Network pharmacology, molecular docking and In vivo validation reveal the neuroprotective effects of TT-TeMac™ against cholinergic dysfunction and hippocampal lesions.

AD is a neurodegenerative disorder marked by progressive cognitive decline, particularly memory impairment, largely driven by cholinergic dysfunction. This study aimed to investigate the pharmacological basis and modes of action of the identified TT-TeMac™ compounds againsts cholinergic dysfunction associated with memory loss. TT-TeMac™ compounds targets which were identified by LC-MS were extracted from SwissTarget Prediction and PharmMapper, while cholinergic dysfunction-related targets were obtained from DisGeNET and GeneCards. Fifteen common targets were identified, with nine key targets highlighted by STRING, Cytoscape, and Venny analyses and confirmed by molecular docking using MOE. The in vivo validation used a rat model with scopolamine-induced cholinergic dysfunction (1 mg/kg bw/day, ip) for 7 days. Subsequently, behavioral (NOR and MWM), biochemical (AchE and BuhE) and histological (H&E and CV) analyses were performed. Seven compounds were identified in TT-TeMac™ (terminolic acid, sericic acid, arjunolic acid, gallic acid, ellagic acid, 3-O-methyl ellagic acid and 3,3'-di-O-methyl ellagic acid). Network pharmacological analysis showed that TT-TeMac™ acted on 15 common targets of which ACHE, IL6, TNF, SNCA, AKT1, SERPINE1, STAT3, ACE, and ALB were the pivotal genes. Also, docking studies confirmed the involvement of the target within the network with meaningful binding energies. Furthermore, TT-TeMac™ prevented cholinergic dysfunction associated with memory loss in rats by significantly reducing cholinesterase activity and protecting against morphological alterations and neuronal loss in the hippocampus. Our study shows that the ingredient TT-TeMac™ has a multi-targeted mode of action on protein targets involved in cholinergic dysfunction and counteracts this dysfunction in scopolamine-treated rats.