Optimized extraction, isolation and identification of bioactive sesquiterpenes from Inula racemosa

Alantolactone (AL) and isoalantolactone (IAL) are the bioactive components of Inula racemosa Hook. f. and constitute a significant portion in this herb. These two compounds are widely utilized in pharmaceuticals, cosmetics, agriculture and other fields due to their extensive biological activities, exhibiting remarkable market potential. However, their current acquirement remains primarily reliant on traditional Chinese medicine, which makes it necessary to optimize the extraction process. Additionally, the minor components, particularly sesquiterpenes, are often overlooked due to the high contents of AL and IAL, which makes their chemical components still unclear. This study developed an HPLC detection method and optimized an ultrasound-assisted extraction process with response surface methodology (RSM), achieving optimal parameters: liquid-solid ratio of 15.34: 1, 88.92 % ethanol, and 49.21 °C. Furthermore, to identify the minor components, a novel combination of Feature-based Molecular Networking (FBMN), Network Annotation Propagation (NAP), and ClassyFire was employed. The results revealed that the minor sesquiterpenes still accounted for a high proportion. Systematic separation yielded 22 sesquiterpenes, including three novel compounds (1-3) and 19 known ones (4-22). Biological evaluation demonstrated that some compounds exhibited stronger cytotoxicity than AL and IAL against HepG2, SKOV3 and HeLa cells, and compound 14 showed the best cytotoxicity via apoptosis induction. Moreover, four compounds exhibited synergistic effects with cisplatin (CDDP) on SKOV3 cells. Compounds 14 and 15 displayed potent anti-inflammatory effects with IC₅₀ < 3 μM. These findings indicated that the neglected part still contain bioactive sesquiterpenes, deserving for further exploration.