Efflux pump inhibitory activity of flavonoids isolated from Alpinia calcarata against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for several infections in humans. The infections caused by this bacterial strain are difficult to treat due to the resistance of MRSA to clinically used antibiotics. Several medicinal plants extracts and their phytoconstituents have been reported to possess modulation and efflux pump inhibitory (EPI) activity against MRSA strains. Alpinia calcarata rhizomes have been reported to be used in Ayurveda for several ailments including fungal infections. Based on this information and in continuation with our efforts to discover EPIs from Indian medicinal plants, we describe EPI activity of flavonoids isolated from A. calcarata. Galangin and kaempferol showed >= 32-fold modulation in minimum inhibitory concentration (MIC) of ethidium bromide (EtBr) as well as norfloxacin in NorA-overexpressed S. aureus (SA-1199B) strain. Pinocembrin showed 32-fold modulation of EtBr MIC in SA-1199 strain, but not in SA-1199B and K1758 strains. A significant difference was not observed in the modulation of norfloxacin MIC by galangin in SA-1199 and SA-1199B strains, which may be due to non-specific nature of galangin as modulator or EPI. However, kaempferol modulated the MIC of EtBr as well as norfloxacin 64-fold and 32-fold, respectively. Also, the best modulatory effect of kaempferol was observed only in SA-1199B strain compared to two other strains. The EPI activity of kaempferol and galangin were found to be competitive with respect to verapamil. In dose-response assay, kaempferol at 31.25 mu g/mL concentration was found to be better EPI by inhibiting NorA pump in SA-1199B strain and also demonstrated further confocal microscopy.