Thiol-reactive compound depletion reveals electrophile-dependent and independent anti-inflammatory constituents in Saussurea costus

Background Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and joint damage. Despite available treatments, many patients fail to achieve adequate disease control, prompting interest in complementary approaches. In traditional Chinese medicine, multi-herbal formulations such as Formulation C (Qushi-Juanbi Granules) are used clinically to treat RA. Among its component plants, S. costus ( S. costus ) has long been recognized for its anti-inflammatory properties that have largely been attributed to reactive α -methylene- γ -lactone-containing sesquiterpene lactones. However, comparatively little is known about the activities of non-reactive compounds. Objective This study aimed to investigate the anti-inflammatory and anti-arthritic potential of S. costus and to determine the contribution of reactive sesquiterpene lactones to this effect. Materials and methods Extracts from Formulation C plants were screened in cell-based assays targeting RA-relevant pathways (NF-κB, NFAT, STAT3/STAT5), prostanoid formation in a synovial fibroblast cell line, and cytokine production in primary B cells. The aqueous extract of S. costus and an electrophile-depleted version were prepared by elution through thiol-bonded silica and tested in vitro and in the KRN T cell transfer arthritis model in vivo . Seven compounds were isolated from S. costus and their activity characterized in vitro . Results Screening identified the aqueous extract of S. costus with potent in vitro activities, mainly affecting the NF-κB and NFAT pathways, and the B cell assay. This activity was lost upon electrophilic-compound depletion, and the sesquiterpene lactones costunolide and dehydrocostus lactone were identified as major active constituents. In vivo , the aqueous extract modestly reduced paw swelling during early disease phase, whereas the electrophile-depleted extract showed minimal activity. Conclusion This study applies a straightforward thiol-reactive compound depletion method to explore the bioactive constituents of S. costus , a strategy that can be broadly applied to other natural product extracts. Our findings indicate that sesquiterpene lactones are major contributors to the overall anti-inflammatory activity of S. costus , while other, non-thiol-reactive compounds may also play a role for in vivo activity. Overall, these results provide a more detailed characterization of the bioactive profile of S. costus and support its future mechanistic and preclinical investigation for RA research.