Introduction

Epilepsy affects ≈ 50 million people worldwide and is characterized by recurrent, unprovoked seizures that arise when the delicate equilibrium between excitatory and inhibitory neurotransmission is disrupted1. Over the past decade, convergent evidence from epidemiological surveys, clinical trials, and rodent studies has revealed that dietary phytochemicals, particularly polyphenolic flavonoids, can modulate neuronal excitability by coordinating redox homeostasis, neuro-immune signaling and neurotrophin pathways2. In a recent study, flavanone-rich Citrus reticulata peel extract and the ω−9 fatty acid erucic acid have each curtailed pentylenetetrazole- (PTZ-) induced seizures in mice while simultaneously restoring antioxidant defenses and brain-derived neurotrophic factor (BDNF) signaling3. These observations highlight the therapeutic potential of food-borne bioactives to complement, or even surpass, conventional antiepileptic drugs (AEDs) that primarily target ion channels but leave oxidative–inflammatory dysregulation unchecked.

PTZ is a widely used GABAA​ receptor antagonist that recapitulates key molecular features of epileptogenesis, including surges in reactive oxygen/nitrogen species (ROS/RNS), up-regulation of pro-inflammatory cytokines [Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)] and activation of the intrinsic apoptotic cascade via caspase-34. These processes feed into a vicious cycle of mitochondrial dysfunction, neurodegeneration and heightened seizure susceptibility. An ideal neuro-nutraceutical would therefore (i) restore the oxidant-antioxidant balance, (ii) dampen cytokine-driven neuroinflammation, (iii) preserve neurotrophic support and (iv) rebalance neurotransmitter tone5,6.

Sakuranetin (4′-methoxy-naringenin) is an O-methylated flavanone naturally concentrated in edible Prunus species cherry blossoms, plums and almonds as well as in honey and propolis7. Pre-clinical studies have attributed to sakuranetin’s potent antioxidant, anti-inflammatory, and antiviral activities, yet its impact on central nervous system disorders remains unexplored, mainly8. Its low molecular mass (286 Da) and moderate lipophilicity (cLogP ≈ 3.1) satisfy accepted blood-brain-barrier permeability criteria, suggesting that the molecule can reach neuronal targets in vivo. Importantly, the average sakuranetin intake from traditional Japanese cherry-leaf confections has been estimated at 1–3 mg/day in adults; thus, the compound is already part of the human diet9.

Building on the nutritional-neuroscience paradigm, the present study combines in vivo pharmacology with in silico molecular modelling to test the hypothesis that sakuranetin exerts multi-target anticonvulsant effects in the repeated-PTZ mouse model. We (i) quantify alterations in major neurotransmitters, antioxidant enzymes, lipid peroxidation and nitric-oxide load; (ii) profile cytokines alongside BDNF and its high-affinity receptor tropomyosin receptor kinase B (TrkB); (iii) assess apoptosis via caspase-3; and (iv) use molecular docking plus 100-ns molecular-dynamics simulations to validate sakuranetin’s binding affinity and stability at BDNF, TrkB and dopamine D2_22​-like receptor sites—targets previously linked to flavonoid-mediated neuroprotection10,11. By integrating nutritional biochemistry, behavioural neuropharmacology and computational chemistry, this work aims to position sakuranetin as a promising functional-food-derived neuro-nutraceutical for the dietary management of epilepsy (Fig. 1).​.

Fig. 1
figure 1

Experimental Design.

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Methodology

Animals

Adult Swiss albino mice (6–7 weeks; 22 ± 2 g) were obtained from the Central Animal Facility, University of Ha’il. Animals were group-housed (4 per cage) in polypropylene cages with corn-cob bedding under controlled conditions (22 ± 2 °C; 55 ± 5% RH; 12 h light/dark, lights on 06:00 h). Standard rodent chow and water were provided ad libitum. All procedures conformed to the National Committee of Bio-Ethics (Saudi Arabia) and the EU Directive 2010/63/EU and were approved by the University of Ha’il Research Ethics Committee (H-2025-583; 21 Jan 2025).

Chemicals

PTZ and sakuranetin (≥ 98.0%) were obtained from Sigma-Aldrich (St. Louis, USA) and used in analytical grade. ELISA kits were employed to quantify neuroinflammatory and apoptotic markers. Interleukin-1 beta (IL-1β, RAB0572), interleukin-6 (IL-6, RAB0309), and caspase-3 (CASP3C) kits were procured from Sigma-Aldrich (St. Louis, USA). Brain-derived neurotrophic factor (BDNF, MBS3805629) and TrkB (MBS355242) kits were obtained from MyBioSource, USA.

Acute toxicity studies and prediction of ADMET by computational analysis

In line with the OECD ANNEX-423 guidelines, sakuranetin underwent a thorough evaluation of its acute oral toxicity. The results showed that orally administered sakuranetin to mice at a dosage of 10 and 20 mg/kg did not induce any observable signs of toxicity or mortality, indicating a favorable safety profile at this dose12.The pharmacokinetic properties of sakuranetin, including absorption, distribution, metabolism, excretion, and toxicity (ADMET), were evaluated using pkCSM ADMET descriptors13.

Experimental design

The animals were randomized and categorized into the following groups (n = 6).

\({\rm Group}\left\{ \begin{aligned}&{\rm I - Control (saline)} \\&{\rm II - PTZcontrol ({35 mg/kg, i.p.})} \\& {\rm III - PTZ+10 mg/kg, p.o. sakuranetin} \\& {\rm IV - PTZ+20 mg/kg, p.o. sakuranetin}\\ \end{aligned} \right.\)

Swiss albino mice (n = 6 per group) were randomly assigned to four groups: Group I (Control, saline), Group II (PTZ control, 35 mg/kg, i.p.), Group III (PTZ + sakuranetin 10 mg/kg, p.o.), and Group IV (PTZ + sakuranetin 20 mg/kg, p.o.)14,15,16. PTZ was administered intraperitoneally on alternate days for 28 days to induce seizures. Sakuranetin was administered orally 30 min prior to each PTZ injection. Seizure severity was evaluated according to a modified Racine’s scale: stage 0, no response; stage 1, facial and ear twitching; stage 2, myoclonic jerks; stage 3, clonic convulsions of forelimbs; stage 4, rearing with forelimb clonus; and stage 5, generalized tonic–clonic seizures with loss of posture17. Seizure behavior was assessed 30 min following each PTZ administration. Time-course parameters, including latency to seizure onset, seizure severity scores, and mortality rate, were evaluated on day 1, 7, 14, and 28 to monitor the progression and cumulative effects of repeated PTZ exposure. After the seizure test, biochemical parameters were performed.

PTZ-induced seizures test

Following treatment administration, subjects were individually housed and observed for 30 min. The parameters recorded included latency to first convulsion, seizure score, and survival rate. All observations were conducted by an experimenter blinded to treatment groups. The control group received a physiological saline solution instead of sakuranetin18,19.

Rotarod test

The ataxic behavior of mice was assessed using the accelerating rotarod test, following previously described protocols20. Mice were placed with all four limbs on a rotating rod (6 cm diameter) set at a constant speed of 12 rpm, positioned 25 cm above the surface. After 30 min of dosing, motor coordination was evaluated by placing each mouse on the rod for 1 min. The percentage of animals exhibiting motor impairment was subsequently calculated.

Homogenization of brain tissue

Following the seizure assessment, animals were anesthetized using intraperitoneal administration of ketamine (75 mg/kg) and xylazine (10 mg/kg) and sacrificed by cervical dislocation. The brains were rapidly removed, and the hippocampal region was dissected on an ice-cold surface. Tissue samples were homogenized in ice-cold phosphate-buffered saline (PBS, pH 7.4) and centrifuged at 12,000 × g for 10 min at 4 °C. The resulting supernatants were collected and stored at − 80 °C until used for enzymatic and cytokine assays.

Determination of neurotransmitters

Neurotransmitters, including dopamine, serotonin, GABA, acetylcholine, and norepinephrine, were analysed using an isocratic reverse-phase HPLC system (Waters, USA) equipped with a photodiode array (PDA) detector. Separation was achieved on a Sunfire C18 column (250 mm × 4.6 mm, 5 μm particle size). The mobile phase comprised 20 mM ammonium acetate buffer (pH 4.6, adjusted with formic acid), methanol, and acetonitrile in a ratio of 70:20:10 (v/v/v). The flow rate was maintained at 0.6 mL/min, and the total runtime was 15 min. A 20 µL volume of each sample was injected. Detection was carried out at + 320 mV, and identification was based on comparison of retention times with known standards prepared in 0.02 N perchloric acid. Quantification was performed using Empower 3 software (Waters, USA), and results were expressed as mean ± SEM for each group21,22.

Determination of antioxidant enzymes

Several antioxidant enzymes were measured with a modification of the established methodology23,24,25.

Superoxide dismutase

Superoxide dismutase (SOD) activity in hippocampal tissue was estimated using the method of Misra and Fridovich (1972). This method is based on the ability of SOD to inhibit the autoxidation of epinephrine to adrenochrome at alkaline pH. In brief, the assay mixture contained 0.05 M sodium carbonate buffer (pH 10.2), 0.1 mM EDTA, and 10 µL of tissue homogenate. The reaction was initiated by adding freshly prepared 30 µM epinephrine, and the increase in absorbance was measured at 480 nm for 4 min. One unit of SOD activity was defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50%. Results were expressed as units per milligram of protein23. The method was modified by adjusting the reaction volume for microcuvettes and substituting erythrocyte lysates with brain homogenates.

Glutathione

Glutathione (GSH) levels were measured using the method developed by Ellman (1959), based on the formation of a yellow-colored complex with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). Briefly, 100 µL of 10% brain tissue homogenate was mixed with 100 µL of 5% trichloroacetic acid (TCA) and centrifuged at 10,000 × g for 10 min. The supernatant was added to 2 mL of 0.3 M phosphate buffer (pH 8.4) and 0.5 mL of 0.04% DTNB. After 10 min of incubation, the absorbance was recorded at 412 nm. GSH concentrations were expressed as µmol of GSH per g protein24. The original method was adapted using brain homogenates and scaling the reaction volume to 3 mL.

Catalase

Catalase activity (CAT) was determined according to the method of Aebi (1984), which measures the decomposition rate of hydrogen peroxide (H₂O₂). The assay mixture consisted of 1.95 mL of 50 mM phosphate buffer (pH 7.0), 1 mL of freshly prepared 30 mM H₂O₂, and 50 µL of brain tissue homogenate. The decrease in absorbance was recorded at 240 nm for 1 min at room temperature. Catalase activity was calculated using the molar extinction coefficient for H₂O₂ (43.6 M⁻¹ cm⁻¹) and expressed as units per milligram of protein25.

Determination of oxidative and nitrative stress markers

The concentration of MDA, a biomarker of oxidative stress, was determined spectrophotometrically. Tissue homogenate was reacted with trichloroacetic acid, and the supernatant was subsequently reacted with thiobarbituric acid. Following incubation at elevated temperatures, the absorbance was measured at 535 nm26,27,28. Nitric oxide (NO), a marker of nitrative stress, was quantified by spectrophotometry based on the nitrate reductase assay29,30,31.

Estimation of neuroinflammatory markers

The concentrations of pro-inflammatory cytokines were determined via ELISA following the manufacturer’s protocol. ELISA, a microplate-based assay, facilitates the detection and quantification of target analytes. In this application, the cytokines served as the target antigens. Pre-coated antibodies specific to each cytokine were employed to capture the respective antigens from the samples. Cytokine concentrations (IL-6, IL-1β and TNF-α) were quantified and expressed as picograms per millilitre (pg/mL).

Estimation of BDNF and TrkB

The levels of BDNF and its corresponding receptor, TrkB, were assessed by employing a reliable ELISA kit per the manufacturer’s prescribed guidelines. The concentration of BDNF was quantified in picograms per millilitre (pg/mL).

Determination of caspase-3

Caspase-3 levels, a marker of apoptosis, were quantitatively assessed via ELISA following the manufacturer’s instructions. The concentration of caspase-3 was calculated in nanograms per milliliter (ng/mL).

Molecular docking

Ligand preparation involved retrieving the 3D conformer of sakuranetin from PubChem and optimizing its structure using MarvinSketch (ChemAxon, Version 22.13). This process included adding hydrogens and refining the structures in 2D and 3D formats, generating multiple conformers to select the lowest energy configuration for further analysis. The DockPrep module in Chimera version 1.17.1 (build 42449) was utilized to process the Mol2 files of the optimized 3D structures, employing default parameters such as protonation states with AM1-BCC during conjugate gradient optimization. For protein preparation, crystal structures of BDNF (PDB ID 1B8M), TRKB (PDB ID 4AT3), and DOPAMINE (PDB ID 6CM4) were sourced from the RCSB Protein Data Bank. The selected protein structures were validated by assessing resolution metrics and wwPDB scores, checking for missing residues in binding sites via PDBsum, and analyzing Ramachandran plot data. The raw PDB files were optimized in Chimera by removing extraneous residues and adding hydrogen atoms to prepare them for the AMBER force field and charge adjustments, subsequently exporting the refined structures to PDB format. AutoDock Tools version 1.5.6 was employed to convert the protein structures into PDBQT format. Molecular docking studies utilized AutoDock Tools 1.5.6, Chimera, and Maestro for grid generation and validation, with grid parameters derived from the orientation of co-crystal ligands or using the CASTp server for proteins in their apo state, maintaining a grid point spacing of 0.375 Å and specific centre dimensions. The grid parameters are detailed in Table 1. The comparison between standard values and retrieved protein from the RCSB Protein Bank for validation of proteins selected for the docking study is shown in Table 2. Also, the active sites of amino acids are shown in Table 3.

Table 1 Grid parameters.
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Table 2 Comparison between standard values and retrieved protein from RCSB protein bank for validation of proteins selected for the Docking study.
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Table 3 The active sites amino acids.
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Molecular dynamic simulation

MD simulations were conducted on the docked complexes of 1B8M and 6CM4 using Desmond 2020.1 (Schrödinger, LLC). Within the simulation framework, Sakurantin was designated as 1B8M_Sakurantin and 6CM4_Sakurantin. The system was parameterized using the Optimized Potentials for Liquid Simulations (OPLS-2005) force field, and an explicit solvent model incorporating TIP3P water molecules was employed within a periodic boundary solvation box of dimensions 10 × 10 × 10 Å. To mimic physiological conditions, NaCl solutions were introduced into the system, and Na⁺ ions were added to neutralize the 0.15 M charge32,33,34,35.

The biomolecular system containing protein-ligand complexes underwent comprehensive equilibration protocols to achieve optimal conformational stability. Initial thermal equilibration was executed for 10 nanoseconds utilizing an NVT (canonical) ensemble, maintaining constant particle number, volume, and temperature parameters. Subsequently, the system was subjected to a 12 ns equilibration and energy minimization phase under NPT (isothermal-isobaric) conditions, facilitating pressure and temperature regulation. Temperature and pressure coupling was implemented via the Nosé-Hoover chain algorithm, ensuring the maintenance of thermodynamic equilibrium throughout the ensemble36. The simulations were conducted under varying temperature conditions. The system’s relaxation time was set to 1.0 ps, and the target pressure was maintained at 1 bar. A 2 fs time step was implemented to ensure computational accuracy. For precise pressure regulation, a Martyna-Tuckerman-Klein chain coupling barostat with a 2 ps relaxation time was employed37. A 9 Å cutoff radius was implemented for the truncation of short-range Coulombic interactions. Long-range electrostatic interactions were treated using the Particle Mesh Ewald (PME) technique38. The final stage of the simulations, termed production run, was conducted for 100 nanoseconds. Critical parameters were computed systematically and analysed to ensure the accurate assessment of MD simulation stability.

Binding free energy analysis

The approach used in this research included calculating binding free energies for ligand-protein complexes through the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method with molecular mechanics. The evaluation was done by calculating the Prime MM-GBSA binding free energy via a structured process. A one-step sampling method was utilized, where data were gathered from the last 50 frames of the simulation path. The Python software thermal_mmgbsa.py was employed to calculate the prime MM-GBSA binding free energy by consolidating various energy factors, such as Coulombic, covalent, hydrogen bonding, Van Der Waals, self-contact, lipophilic, and solvation energies for the protein and ligand. Ultimately, the prime MM-GBSA binding free energy was obtained using the principle of additivity, following the equation:

$$\:{\varDelta\:G}_{bind}={\varDelta\:G}_{MM}+{\varDelta\:G}_{Solv}-{\varDelta\:G}_{SA}$$

Where,

  • ΔGbind represents the binding free energy,

  • ΔGMM denotes the energy difference between the ligand-protein complex and the sum of the total energies of the isolated protein along with the ligand.

  • ΔGSolv corresponds to the difference in generalized Born surface area (GSA) solvation energies between the ligand-receptor complex and the combined energies of the unbound receptor and ligand.

  • ΔGSA indicates the variation in surface area energies of the protein and ligand.

Statistical analysis

A statistical analysis was performed using GraphPad Prism software (version 8.0.2). All data are presented as the mean ± standard error of the mean (SEM). Results indicate a normal distribution for the biochemical parameters analyzed. According to the Shapiro-Wilk test, all variables yielded p-values greater than 0.05, which indicates a normal distribution. A one-way ANOVA was conducted for multiple comparisons, followed by Tukey’s post hoc test. For the seizure test, a two-way analysis of variance (ANOVA) test was followed by the Bonferroni post hoc test. Statistical significance was defined as a P-value below 0.05.

Result

Acute toxicity assessment

Sakuranetin exhibited a favorable safety profile in acute oral toxicity assessments conducted in rats. Throughout the 28-day observation period, no mortality, signs of illness, or observable clinical symptoms were recorded. The doses of 10 and 20 mg/kg selected for the present study were based on findings from this acute toxicity evaluation. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of sakuranetin are summarized in Table 4.

Table 4 Predicted molecule properties of sakuranetin.
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Effect of Sakuranetin on the PTZ-induced seizures test

The administration of pentylenetetrazol (PTZ) induced generalized tonic-clonic seizures, which were meticulously assessed through seizure testing (P < 0.001). Detailed data analysis from Fig. 2A–C revealed that a 28-day regimen of sakuranetin treatment significantly reduced seizure duration, which was systematically assessed at multiple time points (Day 1, 7, 14, and 28) following each PTZ injection. Furthermore, the sakuranetin-treated groups displayed a considerable extension in the latent period preceding the onset of seizures [F (3, 80) = 413.2; P < 0.0001] in comparison to the control mice that received only PTZ. The seizure score was high in the PTZ group, with notable improvements seen in the sakuranetin + PTZ group. Sakuranetin treatment consistently reduced seizure scores after each dose compared to the PTZ group [F (3, 280) = 1048; P < 0.0001]. Notably, there was a significant decrease in the mortality rate among the treated groups relative to the PTZ control group [F (3, 80) = 47.44; P < 0.0001]. These findings strongly suggest that sakuranetin exhibits potent anticonvulsant properties against PTZ-induced seizures.

Fig. 2
figure 2

Effect of sakuranetin on A) latency to convulsion, B) seizure score, C) animal survival, and D) latency to fall off the rod in the rotarod test in PTZ-induced mice. Seizure severity was evaluated according to a modified Racine’s scale: stage 0, no response; stage 1, facial and ear twitching; stage 2, myoclonic jerks; stage 3, clonic convulsions of forelimbs; stage 4, rearing with forelimb clonus; and stage 5, generalized tonic–clonic seizures with loss of posture. Values are expressed as mean ± SEM (n = 6). Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s post hoc test for panels A–C (#P < 0.001 PTZ vs. control; *P < 0.05, **P < 0.01, ***P < 0.001 vs. PTZ) and one-way ANOVA followed by Tukey’s post hoc test for panel D (#P < 0.001 PTZ vs. control).

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Effect of Sakuranetin on Rotarod test

Treatment with sakuranetin at doses of 10 and 20 mg/kg did not affect the rotarod performance in mice (P > 0.05), suggesting that these treatments did not produce ataxic effects (Fig. 2D).

Outcomes of neurotransmitters

Figure 3A-E illustrates the concentration of various neurotransmitters in mice with PTZ-induced epilepsy. Following PTZ administration, there is a reduction in the levels of several neurotransmitters, specifically Ach, GABA, DA, NE, and 5HT, compared to the control (P < 0.001). However, treatment with sakuranetin remarkably reversed these reductions in Ach [F (3, 20) = 11.20; P = 0.0002], GABA [F (3, 20) = 15.78; P < 0.0001], DA [F (3, 20) = 9.141; P < 0.0001], NE [F (3, 20) = 10.07; P = 0.0003], and 5HT [F (3, 20) = 10.84; P = 0.0002] levels in the PTZ-induced mice, indicating partial restoration.

Fig. 3
figure 3

Effect of sakuranetin on neurotransmitter levels in the brain: A) acetylcholine (ACh), B) γ-aminobutyric acid (GABA), C) dopamine (DA), D) norepinephrine (NE), and E) serotonin (5-HT) in PTZ-induced mice. Values are expressed as mean ± SEM (n = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (#P < 0.001 PTZ vs. control; *P < 0.05, **P < 0.01 vs. PTZ).

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Outcomes of antioxidant enzymes

Figure 4A-C illustrates the effects of PTZ-induced epilepsy and subsequent sakuranetin treatment on key antioxidant enzyme concentrations in mice. PTZ administration resulted in a significant reduction in SOD, GSH, and CAT levels compared to the control group, reflecting impaired antioxidant defence (P < 0.001). However, sakuranetin treatment at doses of 10 mg and 20 mg effectively counteracted this decline, enhance SOD [F (3, 20) = 13.03; P < 0.0001], GSH [F (3, 20) = 13.96; P < 0.0001], and CAT [F (3, 20) = 19.92; P < 0.0001] levels in PTZ-exposed mice. These results indicate that sakuranetin may offer protective effects against PTZ-induced oxidative stress by regulating the activity of essential antioxidant enzymes.

Fig. 4
figure 4

Effect of sakuranetin on antioxidant enzyme activities in the brain: A) superoxide dismutase (SOD), B) reduced glutathione (GSH), and C) catalase (CAT) in PTZ-induced mice. Values are expressed as mean ± SEM (n = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (#P < 0.001 PTZ vs. control; *P < 0.05, **P < 0.01 vs. PTZ).

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Outcomes of oxidative and nitrosative stress markers

Figure 5A-B illustrates the MDA and NO in mice exhibiting PTZ-induced epilepsy. PTZ administration significantly increased MDA and NO levels compared to control (P < 0.001). Sakuranetin treatment at 10 mg and 20 mg significantly reversed these reductions in MDA [F (3, 20) = 21.23; P < 0.0001] and NO [F (3, 20) = 26.42; P < 0.0001] levels as compared to PTZ-induced mice.

Fig. 5
figure 5

Effect of sakuranetin on oxidative stress markers in the brain: A) malondialdehyde (MDA) and B) nitric oxide (NO) levels in PTZ-induced mice. Values are expressed as mean ± SEM (n = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (#P < 0.001 PTZ vs. control; *P < 0.05, **P < 0.01, ***P < 0.001 vs. PTZ).

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Outcomes of neuroinflammatory markers

Figure 6A-C illustrates the concentration of various neuroinflammatory markers in mice with PTZ-induced epilepsy. In these mice, PTZ significantly elevated the levels of IL-6, IL-1β, and TNF-α of several neuroinflammatory markers, compared to the control (P < 0.001). However, treatment with sakuranetin significantly reduced these elevated cytokine levels IL-6 [F (3, 20) = 57.82; P < 0.0001], IL-1β [F (3, 20) = 48.66; P < 0.0001], and TNF-α [F (3, 20) = 38.05; P < 0.0001] levels in PTZ-induced mice.

Fig. 6
figure 6

Effect of sakuranetin on pro-inflammatory cytokines in the brain: A) interleukin-6 (IL-6), B) interleukin-1β (IL-1β), and C) tumor necrosis factor-alpha (TNF-α) in PTZ-induced mice using enzyme-linked immunosorbent assay (ELISA) kits. Values are expressed as mean ± SEM (n = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (#P < 0.001 PTZ vs. control; *P < 0.05, **P < 0.01 vs. PTZ).

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Outcomes of BDNF and TrkB

Figure 7A-B illustrates the concentrations of BDNF and TrkB in mice with PTZ-induced epilepsy. PTZ administration caused a reduction of both BDNF and TrkB levels compared to the control (P < 0.001). Sakuranetin treatment significantly attenuated the PTZ-induced reduction in BDNF [F (3, 20) = 11.24; P = 0.0002] and TrkB [F (3, 20) = 10.10; P = 0.0003], indicating partial restoration.

Fig. 7
figure 7

Effect of sakuranetin on neurotrophic factors in the brain: A) Brain-derived neurotrophic factor (BDNF) and B) tropomyosin receptor kinase B (TrkB) in PTZ-induced mice using enzyme-linked immunosorbent assay (ELISA) kits. Values are expressed as mean ± SEM (n = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (#P < 0.001 PTZ vs. control; *P < 0.05, **P < 0.01 vs. PTZ).

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Outcomes of caspase-3

Figure 8 illustrates the concentration of caspase-3, an apoptotic marker, in mice with PTZ-induced epilepsy. PTZ administration significantly (P < 0.001) increased caspase-3 expression, reflecting activation of apoptotic pathways associated with seizure-induced neuronal injury. Treatment with sakuranetin markedly reduced this PTZ-induced elevation in caspase-3 levels [F (3, 20) = 20.86; P < 0.0001], suggesting its neuroprotective effect involves attenuating neuronal apoptosis.

Fig. 8
figure 8

Effect of sakuranetin on Caspase-3 in PTZ-induced mice using enzyme-linked immunosorbent assay (ELISA) kits. Values are expressed as mean ± SEM (n = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (#P < 0.001 PTZ vs. control; **P < 0.01, ***P < 0.001 vs. PTZ).

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Molecular Docking studies

A Ramachandran plot was used to analyze the protein structure and conformation of 1B8M, 4AT3, and 6CM4, depicted in Fig. 9. The affinity of sakuranetin was studied against the protein BDNF (AB8M), TRKb (4AT3), and dopamine (6CM4). Figure 10 depicts the binding of sakuranetin to the active site with different intermolecular interactions. Among these 1B8M, sakuranetin had the most potent interaction (energy=−10.704 Kcal/Mol), followed by the favourable effect of 4AT3 sakuranetin (energy= −9.113 Kcal/Mol), and 6CM4 sakuranetin (energy= −9.179 Kcal/Mol). Interactions between protein & ligand were identified using the PLIP-server (Table 5).

Fig. 9
figure 9

BDNF (PDB ID 1B8M), TRKB (4AT3), and DOPAMINE (6CM4) were validated by resolution metrics, wwPDB scores, PDBsum for missing residues, and Ramachandran plots.

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Fig. 10
figure 10

Molecular docking 3D images of the sakuranetin with Proteins Caspase-3 1B8M, 4AT3, 6CM4 using LigPlot v1.4.5 Maestro V12.8 software.

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Table 5 Docking score and intermolecular interactions of monomethyl fumarate and Co crystal ligands with BDNF (PDB ID 1B8M), TRKB (PDB ID 4AT3), DOPAMINE (PDB ID 6CM4) from PLIP.
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Molecular dynamics simulation

To assess the stability and convergence of complexes for 1B8M and 6CM4 with Sakurantin, which are denoted as 1B8M_Sakurantin and 6CM4_Sakurantin, respectively, molecular dynamics and simulation (MD) investigations were conducted. A Root Mean Square Deviation (RMSD) analysis of the 1B8M_Sakurantin is shown in Fig. 11A. It reveals that RMSD values first rise during the first 5 ns before stabilising at about 3–4 Å. The complex experiences slight conformational changes before stabilising, as indicated by the reduced average RMSD of 3.68 Å. The observed variations maintain a reasonable level of stability and indicate some flexibility. The RMSD curve of 6CM4_Sakurantin, shown in Fig. 11B, shows noticeably higher RMSD values, stabilising between 6 and 8 Å with peaks rising over 10 Å. This implies that more significant conformational changes occur in the complex. More binding site flexibility or weaker interactions between Sakurantin and 6CM4 resulted in a less stable complex, as indicated by the higher RMSD (average up to 6.90 Å). 1B8M_Sakurantin is more stable than 6CM4_Sakurantin according to RMSD. The Root Mean Square Fluctuation (RMSF) analysis of 1B8M_Sakurantin is shown in Fig. 12A. It shows that most of the protein residues have low variations, with values primarily below 3 Å. The strong peaks are visible around residues 38, 161–168, and 219–223—where RMSF values exceed ⁓9 Å. These oscillations may represent the loop region. The RMSF plot for the 6CM4_Sakurantin complex, shown in Fig. 12B, shows more variations than 1B8M. A high degree of flexibility is suggested by the RMSF values, which continuously stay above 3 Å and have multiple peaks above 5 Å, especially in the 57–74, 95–152, 167–194, 218–231, 278–286, 294–322, 340–351, and 374–404 ranges. Compared to 6CM4_Sakurantin, the 1B8M_Sakurantin exhibits greater structural stability and fewer variations outside specific locations. In contrast to 6CM4_Sakurantin, the Radius of Gyration (Rg) study for the 1B8M_Sakurantin shows a more stable structural behaviour (Fig. 13A). The Rg values, with sporadic spikes, mostly stay between 19.67 Å and 23.44 Å. While these oscillations indicate conformational alterations, substantial structural destabilisation is not suggested. Figure 13B shows the Rg plot for the 6CM4_Sakurantin. The Rg values range from 26.71 Å to 42.28 Å, with notable variations in the first 50 ns. The peaks seen at 10 and 50 ns point to times of structural expansion, which denote brief conformational shifts or flexibility brought on by ligands. According to the Rg study, the 1B8M_Sakurantin is more compact and stable than the 6CM4_Sakurantin. The structural stability of the 1B8M_Sakurantin is revealed by the variation in hydrogen bonds (H-bonds) created throughout time, as shown in Fig. 14A. In contrast to Fig. 14B, which shows hydrogen bonds for 6CM4_Sakurantin ranging from 0 to 3, which suggests weaker interactions in comparison to 1B8M, the hydrogen bond range of 0 to 4 indicates stronger stability. When Sakurantin binds to the proteins 1B8M and 6CM4, its conformational changes and stability are depicted by the solvent-accessible surface area (SASA) study in Fig. 15A-B. The unbound receptor state in complex 1B8M_Sakurantin continuously shows a greater SASA than the receptor-ligand complex. A similar pattern is seen in complex 6CM4_Sakurantin, where the unbound receptor state keeps the SASA value higher. However, the SASA is lower for the Sakurantin-bound complex.

Fig. 11 
figure 11

MD simulation analysis of 100 ns trajectories of RMSD of Cα backbone of A) 1B8M_Sakurantin and B) 6CM4_Sakurantin.

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Fig. 12 
figure 12

MD simulation analysis of 100 ns trajectories of RMSF of Cα backbone of A) 1B8M_Sakurantin and B) 6CM4_Sakurantin.

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Fig. 13
figure 13

MD simulation analysis of 100 ns trajectories of radius of Gyration (Rg) of Cα backbone of A) 1B8M_Sakurantin and B) 6CM4_Sakurantin.

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Fig. 14 
figure 14

MD simulation analysis: Formation of hydrogen bonds in A) 1B8M_Sakurantin and B) 6CM4_Sakurantin.

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Fig. 15 
figure 15

MD simulation analysis of 1000 Frame work of (A) Solvent accessible surface area of A) 1B8M_Sakurantin and B) 6CM4_Sakurantin.

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Simulation experiments that tracked protein-ligand interactions between the complexes during a 100 ns period were summarised by stacked coloured bars that were normalised throughout trajectories. These interactions are classified as water bridges, hydrophobic interactions, ionic interactions, and H-bonds. The value stays > 1 if the protein-ligand complex has more than one contact for a certain interaction. Different bar graphs from other types of interaction percentages against residues present over a 100 ns period are shown in Fig. 16A-B. Figure 17A-B show that water bridges, ionic interactions, and hydrogen bonds produced the high interaction fractions for the protein-ligand complexes 1B8M_Sakurantin and 6CM4_Sakurantin.

Fig. 16 
figure 16

Bar graph of Protein-ligand contacts of A) 1B8M_Sakurantin and B) 6CM4_Sakurantin showing interaction fraction of amino acid residues over the period of simulation.

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Fig. 17 
figure 17

Protein-ligand percent interactions of (A) 1B8M_Sakurantin and (B) 6CM4_Sakurantin.

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In the computational analysis, the binding free energy, along with the extra contributing energy from MM-GBSA, was meticulously determined for both the 1B8M_Sakurantin and 6CM4_Sakurantin compounds by thorough examination of the MD simulation trajectory. The total binding free energy (ΔGbind) for 6CM4_Sakurantin was marginally lower at −59.19 kcal/mol than it was for 1B8M_Sakurantin, which suggests a more favourable binding interaction with 6CM4 (Table 6). The enhanced electrostatic, packing, and Vander Waals interactions are responsible for Sakurantin’s higher binding affinity to 6CM4 than 1B8M. Stronger coulombic and packing results in a more stable complex, even if 6CM4 has a somewhat greater solvation penalty. In both compounds, hydrogen bonding was comparatively weak. This suggests that although hydrogen bonds exist, their contribution to overall stability is moderate.

Table 6 Binding free energy components for the 1B8M_Sakurantin and 6CM4_Sakurantin calculated by MM-GBSA.
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Discussion

The findings of this study provided significant insights into the neuroprotective effect of sakuranetin on PTZ-induced epilepsy in mice. This study demonstrated that PTZ administration markedly dysregulated neurotransmitters, oxidative stress markers, antioxidant enzymes, neuroinflammatory markers, and caspase-3 activity. Sakuranetin treatment, however, significantly mitigated these effects, suggesting its potential as a novel therapeutic option for epilepsy. These results are aligned with previously reported studies39,40. The pkCSM ADMET prediction server was utilized as a reliable tool for toxicity assessment in previous research13. The predicted results indicate that sakuranetin has an intestinal absorbance of 92.60% and a blood-brain barrier (BBB) penetration of less than 1.

In the PTZ-induced seizure model, administration of sakuranetin demonstrated anticonvulsant properties, characterized by prolonged seizure latency, attenuated convulsive activity, and enhanced survival rates. The behavioral assessment indicated that PTZ and sakuranetin intervention did not significantly alter locomotor or exploratory parameters. The pathophysiology of epilepsy is intrinsically linked to neurotransmitter dysregulation, where neuronal signaling homeostasis depends on the precise equilibrium between excitatory and inhibitory neurotransmission. Seizure generation is attributed to perturbations in neural excitability, specifically manifesting as an imbalance between glutamatergic (excitatory) and GABAergic (inhibitory) neurotransmitter systems. Preclinical in vitro and in vivo seizure models demonstrate a shift towards excitation, characterized by a disrupted GABA/glutamate balance, ultimately leading to seizure activity. DA, a catecholamine and phenethylamine neuromodulator present in cortical circuits and basal ganglia, influences seizure susceptibility. Down-regulation of hippocampal DA receptors may disinhibit thalamocortical pathways, thereby potentiating cortical hyperexcitability and increasing the likelihood of seizure occurrence41. The present investigation evaluated the modulatory effects of PTZ administration and sakuranetin intervention on neurotransmitter concentrations in the rodent model. Quantitative analysis revealed that PTZ exposure induced significant depletion of ACh, GABA, DA, NE, and 5-HT relative to control specimens. However, sakuranetin administration at both dosage regimens effectively ameliorated these PTZ-administration perturbations, restoring neurotransmitter concentrations to baseline levels. These outcomes combine with previous investigations demonstrating the neuroprotective efficacy of sakuranetin42. In addition to its GABAergic influence, sakuranetin also appeared to modulate central monoaminergic systems. NE and 5-HT are critical regulators of neuronal excitability and seizure threshold, and their dysregulation is a hallmark of PTZ-induced seizure models. Decreased levels of these monoamines have been associated with heightened excitatory signaling and seizure susceptibility. In the present study, sakuranetin administration restored NE and 5-HT concentrations, suggesting that its anticonvulsant efficacy may be mediated, at least in part, through rebalancing monoaminergic neurotransmission. This restoration may enhance inhibitory tone and contribute to neural circuit stabilization, further supporting sakuranetin’s neuromodulatory and therapeutic potential in seizure conditions43,.

Oxidative and nitrosative stress are involved in the onset of epilepsy. Oxidative stress occurs due to a disparity between pro-oxidants and antioxidants in an abundance of reactive oxygen species44. Evidence for OS in epilepsy stems from both experimental and clinical studies, which reveal a disruption in redox equilibrium through alterations in specific biomarkers. These biomarkers include those directly impacted by ROS for DNA oxidative damage, MDA for lipid peroxidation, and NO. Furthermore, markers of antioxidant defence, including SOD, GSH, and CAT, are also relevant45. In this study, PTZ administration significantly reduced antioxidant levels compared to the control group, indicating impaired antioxidant defence mechanisms. However, treatment with sakuranetin at both tested doses effectively attenuated this decline, restoring SOD, GSH, and CAT levels in PTZ-treated mice, indicating partial restoration. Additionally, PTZ administration led to a lowering of MDA and NO concentrations compared to the control group. Remarkably, sakuranetin treatment successfully counteracted this lowering, restoring MDA and NO levels to values comparable to those observed in healthy mice. These findings suggest that sakuranetin confers protection against PTZ-induced oxidative stress by modulating the activity of key antioxidant enzymes46.

Oxidative post-translational modifications significantly impact the activity of key neuroinflammatory mediators. Neuroinflammation, characterized by inflammation of nervous tissue, can be triggered by numerous external and internal factors, including infections, traumatic brain injuries, toxic metabolites, autoimmune disorders, aging, environmental toxins, exposure to secondhand smoke, and spinal cord injuries. This activation results in the secretion of cytokines and chemokines, enhancing cell survival and growth. Neuroinflammation is regulated at the transcriptional level by NF-κB, both of which are triggered by ROS47,48. Additionally, it is essential to note that the induction of heightened expression and activity of NADPH oxidase creates a highly oxidative atmosphere and dramatically influences the mitochondrial function and metabolic state of neurons and glial cells. These types of cells are already experiencing metabolic stress in epilepsy because of these processes. Moreover, the activation of NF-κB is crucial in initiating the synthesis of pro-inflammatory cytokines. These cytokines play a role in gliosis, mitochondrial impairment, and GSH. Reducing GSH levels also weakens cellular and mitochondrial antioxidant defences, disrupting regular oxidative signaling pathways and subsequent cellular injury. In epilepsy, neuroinflammation is frequently linked to BBB impairment, permitting the entry of peripheral immune cells and serum albumin into the brain tissue, which leads to seizures. Pro-inflammatory cytokines play a role in elevating neuronal excitability and initiating ictogenic occurrences48. This study identified the effects of sakuranetin on neuroinflammation in mice induced with seizures. The results showed that PTZ administration decreased the IL-6, IL-1β and TNF-α levels of pro-inflammatory markers. However, sakuranetin treatment significantly reversed this effect and restored the IL-6, IL-1β and TNF-α levels of these neuroinflammatory markers to those observed in the control group. This outcome is consistent with previous research on the anti-inflammatory properties of sakuranetin49.

BDNF plays a crucial role in neuronal survival, growth, and neurogenesis. Apart from these functions, BDNF signaling is also implicated in initiating long-term potentiation within excitatory neurotransmission, a process relevant to the pathophysiology of epilepsy. As the brain’s most abundant neurotrophic growth factor, it remarkably influences development, cellular growth, synaptic activity, and survival. These effects are mediated through the selective binding of BDNF to its TrkB receptor. Despite its recognized importance, the precise role of BDNF in epilepsy remains a subject of ongoing research and debate. While TrkB signaling is known to participate in epileptogenic processes27. This study observed a decline in BDNF and TrkB levels following PTZ administration, contrasting with some previous outcomes. Remarkably, sakuranetin treatment effectively attenuated the PTZ-induced reduction in BDNF and TrkB, restoring levels closer to those of the control group. These observations are in line with results reported in a prior study, suggesting a potential therapeutic role for sakuranetin in modulating BDNF and TrkB levels in the context of seizures39,50.

Although BDNF–TrkB signaling promotes neuronal survival and synaptic plasticity, accumulating evidence suggests that overactivation of TrkB, particularly in chronic epilepsy, can exacerbate excitatory neurotransmission, contribute to mossy fiber sprouting, and facilitate epileptogenesis. Previously reported that selective inhibition of TrkB, such as using dominant-negative TrkB constructs or pharmacological blockers like ANA-12, reduces seizure burden in chronic epilepsy models51. This underscores the context-dependent duality of TrkB signaling, where controlled upregulation may be neuroprotective in early or acute stages, while excessive activation could worsen chronic pathology.

Caspase-3 serves as a crucial executioner protein within the apoptotic cascade, with its activation being strongly associated with neuronal degeneration following epileptic seizures. Experimental studies have reported elevated expression and activation of caspase-3 in multiple brain regions, including the hippocampus, amygdala, and cortex, following induced status epilepticus. This activation plays a pivotal role in mediating seizure-induced neuronal apoptosis. In human temporal lobe epilepsy, increased levels of cleaved caspase-3, along with the nuclear translocation of caspase-activated DNase, have been documented, indicating the persistence of caspase-dependent apoptotic signaling52. In the current research, the administration of PTZ led to a decrease in caspase-3 levels when compared to the control group. Nonetheless, administering sakuranetin at both evaluated doses significantly reinstated caspase-3 levels in PTZ-induced mice, indicating partial restoration. These results are consistent with earlier reported studies50. In silico molecular docking analyses and MD simulations elucidate potential protein-ligand interaction mechanisms, providing mechanistic insights into the molecular basis of the observed biological processes. These computational methodologies enhance our understanding of the structure-function relationships governing these biomolecular systems. This study evaluated Sakuranentin’s binding affinity and stability with BDNF (1B8M), TRKB (4AT3), and dopamine receptor (6CM4). A docking study found the strongest interaction to be with 1B8M (−10.704 kcal/mol), followed by 6cm4 (−9.517 kcal/mol) and 4at3 (−9.5113 kcal/mol). Molecular dynamics (MD) simulations over 100 ns indicate that 1B8M_SAKURANTIN has a higher stability (RMSD ~ 3.68 RAW, lower RMSF, compact RG values) than 6CM4_Sakurantin (RMSD ~ 6.90, higher RMSF and structural flexibility). The hydrogen bond analysis of 1b8m_sakarantin also confirmed its greater stability. MM-GBSA calculations showed that 6cm4_sakurantin had a slightly higher unbound energy (−59.19 kcal/mol). Overall, Sakuranentin showed strong binding to 1B8M. This illustrates a potential role in modulating relatives of BDNF. The interaction with 6cm4 highlights the possible effects on dopamine signaling. Although prediction from docking suggests that Sakuranetin has the strongest binding affinity toward 1B8M (–10.7 kcal/mol), the MM-GBSA analysis revealed a slightly more favorable binding free energy with 6CM4 (–59.19 kcal/mol). This discrepancy is found in the results because docking scores are based on empirical scoring functions optimized for pose prediction and ranking. At the same time, MM-GBSA incorporates solvation effects, electrostatics, and many post-minimization energy components, providing a more realistic estimate of the binding stability of the drug with protein53,54,55. Such differences are commonly observed in computational studies and highlight the complementary nature of using both approaches to assess ligand–protein interactions.

In the present study, sakuranetin significantly restored GABA levels in PTZ-treated mice, supporting its role in enhancing inhibitory neurotransmission. However, this inference is based on indirect evidence, including molecular docking analyses suggesting possible interactions with GABAergic targets. In the absence of direct receptor binding or electrophysiological data, the proposed restoration of GABAergic tone should be interpreted cautiously. However, based on structural similarities to other flavonoids such as apigenin and naringenin, which are known to act as positive allosteric modulators of GABA-A receptors56, it is plausible that sakuranetin may also interact with GABA-A receptor subunits, possibly at the benzodiazepine binding site. Such interaction could enhance chloride ion influx, promoting hyperpolarization and seizure suppression. Further studies involving receptor binding assays or expression profiling of GABA-A receptor subunits and electrophysiological approaches will be essential to confirm the precise mechanism. Moreover, antioxidant enzyme activity is enhanced, reducing oxidative stress markers like MDA and protecting neurons from damage caused by oxidative stress. Additionally, it likely suppresses neuroinflammation by downregulating pro-inflammatory cytokines, while its anti-apoptotic action, evidenced by reduced caspase-3 activity, prevents neuronal cell death, contributing to its neuroprotective and therapeutic properties in epilepsy. While the restoration of neurotransmitter levels observed in sakuranetin-treated animals is compelling, it is important to consider that this effect may not solely represent a direct pharmacodynamic action. Chronic seizure activity disrupts synaptic neurotransmission through mechanisms such as excitotoxicity, oxidative damage, and neuroinflammation, leading to reductions in inhibitory transmitters like GABA and alterations in monoaminergic tone57. Therefore, the normalization of GABA, dopamine, norepinephrine, serotonin, and acetylcholine levels in our study could partially reflect a secondary response to the attenuation of seizure burden and subsequent restoration of neuronal homeostasis. However, several lines of evidence suggest that sakuranetin may exert direct or upstream neuromodulatory effects. Notably, molecular docking and 100 ns molecular dynamics simulations identified brain-derived neurotrophic factor (BDNF) as a high-affinity target of sakuranetin (ΔG ≈ − 57 kcal/mol), with stable binding throughout the simulation. In vivo, sakuranetin treatment restored hippocampal BDNF and TrkB levels to 92% and 88% of control values, respectively, implying activation of neurotrophin signaling pathways known to influence synaptic plasticity and neurotransmitter regulation. Moreover, the dose-dependent recovery of GABA and other neurotransmitters, even in the context of partially persistent seizure activity, further supports a possible pharmacological effect on neurotransmitter systems. These findings indicate that sakuranetin’s beneficial impact on neurotransmitter homeostasis may result from a combination of indirect neuroprotective effects and direct engagement with molecular pathways involved in neuromodulation. This highlights sakuranetin’s comprehensive role in protecting against PTZ-induced epilepsy. Considering the potential translational relevance of sakuranetin, its dietary origin warrants attention. Sakuranetin, a naturally occurring flavonoid in edible Prunus species, may provide neuroprotective benefits and has potential as a nutraceutical compound. Nevertheless, the estimated human-equivalent doses applied in this study (~ 0.8–1.6 g/day) substantially exceed typical dietary flavonoid intake and are unlikely to be achieved through regular consumption of sakuranetin-rich foods. Therefore, any prospective therapeutic use of sakuranetin would likely necessitate its development into concentrated nutraceutical or pharmacological formulations to attain efficacious plasma concentrations. While no adverse effects were observed in the treated animals during the study period, comprehensive toxicological assessments and safety evaluations will be essential before clinical translation can be considered.

Limitations

Despite the promising findings, this study has several limitations. The limited sample size (n = 6 per group) may reduce the statistical power and sensitivity for detecting subtle treatment effects. The study also relied on a single animal model (PTZ-induced seizures in mice), which may not fully represent the complexity and heterogeneity of human epilepsy. Moreover, a significant limitation is the absence of a positive control group treated with a standard anticonvulsant agent. Including such a group would have allowed for the direct benchmarking of sakuranetin’s efficacy against established therapies and further validation of the PTZ-induced seizure model. Future studies should incorporate a standard anticonvulsant, such as diazepam or valproate, for proper comparative evaluation.

Additionally, long-term toxicity and efficacy data for sakuranetin were not evaluated, limiting our understanding of its overall safety profile and sustained therapeutic potential. Mechanistic interpretations were based on biochemical markers without detailed molecular or pathway-level explorations, and receptor-binding or electrophysiological assessments were not conducted, restricting deeper insights into its mode of action. Potential drug interactions were also not assessed.

In the present study, no signs of toxicity, weight loss, or abnormal behavior were observed in sakuranetin-treated animals throughout the experimental period, aligning with previous reports that demonstrate its favorable tolerability at similar doses in vivo58. Although liver and kidney biomarkers or chronic toxicity profiles were not measured, these findings suggest that sakuranetin is likely safe within the tested dose range. Nonetheless, future studies should include exhaustive toxicological, mechanistic, and comparative efficacy investigations to support the translational relevance of these findings.

Conclusion

Sakuranetin, an O-methylated flavanone naturally enriched in edible Prunus species, exerted robust anticonvulsant and neuroprotective actions in the repeated-PTZ mouse model. Behaviourally, the flavonoid extended seizure latency, shortened generalised tonic-clonic duration and reduced mortality. Mechanistically, it (i) restored inhibitory and monoaminergic neurotransmitters, (ii) reinstated endogenous antioxidant enzymes while curbing lipid peroxidation and nitrosative stress, (iii) suppressed IL-1β/IL-6/TNF-α signaling, and (iv) re-elevated BDNF/TrkB with concurrent caspase-3 down-regulation. In silico docking and 100-ns, molecular-dynamics simulations corroborated a high-affinity, energetically stable interaction between sakuranetin and the BDNF–TrkB axis, providing a molecular rationale for the in vivo. Nonetheless, future work must address (i) oral bioavailability and blood-brain-barrier penetration in higher species, (ii) long-term safety/toxicology, and (iii) synergistic or antagonistic interactions with standard AEDs. Clinical translation will also require controlled dietary intervention studies to validate sakuranetin’s seizure-modifying potential in humans.

Overall, the integrative in vivo and in silico investigation positions sakuranetin as a promising, multi-target neuro-nutraceutical candidate capable of synchronising redox, inflammatory and neurotrophic pathways to mitigate epileptogenesis.