The antimicrobial and chemical properties of South African propolis
Propolis is a sticky resin produced worldwide by honeybees (Apis mellifera). It is used to seal off holes in the hive from intruders, prevent putrefaction and thus prevent infections of the colony. The use of propolis as a natural or traditional product which dates as far back as 300 B.C. Globally, research has been extensively dedicated to studying the antimicrobial properties of propolis from various geographical and climatic regions. Brazilian propolis has, however, become a subject of increasing interest due to its characteristic favourable biological activities and is thus considered the “gold standard” of all propolis. This has resulted in the increased global demand for propolis. Despite this global outlook, research on the antimicrobial and chemical properties of propolis specifically from South Africa (SA) has been sorely neglected. The aim of this study was to evaluate the antimicrobial activity of 39 SA ethanolic extracts of propolis (EEP) and three Brazilian EEP’s(used as controls). The antimicrobial activities of EEP samples were evaluated using the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays against two yeasts, two Gram-positive and two Gram-negative bacteria. Interactive efficacies of the ten most active propolis samples, combined with conventional antimicrobials and honey, were evaluated using the fractional inhibitory concentration (ΣFIC) assessment. The chemical profile and composition of propolis was determined using high performance thin layer chromatography (HPTLC) and ultra-performance liquid chromatography coupled to photodiode array detectorquadrupole/ time-of-flight mass spectrometry (UPLC-PDA-qTOF-MS/MS) analysis. All strains of bacteria and yeasts tested showed susceptibility to the 39South African EEP samples. Some noteworthy activities were observed with some samples (GP9, GP11 and WC8) displaying an MIC and MBC value as low as 6 μg/ml against Staphylococcus aureus. In this study it was found that the majority (56%) of South African EEP samples displayed average antimicrobial activity better than that of the Brazilian control samples; 71% of samples displayed noteworthy activity against S. aureus and 79% against Cryptococcus neoformans. Notable interactions were identified, such as the combination of EEP’s with gentamicin where synergistic profiles were most often observed against Pseudomonas aeruginosa with ΣFIC ranging from 0.19 to 0.37. The Brazilian EEP sample was the only sample found to display antagonism when combined with the antifungals; amphotericin B and nystatin. Chemical analysis led to the identification six compounds namely; quercetin, galangin-5- methyl ether, pinobanksin-3-O-propionate, pinobanksin-3-O-butyrate or isobutyrate, pinobankin-3-O-pentanoate or 2-methylbutyrate and pinobanksin-3-O-hexanoatewhich were identified for the first time in SA propolis in this study. Chemometric analysis of LC-MS data revealed two distinct clusters and confirmed that the South African propolis is chemically distinct from Brazilian propolis. Furthermore, chemometric analysis was used to compare chemical data to antimicrobial activity. Orthogonal projections to latent structures (OPLS) models were created for the two Gram-positive bacteria (Enterococcus faecalis and S. aureus) and Candida albicans. Using the S-plot function, it was possible to identify the bioactive constituents in propolis as chrysin, pinocembrin, galangin and pinobanksin-3-Oacetate. South African propolis displayed noteworthy antimicrobial activity, favourably comparable to that of the Brazilian control and global “gold standard”. Interactive efficacy studies demonstrated notable synergistic profiles when combined with ciprofloxacin and gentamicin against Gram-negative bacteria. This could possibly have an impact on the future use of conventional antimicrobials with alternative therapies including propolis. Furthermore, SA propolis displayed not only superior activity in comparison to the Brazilian propolis but also exhibited superior antimicrobial activity in comparison to other extensively studied propolis from South America, Europe and Asia.
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Pharmacy Johannesburg, 2015