LC-MS/MS Analysis of crude Flavonoid Compounds from <i>Justicia secunda</i> from Democratic Republic of the Congo and evaluation of their antisickling Activity

<i>Justicia secunda</i> Vahl (Acanthaceae) is a tropical herbaceous plant traditionally used by indigenous peoples of the Democratic Republic of the Congo (DRC) to treat symptoms associated with sickle cell disease (SCD), a genetic condition with life-threatening complications. Given the lack of side-effect-free drugs, alternative herbal therapies that can reduce or reverse red blood cell sickling may serve as safe and effective treatments. The primary objective of this study is to conduct a phytochemical screening of crude flavonoids extracted from <i>J. secunda</i> using LC-ESI/MS/MS. <i>J. secunda</i> extracts were evaluated for their phytoconstituents and anti-sickling properties. The anti-sickling properties were assessed through reverse sickling estimation, polymerization and oxidation inhibition, and osmotic fragility assays. Fractionation of the methanol extract and chemical profiling were performed by flash chromatography and high-performance thin-layer chromatography analysis respectively. Characterization and identification of the crude flavonoids were done using chromatography and spectroscopy methods. Chemical screening of crude flavonoids revealed the presence of 12 flavonoid O-glycosides based on one flavonol (methoxy-kaempferol) and two flavones (luteolin and apigenin). The crude flavonoid extracts demonstrated maximum reversal of sickled red blood cells (91.9 ± 0.8%), significant inhibition of hemoglobin polymerization (77.8 ± 7.6%) and hemoglobin S auto-oxidation (96.3 ± 0.2), and provided maximum protection of red blood cells against hyperhemolysis. The results validate the traditional use of <i>J. secunda</i> and identify potential compounds for the development of novel anti-sickling agents.