LC-MS/MS-Based Metabolomic Profiling and Antioxidant Potential of Combretum mellifluum Flower Extracts

Introduction: Plant metabolomics is a valuable strategy for investigating the chemical diversity of medicinal species and supporting the annotation of specialized metabolites. Species of the genus Combretum are recognized for producing phenolic compounds, flavonoids, tannins, and triterpenoids with biological relevance. However, LC-MS/MS-based studies focused on Combretum mellifluum flowers remain scarce. Therefore, this study investigated flower extracts of C. mellifluum using HPLC-DAD and LC-MS/MS, combined with antioxidant, total phenolic, and flavonoid content assays.

Methods: Flowers were subjected to ultrasound-assisted extraction using methanol, acetonitrile, acetone, and a ternary mixture of methanol:acetonitrile:acetone. The extracts were analyzed by HPLC-DAD and LC-MS/MS in positive ionization mode. Metabolite annotation was supported by retention time, precursor ions, MS/MS fragmentation patterns, mass accuracy, spectral similarity scores, DAD responses, and literature data. Antioxidant activity was evaluated by DPPH and ABTS assays, while total phenolic and flavonoid contents were determined by Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively.

Results: Three representative metabolites were putatively annotated: betulin, rutin/rutoside, and pedunculagin. Betulin was assigned as a lupane-type pentacyclic triterpenoid based on the dehydrated ion [M+H−H₂O]⁺ at m/z 425.3777 and product ions related to dehydration and skeletal cleavages. Rutin/rutoside showed [M+H]⁺ at m/z 611.1614 and diagnostic fragments at m/z 465, 303, and 153. Pedunculagin was annotated through the ammonium adduct [M+NH4]⁺ at m/z 802.1103 and product ions consistent with ellagitannin fragmentation. The ternary extract showed the strongest antioxidant performance among C. mellifluum extracts, with IC50 values of 14.90 µg/mL for DPPH and 57.69 µg/mL for ABTS, as well as the highest flavonoid content, 73.46 mg RE ^g−1.

Conclusion: These preliminary findings suggest that C. mellifluum flowers contain specialized metabolites with antioxidant potential and support further LC-MS/MS-based studies to expand the chemical characterization of this poorly explored plant matrix.