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 IC₅₀ 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

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 IC₅₀ 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

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 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.</p></div

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 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