28 research outputs found
Biochemometrics to Identify Synergists and Additives from Botanical Medicines: A Case Study with <i>Hydrastis canadensis</i> (Goldenseal)
A critical challenge in the study
of botanical natural products
is the difficulty of identifying multiple compounds that may contribute
additively, synergistically, or antagonistically to biological activity.
Herein, it is demonstrated how combining untargeted metabolomics with
synergy-directed fractionation can be effective toward accomplishing
this goal. To demonstrate this approach, an extract of the botanical
goldenseal (<i>Hydrastis canadensis)</i> was fractionated
and tested for its ability to enhance the antimicrobial activity of
the alkaloid berberine (<b>4</b>) against the pathogenic bacterium <i>Staphylococcus aureus</i>. Bioassay data were combined with
untargeted mass spectrometry-based metabolomics data sets (biochemometrics)
to produce selectivity ratio (SR) plots, which visually show which
extract components are most strongly associated with the biological
effect. Using this approach, the new flavonoid 3,3′-dihydroxy-5,7,4′-trimethoxy-6,8-<i>C</i>-dimethylflavone (<b>29</b>) was identified, as were
several flavonoids known to be active. When tested in combination
with <b>4</b>, <b>29</b> lowered the IC<sub>50</sub> of <b>4</b> from 132.2 ± 1.1 μM to 91.5 ± 1.1 μM.
In isolation, <b>29</b> did not demonstrate antimicrobial activity.
The current study highlights the importance of fractionation when
utilizing metabolomics for identifying bioactive components from botanical
extracts and demonstrates the power of SR plots to help merge and
interpret complex biological and chemical data sets
Investigations of Analyte-Specific Response Saturation and Dynamic Range Limitations in Atmospheric Pressure Ionization Mass Spectrometry
With this study, we investigated
why some small molecules demonstrate
narrow dynamic ranges in electrospray ionization-mass spectrometry
(ESI-MS) and sought to establish conditions under which the dynamic
range could be extended. Working curves were compared for eight flavonoids
and two alkaloids using ESI, atmospheric pressure chemical ionization
(APCI), and heated electrospray ionization (HESI) sources. Relative
to reserpine, the flavonoids exhibited narrower linear dynamic ranges
with ESI-MS, primarily due to saturation in response at relatively
low concentrations. Saturation was overcome by switching from ESI
to APCI, and our experiments utilizing a combination HESI/APCI source
suggest that this is due in part to the ability of APCI to protonate
neutral quercetin molecules in the gas phase. Thermodynamic equilibrium
calculations indicate that quercetin should be fully protonated in
solution, and thus, it appears that some factor inherent in the ESI
process favors the formation of neutral quercetin at high concentration.
The flavonoid saturation concentration was increased with HESI as
compared to ESI, suggesting that inefficient transfer of ions to the
gas phase can also contribute to saturation in ESI-MS response. In
support of this conclusion, increasing auxiliary gas pressure or switching
to a more volatile spray solvent also increased the ESI dynamic range.
Among the sources investigated herein, the HESI source achieved the
best analytical performance (widest linear dynamic range, lowest LOD),
but the APCI source was less subject to saturation in response at
high concentration
High-Resolution MS, MS/MS, and UV Database of Fungal Secondary Metabolites as a Dereplication Protocol for Bioactive Natural Products
A major problem in the discovery
of new biologically active compounds
from natural products is the reisolation of known compounds. Such
reisolations waste time and resources, distracting chemists from more
promising leads. To address this problem, dereplication strategies
are needed that enable crude extracts to be screened for the presence
of known compounds before isolation efforts are initiated. In a project
to identify anticancer drug leads from filamentous fungi, a significant
dereplication challenge arises, as the taxonomy of the source materials
is rarely known, and, thus, the literature cannot be probed to identify
likely known compounds. An ultraperformance liquid chromatography–photodiode
array–high-resolution tandem mass spectrometric (UPLC-PDA-HRMS-MS/MS)
method was developed for dereplication of fungal secondary metabolites
in crude culture extracts. A database was constructed by recording
HRMS and MS/MS spectra of fungal metabolites, utilizing both positive-
and negative-ionization modes. Additional details, such as UV-absorption
maxima and retention times, were also recorded. Small-scale cultures
that showed cytotoxic activities were dereplicated before engaging
in the scale-up or purification processes. Using these methods, approximately
50% of the cytotoxic extracts could be eliminated from further study
after the confident identification of known compounds. The specific
attributes of this dereplication methodology include a focus on bioactive
secondary metabolites from fungi, the use of a 10 min chromatographic
method, and the inclusion of both HRMS and MS/MS data
Flavonolignans from <i>Aspergillus iizukae</i>, a Fungal Endophyte of Milk Thistle (<i>Silybum marianum</i>)
Silybin A (<b>1</b>), silybin
B (<b>2</b>), and isosilybin
A (<b>3</b>), three of the seven flavonolignans that constitute
silymarin, an extract of the fruits of milk thistle (<i>Silybum
marianum</i>), were detected for the first time from a fungal
endophyte, <i>Aspergillus iizukae</i>, isolated from the
surface-sterilized leaves of <i>S. marianum</i>. The flavonolignans
were identified using a UPLC-PDA-HRMS-MS/MS method by matching retention
times, HRMS, and MS/MS data with authentic reference compounds. Attenuation
of flavonolignan production was observed following successive subculturing
of the original flavonolignan-producing culture, as is often the case
with endophytes that produce plant-based secondary metabolites. However,
production of <b>1</b> and <b>2</b> resumed when attenuated
spores were harvested from cultures grown on a medium to which autoclaved
leaves of <i>S. marianum</i> were added. The cycle of attenuation
followed by resumed biosynthesis of these flavonolignans was replicated
in triplicate
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
Interaction Metabolomics to Discover Synergists in Natural Product Mixtures
Mass spectrometry metabolomics has become increasingly
popular
as an integral aspect of studies to identify active compounds from
natural product mixtures. Classical metabolomics data analysis approaches
do not consider the possibility that interactions (such as synergy)
could occur between mixture components. With this study, we developed
“interaction metabolomics” to overcome this limitation.
The innovation of interaction metabolomics is the inclusion of compound
interaction terms (CITs), which are calculated as the product of the
intensities of each pair of features (detected ions) in the data matrix.
Herein, we tested the utility of interaction metabolomics by spiking
known concentrations of an antimicrobial compound (berberine) and
a synergist (piperine) into a set of inactive matrices. We measured
the antimicrobial activity for each of the resulting mixtures against Staphylococcus aureus and analyzed the mixtures with liquid
chromatography coupled to high-resolution mass spectrometry. When
the data set was processed without CITs (classical metabolomics),
statistical analysis yielded a pattern of false positives. However,
interaction metabolomics correctly identified berberine and piperine
as the compounds responsible for the synergistic activity. To further
validate the interaction metabolomics approach, we prepared mixtures
from extracts of goldenseal (Hydrastis canadensis) and habañero pepper (Capsicum chinense)
and correctly correlated synergistic activity of these mixtures to
the combined action of berberine and several capsaicinoids. Our results
demonstrate the utility of a conceptually new approach for identifying
synergists in mixtures that may be useful for applications in natural
products research and other research areas that require comprehensive
mixture analysis
Polyhydroxyanthraquinones as Quorum Sensing Inhibitors from the Guttates of <i>Penicillium restrictum</i> and Their Analysis by Desorption Electrospray Ionization Mass Spectrometry
The endophytic fungus <i>Penicillium
restrictum</i> was
isolated from the stems of a milk thistle (<i>Silybum marianum</i>) plant. In culture, the fungus produced distinct red guttates, which
have been virtually uninvestigated, particularly from the standpoint
of chemistry. Hence, this study examined the chemical mycology of <i>P. restrictum</i> and, in doing so, uncovered a series of both
known and new polyhydroxyanthraquinones (<b>1</b>–<b>9</b>). These compounds were quorum sensing inhibitors in a clinical
isolate of methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), with IC<sub>50</sub> values ranging from 8 to 120 μM,
suggesting antivirulence potential for the compounds. Moreover, the
spatial and temporal distribution of the polyhydroxyanthraquinones
was examined <i>in situ</i> via desorption electrospray
ionization–mass spectrometry (DESI-MS) imaging, demonstrating
the first application of this technique to a guttate-forming fungus
and revealing both the concentration of secondary metabolites at the
ventral surface of the fungus and their variance in colonies of differing
ages
Phylogenetic and chemical diversity of fungal endophytes isolated from <i>Silybum marianum</i> (L) Gaertn. (milk thistle)
<div><p>Use of the herb milk thistle (<i>Silybum marianum</i>) is widespread, and its chemistry has been studied for over 50 years. However, milk thistle endophytes have not been studied previously for their fungal and chemical diversity. We examined the fungal endophytes inhabiting this medicinal herb to determine: (1) species composition and phylogenetic diversity of fungal endophytes; (2) chemical diversity of secondary metabolites produced by these organisms; and (3) cytotoxicity of the pure compounds against the human prostate carcinoma (PC-3) cell line. Forty-one fungal isolates were identified from milk thistle comprising 25 operational taxonomic units based on BLAST search via GenBank using published authentic sequences from nuclear ribosomal internal transcribed spacer sequence data. Maximum likelihood analyses of partial 28S rRNA gene showed that these endophytes had phylogenetic affinities to four major classes of Ascomycota, the Dothideomycetes, Sordariomycetes, Eurotiomycetes, and Leotiomycetes. Chemical studies of solid–substrate fermentation cultures led to the isolation of four new natural products. In addition, 58 known secondary metabolites, representing diverse biosynthetic classes, were isolated and characterized using a suite of nuclear magnetic resonance and mass spectrometry techniques. Selected pure compounds were tested against the PC-3 cell line, where six compounds displayed cytotoxicity.</p></div
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