In the present study, G. tiliaefolia methanolic extract (400 mg/kg) showed an antiepileptic effect manifested by increased latency to onset of myoclonic jerks and GTCS, decreased GTCS duration and seizure severity score. The observed antiepileptic potential might be due to the antioxidant potential owing to the presence of different polyphenols as revealed by the UHPLC. In silico studies also revealed that gallic acid and kaempferol had shown similar interaction with GABA receptor as that of Diazepam (GABA agonist) and interaction with Glutamate AMPA receptors was similar to that of parempenal (Glutamate AMPA antagonist), which might explain its antiepileptic activity. The justification of the adopted methodology and outcome of the study is discussed herewith.
In vitro studies
Oxidative stress is defined as an imbalance between cellular antioxidant defense mechanisms and reactive oxygen species (ROS), such as superoxide radical (O2−), hydrogen peroxide (H2O2), nitric oxide (NO) (Geronzi et al., 2018). Mitochondrial O2 is a primary cause of cellular oxidative injury since it is a precursor to more harmful species such as hydrogen peroxide, hydroxyl radical, and peroxynitrite. Excessive mitochondrial superoxide radical and its more hazardous byproducts have been reported in the genesis of neurodegenerative diseases such as epilepsy (Aguiar et al., 2012; Maier and Chan, 2002). The role of oxidative stress in the pathogenesis of epilepsy has been suggested by several experimental and clinical findings (Eastman et al., 2020; Olowe et al., 2020; Parsons et al., 2022; Lin et al., 2020). Therefore, in this regard natural products can be very beneficial as in addition to anticonvusant activity, they also possess potent antioxidant potential (Geronzi et al., 2018; Abdel-Salam et al., 2022; Chen et al., 2022; Verma et al., 2021). Therefore, as an initial part of our screening, different extracts of G. tiliaefolia were evaluated for antioxidant potential by in vitro DPPH, total antioxidant capacity, reducing power, and DNA nicking assays. In DPPH radical scavenging activity, G. tiliaefolia methanolic extract was found to be most potent as compared to the G. tiliaefolia hexane and G. tiliaefolia chloroform extract. Similarly, in the total antioxidant capacity assay, the ability to reduce the molybdenum (VI) to molybdenum (V) was observed in which G. tiliaefolia methanol extract had shown higher activity as compared to G. tiliaefolia hexane and G. tiliaefolia chloroform extract. In reducing power assay, G. tiliaefolia methanolic extract has shown higher potential to reduce the ferricyanide complex to ferrocyanide as compared to other fractions G. tiliaefolia hexane extract and G. tiliaefolia chloroform extract. Previous reports have demonstrated that various natural products such as Termimalia tomentosa (Pasha et al., 2014), Asparagus racemosus (Shastry et al., 2015; Pahwa and Goel., 2016) possess antiepileptic activity due to their marked antioxidant potential which explained our interest to elucidate the antioxidant potential for different extracts of G. tiliaefolia.
In addition, DNA nicking assay was carried out, which indicates DNA damage as a result of increased production of ROS (Han and Chen, 2013). G. tiliaefolia methanolic extract was found to be most potent amongst all the extracts possibly due to its higher antioxidant potential. As per the present literature, phenolic and flavonoid compounds have been found to exhibit marked antiepileptic effects too (Dhir, 2020). Therefore, total phenolic content (TPC) and total flavonoid content (TFC) were also estimated in different extracts. G. tiliaefolia methanolic extract possessed higher TPC and TFC as compared to other extracts. Previous studies have also shown that extracts with higher TPC and TFC were found to possess remarkable antioxidant and antiepileptic potential (Qneibi et al., 2021; Ojong et al., 2016; Ferreira et al., 2018; Cárdenas-Rodríguez et al., 2014).
Furthermore, as oxidative stress is at the core of a variety of disorders, including neurological diseases, epilepsy, cancer, cardiovascular diseases, liver disorders, and aging, a plant with higher antioxidant activity may be a viable choice for treating these conditions(Qneibi et al., 2021; Ferreira et al., 2018). The methanolic extract of a plant used in the present study had shown significant antioxidant activity, DNA protective activity, and higher TPC and TFC. Keeping in view the traditional uses and findings of this study, methanolic extract of G. tiliaefolia was further evaluated for antiepileptic activity.
In vivo analysis
The anticonvulsant effect of G. tiliaefolia methanolic extract was evaluated using an acute epilepsy model induced by PTZ (GABA antagonist) in mice.G. tiliaefolia methanolic extract increased the latency to onset of myoclonic jerks and GTCS and reduced the duration of GTCS as well as seizure severity score in a dose-dependent manner. Novel drugs delaying the onset of myoclonic jerks and GTCS, shortening GTCS duration with ameliorative effect on seizure severity are said to be effective antiepileptics (Rogawski and Löscher, 2004). According to published reports, novel molecules have been reported to improve all these parameters such as agmatine (Singh et al., 2017), lobeline (Tamboli et al., 2012), and Urtica dioica (Loshali et al., 2021) which supported the antiepileptic effect of methanolic extract of G. tiliaefolia.
To further characterize the active fraction, UHPLC fingerprinting of the methanolic extract was carried out which revealed the presence of various polyphenolic compounds such as gallic acid, catechin, chlorogenic acid, epicatechin, caffeic acid, umbelliferone, coumaric acid, ellagic acid, quercetin, and kaempferol. Quercetin, caffeic acid, kaempferol, epicatechin, ellagic acid, chlorogenic acid, and gallic acid have already been reported to possess antiepileptic activity (Nieoczym et al., 2014; Coelho et al., 2015; Silva dos Santos et al., 2021; Alatorre et al., 2015; Dhingra and Jangra, 2014; Aseervatham et al., 2016; Kumara et al., 2014; Singh et al., 2017). The presence of these phytoconstituents in G. tiliaefolia methanol extract possibly explained its anticonvulsant potential.
In silico analysis
In silico analysis further justified the anticonvulsant effect of G. tiliaefolia. The in silico method's remarkable capability in prospectively predicting the therapeutic potential of drug targets stems from its integrated analysis of multiple protein-ligand interactions properties reported in the literature, such as sequence similarity to pre-existing targets, binding-site geometric and energetic features, and structural similarity to pre-existing targets, and also the physicochemical characteristics learned from computational studies of the pre-existing targets (Zhu et al., 2018). Among all phytoconstituents, gallic acid and kaempferol were present in higher amounts in G. tiliaefolia methanol extract. Based on that these compounds were selected for docking with GABA and Glutamate AMPA receptors. The lesser the value of binding energy, the stronger is the interaction between the ligand and receptor (Sharma et al., 2021). To predict the mechanistic action of the gallic acid, kaempferol and diazepam, we docked these ligands on the diazepam binding site of GABA receptor (PDB: 6X3X). On successful completion of the docking studies (Docking protocol RMSD = 0.6397) various poses of ligands were generated and were evaluated by sorting them according to their binding energies. Diazepam elicits its antiepileptic effect by agnostic interaction with GABA receptors. Docking suggests that both gallic acid and kaempferol have shown interaction at the diazepam binding site of GABA receptor. Hence it can be concluded that both compounds might have shown the antiepileptic effect by the same mechanism as that of diazepam. Additionally, gallic acid and kaempferol were also docked on the Glutamate AMPA receptor along with perampenal. Parempenal was docked to reveal the other possible mechanism of antiepileptic activity of gallic acid and kaempferol. Hence the perampenal binding site of the Glutamate AMPA receptor was selected to dock these phytoconstituents. It was revealed that the compounds were also good inhibitors of the action of glutamate AMPA receptors as the energies of ligands were much lower than parempenal, indicating the same mechanism of eliciting anticonvulsant effect.
As both gallic acid and kaempferol were present in higher quantities as well as they have both have shown agnostic action on GABA receptor as well as antagonistic action on Glutamate AMPA receptor, it can be hypothesized that their presence in bioactive extract might be responsible for antiepileptic potential.