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Antioxidant activity of ethanolic extract of Desmodium adscendens leaves was assessed.Activity-guided fractionation allowed separation of most potent fraction (50% MeOH).Two major compounds were identified as isovitexin- and vitexin-2″-O-xylosides.Hybrid HSQC-DEPT technique provided fast determination of flavonoid isomers.D. adscendens has been recognized as a rich source of flavone C-glycosides.The antioxidant activity of the crude extract (60% ethanol) from the leaves of Desmodium adscendens (Sw.) DC. (Fabaceae) was observed in DPPH, xanthine/xanthine oxidase, lipid peroxydation and neutrophils burst tests. Further activity-guided fractionation on C18 column (water, 20% methanol, 50% methanol and 100% methanol) resulted in the separation of the fraction (50% methanol) with the highest antioxidant capacity. HPLC-DAD analysis of biologically active fraction revealed the presence of two pairs of flavonoid isomers as the dominant constituents. Those compounds were isolated and purified by multi-step liquid column chromatography (Sephadex LH20). Their structures were elucidated by various spectroscopic techniques, including NMR, UV and MS. Based on 1D and 2D NMR spectra as well as ion fragmentation, flavonoids were identified as: isovitexin 2″-O-xyloside (1), vitexin 2″-O-xyloside (2), vitexin (3) and isovitexin (4). The hybrid HSQC-DEPT technique provided very fast determination of the glycosylation positions in aglycone and the type of glycosidic bond in the flavonoid isomers. This study provides novel information concerning identity of the major compounds present in the leaves of D. adscendens cultivated in Ghana, which broadens the knowledge about anti-inflammatory, antiallergic and antioxidant properties of their extracts.