Red Clover Isoflavones as Effective Longevity Agents for Anti-Aging and Regenerative Skin Applications

Red clover (Trifolium pratense L.) is a rich source of isoflavones with documented antioxidant and skin-protective properties, yet substantial differences in phytochemical composition exist among cultivars. In this study, fourteen T. pratense cultivars were compared with respect to formononetin and biochanin A contents (Milena, Pasieka, Pyza, Milvus, Nemaro, Maro, Larus, Hammon, Vesna, Fregata, Carbo, Forelia, Osimia, and Elanus), and the relationship between isoflavone profiles and skin-related biological activity was evaluated. High-performance liquid chromatography revealed pronounced cultivar-dependent variability with formononetin and biochanin A contents ranging from 1.60 to 7.80 mg/g DW and from 0.69 to 6.44 mg/g DW, respectively. The observed variability was further visualized by principal component analysis. The cultivar with the highest total isoflavone content-Hammon, was selected for biological assessment. Its extract exhibited antioxidant (DPPH IC₅₀ = 0.619 mg/mL; FRAP IC_0.5 = 0.302 mg/mL) and enzyme inhibitory activities (elastase IC₅₀ = 0.602 mg/mL, hyaluronidase IC₅₀ = 22.44 mg/mL), and it significantly enhanced fibroblast migration in an in vitro scratch assay, indicating anti-aging and regenerative potential. These results demonstrate that red clover cultivars differ significantly in their suitability as sources of bioactive isoflavones and highlight the importance of cultivar selection for the development of standardized plant-derived anti-aging ingredients. However, it is worth emphasizing that isoflavones derived from red clover are a valuable group of active compounds with significant potential for topical application as anti-aging and regenerative agents, warranting further formulation development and in vivo validation.