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

What Does It Take to Synergistically Combine Sub-Potent Natural Products into Drug-Level Potent Combinations?

10.1371/journal.pone.0049969PLoS ONE711

Oviposition Cues for a Specialist Butterfly–Plant Chemistry and Size

The oviposition choice of an insect herbivore is based on a complex set of stimuli and responses. In this study, we examined the effect of plant secondary chemistry (the iridoid glycosides aucubin and catalpol) and aspects of size of the plant Plantago lanceolata, on the oviposition behavior of the specialist butterfly Melitaea cinxia. Iridoid glycosides are known to deter feeding or decrease the growth rate of generalist insect herbivores, but can act as oviposition cues and feeding stimulants for specialized herbivores. In a previous observational study of M. cinxia in the field, oviposition was associated with high levels of aucubin. However, this association could have been the cause (butterfly choice) or consequence (plant induction) of oviposition. We conducted a set of dual- and multiple-choice experiments in cages and in the field. In the cages, we found a positive association between the pre-oviposition level of aucubin and the number of ovipositions. The association reflects the butterfly oviposition selection rather than plant induction that follows oviposition. Our results also suggest a threshold concentration below which females do not distinguish between levels of iridoid glycosides. In the field, the size of the plant appeared to be a more important stimulus than iridoid glycoside content, with bigger plants receiving more oviposition than smaller plants, regardless of their secondary chemistry. Our results illustrate that the rank of a cue used for oviposition may be dependent on environmental context

Molecular Interactions of Pyrrolizidine Alkaloids with Critical Cellular Targets

Pyrrolizidine alkaloids (PAs) are common plant toxins produced by several genera of flowering plants, including Senecio, Crotalaria and Cynoglossum. Pyrrolizidine alkaloid-containing plants pose significant health hazards to animals and to people who consume natural herbal teas and traditional folk remedies

Comparative DNA Cross-Linking by Activated Pyrrolizidine Alkaloids

The toxicity and bioactivity of pyrrolizidine alkaloids (PAs), common constituents of hundreds of plant species, and in herbal remedies and folk medicines prepared thereof, are probably due to their ability to form DNA cross-linking. We investigated DNA cross-linking activity by chemically-activated PAs from four different structural classes in Madin–Darby bovine kidney (MDBK) cells and in pBR322 DNA. In cell culture, α,β-unsaturated macrocyclic diester pyrroles dehydrosenecionine (DHSN), dehydroriddelliine (DHRD) and the saturated macrocyclic diester pyrrole dehydromonocrotaline (DHMO) were significantly more potent cross-linkers than the simple necine base (retronecine) and an N-oxide (indicine N-oxide; INO) as determined by alkaline elution. The proportion of total DNA cross-links that were proteinase K-resistant (DNA–DNA cross-links) induced by the various pyrroles ranged from 0.08 (DHRN) to 0.67 (DHSN). Those pyrroles that were potent cross-linkers of cellular DNA also cross-linked, in a dose-dependent manner, Bam H1-digested pBR322 DNA as assessed by a gel retardation assay. The possible functional relevance of pyrrole–DNA cross-links was determined by their ability to interrupt PCR amplification of a 1.129 kb segment of pBR322. Dehydrosenecionine completely inhibited amplification, while DHMO was of intermediate potency, while DHRN and INO had no effect. Taken together, these studies suggest that structural features, most notably the presence of a macrocyclic diester, confer potent cross-link activity to PAs. In any event, DNA–DNA cross-linking is probably biologically relevant as indicated by their interference with DNA replication

DNA Cross-Linking in Mammalian Cells by Pyrrolizidine Alkaloids: Structure-Activity Relationships

Pyrrolizidine alkaloids (PAs) are common constituents of many species of flowering plants which posses carcinogenic as well as anticarcinogenic activityin vivo. Pyrrolizidine alkaloids are genotoxic in various short-term assays. The mechanisms by which these compounds exert these effects is still unclear. In this study, we characterized the ability of eight bifunctional PAs, with differing stereochemistry and functional groups, to cross-link cellular DNA in cultured bovine kidney epithelial cells. PAs representative of three major structural classes, the macrocycles (seneciphylline, riddelline, retrorsine, senecionine, monocrotaline), the open diesters (heliosupine, latifoline), and pyrrolizidine base (retronecine) were cultured for 2 hr with cells and an external metabolizing system. Every PA induced DNA cross-links which consisted primarily of proteinase-sensitive cross-links (DPC), but also to a smaller extent, DNA interstrand cross-links (ISC). None of the PAs induced detectable amounts of DNA single-strand breaks. The PAs which produced DPC and/or ISC (ranked from most potent to least) were: seneciphylline (DPC \u3e ISC); riddelline (DPC \u3e ISC); retrorsine (DPC \u3e ISC); senecionine (DPC \u3e ISC); heliosupine (DPC \u3e ISC); monocrotaline (ISC = DPC); latifoline (DPC \u3e ISC); and retronecine (ISC \u3e DPC). Although the PAs induced DNA cross-linking to varying degrees, cell viabilities for all treatment groups were \u3e90% as determined by trypan blue dye exclusion. Since the cross-linking ability of these PAs paralleled their ability to inhibit colony formation, cross-link formation may be involved in the biological activity of these compounds. Two structural determinants of biological activity appear to be the presence of both a macrocyclic necic acid ester and an ά,β-unsaturated ester function since the cross-linking ability of seneciphylline, riddelline, retrorsine, and senecionine far exceeded that of monocrotaline, heliosupine, latifoline, and retronecine. In addition, the stereochemical orientation of the ester linkage was found to have no effect on bm logicaI activia

Structural Influences on Pyrrolizidine Alkaloid Induced Cytopathology

Pyrrolizidine alkaloids (PAs), which are common constituents of hundreds of plant species around the world, have been reported to have cytotoxic, carcinogenic, antineoplastic, and genotoxic activity in vivo and in vitro. The exact mechanism of these biological toxicities is not yet clear. The ability of eight PA congeners to inhibit mitosis and induce megalocyte formation in cultured bovine kidney epithelial cells was studied to examine possible structural influences on these endpoints. Representatives of the three PA structural groups, the macrocycles (seneciphylline, senecionine, riddelliine, retrorsine, monocrotaline), open diesters (heliosupine, latifoline), and a necine base (retronecine), were cocultured for 2 hr with a NADPH-generating system and rat liver S9. Macrocylic PAs with αβ-unsaturation (seneciphylline, senecionine, riddelliine, retrorsine) showed a dose-dependent inhibition of colony formation at 50, 100, and 300 μM and induction of megalocytosis at 500 μM. Colony growth resumed 3 weeks after removal of PAs at 50 and 100 μM, and normal cellular morphology returned 5 or 6 weeks after removal of PAs at 500 μM. The saturated macrocyclic (monocrotaline) and open diesters (heliosupine, latifoline), elicited only a slight inhibition of colony formation and had no effect on cellular morphology at 500 μM. The necine base (retronecine) had no effect on either colony formation or cell morphology. Pyrrolic PAs (dehydrosenecionine, dehydromonocrotaline, dehydroretronecine) were more active in inhibition of colony formation than their parent compounds and were potent inducers of abnormal cellular morphology at 500 μM. An N-oxide metabolite, indicine N-oxide, was completely inactive. The results support previous studies showing that there are structural influences on PA-induced cytopathological effects