ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Secondary metabolites play a critical role in plant defense against disease and are of great importance to ethnomedicine. Bacterial efflux pumps are active transport proteins that bacterial cells use to protect themselves against multiple toxic compounds, including many antimicrobials. Efflux pump inhibitors from plants can block these efflux pumps, increasing the potency of antimicrobial compounds. This study demonstrates that efflux pump inhibition against the Gram-positive bacterial pathogen Staphylococcus aureus is widespread in extracts prepared from individual species throughout the land plant lineage. It therefore suggests a general mechanism by which plants used by indigenous species may be effective as a topical treatment for some bacterial infections. AIM OF THE STUDY: The goal of this research was to evaluate the distribution of efflux pump inhibitors in nine plant extracts with an ethnobotanical use suggestive of an antimicrobial function for the presence of efflux pump inhibitory activity against Staphylococcus aureus. MATERIALS AND METHODS: Plants were collected, dried, extracted, and vouchers submitted to the Herbarium of the University of North Carolina Chapel Hill (NCU). The extracts were analyzed by quantitative mass spectrometry (UPLC-MS) to determine the presence and concentration of flavonoids with known efflux pump inhibitory activity. A mass spectrometry-based assay was employed to measure efflux pump inhibition for all extracts against Staphylococcus aureus. The assay relies on UPLC-MS measurement of changes in ethidium concentration in the spent culture broth when extracts are incubated with bacteria. RESULTS: Eight of these nine plant extracts inhibited toxic compound efflux at concentrations below the MIC (minimum inhibitory concentration) value for the same extract. The most active extracts were those prepared from Osmunda claytoniana L. and Pinus strobes L., which both demonstrated IC50 values for efflux inhibition of 19 ppm. CONCLUSIONS: Our findings indicate that efflux pump inhibitors active against Staphylococcus aureus are common in land plants. By extension, this activity is likely to be important in many plant-derived antimicrobial extracts, including those used in traditional medicine, and evaluation of efflux pump inhibition may often be valuable when studying natural product efficacy.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Secretion Systems/drug effects , Membrane Transport Modulators/pharmacology , Membrane Transport Proteins/drug effects , Plants, Medicinal , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/isolation & purification , Bacterial Proteins/metabolism , Membrane Transport Modulators/isolation & purification , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Phytotherapy , Plants, Medicinal/chemistry , Plants, Medicinal/classification , Staphylococcus aureus/metabolismABSTRACT
Drug resistant bacterial infections cause significant morbidity and mortality worldwide, and new strategies are needed for the treatment of these infections. The anti-virulence approach, which targets non-essential virulence factors in bacteria, has been proposed as one way to combat the problem of antibiotic resistance. Virulence in methicillin-resistant Staphylococcus aureus (MRSA) and many other Gram-positive bacterial pathogens is controlled by the quorum sensing system. Thus, there is excellent therapeutic potential for compounds that target this system. With this project, we have developed and validated a novel approach for measuring quorum sensing inhibition in vitro. Ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS) was employed to directly measure one of the important outputs of the quorum sensing system in MRSA, auto-inducing peptide I (AIP I) in bacterial cultures. The method for AIP detection was validated and demonstrated limits of detection and quantification of range of 0.0035µM and 0.10µM, respectively. It was shown that the known quorum sensing inhibitor ambuic acid inhibited AIP I production by a clinically relevant strain of MRSA, with an IC50 value of 2.6±0.2µM. The new method performed similarly to previously published methods using GFP reporter assays, but has the advantage of being applicable without the need for engineering of a reporter strain. Additionally, the mass spectrometry-based method could be applicable in situations where interference by the inhibitor prevents the application of fluorescence-based methods.
Subject(s)
Bacterial Proteins/isolation & purification , Cyclohexanones/pharmacology , Mass Spectrometry/methods , Methicillin-Resistant Staphylococcus aureus/drug effects , Peptides, Cyclic/isolation & purification , Quorum Sensing , Gene Expression Regulation, Bacterial/drug effects , Green Fluorescent Proteins , Inhibitory Concentration 50 , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Methicillin-Resistant Staphylococcus aureus/physiology , Staphylococcal Infections/diagnosis , Staphylococcal Infections/microbiology , Virulence FactorsABSTRACT
Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 µg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 µg/mL to 75 µg/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.
Subject(s)
Biological Transport/drug effects , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/metabolism , Mass Spectrometry , Chromatography, Liquid , Dose-Response Relationship, Drug , Ethidium/metabolism , Inhibitory Concentration 50 , Plant Extracts/pharmacology , Spectrometry, Fluorescence/methods , Staphylococcus aureus/drug effects , Staphylococcus aureus/metabolismABSTRACT
Staphylococcus aureus infections acquired in hospitals now cause more deaths per annum in the US than does HIV/AIDS. Perhaps even more alarming is the rise in community associated methicillin-resistant S. aureus (CA-MRSA) infections, which have spread out of hospital settings and are infecting otherwise healthy individuals. The mechanism of enhanced pathogenesis in CA-MRSA remains unclear, but it has been postulated that high activity in the agr quorum-sensing system could be a contributing factor. The purpose of this study was to develop a quantitative method for analysis of autoinducing peptide I (AIP-I), the activating signal for the agr system in S. aureus. An effective method was developed using ultrahigh performance liquid chromatography (UHPLC) coupled to electrospray ionization mass spectrometry with an LTQ Orbitrap mass spectrometer. Relying on the exceptional resolving power and mass accuracy of this instrument configuration, it was possible to quantify AIP-I directly from the complex growth media of S. aureus cultures with a limit of detection (LOD) of 0.25µM and a linear dynamic range of 2.6 to 63µM. The method was then employed to monitor time-dependent production of AIP-I by S. aureus cultures, and it was observed that AIP-I production reached a maximum and leveled off after approximately 16h. Finally, it was determined that virulence of S. aureus was correlated with AIP-I production in some (but not all) strains analyzed.
Subject(s)
Bacterial Proteins/analysis , Chromatography, High Pressure Liquid/methods , Mass Spectrometry/methods , Peptides, Cyclic/analysis , Staphylococcus aureus/chemistry , Animals , Bacterial Proteins/metabolism , Bacterial Proteins/toxicity , Hemolysis/drug effects , Peptides, Cyclic/metabolism , Peptides, Cyclic/toxicity , Rabbits , Reproducibility of Results , Staphylococcus aureus/metabolismABSTRACT
It is often argued that the efficacy of herbal medicines is a result of the combined action of multiple constituents that work synergistically or additively. Determining the bioactive constituents in these mixtures poses a significant challenge. We have developed an approach to address this challenge, synergy-directed fractionation, which combines comprehensive mass spectrometry profiling with synergy assays and natural products isolation. The applicability of synergy-directed fractionation was demonstrated using the botanical medicine goldenseal (Hydrastis canadensis) as a case study. Three synergists from goldenseal were identified, sideroxylin, 8-desmethyl-sideroxylin, and 6-desmethyl-sideroxylin. These flavonoids synergistically enhance the antimicrobial activity of the alkaloid berberine (also a constituent of H. canadensis) against Staphylococcus aureus by inhibition of the NorA multidrug resistance pump. The flavonoids possess no inherent antimicrobial activity against S. aureus; therefore, they could have been missed using traditional bioactivity-directed fractionation. The flavonoid synergists are present at higher concentration in extracts from H. canadensis leaves, while the antimicrobial alkaloid berberine is present at higher levels in H. canadensis roots. Thus, it may be possible to produce an extract with optimal activity against S. aureus using a combination of goldenseal roots and leaves.
Subject(s)
Flavonoids/isolation & purification , Hydrastis/chemistry , Plants, Medicinal/chemistry , Staphylococcus aureus/drug effects , Bacterial Proteins/drug effects , Berberine Alkaloids/pharmacology , Flavonoids/chemistry , Flavonoids/pharmacology , Multidrug Resistance-Associated Proteins/drug effects , North Carolina , Plant Leaves/chemistry , Plant Roots/chemistryABSTRACT
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.
Subject(s)
Alkaloids/pharmacology , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Hydrastis/chemistry , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Plant Extracts/pharmacology , Staphylococcus aureus/drug effects , Alkaloids/isolation & purification , Anti-Bacterial Agents/isolation & purification , Benzylisoquinolines/isolation & purification , Benzylisoquinolines/pharmacology , Berberine/analogs & derivatives , Berberine/chemistry , Berberine/isolation & purification , Berberine/pharmacokinetics , Berberine/pharmacology , Drug Synergism , Ethidium/pharmacokinetics , Ethidium/pharmacology , Plant Components, Aerial/chemistry , Plant Extracts/isolation & purification , Plant Roots/chemistry , Staphylococcus aureus/metabolismABSTRACT
INTRODUCTION: The medicinal plant Spilanthes acmella (L.) Murr. has demonstrated an array of biological activities that are generally attributed to the presence of spilanthol and other alkylamides. Recently this plant has been of interest due to its potential for the treatment and prevention of malaria. OBJECTIVE: The aim of this study was to develop a liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-esiMS) method for rapid identification and quantification of the alkylamide spilanthol from S. acmella. METHODOLOGY: Hydroethanolic extracts were prepared from fresh S. acmella using different percentages of ethanol and were stored at -80, -20 and 25°C. Spilanthol was isolated and used as a standard for quantitative analysis. RESULTS: Validation parameters for the HPLC-esiMS analysis of spilanthol were as follows: repeatability, ≤ 6%; intermediate precision, ≤ 2%; range, 0.45-450 µm; limit of detection, 0.27 µm; and limit of quantification, 0.45 µm. Eight alkylamides in the S. acmella extract were identified based on MS-MS fragmentation patterns, and NMR analysis confirmed the identity of the most abundant of these as spilanthol. Spilanthol was extracted most efficiently in solvents containing >75% ethanol, and was stable in ethanolic extracts stored at all three temperatures. CONCLUSION: These results demonstrate the effectiveness of HPLC-esiMS for quantitative and qualitative analysis of spilanthol. We show that spilanthol is effectively extracted in ethanol, and is stable in ethanol extracts for over 6 months, even at room temperature.