Comparison of Three Different Extraction Methods and HPLC Determination of the Anthraquinones Aloe-emodine, Emodine, Rheine, Chrysophanol and Physcione in the Bark of Rhamnus alpinus L. (Rhamnaceae)

Introduction – Rhamnus alpinus L. (Rhamnaceae), a traditional plants in the fl ora of the Abruzzo region, is known to contain active anthraquinone secondary metabolites. However, the content of anthraquinones varies among R. alpinus samples depending on collection season and site. Thus, using simple, reliable and accurate analytical methods for the determination of anthraquinones in R. alpinus extracts allows comparative study of diff erent methods of extraction. Objective – After a partial validation of an HPLC method for the simultaneous determination of fi ve anthraquinones, aloeemodine, rheine, emodine, chrysophanol and physcione, in the bark of R. alpinus, we compared three diff erent methods of extraction. Methodology – Anthraquinones were extracted from the bark of R. alpinus using diff erent techniques (methanol maceration, ultrasonic and supercritical CO2 extraction). Separation and quantifi cation of anthraquinones were accomplished using a reversed-phase C18 column with the mobile phase of H2O–methanol (40 : 60, v/v, 1% formic acid) at a wavelength of 254 nm. The qualitative analyses were also achieved at wavelength of 435 nm. Results – All calibration curves were linear over the concentration range tested (10–200 mM) with the determination coeffi cients ≥0.991. The detection limits (S/N = 3) were 5 mM for each analytes. All fi ve anthraquinones were found in the samples tested at concentrations reported in experimental data. Conclusion – The described HPLC method and optimised extraction procedure are simple, accurate and selective for separation and quantifi cation of anthraquinones in the bark of R. alpinus and allow evaluation of the best extraction procedure between the tested assays.