[1] 69w Anti-epileptic activity of daidzin in PTZ-induced mice model by targeting oxidative stress and BDNF/VEGF signaling Zartashia Kazmi (Conceptualization) (Investigation) (Methodology) (Visualization), Sara Zeeshan (Investigation) (Methodology) (Writing -original draft)<ce:contributor-role>Writing -review editing), Adnan Khan (Investigation) (Software) (Writing -original draft)<ce:contributor-role>Writing -review editing), Sumra Malik (Investigation) (Software), Adeeb Shehzad (Resources) (Validation), Eun Kyoung Seo (Funding acquisition) (Resources) (Validation), Salman Khan (Conceptualization) (Data curation) (Formal analysis) (Project administration) (Software) (Supervision) (Validation) (Visualization)<ce:contributor-role>Writing -review editing

[1] 94w This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

[1] 126w Epilepsy is a relentless neurodegenerative disorder of which the most prominent feature is recurrent seizures (Sun et al., 2019). Globally, It affects 50-70 million individuals and awaiting for safer profile drugs with better anticonvulsant activity (Yu et al., 2019). Several classes of antiepileptic drugs (AEDs) are available for the management of epilepsy. However, approximately 1/3 of epileptic patients fail to achieve complete remission (Sharma et al., 2018). Moreover, the clinical uses of these medications are associated with addiction, tolerance, and severe adverse effects, such as paresthesia, agranulocytosis teratogenicity, and glaucoma (Sun et al., 2019). Therefore, the J o u r n a l P r e -p r o o f development of novel antiepileptic agents that suppress the epileptogenesis effectively and safely is urgently required.

[2] 17w For the last two decades, research has been carried out to explore the underlying mechanisms of epileptogenesis.

[3] 175w The role of BDNF/VEGF, oxidative stress, and apoptosis in the pathophysiology of epilepsy are well established (Singh et al., 2019). It is well accepted that BDNF mediating various neuronal processes in the brain, including survival of existing neurons, neuronal differentiation, synaptogenesis, synaptic transmission, neuronal plasticity, as well as higher cognitive functions (Chen et al., 2017). Several lines of evidence have indicated the upregulation of BDNF shown a protective response against seizures, which is ultimately capable of counteracting the hippocampal epileptogenesis (Yu et al., 2019). Moreover, a recent study indicated that the down-regulation of BDNF in PTZinduced seizures contributes to seizure-induced neuronal apoptosis (Yu et al., 2019). It is well accepted that excessive production of angiogenic factors such as vascular endothelial growth factor (VEGF) plays a crucial role in epilepsy (Tawfik et al., 2018). It has been revealed that seizures promote BBB disruption by upregulation of VEGF which enhanced angiogenesis and vascular permeability within the epileptic tissue (Morin-Brureau et al., 2012). Thus, modulation of the BDNF/VEGF signaling appears to be a promising strategy to suppress epilepsy.

[4] 132w Numerous studies reported that oxidative stress plays a significant role in the pathogenesis and progression of epilepsy in various kindling models (Etemad et al., 2019;Khodayar et al., 2019). The evidence from experimental studies reveals that oxidative stress results in depletion of antioxidants (GSH, GST, SOD, CAT) while increased MDA and NO levels which leads to alterations in the structure of lipids membrane, cellular proteins, and DNA (Aguiar et al., 2012;Liu et al., 2019). Heme oxygenase-1 (HO-1) is an important antioxidant system that exerts protection against oxidative stress, inflammation, and apoptosis (Han et al., 2019). Moreover, the HO-1 pathway has been recently shown to exhibit significant neuroprotection in epilepsy by counteracting oxidative stress (Chen, 2014). It has been speculated that HO-1 might be a new therapeutic target for neuroprotection (Jazwa and Cuadrado, 2010).

[5] 142w Natural products are a better alternative for the management of epileptogenesis due to their multi-targeted nature (Sharma et al., 2018). Daidzin is an isoflavone extracted from Pueraria lobata (Fabaceae) (Yang et al., 2020). The structure of daidzin is given in (Fig. 1A). Daidzin exhibits several pharmacological activities including anti-cancer, cholesterol-lowering effect, anti-inflammatory, anti-oxidant, cardio-protective, and neuroprotective effects (Leggio et al., 2010;Soumyakrishnan et al., 2014). The antiepileptic potential daidzin has not been reported yet. Different kindling models have been established to screen potential anti-convulsant drugs. PTZ-kindling model has been used to evaluate the antiepileptic potential of drugs (Tekgul et al., 2019). Therefore, in the current study, the anti-epileptic J o u r n a l P r e -p r o o f potential of daidzin was evaluated against the model of PTZ-induced epilepsy via regulation of the oxidative stress and BDNF/VEGF signaling.

[1] 12w Vehicle control (was injected 0.9% saline and 2% DMSO, via intraperitoneal route)

[2] 43w (II) Negative control (was administered PTZ, 35 mg/kg, via intraperitoneal route) (III) Positive control (diazepam (4mg/kg) was administered 30 min before PTZ, via intraperitoneal route) (IV) Treatment group (optimized dose of daidzin (10 mg/kg) was injected 30 min before PTZ, via intraperitoneal route)

[3] 98w Daidzin was dissolved in 0.9 % normal saline and 2% DMSO. Diazepam and PTZ were also dissolved in pyrogenfree 0.9 % saline. All the experimental mice injected with sub-chronic dose 35 mg/ kg of PTZ by intraperitoneal route, on alternate days for 21 days excluding the mice of the vehicle control group. The experiment design of this study was according to the established epileptic model with minor amendments (Tawfik et al., 2018). Mice were pretreated with daidzin and diazepam 30 min before induction on alternate days for 21 days. The study plan is shown diagrammatically in (Fig. 1B).

[4] 49w Initially, an acute study was performed for dose optimization. Three different doses of daidzin were used (1 mg/kg, 5mg/kg, and 10 mg/kg). There was a remarkable reduction in epileptogenesis in a dose-dependent manner. Daidzin 10mg/kg dose was selected with maximum efficacy for the chronic study of epilepsy (Fig. 2A).

[5] 84w There were decreased levels of GST, GSH, catalase, and SOD observed in the PFC, hippocampus, and thalamus of PTZ-kindled mice compared to the vehicle control group. The difference was statistically significant. Daidzin and diazepam treatment have reversed this suppression of antioxidant enzymes (Figure . 3). Similarly, the MDA level was also measured to support the additional antioxidant potential of daidzin. Kindling with PTZ remarkably (p < 0.05) increased the MDA level in the PFC, hippocampus, and thalamus as compared to the vehicle control group.

[6] 68w Pretreatment with daidzin and diazepam significantly (p < 0.05) reduced the MDA level compared with PTZkindled group (p < 0.001). Moreover, NO (marker of nitrative stress) production was also measured in the PFC, hippocampus, and thalamus. Kindling with PTZ significantly (p < 0.05) increased the production of NO. Daidzin pretreatment remarkably (p < 0.05) inhibited NO production. Similarly, results were shown by diazepam as shown in (Fig. 3).

[7] 51w To explore further, HO-1 (endogenous antioxidant enzymes) was measured in the brain section particularly the hippocampus. Immunohistochemical analysis revealed that the PTZ-kindled group remarkably reduced hippocampal HO-1 expressions compared to the vehicle control group. However, pretreatment with daidzin strikingly amplified the HO-1 expressions in PTZ-insulted mice as shown in (Fig. 4).

[8] 16w VEGF, the key mediator for angiogenesis and BBB permeability, was determined in the current kindled model.

[9] 40w Immunohistochemistry of the hippocampus section demonstrated that the PTZ-kindled group significantly amplified the VEGF expressions in the hippocampus compared to the vehicle control group. However, pretreatment with daidzin reduced the VEGF expressions in PTZ-insulted mice as shown in (Fig. 4).

[10] 49w To investigate further, Evans blue assay was performed to evaluate BBB permeability. PTZ-kindled mice showed remarkably (p <0.001) increased the concentration of Evans blue as compared to the vehicle control group. The pretreatment of daidzin in PTZ-kindled mice reduces the concentration of Evans blue in brain tissue (Fig. 5).

[11] 61w Protein misfolding and aggregations were investigated in the mouse brain by FTIR spectroscopy. Results demonstrated that the vehicle control group exhibit normal protein structure. Marked protein structural changes and increased protein aggregation were observed in the brain of the PTZ-kindled group. Pretreatment with Daidzin for 21 days preserved the PTZ induced morphological alterations of proteins and inhibit the aggregation of proteins.

[12] 9w Similarly, diazepam significantly reduced protein structural deformities (Fig. 8).

[13] 124w Similarly, DSC was also used to investigate the effects of epileptic seizures on the denaturation of proteins. In DSC, a thermogram of a brain supernatant of mice was taken. In the first heating scan, there were many peaks observed in the region of 65-110 0 C which represents a complex exothermic-endothermic denaturation transition. These events were permanent and did not reappear in the next heating scans. It demonstrates the thermal properties of the denatured samples which provide a sufficient measure of the degree of the protein damage taking place in the 1 st cycle of heat. There was significant protein damage in the brain of PTZ-kindled mice. However, daidzin and diazepam pretreatment in PTZ-kindled mice brain preserved the secondary structure of proteins (Fig. 8).

[14] 119w Uptake of daidzin into the brain tissue was evaluated by HPLC-UV-spectrophotometry. The basic protocol requires 60 min of running time for HPLC analysis of daidzin and aglycone daidzein. The chromatogram of the daidzin standard and brain samples of daidzin treated group were presented in (Fig. 10). The peak which was observed in the chromatogram of the daidzin treated mice brain sample was similar to the peak of pure daidzin compound. They both have similar retention time and absorption areas at a wavelength of 260 nm as shown in (Table -2). There was no peak of aglycone daidzein in the brain sample chromatogram. Therefore, the anticonvulsant effect of the compound is due to its direct entry into the brain tissue.

[15] 105w To understand the mechanism of interaction between daidzin and protein targets, a molecular docking study was performed. Daidzin showed significant interaction with various protein targets such as ALDH2, estrogen receptorβ, P13k, AKT2, mTORC1, and HIF-1-α. To elucidate the interaction of daidzin with these proteins at the active site, the binding affinities were calculated. The binding energies of the daidzin with various proteins targets were -8.5 Kcal/mol for 2vle, -8.8 Kcal/mol for 2qtu, -9.3 Kcal/mol for 5xgj, -8.5 Kcal/mol for 3d0e, -8.1 Kcal/mol for J o u r n a l P r e -p r o o f 5gs9, and -8.9 Kcal/mol for 1h2n (

[16] 59w Table.2). Binding interactions of daidzin and protein targets exhibited hydrogenbonding interactions at Lys 352 and Lys 192 for 2vle, at Arg 346, Asp 349, and His 394 for 2qtu, at Leu 1006 and Gly 1007 for 5xgj, at Lys 191, Thr 197 and Asp 293 for 3d0e, at Gln 181, Asn 182, Arg 183, Val 204 and Glu 046

[17] 29w for 5gs9, at Thr 149, Ser 184, and Gln 239 for 1h2n (Table . 3). The two-dimensional (2D) and three-dimensional (3D) images were presented in (Fig. 11, 12) respectively.

[1] 121w Epilepsy is one of the common neurological disorders which is characterized by a sudden onset of epileptic seizures (Owoalade et al., 2019). Epileptic seizures may result in unusual sensations, behavior, and sometimes, muscle spasms, loss of consciousness (Saleh, 2019). In this experimental study, the neuroprotective potential of daidzin was evaluated against the PTZ-induced mice model. Results of the current experimental data reveal that daidzin has robust protective potential in PTZ-kindled mice as evident from its preventive effects on biochemical, behavioral, and histological parameters. Importantly, daidzin treatment suppressed neuronal loss through the regulation of oxidative stress, neurotrophins, and neuronal apoptosis. Thus, we conclude that daidzin exhibits anticonvulsant effects through modulation of the oxidative stress, BDNF/VEGF signaling, and apoptosis pathway in PTZ-kindled mice.

[2] 148w The current research outcomes revealed that there was a remarkable increase in seizure score in the PTZ-kindled group. Pretreatment with daidzin significantly decreased the incidence of PTZ-induced seizures in a dose-dependent manner. Therefore, daidzin could be effective in improving PTZ-induced epilepsy and associated symptoms. In addition, the histopathological analysis demonstrated that there was damage in the granular cell layer of the hippocampus in the PTZ-treated mice which eventually reduced the thickness of the dentate gyrus of the hippocampus. However, daidzin pretreatment preserved the normal morphology of the hippocampus and its granular layer was also intact likewise positive control. Findings of the histopathological analysis reinforced the anti-epileptic potential of daidzin in the reduction of PTZ-induced damage to brain tissue. Our results were in accordance with previous reports that the administration of PTZ induced seizures and cause neurodegeneration in hippocampal neurons of rodents (Anissian et al., 2018;Singh et al., 2019).

[3] 271w Oxidative stress is a key factor in the pathophysiology of epilepsy and signifies an intrinsic risk of aggravating neurodegeneration (E. Mansour and Ibrahim, 2015). Previous studies investigated that seizures induced in the PTZkindling model are associated with pathological changes in the brain (hippocampus region) (Tawfik et al., 2018; J o u r n a l P r e -p r o o f Zhu et al., 2017). These changes are accompanied by amplified production of reactive oxygen species (ROS), diminished ROS scavenging capacity, and accumulation of NO centrally, thus generating status of oxidative and nitrative stress (Shin et al., 2011;Zhen et al., 2016). In oxidative stress, inactivation of enzymes, oxidation of lipids, and disruption of nucleic acids take place due to free radicals (Koskenkorva-Frank et al., 2013). Moreover, antioxidant such as heme oxygenase-1 (HO-1) pathway has been recently shown to exhibit significant neuroprotection in epilepsy by counteracting oxidative stress (Chen, 2014). The present study revealed PTZinduced kindling increased oxidative stress by depletion of antioxidants (GSH, GST, CAT, SOD, and HO-1) and increasing MDA and NO level in the mice brain and thus resulted in neuronal damage. Daidzin diminished the oxidative stress by elevates antioxidants levels and decreased MDA and NO level in PTZ-kindled mice. It is well accepted that PTZ-induced kindling provoked oxidative damage mainly in the hippocampus by depletion of antioxidants while increase lipid peroxidation (Ilhan et al., 2005). The present results were consistent with previous studies that also demonstrated that antiepileptic drugs contribute to neuroprotection against seizures by suppression of oxidative stress and thus mitigated hippocampal neuronal damage in PTZ-induced kindling model (Shi et al., 2018;Zhen et al., 2014).

[4] 82w Mounting evidence reported that BDNF plays a critical role in the survival of neuron including hippocampal and cortical neurons (Chen et al., 2017). Studies have been reported that epilepsy has been associated with the downregulation of BDNF in the hippocampus (Yu et al., 2019). In the present study, immunohistochemical analysis revealed that expression of BDNF is reduced in PTZ-treated mice, suggesting that BDNF has a protective role against seizure-mediated excitotoxicity. However, daidzin has preserved the expression of BDNF in the mice brain.

[5] 125w The present results were in accordance with a previous study that revealed that antiepileptic drugs contribute to neuroprotection by increasing the BDNF expression in the hippocampus (Zhen et al., 2016). In addition to BDNF, dysregulation of vascular endothelial growth factor (VEGF) plays a crucial role in the pathogenesis of epilepsy (Tawfik et al., 2018). VEGF is one of the prominent mediators of angiogenesis and also important in promoting BBB permeability (Chi et al., 2004). Several lines of evidence indicated that VEGF expression is up-regulated in PTZ-induced epilepsy which altered vascular permeability and promoting BBB disruption (Morin-Brureau et al., 2012). In the current study, immunohistochemistry revealed that there was significant up-regulation of VEGF expressions in PTZ-insulted mice. Whereas, pre-treatment with daidzin, downregulated the expression of VEGF.

[6] 103w These results were consistent with the previous studies that suggested that anticonvulsant drugs downregulated the expression of VEGF (Ogaki et al., 2020). In addition, Evans blue assay was also performed to evaluate BBB permeability (Harford-Wright et al., 2014). The data of the current study shows that BBB permeability was significantly enhanced in PTZ-treated mice. Whereas, pre-treatment with daidzin, significantly decreased BBB J o u r n a l P r e -p r o o f permeability in PTZ-insulted mice. Our results are corroborated by an earlier study which showed that daidzin reduced BBB permeability in the ischemic model (Liu et al., 2017).

[7] 32w It is well recognized hypoxia-inducible factor 1-alpha (HIF-1-α) is the major transcription factor that activates the downstream target gene (VEGF) and regulates VEGF expression in PTZ kindling model (Feast et al., 2012).

[8] 206w Numerous studies reported that HIF-1-α is responsible for the up-regulation of VEGF in the hippocampal neuron following seizures (Tawfik et al., 2018). Recently a study has been indicated that overexpression of HIF-1α induces hippocampal apoptosis in the epilepsy model (Wu et al., 2018). It is well established that HIF-1-α is regulated by mTOR signaling and activation of mTOR potently enhances the activity of HIF-1-α and VEGF (Land and Tee, 2007). Hyperactive PI3K/Akt/mTOR signaling has been well characterized in epilepsy. It is well recognized that compounds that inhibit mTOR complex 1 (mTORC1) decrease seizures and prevent epileptogenesis in animal models seizures (Nguyen et al., 2015;Ostendorf and Wong, 2015;Ryther and Wong, 2012). To investigate a direct interaction of daidzin with biological targets that regulate VEGF signaling, molecular docking of daidzin was carried out against protein targets such as P13k, AKT2, mTORC1, and HIF-1-α. The binding energies of the daidzin with mentioned protein targets were -9.3 Kcal/mol for 5xgj, -8.5 Kcal/mol for 3d0e, -8.1 Kcal/mol for 5gs9, and -8.9 Kcal/mol for 1h2n. The lower value reflected the more stable complex between the ligand and the target protein. Analysis of the binding interactions of daidzin revealed that the compound was efficiently docked inside the active site of the target structures.

[9] 176w Numerous studies reported that daidzin is a specific inhibitor of ALDH2 and a well-described phytoestrogen which modulates its biological effects by binding to estrogen receptor (ER) (Isoda et al., 2002;Kostelac et al., 2003;Lowe et al., 2008). Several lines of evidence indicated that phytoestrogen downregulated the expression of VEGF through inhibition of estrogen receptor-β (Sutrisno et al., 2018). Recently a study has been indicated that inhibition of ALDH2 downregulated the expressions of VEGF (Roy and Palaniyandi, 2020). These results specify that phytoestrogen regulates the VEGF expression through inhibition of estrogen receptor-β and ALDH2. In the present study, we investigated the interaction of ALDH2 and estrogen receptor-β with daidzin by performing molecular docking. The binding energies of the daidzin with the mentioned protein targets were -8.5 Kcal/mol for 2vle and -8.8 Kcal/mol for 2qtu. The analysis shows that daidzin bind to ALDH2 and estrogen receptor-β with high binding affinity. In this regard, it is concluded from the current study that daidzin inhibits VEGF signaling through modulation of biological targets such as ALDH2/estrogen receptor-β, P13k, AKT2, mTORC1, and HIF-1-α.

[10] 173w It has been reported recurrent seizures producing various changes which can lead to neuronal death (Singh et al., 2019). PTZ-induced kindling increasing DNA damage in the brain tissue (De Oliveira et al., 2008). The comet J o u r n a l P r e -p r o o f assay is useful for investigating the DNA damage in the neuron of the brain tissue (Khan et al., 2019). In the current study, increased DNA damage in the neurons of the brain tissue of PTZ-kindled mice was observed. However, daidzin pretreatment remarkably inhibited damage to DNA in the neurons of the hippocampus. The results of the current study are comparable with the previous studies that showed that antiepileptic drugs decreased DNA damage in the brain tissue of the PTZ-kindled mice (De Oliveira et al., 2008). Furthermore, it has been reported that seizureinduced neuronal apoptosis and was accompanied by down-regulation of BDNF in the hippocampi which recommend that inactivation of the BDNF signaling contributes to neuronal apoptosis in the seizures (Yu et al., 2019).

[11] 158w Protein aggregation and misfolding is a hallmark of many neurodegenerative diseases including epilepsy (Li et al., 2013). In the present study spectroscopic analysis of the protein damage was done to evaluate the neuroprotective potential of daidzin against PTZ-induced epilepsy by using FTIR and DSC analysis. The spectroscopic analysis demonstrated that there were remarkable changes in the secondary structure of the protein of the hippocampus of PTZ-treated mice which eventually prompts protein aggregation and misfolding. However, daidzin pretreatment remarkably preserved the secondary structure of proteins. Results of the current study elucidate that daidzin inhibits the protein damage in the PTZ-kindled mice brain. Results of the current study are comparable with previous studies which reported that PTZ induced epilepsy is associated with the changes in protein structure (Görgülü Türker, 2009;Turker et al., 2014). Overall results of the present study give significant evidence of the ameliorative potential of daidzin against PTZ-induced epilepsy by regulation of oxidative stress, BDNF/VEGF, and neuronal damage.

[1] 42w The current work provides considerable evidence that daidzin inhibited PTZ-induced epilepsy and associated clinical symptoms. The behavioral, biochemical, histological, analytical, in-silico, and molecular findings of this study supported the argument that daidzin has antioxidant and anti-epileptic properties in mice model of epilepsy.

[2] 38w The antiepileptic potential of daidzin was achieved via modulation oxidative stress, BDNF/VEGF signaling, and neuronal apoptosis in brain tissue (Fig. 13). Furthermore, more in-depth investigations are needed to elucidate the detailed molecular mechanism of daidzin in PTZ-induced seizures.

[3] 84w J o u r n a l P r e -p r o o f Figure 1. (A) Structure of daidzin (B) Schematic overview of the experimental study plan. Figure 1 Figure 2 J o u r n a l P r e -p r o o f Figure 3 J o u r n a l P r e -p r o o f Figure 5 Figure 6 J o u r n a l P r e -p r o o f

[1] 80w Daidzin was procured from Sigma-Aldrich (USA). Dimethyl sulfoxide (DMSO), sodium phosphate buffer, normal saline, potassium phosphate buffer, ethylenediaminetetraacetic acid (EDTA), thiobarbituric acid (TBA), formalin, 5,5′-dithio-bis-[2-nitrobenzoic acid] (DTNB), ascorbic acid, 1-chloro-2,4-dinitrobenzene (CDNB), GSH, diaminobenzidine substrate (DAB), hydrogen peroxide (H2O2), trichloroacetic acid (TCA), phenylmethylsulfonylfluoride (PMSF), 0.25M sucrose, per-chloric acid, nitric acid, and Tris-HCL were purchased from Sigma Aldrich (USA). Antibodies such as primary antibodies i.e., rabbit polyclonal (anti-HO-1, anti-BDNF, and anti-VEGF) and anti-rabbit secondary antibody were purchased from Santa Cruz Biotechnology (USA).

[2] 93w Epileptogenesis was evaluated by measuring seizures score and was graded from (1-6) according to previously established protocols (Fischer and Kittner, 1998). Epileptogenesis was induced with a sub-chronic dose of PTZ which is 35 mg/kg. PTZ doses were administered in mice via an intraperitoneal route on every alternative day (overall 11 injections in 21 days) (Inan and Büyükafşar, 2008). Immediately, afterward, each induction with PTZ, animals in all groups was examined for the next 30 min for seizure scoring. Mice were blindly evaluated by the observer for seizures as mentioned in (Table . 1).

[3] 51w All experimental animals were sacrificed by euthanization with CO2 on the 21 st day of the experiment. Brains were dissected from all groups and chill saline (0.9%) was used for washing. The weight of each mouse's brain was measured. For histopathological observation, all brain tissues were preserved in phosphate-buffer with 10%

[4] 38w J o u r n a l P r e -p r o o f formalin. Specimens of blood were collected and centrifuged at 2500 rpm and for biochemical analysis, serum specimens were separated (Khan et al., 2014).

[5] 54w GSH levels in the hippocampus, PFC, and thalamus were evaluated according to a previously reported method (Zeeshan et al., 2019). In this assay, a yellow chromophore was generated by the conjugation reaction between free thiol groups of GSH and DTNB. The intensity of this chromophore was measured at 412 nm by a UV-visible spectrophotometer.

[6] 44w GST levels in the hippocampus, PFC, and thalamus were investigated by using an established protocol (Atiq et al., 2019;Khalid et al., 2019). In this assay, the conjugation reaction has occurred between GSH and CDNB. The OD was recorded at 340nm by a UV-visible spectrophotometer.

[7] 40w Catalase activity in the hippocampus, PFC, and thalamus was analyzed by using a reported standard protocol (Ullah et al., 2018). In this assay, H2O2 is decomposed by catalase. OD was recorded in triplicates at 240nm by using a UV-visible spectrophotometer.

[8] 30w SOD level was determined in the hippocampus, PFC, and thalamus by using a reported standard protocol (Ali et al., 2019). The difference in OD was recorded in a UV-visible spectrophotometer.

[9] 45w Oxidative damage to lipids was evaluated in the hippocampus, PFC, and thalamus by measuring the MDA level with few modifications in the previously reported protocol (Atiq et al., 2019). The absorbance was taken at 535 nm to determine the amount of thiobarbituric acid reactive substances.

[10] 106w The molecular docking interaction was performed to determine the binding affinity between daidzin with various protein targets such as aldehyde dehydrogenase-2 (ALDH2) (PDB ID: 2vle), Estrogen receptor-β (PDB ID: 2qtu), Phosphatidylinositol-3-kinase (P13k) (PDB ID: 5xgj), AKT2 (PDB ID: 3d0e), mammalian target of rapamycin complex 1 (mTORC1) (PDB ID: 5gs9), and hypoxia-inducible factor 1-alpha (HIF-1-α) (PDB ID: 1h2n). These proteins were downloaded from the protein data bank RCSB and were saved as the PDB format. The Autodockvina was used to assess the docking interaction of daidzin with these proteins. The results obtained were expressed in the form of the number of hydrogen bonds and binding energy (Kcal/mol).

[11] 74w Results were shown as the means (n=10) ± standard deviations (S.D) in triplicate experiments. One-way analysis of variance (ANOVA) was used for statistical analysis of data. Statistical significance between the groups was J o u r n a l P r e -p r o o f determined by using a Dunnett's t-test. A value of p < 0.05 was considered statistically significant. All the results were analyzed by using Sigma plot version 12.5.

[1] 34w The animal experimentations were performed on male mice (albino, BALB/c) weighing (30-35 g) and having the age of 8 weeks. Male BALB/c mice were acquired from the National Institute of Health (NIH), Islamabad, Pakistan.

[2] 84w All mice were acclimatized under standard laboratory conditions of humidity (55 ± 5 %), temperature (22 ± 1ºC), and a light/dark cycle of 12 h was provided with access to food and water as much as required. The animal experimentations were performed according to protocols of National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All protocols of experiments were approved by the bioethical committee of the Quaid-i-Azam University, Islamabad, Pakistan. The (BEC-FBS-QAU2018-124) was the animal ethical approval protocol number.

[3] 16w In this experiment, mice were randomly distributed into four different Groups. Each group contains 10 mice.

[4] 12w Vehicle control (was injected 0.9% saline and 2% DMSO via intraperitoneal route)

[5] 21w Negative control (was administered with PTZ, 90 mg/kg, via intraperitoneal route) The dose with maximum efficacy was chosen for further experiments.

[6] 20w Before starting the chronic model of epilepsy, animals were again randomly distributed into four groups, each group contains 10 mice.

[7] 18w Antioxidant markers such as GST, GSH, SOD, and Catalase levels were evaluated in different parts of the brain.

[8] 13w Similarly, the levels of NO and MDA were also analyzed in brain tissue.

[9] 43w NO level in brain parts was evaluated by following previously established protocols (Khalid et al., 2018;Rasheed et al., 2018). Griess reaction was used for the determination of NO concentrations in brain tissue. OD was recorded at 560 nm in a UV-VIS microplate reader.

[10] 121w Brain tissues were separated for evaluation of histopathological changes. Isolated samples of the brain cut and fixed instantly in a 10% solution of formalin for a period of 24 h. dissected tissues were subjected to dehydration and followed by fixation of the tissue with paraffin. After this, brain samples cut at a thickness of 5μm. Furthermore, hematoxylin and eosin dye were used for the staining of these sections. The histopathological alterations between the different groups were analyzed under a light microscope at a magnification of (20X). To avoid experimenter bias during all phases of image collection and analysis, the experimenter was blinded to the treatment groups. Image-J software (NIH, USA) was used for the interpretation of images (Khan et al., 2019).

[11] 30w Immunohistochemistry of the VEGF, BDNF, and HO-1 was performed using the avidin-biotin-peroxidase complex (ABC) method (Khan et al., 2019). For IHC examinations, 5μm thick brain sections were deparaffinized in xylene.

[12] 112w Briefly, the paraffin-embedded 5μm thick brain sections were treated with xylene and alcohol. Then, antigens were retrieved by the enzymatic method and then treated with PBS. Then, brain sections were treated by 3% H2O2 in menthol to inactivate endogenous peroxidase. Following washing with phosphate buffer saline (PBS) the brain sections were treated with normal goat serum (NGS), primary antibodies i.e., rabbit polyclonal (anti-HO-1 anti-BDNF/VEGF) (Santa Cruz Biotechnology, USA) and anti-rabbit secondary antibody (Santa Cruz Biotechnology, USA). Each marker expression was labeled with peroxidase and colored with DAB for the detection of the antigenantibody complex. Image-J software (NIH, USA) was used for the quantification of relative protein expression of VEGF, BDNF, and HO-1.

[13] 85w BBB Permeability was evaluated by using Evans blue (EB) according to a previously established protocol (Harford-Wright et al., 2014). The concentration of EB dye in different parts of the brain tissue including the hippocampus, J o u r n a l P r e -p r o o f PFC, and thalamus were calculated to quantify the disruption in the BBB and its permeability. The OD of each sample was recorded at 620 nm. The amount of EB extravasation in brain tissue expressed as µg/ml.

[14] 35w DNA damage in the different parts of the brain tissues was measured by COMET assay according to a previously reported method (Shal et al., 2019). CASP 1.2.3.b software was used for quantification of DNA damage.

[15] 53w FTIR and DSC spectroscopy is an advanced analytical technique which gives structural information about protein changes associated with epileptic seizure in mice brain (Dogan et al., 2007). The established protocol was followed for the estimation of protein damage by FTIR and DSC analysis in brain tissue (Tenchov et al., 2017;Turker et al., 2014).

[16] 47w HPLC-UV spectrophotometry is an advanced analytical technique that helps in the detection of compounds that are present in any sample (Iammarino et al., 2019). The established method was followed for the detection of daidzin and its metabolite (aglycone daidzein) in the brain samples (Huang and Xia, 2019).

[17] 69w The vehicle control group exhibited no epileptogenesis. The PTZ-kindled group represents the highest seizure score and symptoms worsen in severity after 5 doses of PTZ (on 11 th day of the experiment) The score of seizures in the daidzin treated mice was significantly less pronounced than the PTZ-kindled group (p < 0.05). In diazepam treated mice the seizure scores were comparable to the daidzin (10mg/kg) treatment group (Fig. 2B).

[18] 31w J o u r n a l P r e -p r o o f BDNF, a critical neurotrophic factor for neuronal survival and was evaluated in the current kindled model.

[19] 38w Immunohistochemistry of the hippocampus section revealed that the PTZ-kindled group remarkably reduced BDNF expressions in the hippocampus compared to the vehicle control group. However, pretreatment with daidzin amplified BDNF expressions in PTZ-insulted mice as shown in (Fig. 4).

[20] 76w The vehicle control group exhibited no histopathological deformities in DG of the hippocampus and the granular cell layer of the DG was intact. In PTZ-kindled group marked histopathological changes were detected in the hippocampus and the width of the granular layer of DG was significantly decreased. Daidzin pretreatment in PTZkindled animals significantly (p < 0.05) preserved histopathological variations. Similarly, diazepam significantly reduced the histopathological deformities (p <0.001) in the granular cell layer of DG (Fig. 6).

[21] 71w DNA damage in the neurons of the PFC, hippocampus, and thalamus was investigated by comet assay. PTZ-kindled mice showed a significant increase in the fragmentation of DNA by an increase in the tail length and high percent (%) of DNA in the tail. Pretreatment with daidzin considerably averts (p <0.001) fragmentation of DNA by inhibiting % DNA in the tail and tail length as compared to the PTZ-kindled group (Fig. 7).