Synergy Directed Fractionation: A Case Study Identifying Efflux Pump Inhibitors from Goldenseal (Hydrastis canadensis)

It is often argued that herbal medicines are effective due to the combined effects of multiple chemical constituents that act synergistically. Identifying the constituents involved in these complex interactions continues to be a major hurdle in research with botanicals. Here we demonstrate an approach to address this problem, synergy directed fractionation. With this technique, crude extracts are screened prospectively for the presence of synergists. These extracts are then fractionated, and the fractions are again submitted to the synergy assay. Comprehensive profiling with liquid chromatography mass spectrometry (LC-MS) is used after each stage of fractionation as a central tool towards identifying possible active constituents. Bioactive compounds and synergists are ultimately isolated and their structures elucidated. This technique was applied to extracts prepared from the leaves of the widely used botanical goldenseal (Hydrastis canadensis L.). As a result, several flavonoids that have not previously been reported as constituents of H. canadensis were identified. The most abundant of these flavonoids, 8-demethly-sideroxylin (1), was shown to act as an inhibitor of the multidrug efflux pump NorA in Staphylococcus aureus. Importantly, this compound was only effective in combination with the antimicrobial alkaloid berberine (also produced by H. canadensis). Because the compound has no inherent antimicrobial activity, it would likely have been missed had the plant been subjected to traditional bioassay guided fractionation.

Acknowledgements: This work was accomplished in collaboration with Dr. Nicholas Oberlies at the University of North Carolina Greensboro, and Dr. Glenn W. Kaatz of the John Dingell VA Medical Center. We gratefully acknowledge funding (1 R15 AT005005–01) from the National Center for Complementary and Alternative Medicine (NCCAM), a component of the National Institutes of Health (NIH).