Development of advanced chromatographic methods for a complete phytochemical characterization of raw propolis polyphenols and volatile compounds

Propolis, or bee glue, is a dark-colored resinous substance collected by honeybees (Apis mellifera L.) from several tree species. Because of its biological and pharmacological properties, such as antibacterial, antiviral, antifungal, anticancer, anti-inflammatory, and immunomodulatory activities, propolis has attracted researchers’ interest in the last decades. Since propolis is used extensively, a rapid and efficient method for the extraction and analysis of propolis is certainly very useful. With the aim to determine polyphenols in propolis we have applied RP-HPLC coupled with DAD and MS detection, using the new fused-core column technology, which allowed a sensitive reduction in the total analysis time in comparison with conventional particulate stationary phases. The HPLC analyses were carried out on an Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm), with a mobile phase composed by 0.1% aqueous formic acid and acetonitrile, under gradient elution. Different extraction methods were compared, including sonication and microwave extraction, in order to obtain a high recovery of polyphenols from raw propolis. The other active component of propolis includes volatile compounds and its analysis was performed by GC coupled with MS detection. A suitable HS-SPME extraction procedure, whose experimental parameters were properly optimized, was developed using a 1 cm PDMS fiber. The GC analyses were carried out on an HP-5 MS cross-linked 5% diphenyl-95% dimethyl polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 m film thickness), under programmed-temperature elution. The volatile compounds extracted from raw propolis were identified by comparing the MS fragmentation patterns with those of pure compounds and by mass spectrum database search using NIST. The chromatographic methods developed in this study were applied to samples of raw propolis collected in different Italian regions and allowed us to determine their metabolite fingerprinting, thus providing new and reliable tools for the complete phytochemical characterization of this material.