Antimicrobial and Efflux Pump Inhibitory Activity of Caffeoylquinic Acids from Artemisia absinthium against Gram-Positive Pathogenic Bacteria

Background: Traditional antibiotics are increasingly suffering from the emergence of multidrug resistance amongst pathogenic bacteria leading to a range of novel approaches to control microbial infections being investigated as potential alternative treatments. One plausible antimicrobial alternative could be the combination of conventional antimicrobial agents/antibiotics with small molecules which block multidrug efflux systems known as efflux pump inhibitors. Bioassay-driven purification and structural determination of compounds from plant sources have yielded a number of pump inhibitors which acted against gram positive bacteria. Methodology/Principal Findings: In this study we report the identification and characterization of 4′,5′-O-dicaffeoylquinic acid (4′,5′-ODCQA) from Artemisia absinthium as a pump inhibitor with a potential of targeting efflux systems in a wide panel of Gram-positive human pathogenic bacteria. Separation and identification of phenolic compounds (chlorogenic acid, 3′,5′-ODCQA, 4′,5′-ODCQA) was based on hyphenated chromatographic techniques such as liquid chromatography with post column solid-phase extraction coupled with nuclear magnetic resonance spectroscopy and mass spectroscopy. Microbial susceptibility testing and potentiation of well know pump substrates revealed at least two active compounds; chlorogenic acid with weak antimicrobial activity and 4′,5′-ODCQA with pump inhibitory activity whereas 3′,5′-ODCQA was ineffective. These intitial findings were further validated with checkerboard, berberine accumulation efflux assays using efflux-related phenotypes and clinical isolates as well as molecular modeling methodology. Conclusions/Significance: These techniques facilitated the direct analysis of the active components from plant extracts, as well as dramatically reduced the time needed to analyze the compounds, without the need for prior isolation. The calculated energetics of the docking poses supported the biological information for the inhibitory capabilities of 4′,5′-ODCQA and furthermore contributed evidence that CQAs show a preferential binding to Major Facilitator Super family efflux systems, a key multidrug resistance determinant in gram-positive bacteria.National Institutes of Health (U.S.) (grant R01GM59903)National Institutes of Health (U.S.) (grant R01AI050875)Netherlands Organization for Scientific Research (VICI grant 700.56.442)Massachusetts Technology Transfer Center (MTTC)National Institutes of Health (U.S.) (grant 5U54MH084690-02

Stalikas, “Study of the behavior of water-soluble vitamins in HILIC on a diol column

Abstract An effort has been made to investigate the chromatographic behavior and to understand the basic mechanisms in HILIC-based separation of water-soluble vitamins with highly varied properties on a diol column. The water content of the mobile phase is of utmost importance because it directly affects the type and extent of interactions of the solutes with the stationary phase and with the buffered mobile phase. A mixed-mode partitioning-surface adsorption mechanism enables most precise description of their chromatographic retention and separation. The point at which surface adsorption becomes apparent, however, depends on the properties of the solutes on the given stationary phase, and on the presence of buffer salt ions. Adjustment of mobile phase pH and use of different buffer salts can be used to modify electrostatic interactions among the solutes, active silanols, and counter-ions. The role of hydrogen bonding was clarified by substitution of ACN by solvents with moderate to strong hydrogen bonding potential. Analytes which are neutral at the working pH start to interact with the stationary phase when the ACN content is increased to 80%. Negatively charged analytes are adsorbed on the stationary phase when the ACN content is approximately 86%, because augmentation of the counter-ions weakens electrostatic repulsion by the active silanol groups. On the other hand, the electrostatic attraction of thiamine contributes significantly to its retention even when using mobile phases with high water content

Stalikas, “Study of the behavior of water-soluble vitamins in HILIC on a diol column

Abstract An effort has been made to investigate the chromatographic behavior and to understand the basic mechanisms in HILIC-based separation of water-soluble vitamins with highly varied properties on a diol column. The water content of the mobile phase is of utmost importance because it directly affects the type and extent of interactions of the solutes with the stationary phase and with the buffered mobile phase. A mixed-mode partitioning-surface adsorption mechanism enables most precise description of their chromatographic retention and separation. The point at which surface adsorption becomes apparent, however, depends on the properties of the solutes on the given stationary phase, and on the presence of buffer salt ions. Adjustment of mobile phase pH and use of different buffer salts can be used to modify electrostatic interactions among the solutes, active silanols, and counter-ions. The role of hydrogen bonding was clarified by substitution of ACN by solvents with moderate to strong hydrogen bonding potential. Analytes which are neutral at the working pH start to interact with the stationary phase when the ACN content is increased to 80%. Negatively charged analytes are adsorbed on the stationary phase when the ACN content is approximately 86%, because augmentation of the counter-ions weakens electrostatic repulsion by the active silanol groups. On the other hand, the electrostatic attraction of thiamine contributes significantly to its retention even when using mobile phases with high water content