Profile of phenolic compounds in Trifolium pratense L. extracts at different growth stages and their biological activities

Although Trifolium pratense (Red Clover) is considered to be one of the leading crops for livestock grazing, it could also be used as a potential source of bioactive compounds in phytopharmacy. The aim of this study was to investigate the phenolic content and its biological activity at the growth phases (30 cm, 50 cm, and bud) of this plant. The phenolic compounds in methanolic extracts of T. pratense leaves at three growth stages, obtained by Microwave Assisted Extraction, were quantified using the HPLC-ESI-MS/MS technique, and their antioxidant and antimicrobial activity were assessed. Isoflavonoids, genistein, and daidzein, as well as other phenols, p-hydroxybenzoic and caffeic acids, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, and hyperoside were found in all the extracts, but the content of these compounds was the highest in the extract of the plant at the lowest growth stage (30 cm, vegetative). Therefore, this extract showed the best antioxidant potential and it was most effective against bacterial strains such as Escherichia coli and Enterobacter aerogenes. These results indicated that red clover has potential health benefits, and that growth phase contributes to its biological activity. The extract of red clover at the growth stage of 30 cm is a great source of bioactive compounds and could be used in phytotherapy and nutrition.