| Summary: | A simple and rapid analytical UHPLC methodology with spectrophotometric (UV/Vis) detection, coupled with different extraction procedures, has been perfected to investigate the presence of biologically active <i>O</i>-prenylated umbelliferone derivatives, such as auraptene and umbelliprenin, in pomegranate (<i>Punica granatum</i> L.) seed extracts. Absolute ethanol was the most efficient extraction solvent in terms of yields, after a short ultrasound-assisted. The highest concentration values recorded under these experimental conditions were 1.99 μg/g of dry extract and 6.53 μg/g for auraptene and umbelliprenin, respectively. The parent metabolite umbelliferone was also detected (0.67 μg/g). The extraction and UHPLC analytical methodology set up in the present study proved to be an efficient, powerful, and versatile technique for the simultaneous qualitative analysis and quantification of oxyprenylated coumarins in pomegranate seed extracts. The characterization of such secondary metabolites in the mentioned phytopreparation represents, to the best of our knowledge, the first example in the literature.
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