Comparative phytochemical and biological profiling of Trifolium rubens L. leaves, flowers, and callus cultures for cosmetic applications

Abstract

Abstract Trifolium rubens (Fabaceae) is a rare and regionally endangered European species with limited phytochemical and biological data. This study compares extracts obtained from leaves, flowers, and in vitro derived callus cultures. HPLC–DAD analysis confirmed three major groups of metabolites: flavonoids, isoflavonoids, and phenolic acids, with leaves showing the highest polyphenol levels, including trifolin (1257.13 mg/100gDW) and myricetin (1068.20 mg/100gDW). Callus cultures exhibited a distinct isoflavonoid profile dominated by calycosin-7- O -glucoside (516.01 mg/100gDW) and formononetin (103.26 mg/100gDW), accompanied by reduced genistein content compared with parent tissues. All extracts demonstrated antioxidant activity (ABTS IC₅₀: flower 0.02 mg/mL, leaf 0.17 mg/mL, callus 1.51 mg/mL; DPPH IC₅₀: 3.21, 3.93, and 6.16 mg/mL, respectively), moderate inhibition of tyrosinase and elastase, and no cytotoxicity toward HaCaT keratinocytes or A375 melanoma cells. In ex vivo Franz diffusion studies, genistein showed limited permeation but substantial skin accumulation (leaf: 17.07 µg/g; flower: 16.55 µg/g; callus: 2.58 µg/g), supporting localized antioxidant effects. Despite lower total polyphenol content, callus cultures retained relevant bioactivity and accumulated unique isoflavonoids, highlighting their potential as a sustainable alternative source of bioactive compounds. These findings support the cosmetic and dermatological relevance of T. rubens and emphasize the importance of optimizing in vitro cultures to enhance metabolite production.