A New Approach for Identifying Synergists in Natural Product Mixtures Proves Useful

A study published in 2023 in the Journal of Natural Products introduced a new approach, called interaction metabolomics, for identifying active synergists in natural product mixtures. The approach may be useful in research on natural products and in other areas of research that involve comprehensive mixture analysis. The study, which was jointly funded by the National Center for Complementary and Integrative Health (NCCIH) and the Office of Dietary Supplements through the National Institutes of Health (NIH) Consortium for Advancing Research on Botanical and Other Natural Products (CARBON) Program, was led by researchers from the University of North Carolina at Greensboro. 

The standard approach to identifying biologically active compounds in natural products involves a process called bioassay-guided fractionation. Some therapeutically important natural products, such as artemisinin and penicillin, were discovered through this process. But this approach focuses on isolating single biologically active compounds and does not account for the possibility of combined actions of multiple compounds within a mixture.

Past research suggests the occurrence of synergistic biological effects in some natural products. For instance, some plants have been found to contain not only berberine, a substance with antimicrobial properties, but also molecules that enhance its activity. Artemisinin is another example—it has been shown to be more potent against malaria in the form of a complex tea than as an isolated molecule. These examples show that analytic approaches that focus on isolating and purifying only single compounds in natural product mixtures may miss important synergistic interactions playing out between multiple constituents of the mixture.

In this study, the researchers first designed an experimental system in which the observed biological activity—antimicrobial activity against the bacterium Staphylococcus aureus—was a result of the interaction between known synergists, the antimicrobial berberine and the synergist piperine. The researchers measured the antimicrobial activity of mixtures containing different known concentrations of berberine and piperine. They also collected mass spectrometry metabolomics data for all the mixtures and calculated a set of synthetic features from the data to determine what they referred to as compound interaction terms, or CITs.

Two data analysis workflows were then tested, one that included CITs (representing interaction metabolomics) and one without CITs (representing classical metabolomics). The workflow testing was done using both the mixtures containing berberine and piperine and actual botanical extracts from goldenseal (Hydrastis canadensis), a plant that produces berberine, and habañero pepper (Capsicum chinense), a plant that produces capsaicin, a substance that has synergistic antimicrobial activity with berberine. The goldenseal and habañero pepper extracts were first partitioned with liquid-liquid partitioning and then separated by normal phase flash chromatography and reversed-phase preparative high-performance liquid chromatography, resulting in different fractions. To create mixtures for interaction metabolomics analysis, the single subfraction of goldenseal with the most potent antimicrobial activity was combined with each of the habañero pepper subfractions.

In the experiments on the berberine/piperine combination, interaction metabolomics (with CITs) correctly identified these two substances as the compounds responsible for the synergistic activity against the bacteria, but classical metabolomics (without CITs) did not. In experiments on the goldenseal/habañero pepper extract mixtures, interaction metabolomics correctly correlated synergistic activity of the mixtures with the combined actions of berberine (from goldenseal) and capsaicinoids (from habañero pepper).   

The results from this study demonstrate the usefulness of interaction metabolomics for predicting natural product constituents that interact to produce a combined biological effect. The new approach successfully identified synergists both in a simulated extract containing berberine and piperine and in combined fractions from actual botanical extracts, something that the classical metabolomics workflow could not do. Although the study was focused on synergistic interactions, the researchers indicated that interaction metabolomics might also be useful for identifying additive and antagonistic interactions, which could be a potential focus of future research. This paper won the American Society of Pharmacognosy 2024 Schwarting Award, after Arthur E. Schwarting, a former distinguished editor of the American Chemical Society Journal, Journal of Natural Products.