Targeted isolation of dimeric sesquiterpene lactones and sesquiterpene derivatives from the roots of Inula helenium.

LC-HRMS/MS-based molecular networking was applied to investigate the bioactive sesquiterpene lactones and their analogs contained in Inula helenium, enabling the isolation of four undescribed eudesmane-eudesmane sesquiterpene dimers (1-4), a sesquiterpene-amino acid adduct (5), and 17 known sesquiterpenes (6-22). The structures were determined based on 1D, 2D NMR, and HRESIMS data analysis, as well as DP4+ probability analyses and ECD calculations. The biosynthetic pathway of the sesquiterpene dimers is postulated to proceed via the Diels-Alder reaction as the key step. The inhibitory activity of all isolates on LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages was evaluated. Compounds 4, 17, and 20 exhibited significant inhibitory activity against NO production, with IC50 values of 8.4, 5.5, and 9.1 μM, respectively.