Triterpenoid saponins from the roots and rhizomes of Clematis terniflora var. manshurica (Rupr.) Ohwi and their anti-inflammatory activities.

The 60 % EtOH-eluted fraction (60 %EF) from the roots and rhizomes of Clematis terniflora var. manshurica (Rupr.) Ohwi exhibited potent anti-inflammatory activity with minimal cytotoxicity compared to other fractions. A total of 88 compounds were identified from this fraction by UPLC-Q-TOF-MS analysis, with triterpenoid saponins found to be the most abundant class. Subsequently, nine previously undescribed oleanane-type triterpenoid saponins (1-9), along with twenty known analogues (10-29), were obtained from the 60 % EF. Their structures were elucidated through comprehensive spectroscopic analysis, employing HRESIMS, 1D and 2D NMR techniques. The plausible biosynthetic pathways of the compounds 1-9 were also proposed. All isolates were assessed for anti-inflammatory activity in LPS-stimulated RAW264.7 macrophages, among which 4 showed the strongest inhibition of NO and TNF-α production. Mechanistic studies using western blotting, molecular docking, and molecular dynamics simulations revealed that 4 suppresses NF-κB activation mainly by inhibiting IκBα phosphorylation and stabilizing IκBα protein levels, while also directly interacting with p65 to attenuate its phosphorylation. These findings provide new insights into the anti-inflammatory potential of triterpenoid saponins from C. terniflora var. mandshurica and highlight 4 as a promising NF-κB pathway modulator.