Multidrug resistance pumps (MDRs) protect microbial cells from both synthetic and natural antimicrobials. Amphipathic cations are preferred substrates of MDRs. Berberine alkaloids, which are cationic antimicrobials produced by a variety of plants, are readily extruded by MDRs. Several Berberis medicinal plants producing berberine were found also to synthesize an inhibitor of the NorA MDR pump of a human pathogen Staphylococcus aureus. The inhibitor was identified as 5-methoxyhydnocarpin (5-MHC), previously reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy. 5-MHC is an amphipathic weak acid and is distinctly different from the cationic substrates of NorA. 5-MHC had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of ethidium bromide and berberine from S. aureus cells was completely inhibited by 5-MHC. The level of accumulation of berberine in the cells was increased strongly in the presence of 5-MHC, indicating that this plant compound effectively disabled the bacterial resistance mechanism against the berberine antimicrobial.multidrug resistance ͉ efflux inhibitor B acteria have evolved numerous defenses against antimicrobial agents, and drug-resistant pathogens are on the rise (1). A general and effective defense is conferred by ubiquitous multidrug resistance pumps (MDRs), membrane translocases that extrude structurally unrelated toxins from the cell (2-5). Preferred substrates of most MDRs are synthetic hydrophobic cations such as quaternary ammonium antiseptics (6, 7). We have identified a group of cationic berberine alkaloids as natural substrates of MDR pumps (6). We suggested that berberine alkaloids represent a possibly larger group of cationic toxins that fueled the evolution of MDRs (7). Considering that microbial MDRs can render berberine alkaloids essentially ineffective, we reasoned that plants would benefit from making an MDR inhibitor. Here we show that Berberis fremontii, a berberine producer (8) used in Native American traditional medicine (9, 10), synthesizes a potent MDR inhibitor. Structural determination identified the substance as 5Ј-methoxyhydnocarpin (5Ј-MHC). Efflux of berberine from pathogenic Staphylococcus aureus expressing the NorA MDR pump that confers resistance to quinolones and antiseptics (6,11,12) was inhibited completely by 5Ј-MHC. This is a clear example of synergy between components of a medicinal plant described at a molecular level. Materials and MethodsCell Culturing and Susceptibility Testing. S. aureus 4222 parent strain and the norA mutant KLE 820 (6) were cultured in Mueller-Hinton (MH) broth. Cells (10 5 ͞ml) were inoculated into MH broth and dispensed at 0.2 ml͞well in microtiter plates. All tests were done in triplicate by following National Center for Clinical Laboratory Standards recommendations. Briefly, minimal inhibitory concentrations (MIC) were determined by serial 2-fold dilution of test compounds. MIC was defined as a concentr...

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Three trace alkaloids from Colorado blue spruce, Picea pungens, were identified by synthesis and GC-MS comparisons as 4alpha-hydroxy-cis-2-methyl-6-(2-oxopropyl)piperidine (1), 4alpha-hydroxy-cis-2-methyl-6-propylpiperidine (11), and 1-methylgranatanone (15) (N-methyl-9-aza-1-methylbicyclo[3.3. 1]nonane or N-methyleuphococcinine). Alkaloids 1 and 11 are the first among numerous known pine and spruce piperidines to contain a ring-oxygenated substituent.