As the concentration

of water miscible solvent increases, a decrease in the size of particle can be achieved (Mohanraj & Chen, 2006). In preliminary tests, the bixin concentrations tested in the bixin nanocapsule formulations were 100, 58, 37, 16 and 11 μg/mL; these were stored under ambient conditions (25 ± 1 °C) in amber glasses, and the parameter of size distribution was evaluated periodically during three weeks. Based on the nanocapsules stability, an optimal formulation was prepared in triplicate and was characterised in terms of viscosity, bixin content, encapsulation Z-VAD-FMK price efficiency, pH, diameter, zeta-potential and colour. Moreover, the stability of the optimum formulation was studied during storage at ambient temperature. The pH, diameter and bixin concentration were evaluated weekly for 9 weeks; after this period, the evaluation was performed every 2 weeks up to 119 days of storage. The viscosity of the bixin nanocapsule suspension was measured immediately after preparation using a Brookfield rotational viscometer (model DV-II + Pro, spindle LV2, Brookfield Engineering, USA) at 25 °C. Data were analysed

using Brookfield Rheocalc 32 software. The bixin nanocapsule suspension (optimal formulation) (10 mL) and a free bixin solution (10 mL) were analysed using a portable colorimeter (Konica Minolta model CR 400, Singapore). Both samples were prepared buy LY294002 in triplicate in the same bixin concentration (16.92 μg/mL). The free bixin was solubilised in ethanol:water (2:8) due to the low solubility of bixin in pure water. The colorimetric parameters were obtained Farnesyltransferase according to the Comission Internationale de l’Eclairage (CIELAB system); the coordinates

were L∗ (lightness), and the colour coordinates a∗ (red-green component) and b∗ (yellow-blue component), which were measured using the illuminant D65 and an angle of viewing of 0°. The total content of bixin was determined through the extraction of bixin from the bixin nanocapsule suspension. This method consisted of the extraction from an aliquot of 250 μL of formulation with acetonitrile (4.75 mL). This extract was sonicated by ultrasound (30 min) and centrifuged (15 min at 2820×g). The supernatant was injected in the HPLC. The bixin content in the aqueous phase of the bixin nanocapsule suspension was determined through the injection of the filtrate in the HPLC. The filtrate was obtained after the ultrafiltration/centrifugation of an aliquot of bixin nanocapsule suspension (400 μL) using a Ultrafree-MC® (10,000 MW, Millipore, Bedford, USA) in a centrifuge (15 min at 1690×g). The encapsulation efficiency was determined according to the method of Venturini et al.