LC-MS-guided characterization and neuroprotective evaluation of Galium libanoticum in an LPS-induced...

Galium libanoticum is traditionally employed in Mediterranean folk medicine for alleviating headaches, inflammatory ailments, fatigue, and nerve-related discomfort. Despite its long-standing use, the plant has never been scientifically evaluated for neuroprotective or anti-neuroinflammatory properties, nor has its phytochemical composition been comprehensively profiled. Given that several Galium species are known to contain flavonoids, iridoids, and phenolic acids with anti-inflammatory potential, a systematic assessment of G. libanoticum is warranted to validate its ethnomedicinal use and explore its mechanistic actions in neuroinflammation.

This study aimed to characterize the metabolite profile of G. libanoticum using high-resolution UPLC-MS/MS, and to evaluate its neuroprotective and anti-inflammatory effects in an LPS-induced neuroinflammatory rat model. In parallel, we sought to elucidate the molecular mechanism underlying its activity through integrated in vivo assessments and network-pharmacology analyses to determine how its phytoconstituents interact with key neuroinflammatory pathways.

The chemical composition of the hydro-ethanolic extract was analyzed using UPLC-MS/MS, and identified metabolites were subjected to compound-target prediction, interaction-network construction, and pathway enrichment. Male Wistar rats received G. libanoticum (50, 100, 200 mg/kg) after pretreatment with Galium libanoticum and subsequent LPS challenge. Neurobehavioral tests, oxidative-stress markers, cytokine quantification, Western blotting, and immunohistochemistry of hippocampal and cortical tissues were performed to evaluate anti-inflammatory and neuroprotective outcomes. Computational results were aligned with in vivo data to construct a comprehensive mechanistic profile.

UPLC-MS/MS profiling revealed a phytochemical matrix dominated by caffeoylquinic acids, flavonoid glycosides, and iridoids. In vivo, G. libanoticum produced a clear, dose-dependent reduction of LPS-induced neuroinflammation, reflected by suppression of NF-κB p65 activation, downregulation of TNF-α expression, stabilization of IκB-α, and marked restoration of CREB signaling. Immunohistochemical analyses confirmed reduced NF-κB and TNF-α immunopositivity and enhanced CREB expression, particularly at 200 mg/kg. Network-pharmacology analysis revealed that major constituents-such as rutin, chlorogenic acids, and iridoid glycosides-strongly interacted with central neuroinflammatory mediators including TNF, IL-6, IL-1β, NFKB1, RELA, MAPK1, and AKT1. Pathway enrichment indicated significant involvement of NF-κB, TNF cytokine, IL-17, MAPK/ERK, COX-2/iNOS, and cAMP/CREB pathways, mirroring the biochemical and histological outcomes observed experimentally.

The combined experimental and computational findings demonstrate that Galium libanoticum exerts significant neuroprotective and anti-neuroinflammatory activity by simultaneously suppressing pro-inflammatory cascades and activating CREB-mediated neuronal survival pathways. These results validate the plant's traditional use and support its potential as a multi-target natural therapeutic for neuroinflammatory and neurodegenerative conditions. The mechanistic insights gained provide a strong foundation for future isolation studies, synergy assessments, and translational research.