Structural characterization, neuroprotective activity, and CASP3/ERK1 interaction analysis of phytochemicals from Clematis mandshurica

초록

This study isolated phytochemicals from Clematis mandshurica and investigated their possible relevance to Alzheimer's disease (AD)-related neuroprotective mechanisms. Among the solvent fractions, the n-hexane fraction exhibited the strongest protective activity against glutamate-induced oxidative injury in HT22 hippocampal neuronal cells. Accordingly, sixteen compounds were isolated from the bioactive n-hexane fraction and structurally characterized by spectroscopic analysis. Methyl delta,4-dimethyl-1,4-cyclohexadiene-1-pentanoate (MDCP), the most active compound in the cell-based screening, was further evaluated in vitro. MDCP significantly reduced intracellular reactive oxygen species (ROS) and Ca2+ accumulation, preserved mitochondrial membrane potential, and restored glutathione-dependent antioxidant capacity. To explore the possible molecular basis underlying the neuroprotective potential of C. mandshurica constituents, network-based target prediction, molecular docking, induced-fit docking, and molecular dynamics simulations were performed. These analyses prioritized CASP3 and ERK1 as putative AD-related proteins associated with apoptosis-and kinase-related signaling and suggested favorable predicted interactions for the selected stigmasterol glucoside (SG)-and MDCP-bound complexes. However, these findings were obtained only from computational analyses and do not demonstrate direct modulation of CASP3 or ERK1. In a scopolamine-induced mouse model of cognitive impairment, oral administration of C. mandshurica crude extract improved memory-related behavioral performance, reduced hippocampal acetylcholinesterase activity, and increased hippocampal BDNF and CREB mRNA expression. Overall, these findings suggest that C. mandshurica phytochemicals may contribute to antioxidant neuroprotection and provide computationally prioritized candidates for further investigation in relation to AD-related signaling proteins. Further target-validation studies and compound-specific animal experiments are required to clarify the mechanistic and therapeutic relevance of these phytochemicals in AD-related models.

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