Comparative Phenolic Profiling and Antioxidant Activity of Clinopodium nepeta (L.) Kuntze Shoots Cultured In Vitro Under Different Cytokinin Treatments

Clinopodium nepeta (L.) Kuntze is a medicinal and aromatic species of the Lamiaceae family, rich in phenolic compounds; however, studies regarding its in vitro culture, growth regulation properties and secondary metabolism remain limited. The present study investigated the effects of three adenine-type cytokinins: 6-benzylaminopurine (BAP), meta-topolin (m-TOP), and BAP riboside (r-BAP), applied at concentrations of 0.5, 1.0, 2.0 or 4.0 mg L−1, on shoot proliferation, biomass accumulation, phenolic profile, and antioxidant activity in C. nepeta shoot cultures. The phenolic constituents of hydromethanolic shoot extracts were subjected to qualitative profiling using UHPLC–DAD–ESI–MS, while antioxidant potential was evaluated using spectrophotometric assays (DPPH, ABTS, FRAP). All tested cytokinins stimulated shoot proliferation and biomass growth compared with the control; of these, m-TOP demonstrated the most pronounced positive effect, characterized by high multiplication rate and improved shoot morphology. UHPLC–DAD–ESI–MS analysis revealed the presence of numerous caffeic acid derivatives including rosmarinic acid, chlorogenic acids, salvianolic acid derivatives, and flavonoid glycosides; their accumulation was strongly influenced by cytokinin type and concentration. Notably, rosmarinic acid, the dominant phenolic constituent in the treated shoots, reached 23.28 mg g−1 DW under m-TOP treatment, i.e., an approximate 20-fold increase compared with the control. The extracts from shoots cultured on cytokinin-supplemented media exhibited enhanced antioxidant activity, which correlated with increased phenolic content. These relationships were confirmed by principal component analysis (PCA). Hence, C. nepeta shoot cultures represent an efficient in vitro system for biomass production and phenolic compound biosynthesis, and the selection of cytokinin type is a critical factor modulating both morphogenetic and metabolic responses.