High-performance counter-current chromatography separation of extract from Peucedanum cervaria towards the isolation of coumarin derivatives

Abstract

The genus Peucedanum, belonging to the Apiaceae family comprises numerous species widely distributed in Europe. Some of these plants are used in traditional medicine for a long time, for the treatment of different serious diseases such as asthma, angina, epilepsy or gastrointestinal disorders. P. praeruptorum is officially listed in Chinese Pharmacopoeia. Major bioactive components were identified as angular dihydropyranocoumarins, thus their separation and purification is a very important task. In present research a high-performance counter-current chromatography (HPCCC) method was applied. Method, as an efficient preparative technique, offers a series of advantages over conventional chromatography like elimination of irreversible adsorption of sample or easy scaling-up. HPCCC method was applied for the preparative separation and purification of major coumarins from the fruits of Peucedanum cervaria. The Spectrum HPCCC (Dynamic Extractions, UK) equipped with both analytical and semi-preparative columns (22 ml and 137 ml volume, respectively) was used in this study. A scale-up process from analytical to preparative in a very short time was developed. Several solvent systems: mixtures of n-hexane, ethyl acetate, methanol and water in reverse phase system were elaborated. Both rotation speed and flow rate were optimized. After injection of 500 mg of extract four pirano-coumarins were isolated in pure form (4.58 mg; 3.7 mg; 3.14 mg and 1.5 mg) and then analyzed by HPLC-DAD and HPLC/DAD/ESI-TOFMS with accurate mass measurements. Further complete structure elucidation with NMR is in progress.