Discussion

Preventing cognitive decline is essential for combating AD, especially in the context where is there is a lack of curative treatment (Tipton, 2024). Memory loss is the main symptom affecting the quality of life of patients with AD, and treatments do not effectively improve memory loss. This is likely due to the single-target action, yet polypharmacology shows promise for complex diseases. Computational approaches, including network pharmacology and molecular docking have proven to be valuable tools to facilitate the exploration of the pharmacological properties of active ingredients against AD. Recently, Ambamba et al., 2025a, Ambamba et al., 2025b, showed that Terminalia macroptera modulate cholinergic dysfunction associated with memory loss in scopolamine-treated rats. In this work we explored network pharmacology and molecular docking followed by experimental validation to explore the neuroprotective effects of TT-TeMac™ on cholinergic dysfunction associated with memory loss in scopolamine-treated rats.

The LC-MS analysis of the TT-TeMac™ allowed the identification of seven compounds, namely Terminolic acid, Sericic acid, Arjunolic acid, Gallic acid, Ellagic acid, 3-O methyl ellagic acid and 3,3’-di-O-methyl ellagic acid. These compounds have been previously isolated from this plant by Conrad et al., 1998; Conrad et al., 2001; Adey et al., 2021 and Roméo et al., 2024.

Using network pharmacology analysis makes it possible to identify the key targets of bioactive compounds involved in the pathophysiological mechanism, thus guiding more targeted and cost-effective experimental validation. In this study, the network pharmacology analysis revealed nine pivotal genes that are potential targets of TT-TeMac™ compounds in the treatment of cholinergic dysfunction. These genes include ACHE, IL6, TNF, SNCA, AKT1, SERPINE1, STAT3, ACE, and ALB. Scientific evidence suggests the involvement of all proteins through these genes in cholinergic dysfunction (Madziar et al., 2008; Tyagi et al., 2010; Chen et al., 2022). Since a protein is the functional version of a gene, molecular docking study was done to confirm the targets generated by the network pharmacology. Molecular simulation showed that all priority proteins have a high binding affinity with the identified TT-TeMac™ compounds (with binding energies ranging from −3.39 to −7.40 kcal/mol). In addition to good binding energy, these compounds interact with amino acids in the active pockets of important proteins involved in the pharmacological network (Table 4). These results predict that, TT-TeMac™ compounds have multi-target actions on proteins involved in cholinergic dysfunction.

Scopolamine, which induces a cholinergic deficit, experimentally reproducing memory disorders similar to those observed in dementia, has been used to experimentally validate the neuroprotective effect of TT-TeMac™. During the MWM, the learning profile that was modified by scopolamine was preserved by the treating with TT-TeMac™ (Fig. 7A). The mean escape latency increased significantly in the scopolamine-treated group during the acquisition phase. However, this change was significantly reversed by TT-TeMac™ therapy (Fig. 7 B). The number of times that the animals entered the target quadrant during the tests was used to assess their memory of the exact location where the platform had previously been placed. Rats treated with scopolamine were less present in the target quadrant, suggesting a problem with spatial memory and learning. In contrast, treatment with TT-TeMac™ increased their presence in the target quadrant. Furthermore, TT-TeMac™ increased the time spent exploring the novel object compared to the well-known object and increased the RI in the NOR (Fig. 6).

All of these results show that TT-TeMac™ (100 mg/kg bw) protects against cholinergic dysfunction associated with scopolamine-induced memory loss in rats. This is explained firstly by the ability of TT-TeMac™ to protect certain areas of the hippocampus (CA1, CA2, CA3 and DG) against scopolamine-induced neurodegeneration (Fig. 8a, Fig. 8b, Fig. 8c, Fig. 8d) and secondly by preventing the increase in cholinesterase activity (AChE and BuchE) (Fig. 9). The reduction in AChE activity validates the results of molecular docking, which showed that the compounds in TT-TeMac™ have an affinity for AChE and interact with Trp86, Asp74, Tyr341, and Tyr124 residues present in various acyl and anionic pockets. Furthermore, one of the ingredient's components (arjunolic acid) interacts with His447, which is involved in the catalytic triad of the functional AChE mechanism.