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, 77 (8), 835-40

Goldenseal (Hydrastis Canadensis L.) Extracts Synergistically Enhance the Antibacterial Activity of Berberine via Efflux Pump Inhibition

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Goldenseal (Hydrastis Canadensis L.) Extracts Synergistically Enhance the Antibacterial Activity of Berberine via Efflux Pump Inhibition

Keivan A Ettefagh et al. Planta Med.

Abstract

Goldenseal (Hydrastis canadensis L.) is used to combat inflammation and infection. Its antibacterial activity in vitRO has been attributed to its alkaloids, the most abundant of which is berberine. The goal of these studies was to compare the composition, antibacterial activity, and efflux pump inhibitory activity of ethanolic extracts prepared from roots and aerial portions of H. canadensis. Ethanolic extracts were prepared separately from roots and aerial portions of six H. canadensis plants. Extracts were analyzed for alkaloid concentration using LC-MS and tested for antimicrobial activity against Staphylococcus aureus (NCTC 8325-4) and for inhibition of ethidium bromide efflux. Synergistic antibacterial activity was observed between the aerial extract (FIC 0.375) and to a lesser extent the root extract (FIC 0.750) and berberine. The aerial extract inhibited ethidium bromide efflux from wild-type S. aureus but had no effect on the expulsion of this compound from an isogenic derivative deleted for norA. Our studies indicate that the roots of H. canadensis contain higher levels of alkaloids than the aerial portions, but the aerial portions synergize with berberine more significantly than the roots. Furthermore, extracts from the aerial portions of H. canadensis contain efflux pump inhibitors, while efflux pump inhibitory activity was not observed for the root extract. The three most abundant H. canadensis alkaloids, berberine, hydrastine, and canadine, are not responsible for the efflux pump inhibitory activity of the extracts from H. canadensis aerial portions.

Figures

Figure 1
Figure 1
Structures of major alkaloid constituents of Hydrastis canadensis, berberine (1), (1R,9S)-(−)-β-hydrastine (2) and canadine (tetrahydroberberine) (3).
Figure 3
Figure 3
Isobolograms indicating inhibition of bacteria growth by H. canadensis extracts and added berberine. Berberine alone was also included in the assay for validation and to serve as a positive control. Extracts were dissolved in 2% DMSO in Müeller Hinton broth. Assays were performed in triplicate in a 96-well plate with 5 × 105 CFU/mL S. aureus.
Figure 4
Figure 4
Percent fluorescence over time for S. aureus (NCTC 8325-4) loaded with ethidium bromide and treated with various extracts and controls. Treatments included the known efflux pump inhibitor CCCP (positive control), and extracts from the roots and aerial portions of H. canadensis. All extracts and CCCP were dissolved in Müeller Hinton broth containing 2% DMSO. Data points represent the average of three separate experiments (using 3 different pellets of S. aureus). Error bars are +/− standard error.
Figure 5
Figure 5
Percent fluorescence over time for the norA-deleted S. aureus (K1758). Aside from the use of a different strain of bacteria, conditions were identical to those employed for Figure 4.

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