Investigation and Mechanism of Coumarin for Potential Anti-Epileptic Targets using in-vitro SH-SY5Y Cell Line, Molecular Docking, and Network Pharmacology-based Analysis

Epilepsy affects 1-2% of the world population. In about 30% of individuals with epilepsy, the etiology is unknown after ruling out genetic mutations, severe injury, and several other possible causes. In about 20-30% of epilepsy patients, anti-epileptic drugs fail to control the seizures. The general trend in epilepsy genetics research is towards an increasingly powerful genetic platform for investigating genomic sequence and structural variation. This pattern will inevitably result in a quick rate of genetics-related discoveries and have significant effects on our capacity to identify and forecast epilepsy and related illnesses. About one-third of epileptic patients do not receive enough seizure control from the current medications. To close this treatment gap, new alternatives are required. Since phenytoin, a commercially available antiepileptic medicine, has a significant adverse effect called hypoguasia, which results in a diminished sense of taste, coumarin may lessen this side effect in addition to its antiepileptic properties, which are supported by several in-silico and in-vitro studies.

The current study examined the potential anti-epileptic effects of coumarin using network pharmacology and in-vitro studies.

During the initial stage, information about the phytoconstituent and the target genes linked to epilepsy and Coumarin was collected from open-source databases and scholarly literature. These data were then analyzed to identify common targets between the phytoconstituent and epilepsy. A Protein-Protein Interaction (PPI) network was built using the Search Tool for Identifying Interacting Genes and Proteins (STRING) database based on these common targets. Then, the hub genes were identified according to the degree of connectedness by integrating the Protein-Protein Interaction (PPI) network into the Cytoscape software. The networks of disease, genes, and Coumarin were obtained by following the processes of network pharmacology. A cell line investigation included the Cytotoxicity Study (MTT assay), Ca²⁺ Expression assay, and Mitochondrial Membrane Potential (JC-1 dye).

In the intracellular Ca²⁺ expression assay, the intracellular Ca²⁺ rate was highly enhanced in the toxic group and moderately in the co-treatment of the poisonous and sample groups, suggesting the neuroprotective effect of coumarin-containing liposomes (Coumarosome) against the pentylenetetrazol (PTZ) induction on Epilepsy model. Also, a membrane potential dye (JC-1) ratio of pentylenetetrazol (PTZ)-treated cells was very low, 0.61 ± 0.12, whereas untreated cells showed a JC-1 ratio of 68.23 ± 36.37, respectively. It is suggested that coumarin-containing liposomes (Coumarosome) may have a better mitochondrial recovery rate. The evidence that this study exhibits antiepileptic activity comes from cell line research.

To investigate the possible molecular processes of coumarin, the current study combined network pharmacology with bioinformatics techniques as it may function as an anti-epileptic tool, and it contains the TAS2R38 gene, which is involved in the compound-target network of epilepsy during the initial stage. The prepared Coumarin-containing liposomes (Coumarosomes) were well dispersed. These observed results suggest the neuroprotective effect of coumarosomes against the PTZ induction or epilepsy model.

The obtained data demonstrate that coumarin efficiently suppresses epileptic effects produced by pentylenetetrazol (PTZ). Thus, coumarin-containing liposomes (Coumarosome) represent a high potential therapeutic value as an antiepileptic pharmaceutical agent for the treatment of epilepsy.