7 research outputs found
Biochemometrics for Natural Products Research: Comparison of Data Analysis Approaches and Application to Identification of Bioactive Compounds
A central challenge of natural products
research is assigning bioactive
compounds from complex mixtures. The gold standard approach to address
this challenge, bioassay-guided fractionation, is often biased toward
abundant, rather than bioactive, mixture components. This study evaluated
the combination of bioassay-guided fractionation with untargeted metabolite
profiling to improve active component identification early in the
fractionation process. Key to this methodology was statistical modeling
of the integrated biological and chemical data sets (biochemometric
analysis). Three data analysis approaches for biochemometric analysis
were compared, namely, partial least-squares loading vectors, S-plots,
and the selectivity ratio. Extracts from the endophytic fungi <i>Alternaria</i> sp. and <i>Pyrenochaeta</i> sp. with
antimicrobial activity against <i>Staphylococcus aureus</i> served as test cases. Biochemometric analysis incorporating the
selectivity ratio performed best in identifying bioactive ions from
these extracts early in the fractionation process, yielding altersetin
(<b>3</b>, MIC 0.23 μg/mL) and macrosphelide A (<b>4</b>, MIC 75 μg/mL) as antibacterial constituents from <i>Alternaria</i> sp. and <i>Pyrenochaeta</i> sp., respectively.
This study demonstrates the potential of biochemometrics coupled with
bioassay-guided fractionation to identify bioactive mixture components.
A benefit of this approach is the ability to integrate multiple stages
of fractionation and bioassay data into a single analysis
A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition
<div><p>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 <i>Staphylococcus aureus</i>. 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 <i>Hydrastis Canadensis</i>, 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 IC<sub>50</sub> value of 75 μg/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC<sub>50</sub> 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.</p></div
Efflux pump inhibition in <i>S</i>. <i>aureus</i> by a goldenseal (<i>Hydrastis canadensis</i>) extract.
<p>(A) Data collected using the fluorescence-based ethidium accumulation assay for a range of <i>H</i>. <i>canadensis</i> extract concentrations. (B) Data collected using the mass spectrometry-based ethidium accumulation assay. Incubation time was 30 min for both A and B, data represents mean of 3 samples, error bars represent standard deviation.</p
Efflux pump inhibitory activity and antimicrobial activity of flavonoids.
<p>a: Efflux pump inhibition was measured via LC-MS analysis of ethidium in spent, filtered culture supernatant after a 30 min incubation in triplicate wells.</p><p>b: Growth inhibition was measured by optical density at 600nm (in triplicate) after an 18 hr incubation.</p><p>c: CCCP is an abbreviation for the compound carbonyl cyanide m-chloro-phenylhydrazone</p><p>Efflux pump inhibitory activity and antimicrobial activity of flavonoids.</p
Efflux pump inhibitory activity of apigenin, piperine, and quercetin as indicated by LC-MS measurement of residual ethidium bromide in spent broth after a 30 min incubation.
<p>Relative peak area (expressed as a percentage) for ethidium is plotted as a function of concentration of the putative inhibitor. Data points represent the mean of 3 measurements (biological replicates), with error bars representing standard deviation.</p
Representative selected chromatograms of filtered, spent broth showing a peak for the ethidium ion (MS-MS transition of <i>m/z</i> 314 to 286).
<p>Sample 1 is the negative control (<i>S</i>. <i>aureus</i> cultured for 30 min in Mueller Hinton broth with 1.25 μg/mL ethidium bromide and 10% DMSO), samples 2 and 3 were cultured under the same conditions as sample 1 with the addition of 75 μg/mL and 300 μg/mL piperine, respectively. All three peaks are normalized to a signal intensity of 3.45 x 10<sup>6</sup>. As piperine is added, efflux pumps are blocked, trapping the ethidium inside the cells and decreasing the quantity of ethidium (as indicated by the area of the ethidium peak) in the spent broth.</p
Change in absolute fluorescent intensity over time for <i>Staphylococcus aureus</i> exposed to ethidium bromide in the efflux pump inhibitor piperine.
<p>Fluorescence increases over time due to intracellular accumulation of ethidium bromide. The increase is more pronounced in the presence of piperine, which enhances intracellular accumulation of ethidium bromide by blocking efflux. Data points represent the mean of 3 samples, error bars represent standard deviation.</p