2.5.3. Evaluating the Chloroform Fraction and Isolated Metabolites’ Direct Relaxant Effect

Vasodilating capacities were assessed using the isolated artery method, as formerly reported [17,18]. Briefly, the aorta was removed, cleansed of any connective tissue and fats, and sliced into rings (3 mm). Each ring was hung in Krebs Henseleit buffer channels (4.8 mM KCl, 118 mM NaCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 11.1 mM glucose, and 25 mM NaHCO3) at 37 °C, with continuous aeration with gas (5% CO2 and 95% O2). Every 30 min, the channel buffer solution was exchanged. Quantification of the aortic tension was accomplished using an isometric force transducer, and the results were presented through a PowerLab data interface module linked to a PC running Chart software v8 (ADI Instruments).

The aortic rings were set aside for 30 min for equilibration at a 1500 mg ± 50 resting tension. Initial aorta contraction and relaxation were then carried out by the addition of PE, followed by ACh (both at 10 μM). After the tension was reverted to the rest state, accumulative concentrations of 1–10 μg/mL and 1–10 μM for the chloroform fraction or the pure metabolites, respectively, were added to the organ bath precontracted (PE, 10 μM)-isolated aortae. Tension reduction was estimated as a measurement of vasodilating actions. In other sets of experiments for investigating the role of the endothelium in the vasodilating effect, it was mechanically made bare. Additionally, L-NAME (100 μM) was added in the organ bath 15 min before adding the isolated metabolites or chloroform fraction to explore the role of nitric oxide in the vasodilating influence on different sets of experiments.

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