Accelerated characterization of active natural extracts with single-step microfractionation coupled to mass spectrometry and bioassays

Le résumé fourni par la source

Microfractionation is a prominent alternative for the discovery and characterization of bioactive natural products in complex extracts (e.g., plant, fungal, or microbial); however, the method faces limitations as it requires arduous scale-up for complete characterization of molecules. Furthermore, the integration of microfractionation data with metabolomic data is still a complex and tedious process. To circumvent current limitations, we developed a microfractionation method allowing an integrated, reproducible, milligram-scale, 96-deep-well plate framework that combines biological and computational metabolomic readouts. Fractionation is coupled to a single quadrupole mass analyzer, which enables integration with high-resolution metabolomic outputs using adaptable Python scripts. To illustrate the method performances, we applied it to characterize extracts from 7 plants -- Macleya microcarpa, Macleya cordata, Ruta corsica, Thymus camphoratus, Helleborus foetidus, Hypericum hookerianum, and Hypericum olympicum -- spanning different extract complexity and biological activities. Over nine independent experiments, totaling 151 sequential injections, we obtained high reproducibility. Across the study, we utilized no more than 10 mg of crude extract per fractionation and we were able to isolate 17 compounds up to 1H NMR dereplication. Furthermore, we were able to link molecular class and specific molecules to bioactivity found in active extracts. Lastly, to facilitate the adoption of this workflow, we created a user-friendly graphical user interface. Our high-throughput, milligram-scale workflow reduces the need for scale-up and is broadly applicable to natural extracts and biological systems, thus allowing for rapid characterization of natural extracts.

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