Neuroprotective eremophilane sesquiterpenoids from Ligularia species attenuate glutamate-induced oxidative stress via MAPK signaling

Ethnopharmacological relevance Ligularia species (Asteraceae) have long been used in traditional East Asian medicine for the treatment of pain and inflammatory disorders. Although neuroprotection is not among their documented traditional indications, the close relationship among inflammation, oxidative stress, and neuronal injury, together with the reported analgesic and anti-inflammatory activities of these species, provides a rationale for investigating their neuroprotective potential. Aim of the study Eremophilane sesquiterpenoids are the principal and most characteristic constituents of the genus Ligularia , and those from other natural sources have been reported to protect neuronal cells against oxidative stress. Given the close relationship among oxidative stress, inflammation, and neuronal injury, the traditional anti-inflammatory use of Ligularia and the reported neuroprotective activity of eremophilane sesquiterpenoids suggest that the genus may be a source of neuroprotective constituents, which has not yet been investigated. This study aimed to comparatively evaluate the neuroprotective activity and eremophilane sesquiterpenoid profiles of three traditionally used Ligularia species ( L. taquetii , L. fischeri , and L. stenocephala ), and to identify the active constituents and their underlying mechanism. Materials and methods Neuroprotective activity was assessed in a glutamate-induced HT-22 hippocampal neuronal cell model. Non-targeted UHPLC–Orbitrap MS metabolomics with feature-based molecular networking was used to compare metabolite profiles and to guide isolation. Isolated compounds were structurally characterized by spectroscopic and computational analyses and quantified by validated LC–MS/MS. Intracellular ROS and Ca 2+ levels, along with the expression of apoptosis-related and MAPK proteins, were measured, complemented by network pharmacology, molecular docking, and a Caenorhabditis elegans 6-hydroxydopamine model. Results Among the three species, L. taquetii exhibited the highest neuroprotective activity without detectable cytotoxicity and contained a markedly higher relative abundance of eremophilane sesquiterpenoids. Metabolomics-guided isolation yielded 15 eremophilane sesquiterpenoids, including eight previously undescribed compounds (taquetins A–H). Compounds 3 and 7 showed the strongest neuroprotective effects and accumulated at the highest levels in L. taquetii , with compound 7 detected exclusively in this species. Both compounds attenuated glutamate-induced ROS accumulation, Ca 2+ overload, and AIF-mediated apoptotic signaling, and suppressed phosphorylation of p38, ERK, and JNK, consistent with network pharmacology and molecular docking predictions. Compound 7 further attenuated 6-hydroxydopamine-induced locomotor impairment in C. elegans without affecting growth. Conclusions Among the three Ligularia species examined, the highest levels of neuroprotective eremophilane sesquiterpenoids were found in L. taquetii , and taquetins C and G ( 3 and 7 ) attenuated glutamate-induced oxidative neuronal injury through modulation of MAPK signaling. These findings support the traditional anti-inflammatory use of Ligularia species as a rational starting point for the discovery of neuroprotective constituents and identify L. taquetii as a source of eremophilane sesquiterpenoids that merit further investigation as candidates against oxidative stress-related neurodegeneration.