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 (Hydrastis canadensis) was fractionated and tested for its ability to enhance the antimicrobial activity of the alkaloid berberine (4) against the pathogenic bacterium Staphylococcus aureus. 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-C-dimethylflavone (29) was identified, as were several flavonoids known to be active. When tested in combination with 4, 29 lowered the IC50 of 4 from 132.2 ± 1.1 μM to 91.5 ± 1.1 μM. In isolation, 29 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.

Echinacea preparations, which are used for the prevention and treatment of upper respiratory infections, account for 10% of the dietary supplement market in the U.S., with sales totaling more than $100 million annually. In an attempt to shed light on Echinacea's mechanism of action, we evaluated the effects of a 75% ethanolic root extract of Echinacea purpurea, prepared in accord with industry methods, on cytokine and chemokine production from RAW 264.7 macrophage-like cells. We found that the extract displayed dual activities; the extract could itself stimulate production of the cytokine TNF-α, and also suppress production of TNF-α in response to stimulation with exogenous LPS. Liquid:liquid partitioning followed by normal-phase flash chromatography resulted in separation of the stimulatory and inhibitory activities into different fractions, confirming the complex nature of this extract. We also studied the role of alkylamides in the suppressive activity of this E. purpurea extract. Our fractionation method concentrated the alkylamides into a single fraction, which suppressed production of TNF-α, CCL3, and CCL5; however fractions that did not contain detectable alkylamides also displayed similar suppressive effects. Alkylamides, therefore, likely contribute to the suppressive activity of the extract but are not solely responsible for that activity. From the fractions without detectable alkylamides, we purified xanthienopyran, a compound not previously known to be a constituent of the Echinacea genus. Xanthienopyran suppressed production of TNF-α suggesting that it may contribute to the suppressive activity of the crude ethanolic extract. Finally, we show that ethanolic extracts prepared from E. purpurea plants grown under sterile conditions and from sterilized seeds, do not contain LPS and do not stimulate macrophage production of TNF-α, supporting the hypothesis that the macrophage-stimulating activity in E. purpurea extracts can originate from endophytic bacteria. Together, our findings indicate that ethanolic E. purpurea extracts contain multiple constituents that differentially regulate cytokine production by macrophages.

The study presented herein constitutes an extensive investigation of constituents in Hydrastis canadensis L. (Ranunculaceae) leaves. It describes the isolation and identification of two previously unknown compounds, 3,4-dimethoxy-2-(methoxycarbonyl)benzoic acid (1) and 3,5,3′-trihydroxy-7,4′-dimethoxy-6,8-C-dimethyl-flavone (2), along with the known compounds (±)-chilenine (3), (2R)-5,4′-dihydroxy-6-C-methyl-7-methoxy-flavanone (4), 5,4′-dihydroxy-6,8-di-C-methyl-7-methoxy-flavanone (5), noroxyhydrastinine (6), oxyhydrastinine (7) and 4′,5′-dimethoxy-4-methyl-3′-oxo-(1,2,5,6-tetrahydro-4H-1,3-dioxolo-[4′,5′:4,5]-benzo[1,2-e]-1,2-oxazocin)-2-spiro-1′-phtalan (8). Compounds 3-8 have been reported from other sources, but this is the first report of their presence in H. canadensis extracts. A mass spectrometry based assay was employed to demonstrate bacterial efflux pump inhibitory activity against Staphylococcus aureus for 2, with an IC50 value of 180 ± 6 μM. This activity in addition to that of other bioactive compounds such as flavonoids and alkaloids, may explain the purported efficacy of H. canadensis for treatment of bacterial infections. Finally, this report includes high mass accuracy fragmentation spectra for all compounds investigated herein which were uploaded into the Global Natural Products Social molecular networking library and can be used to facilitate their future identification in H. canadensis or other botanicals.

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