FDP) parameter was used according to the Acute Toxic Class Method of 2001 (OECD). 16 In this test, mice fasted for 12 hours prior to the experiment and were divided into 3 groups (n sample = 6, used for convenience): group 1 (control), 2 (dose of 2000 mg/kg) and 3 (dose of 5000 mg/kg), comprising a total number of 18 animals. Only saline solution was administered in group 1, and in group 2, the dose of 2000 mg/kg. In the absence of lethality or toxicity, then the dose of 5000 mg/kg was administered in group 3. The toxicity assessed by observing the following behavioral parameters, described in the hippocratic test of Malone and Robichaud 17 are: attention, alertness, analgesia, spontaneous motor activity, locomotion, lack of appetite, apathy, response to tact, piloerection, ptosis, respiratory rate, cyanosis, stereotypy, contortion, aggressiveness, ataxia, posture, sweating, urination, diarrhea and seizure. The parameters were observed every 60 minutes for 3 hours after the administration of the extract. Subsequently, they were also observed after 24, 48, and 72 hours and on the 15 th day. At the end of the observations all the animals were euthanized according to the euthanasia practice guidelines of the National Council for the Control of Animal Experimentation ( Conselho Nacional de Controle de Experimentação Animal – CONCEA). Traditional barbiturates and anesthetics were used for the euthanasia procedure, with a rapid, gentle effect andwithminimal discomfort to the animals. These drugs are potent Central Nervous Systemdepressants, of which effects are widely known and predictable. Assessment of AECL activity in the annuli of isolated thoracic aorta of rats A total of 14 rats were used in this study, resulting in 42 aortic annuli. Initially the annuli were divided into