Bacterial efflux inhibitors are widely distributed in land plants.

ETHNOPHARMACOLOGICAL RELEVANCE: Secondary metabolites play a critical role in plant defense against disease and are of great importance to ethnomedicine. Bacterial efflux pumps are active transport proteins that bacterial cells use to protect themselves against multiple toxic compounds, including many antimicrobials. Efflux pump inhibitors from plants can block these efflux pumps, increasing the potency of antimicrobial compounds. This study demonstrates that efflux pump inhibition against the Gram-positive bacterial pathogen Staphylococcus aureus is widespread in extracts prepared from individual species throughout the land plant lineage. It therefore suggests a general mechanism by which plants used by indigenous species may be effective as a topical treatment for some bacterial infections. AIM OF THE STUDY: The goal of this research was to evaluate the distribution of efflux pump inhibitors in nine plant extracts with an ethnobotanical use suggestive of an antimicrobial function for the presence of efflux pump inhibitory activity against Staphylococcus aureus. MATERIALS AND METHODS: Plants were collected, dried, extracted, and vouchers submitted to the Herbarium of the University of North Carolina Chapel Hill (NCU). The extracts were analyzed by quantitative mass spectrometry (UPLC-MS) to determine the presence and concentration of flavonoids with known efflux pump inhibitory activity. A mass spectrometry-based assay was employed to measure efflux pump inhibition for all extracts against Staphylococcus aureus. The assay relies on UPLC-MS measurement of changes in ethidium concentration in the spent culture broth when extracts are incubated with bacteria. RESULTS: Eight of these nine plant extracts inhibited toxic compound efflux at concentrations below the MIC (minimum inhibitory concentration) value for the same extract. The most active extracts were those prepared from Osmunda claytoniana L. and Pinus strobes L., which both demonstrated IC50 values for efflux inhibition of 19 ppm. CONCLUSIONS: Our findings indicate that efflux pump inhibitors active against Staphylococcus aureus are common in land plants. By extension, this activity is likely to be important in many plant-derived antimicrobial extracts, including those used in traditional medicine, and evaluation of efflux pump inhibition may often be valuable when studying natural product efficacy.

A mass spectrometry-based assay for improved quantitative measurements of efflux pump inhibition.

Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 µg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 µg/mL to 75 µg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.