Elucidating the Neuroprotective Mechanism of 5,7-Dimethoxyflavone and 5,7’,4’-Trimethoxyflavone Through In Silico Target Prediction and in Memory-Impaired Mice

5,7-dimethoxyflavone (DMF) and 5,7,4'-trimethoxyflavone (TMF) are natural methoxyflavones known for their potential neuroprotective properties. This study investigates their mechanisms of action through in silico target predictions and memory-impaired mice. Ligand-based and proteochemometric models were used to predict potential protein targets, followed by molecular docking using PyRx and Discovery Video Visualiser. To validate these findings, DMF and TMF (10/20/40 mg/kg) were administered to LPS-induced mice for 21 days. Morris Water Maze (MWM) and Open Field Test (OFT) were conducted to assess cognitive functions. Expression levels of predicted targets were determined by RT-PCR, and enzyme-linked immunosorbent assay was conducted to measure BDNF, Aβ, and pro-inflammatory markers. GABRA1, GABRG2, 5-HT_2A, IGF1R, and 5-HT_2C were predicted for DMF. Meanwhile, GABRG2, 5-HT_2A, 5-HT_2B, and 5-HT_2C were predicted for TMF. Molecular docking showed DMF formed strong binding interactions with GABRA1 and GABRG2 (9.40 kcal/mol), interacting with His102 and Tyr160. Meanwhile, TMF formed strong binding interactions with 5-HT_2A (- 9.30 kcal/mol) interacting with Ser242 and Ser159. TMF enhanced spatial memory in MWM, while both compounds reduced anxiety-related measures in OFT. DMF significantly upregulated hippocampal mRNA of GABRA1, 5-HT_2A, and 5-HT_2C, while TMF increased GABRG2, 5-HT_2B, and 5-HT_2C expression. Additionally, both compounds significantly reduced Aβ, IL-1β, IL-6, and TNF-α levels, while DMF-treated groups significantly increased BDNF level. These findings suggest that DMF and TMF exert neuroprotective effects when administered prophylactically, acting through distinct molecular targets involved in neurotransmission and inflammation. Their multi-target activity makes them promising candidates for early intervention in Alzheimer's disease.