Development of a comprehensive approach for the characterization of polar plant decoctions by orthogonal LC-MS reversed phase, HILIC, and NMR metabolite profiling: the case of Combretum micranthum

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Introduction Plant decoctions are especially valued in ethnomedicine for their simplicity, accessibility, and effectiveness in delivering therapeutic compounds. Chemical analysis of such decoctions can be challenging due to their unique composition, predominantly consisting of polar compounds. In this context, an analytical workflow was developed using HILIC and reversed-phase chromatography. The decoction of Combretum micranthum, a medicinal plant widely used in African traditional medicine and particularly in Senegal for the treatment of hypertension, was used for a proof-of-concept. Methods The chemical profile of the decoction was established using UHPLC-PDA-CAD-HRMS/MS with both HILIC and reversed-phase chromatographic techniques, and a molecular network was generated from HR-MS/MS data to establish comprehensive metabolite annotations and make connections between metabolites. Following a butanol/water liquid-liquid partition, compounds were isolated by semi-preparative HPLC-UV with dry load injection, separating polar compounds via HILIC and medium-polarity compounds via reversed-phase chromatography. Major polar constituents were quantified by 1H-NMR. Results and discussion This approach enabled the annotation of 10 metabolites based on high-resolution MS/MS data, with their identities subsequently confirmed through isolation and de novo structural elucidation. In total, the isolation strategy yielded 13 compounds, which were characterized by NMR and HRMS analyses. The phytochemical analysis highlighted the predominance of very polar compounds (choline, stachydrine, betonicine, and quinic acid) alongside nine flavonoid derivatives from the less polar fraction, with triacetiflavan (13.6%) and betonicine (5.1%) as major constituents. Quantitative 1H-NMR analysis allowed choline to be quantified at 0.1%, stachydrine at 1.3%, and betonicine at 5.1% in the traditional water decoction. Notably, several compounds such as stachydrine, vitexin, and isovitexin possess known cardiovascular activities associated with hypotensive effects, which may underline the traditional use of C. micranthum decoction as a hypotensive agent. The workflow proposed here, particularly the complementary use of HILIC and reversed-phase chromatography for the analysis and isolation of polar compounds, offers a valuable approach for advancing knowledge of the chemical composition of traditional plant decoctions.

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