Identification of potential markers of elevated anticandidal activity of propolis extracts

For centuries, propolis has been one of the most important and popular antimicrobial (antibacterial and antifungal) agents used in traditional medicine worldwide, including Central and Eastern Europe. Despite centuries of use of this product, the molecular mechanisms of its activity remain not fully recognized, and the components that determine its biological activity have not been identified.

Hence, the main goal of the present study was to identify propolis ingredients that are crucial for the antifungal activity of this product.

A serial two-fold microdilution method was applied to evaluate the activity of 83 ethanolic extracts of propolis (EEP) samples collected in different regions of Poland. The chemical composition of all EEPs was determined using UHPLC-DAD and UHPLC-QqTOF-MS methods. Advanced chemometric analysis of the correlation between antifungal activity and chemical composition was performed to identify the components related to the increased antifungal potential of propolis. Subsequently, the antifungal activities of pure "active ingredients" and their combinations were determined.

Only seven extracts (8.4 %) exhibited high anticandidal potential with MIC (Minimum Inhibitory Concentration) values between 32 and 256 μg/mL. The identified most important potential markers related to increased antifungal activity of propolis collected in East Europe are: pinocembrin, pinobanksin-3-acetate, chrysin, galangin, pinobanksin, techtochrysin, genkwanin, pinostrobin and sakuranetin isomer. However, the pure compounds did not inhibit the growth of Candida spp. up to a concentration of 256 μg/mL (MIC >256 μg/mL). Much better activity was observed for combinations of these ingredients. The highest activity was observed for a mixture of five compounds: chrysin, galangin, pinocembrin, pinobanksin, and pinobanksin-3-acetate, with MIC and MFC (Minimal Fungicidal Concentration) values 64 and 128 μg/mL (summary concentration of all compounds - 12.8 or 25.6 of each μg/mL), respectively.

The relatively low number of propolis samples collected in Poland exhibit considerable activity against Candida spp. Markers of elevated antifungal potential have been identified. Moreover, it has been proved, that only the composition of these compounds (not pure ingredients alone) is effective in the treatment of Candida spp. Mixtures of these ingredients can be considered as potential antifungal agents (artificial propolis). Moreover, UHPLC-DAD and UHPLC-QqTOF-MS methods of determining the chemical composition of EEPs have been optimized.