Bioactivity-Guided Isolation of Cytoprotective Metabolites from Two Verbascum Species Reveals Differential Modulation of Cigarette Smoke-Induced Inflammation and Oxidative Stress

Verbascum species are traditionally used for respiratory disorders and are rich sources of iridoid glycosides, phenylethanoid glycosides, and triterpenoid saponins. However, V. kurdicum and V. longipedicellatum have not previously been investigated phytochemically or biologically. This study aimed to identify their bioactive constituents and characterize their protective effects against cigarette smoke extract (CSE)-induced bronchial epithelial injury. Bioactivity-guided fractionation of 50% methanol extracts yielded twelve compounds identified by NMR. Cytoprotective potential was evaluated in BEAS-2B cells, and selected compounds were investigated for effects on IL-6, IL-8, HO-1, TLR4 expression and downstream effectors (MyD88 and NF-κB), GSH, 8-OHdG levels, and SOD activity. Ajugol exhibited the strongest cytoprotective activity and significantly restored viability following CSE exposure. Ajugol, aucubin, and 1-O-caffeoyl-β-D-glucopyranose were selected for mechanistic evaluation. Aucubin and 1-O-caffeoyl-β-D-glucopyranose significantly reduced CSE-induced IL-6 release, while all three suppressed IL-8, with ajugol producing the greatest effect. Ajugol also attenuated TLR4 upregulation and reduced 8-OHdG levels and the CSE-induced increase in SOD activity, whereas MyD88, total NF-κB, p-NF-κBSer536, and GSH were not significantly improved. These findings provide the first phytochemical and biological characterization of both species and identify ajugol as a promising lead against CSE-induced bronchial epithelial damage.