A new analytical workflow combining high-resolution mass spectrometry and multilayer molecular networking enables researchers to map how complex plant-derived chemical mixtures are metabolized.
Researchers demonstrated the approach using kratom (Mitragyna speciosa), a Southeast Asian plant whose leaves contain dozens of structurally related alkaloids. While individual kratom compounds – such as the opioid receptor agonist mitragynine – have been studied extensively, the metabolic fate of whole plant mixtures remains difficult to resolve.
“Science is typically reductionist,” explains first author William Crandall, a PhD student in molecular and systems pharmacology at Emory University, in the institution’s press release. “You approach a large system, like a medicinal plant, and try to isolate a compound responsible for that medicinal activity.”
To capture mixture-level metabolism, the team incubated kratom leaf extracts with human liver S9 fractions to simulate hepatic biotransformation. Samples were analyzed using liquid chromatography coupled to high-resolution mass spectrometry, generating MS¹ and MS² datasets.
These were processed using the metabolomics platform MZmine, enabling feature extraction, alignment, and MS² spectral networking. The researchers then integrated multiple layers of evidence – including in silico metabolite predictions from BioTransformer, statistical changes in feature intensity over time, and spectral similarity networks – to construct precursor–metabolite relationships across the extract.
This multilayer molecular networking strategy enabled detection of both phase I and phase II metabolites and helped link dozens of precursor compounds to their metabolic products. The analysis also revealed previously unreported metabolites and showed that metabolic profiles of individual alkaloids can shift when present within complex botanical mixtures.
“This method marks a major, transformative step in natural products research,” adds Dean Jones, co-senior author of the paper. “A process that used to require years of work now takes just days.”
Comparisons between chemically distinct kratom extracts further showed that plant chemotype influences metabolic outcomes, including formation of the bioactive metabolite 7-hydroxymitragynine.
“Our technique does not just look at how one compound in this plant is metabolized,” says Crandall. “It shows how dozens of compounds are metabolized at one time.”
Beyond kratom, the authors suggest the workflow could be applied broadly to dietary supplements, herbal medicines, and other natural product mixtures – helping researchers move beyond single-compound analysis toward a systems-level view of plant chemistry and metabolism.
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