Guaianolide-type sesquiterpenes from Rhaponticum repens (L.) Hidalgo with anti-ischemic stroke activity

Ischemic stroke (IS) is a severe central nervous system disorder with high mortality and disability rates, and microglia-mediated neuroinflammation exacerbates ischemic brain injury. In this study, the traditional ethnic medicine Rhaponticum repens (L.) Hidalgo was found to significantly inhibit lipopolysaccharide (LPS)-induced microglial overactivation and to alleviate cerebral injury in middle cerebral artery occlusion/reperfusion (MCAO/R) rats, suggesting its therapeutic potential against IS. Bioactivity-guided fractionation of its active fraction led to the isolation and identification of 44 compounds, including 22 guaianolide-type sesquiterpenes (1-22) and 22 other types (23-44), among which five were previously undescribed compounds (1-5). Activity screening revealed that most of the guaianolide-type sesquiterpenes markedly suppressed microglial overactivation, representing the principal pharmacologically active constituents responsible for the anti-neuroinflammatory effects of this plant. High-performance liquid chromatography (HPLC) analysis further indicated that cynaropikrin (8), the most active compound (IC₅₀ = 3.8 μM), was the most abundant component in the active fraction, accounting for approximately 14.39% of its content. In vivo experiments confirmed that cynaropikrin at a dose of 5 mg/kg attenuated cerebral ischemia-reperfusion injury in MCAO/R rats, and acute toxicity tests demonstrated its favorable in vivo safety profile. Target prediction, molecular docking, drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR) analyses indicate that cynaropikrin directly targets NLRP3. Western blot analysis further shows that cynaropikrin reduces NLRP3 protein expression, caspase-1 activation and IL-1β maturation, thereby alleviating neuroinflammation and cerebral ischemia-reperfusion injury.