Exploring the anti-inflammatory and cytotoxic effects of Valeriana tuberosa L. constituents: Integrating in vitro and in silico studies

Valeriana tuberosa L. yielded four new iridoids, valtuberoside I-IV (1–3 and 15), along with 13 known secondary metabolites via activity-directed fractionation. Compounds were characterized by NMR and HRESIMS. EtOH extract, fractions, and isolates were evaluated for their inhibition on nitric oxide (NO) release in LPS-induced RAW 264.7 cells. Compounds 3, 4, 6, 8, 9, 11, 13, 16, and 17 exhibited anti-inflammatory activity by inhibiting the release of NO (IC50 43.44–95.71 μM), and their mode of actions were elucidated by ELISA, Western blot, qPCR, immunostaining techniques and supported by molecular modelling studies. Compounds 8, 9, 11, 13, and 17 showed significant reduction in TNF-α, IL-1β, IL-6, PGE2, and COX-2 enzyme production, while 9 and 13 decreased iNOS protein expression in RAW 264.7 cells. Compound 13 exhibited remarkable inhibition on pro-inflammatory markers, cox-2 gene expression and translocation of NF-κB to the nuclear region. Moreover, it had the most favourable interaction (ds: −6.46 kcal/mol) with iNOS in in silico analyses. The cytotoxic activities of the most active isolates against MCF-7, MDA-MB-231, U87, A172, MIA PaCa-2, PANC-1, Mahlavu, and Hep3B cancer cell lines were assessed using CCK8 assay and their cell death mechanisms were unveiled via Apoptosis/Necrosis Assay Kit. Compound 8 had significant cytotoxic activity against MIA PaCa-2 (IC50 23.7 μM) and Hep3B (IC50 25.4 μM) cancer cell lines, via arresting cell cycle especially in G2/M phase and triggering the apoptotic pathway. These findings indicated that 8 and 13 deserve further in vivo assays on the way to discover new potential drug leads.