A Validated HPTLC Densitometric Method for Simultaneous Determination of Evernic and Usnic Acids in Four Usnea Species and Comparison of Their Antioxidant Potential

A simple, sensitive and precise high-performance thin-layer chromatography method with densitometric detection was used for simultaneous determination of evernic (EV) and (+)-usnic acids (USN) in Usnea aciculifera (UA), U. ghattensis (UG), U. longissima (UL) and U. stigmatoides (US). This method was also validated according to the ICH guidelines. Separation and quantification was performed with the mobile phase toluene-1,4-dioxane-formic acid (18:4.5:0.2, v/v/v) on silica gel 60F(254) plates. The linearity for EV and USN was found in the 200-600 ng/band range. The limit of detection for EV and USN was 51.56 and 32.59 ng/band, while the limit of quantification was 156.23 and 98.76 ng/band, respectively. Intra-and interday precisions (n = 6) for EV and USN were 0.70-1.89 and 0.50-0.76 (% RSD), and 1.56-1.60 and 1.54-1.99 (% RSD), respectively. The mean percent recoveries were 99.66 and 99.87%, respectively, for EV and USN. However, USN was estimated in all four Usnea species but EV only in two species with varied quantity. Comparative antioxidant activity revealed that US is a better free radical scavenger in comparison with other three Usnea species. Furthermore, these results indicated that USN and EV are not solely responsible for antioxidant potential, but it may be due to synergistic effect.