The Phytochemical Content and DPPH Activity of Trifolium repens L. Methanol Extract, and In Silico Studies

Trifolium repens L., commonly known as white clover, is an herbaceous perennial plant belonging to the Fabaceae family. It is widely used in traditional medicine to treat diarrhoea, menstrual irregularities, skin damage, coughs, rubella, and stress, irritability and insomnia. In our study, we investigated the phenolic profile of the methanol extract of T. repens by LC-MS/MS and determined the DPPH and total phenolic content (TPC) for the first time using potentiometric biosensor methods. Furthermore, molecular docking was used to theoretically determine the interactions between the dominant compounds and the enzymes responsible for antioxidant activity: thioredoxin reductase (TrxR) and heme oxygenase-1 (HO-1). LC-MS/MS analysis revealed the main components of T. repens to be coumarin (29,673.13 µg/mg extract) and isoquercitrin (21,608.99 µg/mg extract). The extract exhibited lower antioxidant activity than the standard extract, as determined by DPPH (27.43%) and FCR (179.6257 GA/plant extract) analyses. Additionally, isoquercitrin (-7.6, 8.9 kcal/mol) and the proposed coumarin-isoquercitrin complex (-9.2, -9.5 kcal/mol) exhibited significant binding affinity to HO-1 and TrxR enzymes, respectively. In conclusion, the bioactive compounds of T. repens appear to be responsible for its biological activities. Therefore, this application could be widespread in fields such as food, pharmacology and cosmetics.