Studies on the safety and irritability of eco-friendly extracts of Pterocaulon balansae Chodat (Asteraceae) standardized in coumarins aiming at topical application

Abstract

Species of Pterocaulon (Asteraceae) genus have been used in folk medicine to treat skin diseases of different origins. However, only few studies evaluated the toxicity of species of this genus. This study aims to evaluate the safety and irritability of Pterocaulon balansae extracts intended for topical application. Three extracts of P. balansae - aqueous, hydroalcoholic and CO₂ supercritical - were prepared and standardized in coumarins by ultra-fast liquid chromatography. The toxicity profile and cell death mechanism were evaluated in keratinocytes and fibroblasts, the irritability through the hen's egg test on the chorioallantoic membrane (HET-CAM), and the mutagenicity by Salmonella/microsome test. The coumarin profile was similar in all extracts with a higher coumarin content for the supercritical CO₂ extract. Whatever the extract, a reduction in viability and proliferation was observed in human keratinocytes (HaCaT) and fibroblasts (MRC5) by MTT and SRB at the concentrations of coumarins >12.5 μg/mL. Flow cytometry with annexin V-FITC/propidium iodide staining indicated necrosis as the main mechanism of cell death. The HET-CAM assay showed that extracts were non-irritating at concentrations up to 25 μg/mL of coumarins, but the CO₂ supercritical extract was highly irritating at 100 μg/mL. Such extract also exhibited the highest mutagenicity in the Ames test, even after metabolic activation, which may be related to its elevated levels of two key coumarins: 5-methoxy-6,7-methylenedioxy coumarin and 5,6-dimethoxy-7-(2′,3′-epoxy-3′-methylbutyloxy) coumarin. These findings suggest that the concentration and composition of coumarins in P. balansae extracts influence their toxicity, providing insights regarding its safety for developing coumarin-based skin treatments.