Solubility studies for screening of microemulsion components
The solubility of HCA in various oils and surfactants such as eucalyptus oil, clove oil, lemon grass oil, pippermint oil, carvone, D-limonene, citronella, menthone, eugenol, oleic acid, tween 20, isopropyl myristate (IPM), tween 80, polyethylene glycol 400 (PEG 400) were measured. An excess amount of HCA was added to 10mL of oil and the mixture was shaken for 72 h at 25 °C. The resulting suspension was then centrifuged (R-8C REMI equipments, Mumbai) for 10 min at 10,000 rpm. The supernatant was filtered through a membrane filter (0.45µm) and the drug concentration in the filtrate was determined by high performance liquid chromatography (HPLC) analysis after appropriate dilution. The oil phase and surfactant that showed high solubility of HCA was used in the preparation of microemulsion.
Construction of pseudo-ternary phase diagrams
Water titration method was used in constructing pseudo-ternary phase diagrams at ambient temperature by choosing tween 80 as surfactant and ethanol as cosurfactant to obtain the concentration range of components to attain stable microemulsions range. (Vyas et al 2006; Patel et al. 2009)The weight ratio of surfactant to cosurfactant (SMix) varied as 1:1, 1:2, 1:3, 2:1 and 3:1. For each pseudo-ternary phase diagram at a specific surfactant to cosurfactant weight ratio was mixed with oil at a ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9 (w/w). Double distilled water was added drop by drop to each oily-surfactant mixture under magnetic stirring until the mixture became clear at a certain point. The concentrations were recorded in order to complete the pseudo-ternary phase diagrams, and then the contents of oil, surfactant, cosurfactant and water at appropriate weight ratios were selected based on the results obtained.
2.2.3. Development of microemulsions
The criteria for selection of microemulsion formulations in the skin permeation studies were based on the good physicochemical properties and stability of the developed formulations. HCA loaded microemulsions were prepared by dissolving HCA in eucalyptus oil/ clove oil/ lemon grass oil and surfactant mixture, adding the required quantity of water, and stirring to form a clear and transparent liquid. The resulting microemulsion formulations were tightly sealed and stored at ambient temperature, and their physical stability was determined by periodical observation of phase separation.
2.2.4 High performance liquid chromatography (HPLC) analysis
HPLC analysis were performed with shimadzu LC-10AT Prominence (Shimadzu, Japan) apparatus equipped with a Shimadzu SPD-20A UV detector and a shimadzu LC-A pump. A reversed column (Lichrospher C-18, 4.6mm x 250 mm) in conjunction with a security guard cartridge was eluted with a mobile phase composed of methanol and double distilled water (70:30 v/v). The mobile phase was pumped at a flow rate of 1.0mL/min and column temperature was set to 25°C. Calibration...