Epilepsy & Behavior 41 (2014) 98–102

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Naringin ameliorates pentylenetetrazol-induced seizures and associated oxidative stress, inflammation, and cognitive impairment in rats: Possible mechanisms of neuroprotection Mahaveer Golechha a,b, Vikas Sarangal c, Jagriti Bhatia a, Uma Chaudhry d, Daman Saluja d, Dharmveer Singh Arya a,⁎ a

Department of Pharmacology, All India Institute of Medical Sciences, New Delhi 110029, India Public Health Foundation of India, New Delhi 110070, India c Department of Anatomy, All India Institute of Medical Sciences, New Delhi 110029, India d Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi 110007, India b

a r t i c l e

i n f o

Article history: Received 27 June 2014 Revised 22 August 2014 Accepted 20 September 2014 Available online xxxx Keywords: Naringin GABAergic Flumazenil PTZ Cognitive impairment Seizures

a b s t r a c t Oxidative stress and cognitive impairment are associated with PTZ-induced convulsions. Naringin is a bioflavonoid present in the grapefruit. It is a potent antioxidant, and we evaluated its effect on PTZ-induced convulsions. Rats were pretreated with normal saline, naringin (20, 40, and 80 mg/kg, i.p.), or diazepam (5 mg/kg, i.p.) 30 min prior to the administration of PTZ. The administration of PTZ induced myoclonic jerks and generalized tonic– clonic seizures (GTSs). We observed that naringin significantly prolonged the induction of myoclonic jerks dose-dependently. Naringin (80 mg/kg, i.p.) pretreatment protected all rats, and this protective effect was annulled by the GABAA receptor antagonist, flumazenil. In addition, naringin reduced brain MDA and TNF-α levels and conserved GSH. The pretreatment also enhanced the performance of rats in the passive avoidance task. Our observations highlight the antioxidant, antiinflammatory, and anticonvulsant potential of naringin. Also, naringin modulates the GABAA receptor to produce anticonvulsant effects and to ameliorate cognitive impairment. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Epilepsy is one of the most common neurological disorders affecting approximately more than 50 million people globally [1]. Antiepileptic drugs possess a wide range of side effects, and some require continuous monitoring to prevent toxicity [2]. Thus, there is a need for developing safer and more efficacious drugs. Nowadays, many medicinal plants are being screened for their possible antiepileptic effects [3]. Much scientific exploration is underway to identify a new plant-based drug or adjunctive agent to either enhance the efficacy or reduce the adverse effects of currently available antiepileptic drugs. A convulsive episode is usually triggered whenever there is an imbalance between the levels of inhibitory and excitatory neurotransmitters in the central nervous system (CNS) [4]. A decline in the levels of γ-aminobutyric acid (GABA) or an increase in glutamate concentration in the CNS may cause convulsions. One of the animal models used to screen drugs for their potential anticonvulsant properties is the pentylenetetrazole (PTZ) model [5,6]. Pentylenetetrazol triggers seizures in animals by blocking GABA transmission. Pentylenetetrazol also increases reactive oxygen species (ROS) levels in the rat brain ⁎ Corresponding author. Tel.: +91 11 26594266, +91 11 26584121. E-mail address: [email protected] (D.S. Arya).

http://dx.doi.org/10.1016/j.yebeh.2014.09.058 1525-5050/© 2014 Elsevier Inc. All rights reserved.

which further enhances the neurotoxic and convulsant effects of PTZ [7]. In contrast, antioxidants not only minimize convulsions but also limit ROS-induced damage [8]. Vezzani and Granata [9] studied the areas of the brain which were involved in the epileptic activity and exhibited rise in inflammatory mediators. In particular, a rapid onset of inflammatory response occurred in the glia as a result of seizures induced by chemoconvulsants or by electrical stimulation [9,10]. Also, molecular and pharmacological studies of in vivo models showed that cytokines like IL-1β and TNF-α play a significant role and are amongst the first inflammatory mediators to rise following seizures [11]. Thus, the role of cytokines in epilepsy needs to be elucidated and this understanding can potentially be applied to new drug development in epilepsy. Naringin (4′,5,7-trihydroxyflavanone 7-rhamnoglucoside) is a flavanone glycoside that is ubiquitous in citrus herbs and grapefruit. It possesses potent antioxidant, superoxide scavenging, antiapoptotic, antiatherogenic, and metal chelating activities [12–14]. Naringin is hydrolyzed by intestinal microflora to yield naringenin (4′,5,7-trihydroxyflavanone). The latter is readily absorbed and also has good penetration across the blood–brain barrier [15]. A recent study has reported naringin's neuroprotective potential against D-galactose-induced cognitive impairment and oxidative stress in mice [16]. Epilepsy is usually associated with cognitive decline, and use of some antiepileptic drugs is also associated with cognitive impairment.

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Further, our previous work on naringin has demonstrated the antiepileptic potential of naringin against kainic acid-induced status epilepticus in rats [17]. Therefore, it is worthwhile to assess the neuroprotective property of naringin against PTZ-induced seizures in rats in view of its multiple favorable pharmacological activities.

assess the effect of PTZ on learning and memory. The rats were thereafter sacrificed for the estimation of oxidative stress markers and determination of brain levels of TNF-α.

2. Materials and methods

Flumazenil (a benzodiazepine receptor (GABAA) antagonist) was used to assess whether it blocks or reverses naringin ameliorating effects. The rats were pretreated with flumazenil (10 mg/kg, i.p.) and then after 15 min, the animals received an injection of naringin (80 mg/kg, i.p.), vehicle (10 ml/kg, i.p.), or diazepam (1 mg/kg, i.p.). Finally, PTZ (60 mg/kg, i.p.) was administered 30 min after naringin injection to induce seizures. The rats were observed for 30 min for latencies to myoclonic jerks and GTSs as well as duration of GTSs.

2.1. Animals The study was carried out using male Wistar rats weighing 150–200 g obtained from the central animal house facility of Dr. B. R. Ambedkar Center for Biomedical Research (ACBR), University of Delhi, India. The rats were group housed in polyacrylic cages (38 × 23 × 10 cm) with not more than four animals per cage and were maintained under standard laboratory conditions with natural dark and light cycles (approximately 14 h light–10 h dark cycle) and a room temperature of 25 ± 1 °C. They were allowed free access to standard dry diet (Golden Feeds, India) and tap water. All the behavioral procedures were carried out between 0900 and 1300 h. All procedures described were reviewed and approved by the Institutional Committee for Ethical Use of Animals, and care of animals was taken as per guidelines of CPCSEA, Ministry of Forests and Environment, Government of India. 2.2. Drugs and chemicals Naringin, pentylenetetrazole (PTZ), diazepam, flumazenil, radioimmunoprecipitation assay (RIPA) lysis buffer, and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co. (Sigma, St. Louis, MO, U.S.A.). The protease inhibitor cocktail was purchased from Roche Applied Science (Mannheim, Germany). Enzyme-linked immunosorbent assay (ELISA) kits were from Pierce Biotechnology, Inc. (Rockford, IL, U.S.A.). All other materials were of the highest grade available. 2.2.1. Pentylenetetrazole (PTZ)-induced seizures Pentylenetetrazole was dissolved in saline and administered intraperitoneally (i.p.) at the dose of 60 mg/kg. This dose has been standardized previously in our laboratory and is associated with the least mortality [18]. With this dose of PTZ, 99.0% of the animals exhibit a generalized tonic seizure (GTS). In GTSs, there is symmetric forelimb and hindlimb tonus followed by hindlimb clonus and flipping activity. 2.2.2. Experimental design The rats were divided into six groups, and each group consisted of a minimum of six animals. Separate animals were used for each experiment. The naringin dose used in this experiment was determined from previous studies [16,17]. The visual scoring for seizure onset was done unblinded. Group I (control group): The rats were injected with saline, i.p., for 7 days. Group II (vehicle + PTZ group): Vehicle (saline) was administered, i.p., for 7 successive days before PTZ (60 mg/kg, i.p.) administration. Group III (positive control group): Diazepam was administered on the seventh day followed by PTZ (60 mg/kg, i.p.) 30 min after the administration of diazepam. Groups IV, V, and VI (naringin + PTZ group): Naringin in the doses of 20, 40, and 80 mg/kg/day, i.p., was administered for 7 days respectively to rats in different groups prior to PTZ. On the seventh day, 30 min after the drug treatment, 60 mg/kg, i.p., PTZ was administered and the animals were observed for 30 min for latencies to myoclonic jerks and GTSs as well as duration of GTSs. In groups II–VI, 24 h after PTZ administration, initial latency (IL) was noted and 48 h later, i.e., on the ninth day, retention latency (RL) was noted using one trial of the passive avoidance task. This was done to

2.3. GABAergic modulation by naringin in PTZ-induced seizures

2.4. Behavioral test: single-trial passive avoidance test Memory retention deficit was evaluated by a step-through passive avoidance apparatus. The apparatus consisted of equal-sized light and dark compartments (30 × 20 × 30 cm). A 40-W lamp was fixed 30 cm above its floor in the center of the light compartment. The floor consisted of a metal grid connected to a shock scrambler. The two compartments were separated by a trap door that could be raised to 10 cm. Twenty-four hours after the administration of PTZ, the rats were placed in the light compartment and the time lapse before each animal entered the dark compartment and had all four paws inside it was measured in seconds and termed as initial latency (IL). Immediately after the rat entered the dark chamber with all four paws inside the dark chamber, the trap door was closed and an electric foot shock (50 V a.c.) was delivered for 3 s. Five seconds later, the rat was removed from the dark chamber and returned to its home cage. Twenty-four hours after the IL, the latency time was again measured in the same way as in the acquisition trial and was termed as the retention latency (RL). However, during the retention trial, no foot shock was delivered, and the latency time was recorded to a maximum of 600 s. To improve the reliability and validity of the foot shock avoidance test, the grid as well as the rat paw were moistened with water before delivering the foot shock as this is known to reduce the wide interanimal variability in paw skin resistance of the rats [18]. 2.5. Biochemical tests 2.5.1. Tissue preparation Brain tissue samples were thawed and homogenized with 10 times (w/v) ice-cold 0.1 M phosphate buffer (pH 7.4). Aliquots of homogenates from the rat brain were used to determine lipid peroxidation and glutathione. 2.5.2. Estimation of lipid peroxidation Malondialdehyde (MDA), which is a measure of lipid peroxidation, was measured spectrophotometrically [19]. Briefly, brain tissues were homogenized with 10 times (w/v) 0.1 M sodium phosphate buffer (pH 7.4). The reagents 1.5 ml acetic acid (20%), pH 3.5, 1.5 ml thiobarbituric acid (0.8%), and 0.2 ml sodium dodecyl sulfate (8.1%) were added to 0.1 ml of the processed tissue sample. The mixture was then heated at 100 °C for 60 min. The mixture was cooled with tap water, and 5 ml of nbutanol:pyridine (15:1% v/v) and 1 ml of distilled water were added. The mixture was shaken vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was withdrawn and absorbance was measured at 532 nm using a spectrophotometer. 2.5.3. Estimation of reduced glutathione Glutathione (GSH) was measured spectrophotometrically [20]. Briefly, brain tissues were homogenized with 10 times (w/v) 0.1 M sodium phosphate buffer (pH 7.4). This homogenate was then centrifuged with 5% trichloroacetic acid to centrifuge out the proteins. To

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0.1 ml of this homogenate, 2 ml of phosphate buffer (pH 8.4), 0.5 ml of 5′5 dithiobis (2-nitrobenzoic acid) (DTNB), and 0.4 ml of double-distilled water were added. The mixture was vortexed and the absorbance read at 412 nm within 15 min. 2.5.4. TNF-α assay in the rat brain by enzyme-linked immunosorbent assay (ELISA) The brain was homogenized in 1 ml of ice-cold lysis buffer (radioimmunoprecipitation assay, RIPA) containing 50 mM Tris–HCl (pH 8.0), 150 mM sodium chloride, 1.0% Iepal CA-630 (NP-40), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% phosphatase inhibitor cocktail, and a protease inhibitor cocktail. The lysate was centrifuged (15,000 ×g at 4 °C) for 15 min, and the supernatant was added to 96-well ELISA plates. The TNF-α concentration was then determined by reading the ELISA plate. 2.6. Statistical analysis of data Data are expressed as mean ± SEM. Statistical differences between the treatment and control groups were evaluated by one-way ANOVA followed by the Tukey–Kramer post hoc test. The value of p b 0.05 was considered to be significant. 3. Results 3.1. Effect of naringin on the latency of myoclonic jerks All the rats treated for 7 days with naringin (20, 40, and 80 mg/kg, i.p.) exhibited myoclonic jerks following PTZ administration. However, there was a significant increase in the latency of myoclonic jerks in a dose-dependent manner compared to the vehicle-treated PTZ group (Table 1; p b 0.001). Myoclonic jerks were not observed in rats pretreated with diazepam (positive control group).

naringin (80 mg/kg, i.p.)-treated group. Similarly, the antiepileptic effect of diazepam was also significantly blocked by flumazenil pretreatment. Naringin at 80 mg/kg, i.p., abolished the GTSs induced by PTZ, while flumazenil pretreatment significantly reversed it as GTSs occurred in the flumazenil + naringin (80 mg/kg, i.p.)-treated group. The results strongly suggested that naringin acts via modulating GABAA receptors (Table 2). 3.4. Effect of naringin on the levels of GSH in PTZ-induced seizures in rats The brain glutathione levels were estimated in the rat brains in all groups. The brain levels of glutathione showed a significant (p b 0.001) decrease in the vehicle-treated PTZ group (45.83 ± 3.44 μg/g wet tissues) compared to the control group rats (108.68 ± 4.49 μg/g wet tissues). Diazepam pretreatment significantly maintained the GSH levels in the brain. In the groups treated with naringin (20, 40, and 80 mg/kg, i.p.), the values of GSH were 65.56 ± 3.58, 71.05 ± 3.25, and 97.81 ± 4.11 μg/g wet tissue, respectively. The values were significantly higher in groups treated with the doses of 40 and 80 mg/kg, i.p., than the vehicletreated PTZ group (Fig. 1A). 3.5. Effect of naringin on the levels of MDA in PTZ-induced seizures in rats The levels of MDA in the rat brains were significantly raised after PTZ administration (344.81 ± 4.63 nmol/g wet tissue) compared to the control group rats (175.95 ± 4.67 nmol/g wet tissue) (p b 0.001). In the diazepam group, there was no significant rise in MDA levels. In the rats treated with naringin (20, 40, and 80 mg/kg, i.p.), the values of MDA were 311.94 ± 5.83, 251.64 ± 5.18, and 201.33 ± 4.87 nmol/g wet tissue, respectively. The naringin dose-dependently and significantly decreased the levels of MDA compared to the vehicle-treated PTZ group (Fig. 1B). 3.6. Effect of naringin on cognitive impairment induced by PTZ-induced seizures in rats

3.2. Effect of naringin on GTSs and duration of GTSs GTSs were not observed in rats pretreated with diazepam. They were also not observed in the group pretreated with the 80 mg/kg, i.p., dose of naringin for 7 days. The latency of GTSs as well as the duration of GTSs were increased at 20 and 40 mg/kg, i.p., doses in a dosedependent manner (p b 0.001) compared to the vehicle-treated PTZ group (Table 1). 3.3. Effects of flumazenil on the anticonvulsant activity of naringin Flumazenil (10 mg/kg, i.p.) administered 15 min prior to naringin (80 mg/kg, i.p.) caused significant blockade of the antiepileptic effect of naringin. The latency of occurrence of myoclonic jerks was significantly (p b 0.001) reduced in the flumazenil + naringin-treated group compared to rats treated with naringin alone. The mean latency of myoclonic jerks was 120.33 ± 7.17 in the flumazenil + naringin (80 mg/kg, i.p.)-treated group compared to 235.16 ± 9.86 in the

Table 1 Effect of naringin (NAR) on the latency of myoclonic jerks and generalized tonic–clonic seizures (GTSs) in PTZ-induced seizures in rats. Group

Latency of myoclonic jerks (s)

Latency of GTSs (s)

Duration of GTSs (s)

Vehicle-treated PTZ Positive control (DZ) NAR 20 NAR 40 NAR 80

34.5 ± 2.86 Not observeda 115.16 ± 4.80a 172.83 ± 5.72a 250.16 ± 9.75a

38.16 ± 3.17 Not observeda 152.66 ± 8.78a 269.65 ± 6.92a No GTSsa

18.33 ± 1.47 Not observeda 9.16 ± 1.94a 5 ± .96a No GTSsa

Each value represents the mean ± SEM for 6 rats. One-way ANOVA followed by the Tukey–Kramer post hoc test was used to test the difference between groups. a p b 0.001 compared to the vehicle-treated PTZ group.

The mean initial latency recorded 30 min after the administration of PTZ did not differ significantly between any of the groups (Fig. 1C). Retention latency 24 h after the administration of PTZ in the vehicletreated PTZ group was significantly less (p b 0.001) compared with the control group rats. This indicated significant cognitive impairment following PTZ administration. Diazepam significantly prevented the decline in cognitive impairment. The groups treated with naringin (20, 40, and 80 mg/kg, i.p.) showed significant reversal of the PTZinduced cognitive deficit as evidenced by dose-dependent increases in the retention latencies in these groups.

Table 2 Involvement of the GABAergic mechanism in the antiepileptic effect of naringin (NAR) in PTZ-induced seizures in rats. Group

Latency of myoclonic jerks (s)

Latency of GTSs (s)

Duration of GTSs (s)

Vehicle-treated PTZ Positive control (DZ) NAR 80 Fluma Fluma + DZ Flumazenil + NAR 80

39.16 ± 3.43 Not observeda 235.16 ± 9.86a 40 ± 3.77 42.83 ± 4.91b 120.33 ± 7.17c

49.83 ± 3.78 Not observeda Not observeda 42.5 ± 3.27 64.66 ± 4.18b 145.5 ± 9.40c

18.5 ± 2.12 Not observeda Not observeda 19.33 ± 2.59 9.66 ± 1.02d 11.66 ± 1.82e

Each value represents the mean ± SEM for 6 rats. One-way ANOVA followed by the Tukey–Kramer post hoc test was used to test the difference between groups. Fluma— flumazenil; DZ—diazepam. a p b 0.001 compared to the vehicle-treated PTZ group. b p b 0.001 compared to the diazepam-treated group. c p b 0.001 compared to the naringin (80 mg/kg)-treated group. d p b 0.01 compared to the diazepam-treated group. e p b 0.01 compared to the naringin (80 mg/kg)-treated group.

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Fig. 1. A: Effect of naringin (NAR) on levels of glutathione (GSH) in PTZ-induced seizures in rats. B: Effect of naringin (NAR) on levels of malondialdehyde (MDA) in PTZ-induced seizures in rats. C: Effect of naringin (NAR) on cognitive impairment induced by PTZ in rats. D: Effect of naringin (NAR) on the TNF-α level in PTZ-induced seizures in rats. Each value represents the mean ± SEM for 6 rats. ap b 0.001 compared to control and bp b 0.001, cp b 0.01, and dp b 0.05 compared to the vehicle-treated PTZ group. One-way ANOVA followed by the Tukey–Kramer post hoc test was used to test the difference between groups.

3.7. Effect of naringin on the levels of TNF-α in PTZ-induced seizures in rats The brain levels of TNF-α were significantly raised after PTZ administration (749.3 ± 19.3 pg/ml) compared to the control group rats (402.92 ± 13.35 pg/ml) (p b 0.001). Naringin dose-dependently attenuated the high brain levels of TNF-α induced by PTZ (Fig. 1D). The maximum inhibition was observed at the 80 mg/kg dose of naringin (p b 0.001).

4. Discussion Kainic acid (KA), a glutamate agonist, causes severe status epilepticus with motor involvement lasting several hours [21]. These seizures originate in the CA3 region of the hippocampus and spread to other limbic structures and are followed by neuronal loss in selected regions of the brain reminiscent of brain damage seen in patients with temporal lobe epilepsy. Kainic acid activates the kainite class of NMDA (ionotropic glutamate) receptors where it increases synaptic activity, leading to seizures, neurodegeneration, and remodeling, primarily affecting the limbic structures such as the hippocampus [22]. The KA-induced status epilepticus model is quite different from PTZ-induced generalized tonic–clonic seizures in rats on account of the types of seizures, action through different receptors, and neuronal damage. A previous research study by our laboratory reported the antiepileptic effect of naringin against KA-induced status epilepticus [17]. Therefore, we planned the present study to assess whether naringin affords protection against PTZ-induced seizures. Pentylenetetrazol is known to cause the activation of membrane phospholipases, proteases, and nucleases which in turn produce degradation of membrane phospholipids along with the proteolysis of

cytoskeleton proteins and protein phosphorylation [23]. Pentylenetetrazolinduced convulsions also generate neurotoxic reactive oxygen species, and many antioxidative agents have showed potential to mitigate the neurotoxic effect of chemoconvulsants in the animal brain [24,25]. Many plants and their derived extracts or active constituents known to exhibit antioxidant activity also display anticonvulsant activity [26]. In our study, the levels of GSH and lipid peroxidation were measured in the rat brains. Pretreatment of animals with naringin, at all the doses tested, significantly prevented PTZ-induced elevation in lipid peroxidation and conserved the glutathione levels. Such similar findings are also reported by many in which diminution of lipid peroxidation in brain tissue with antioxidants reduces convulsions [27]. Glutathione is an endogenous antioxidant found in all animal cells. It reacts with the free radicals and prevents cellular damage due to singlet oxygen, hydroxide radicals, and superoxide radicals [28]. We observed that administration of PTZ in rats resulted in decreased levels of GSH in the brain, and naringin prevented this decrease in levels of GSH. The ability of naringin to enhance the GSH level is similarly reported also by other researchers [29]. However, further investigation will help to clarify whether the maintenance of GSH levels occurs by ROS depletion or an increase in its synthesis. Thus, our results suggest that, at least in part, the naringin's anticonvulsant and neuroprotective activities may also be related to its antioxidant effects against PTZ-induced oxidative stress in the rat brains. Glial cells play an important role in generating seizures by modulating synaptic transmission and, in the long term, contribute to the epileptic process by inducing inflammatory milieu around neurons [30]. Further, glial cells are well known to produce many proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 in different experimental models of seizures [10,30]. These inflammatory processes are key contributors to acute and chronic neurodegenerative disorders. In

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order to study the antiinflammatory effect of naringin in our experimental model, we measured the brain levels of TNF-α. It was observed that TNF-α levels were elevated in PTZ-treated rats, and naringin (40 and 80 mg/kg, i.p.) significantly attenuated this rise in TNF-α levels (p b 0.001). Naringin's antiinflammatory and antiapoptotic activities have been reported by others also [31]. It also inhibits LPS-induced iNOS expression and nitric oxide production within macrophages [32]. Naringin is one of the most potent flavonoids which suppress the TNF-α secretion. Thus, we can say that naringin's effectiveness in PTZ-induced seizures is also by its virtue of TNF-α suppression. The GABAA receptor plays a pivotal role in mediating inhibitory neurotransmission in the mammalian CNS [33]. It is well known that PTZ produces tonic–clonic seizures by suppressing the inhibitory effects of GABAergic transmission [34]. Naringin exhibited protection against PTZ-induced seizures in our study. Thus, in order to investigate whether the anticonvulsant action of naringin is GABA-dependent or not, the GABAA receptor was blocked with flumazenil, a GABAA antagonist. It was observed that flumazenil pretreatment decreased the prolongation of convulsant latency induced by naringin, suggesting a possible modulation of the benzodiazepine site of the GABAA receptor by naringin to produce its anticonvulsant effect. Pentylenetetrazol induces cognitive impairment in rats. The values of retention latencies measured in the passive avoidance test were reduced in the PTZ group in our study. This conforms to earlier reports of cognitive impairment after a chemoconvulsant challenge [18,35]. Naringin pretreatment significantly increased the retention latencies of rats and reduced the cognitive deficit associated with seizures. The effect was observed even with the low test dose of naringin (20 mg/kg, i.p.). Hence, naringin has significant cognitive deficit-preventing potential. 5. Conclusions In summary, our results demonstrate that naringin possesses significant anticonvulsant effects against PTZ-induced convulsions and also reduces cognitive deficit associated with PTZ-induced seizures. These effects are probably consequences of its positive modulation of GABAA receptors and its antioxidant and antiinflammatory properties. These findings indicate that naringin could prove to be a valuable natural therapeutic agent with anticonvulsant and antioxidant properties. However, further studies are required in order to evaluate the exact mechanism involved in its anticonvulsant action. Acknowledgment The authors are thankful to the Department of Science and Technology (DST), Government of India and Council of Scientific and Industrial Research (CSIR), Government of India for providing Inspire Fellowship to Dr. Mahaveer Golechha and Senior Research Fellowship to Mr. Vikas Sarangal, respectively. Competing interests We have read and understood Epilepsy & Behavior policy on declaration of interests and have no relevant interests to declare. References [1] Savic I. Sex differences in human epilepsy. Exp Neurol 2014;259C:38–43. [2] Beghi E, Schmidt D. When and how to stop antiepileptic drugs. Epileptology 2013; 1(1):17–20. [3] Ding J, Wang JJ, Huang C, Wang L, Deng S, Xu TL, et al. Curcumol from Rhizoma Curcumae suppresses epileptic seizure by facilitation of GABA(A) receptors. Neuropharmacology 2014;81:244–55. [4] Wallace ML, Burette AC, Weinberg RJ, Philpot BD. Maternal loss of Ube3a produces an excitatory/inhibitory imbalance through neuron type-specific synaptic defects. Neuron 2012;74(5):793–800.

[5] Kołosowska K, Maciejak P, Szyndler J, Turzyńska D, Sobolewska A, Płaźnik A. The role of interleukin-1β in the pentylenetetrazole-induced kindling of seizures, in the rat hippocampus. Eur J Pharmacol 2014;731:31–7. [6] Aniol VA, Ivanova-Dyatlova AY, Keren O, Guekht AB, Sarne Y, Gulyaeva NV. A single pentylenetetrazole-induced clonic-tonic seizure episode is accompanied by a slowly developing cognitive decline in rats. Epilepsy Behav 2013;26(2):196–202. [7] Erdoğan F, Gölgeli A, Arman F, Ersoy AÖ. The effects of pentylenetetrazole-induced status epilepticus on behavior, emotional memory, and learning in rats. Epilepsy Behav 2004;5(3):388–93. [8] Waldbaum S, Patel M. Mitochondria, oxidative stress, and temporal lobe epilepsy. Epilepsy Res 2010;88(1):23–45. [9] Vezzani A, Granata T. Brain inflammation in epilepsy: experimental and clinical evidence. Epilepsia 2005;46(11):1724–43. [10] Ravizza T, Balosso S, Vezzani A. Inflammation and prevention of epileptogenesis. Neurosci Lett 2011;497(3):223–30. [11] Auvin S, Mazarati A, Shin D, Sankar R. Inflammation enhances epileptogenesis in the developing rat brain. Neurobiol Dis 2010;40(1):303–10. [12] Rajadurai M, Prince PS. Naringin ameliorates mitochondrial lipid peroxides, antioxidants and lipids in isoproterenol-induced myocardial infarction in Wistar rats. Phytother Res 2009;23(3):358–62. [13] Cavia-Saiz M, Busto MD, Pilar-Izquierdo MC, Ortega N, Perez-Mateos M, Muñiz P. Antioxidant properties, radical scavenging activity and biomolecule protection capacity of flavonoid naringenin and its glycoside naringin: a comparative study. J Sci Food Agric 2010;90(7):1238–44. [14] Jeon SM, Bok SH, Jang MK, Lee MK, Nam KT, Park YB, et al. Antioxidative activity of naringin and lovastatin in high cholesterol-fed rabbits. Life Sci 2001;69(24):2855–66. [15] Zbarsky V, Datla KP, Parkar S, Rai DK, Aruoma OI, Dexter DT. Neuroprotective properties of the natural phenolic antioxidants curcumin and naringenin but not quercetin and fisetin in a 6-OHDA model of Parkinson's disease. Free Radic Res 2005;39(10):1119–25. [16] Kumar A, Prakash A, Dogra S. Naringin alleviates cognitive impairment, mitochondrial dysfunction and oxidative stress induced by D-galactose in mice. Food Chem Toxicol 2010;48(2):626–32. [17] Golechha M, Chaudhry U, Bhatia J, Saluja D, Arya DS. Naringin protects against kainic acid-induced status epilepticus in rats: evidence for an antioxidant, antiinflammatory and neuroprotective intervention. Biol Pharm Bull 2011;34(3):360–5. [18] Golechha M, Bhatia J, Arya DS. Hydroalcoholic extract of Emblica officinalis Gaertn. affords protection against PTZ-induced seizures, oxidative stress and cognitive impairment in rats. Indian J Exp Biol 2010;48(5):474–8. [19] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95(2):351–8. [20] Gl Ellman. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82(1):70–7. [21] Rubio C, Rubio-Osornio M, Retana-Márquez S, Verónica Custodio ML, Paz C. In vivo experimental models of epilepsy. Cent Nerv Syst Agents Med Chem 2010;10(4): 298–309. [22] Hunsberger JG, Bennett AH, Selvanayagam E, Duman RS, Newton SS. Gene profiling the response to kainic acid induced seizures. Brain Res Mol Brain Res 2005;141(1):95–112. [23] De Deyn PP, D'Hooge R, Marescau B, Pei Y-Q. Chemical models of epilepsy with some reference to their applicability in the development of anticonvulsants. Epilepsy Res 1992;12(2):87–110. [24] Ilhan A, Gurel A, Armutcu F, Kamisli S, Iraz M. Antiepileptogenic and antioxidant effects of Nigella sativa oil against pentylenetetrazol-induced kindling in mice. Neuropharmacology 2005;49(4):456–64. [25] González-Ramírez M, Razo-Juárez LI, Sauer-Ramírez JL, González-Trujano ME, Salgado-Ceballos H, Orozco-Suarez S. Anticonvulsive effect of vitamin C on pentylenetetrazol-induced seizures in immature rats. Pharmacol Biochem Behav 2010;97(2):267–72. [26] Chauhan AK, Dobhal MP, Joshi BC. A review of medicinal plants showing anticonvulsant activity. J Ethnopharmacol 1988;22(1):11–23. [27] Bough KJ, Matthews PJ, Eagles DA. A ketogenic diet has different effects upon seizures induced by maximal electroshock and by pentylenetetrazole infusion. Epilepsy Res 2000;38(2–3):105–14. [28] Akbas SH, Yegin A, Ozben T. Effect of pentylenetetrazol-induced epileptic seizure on the antioxidant enzyme activities, glutathione and lipid peroxidation levels in rat erythrocytes and liver tissues. Clin Biochem 2005;38(11):1009–14. [29] Sahu BD, Tatireddy S, Koneru M, Borkar RM, Kumar JM, Kuncha M, et al. Naringin ameliorates gentamicin-induced nephrotoxicity and associated mitochondrial dysfunction, apoptosis and inflammation in rats: possible mechanism of nephroprotection. Toxicol Appl Pharmacol 2014;277(1):8–20. [30] Vezzani A, Moneta D, Richichi C, Perego C, De Simoni MG. Functional role of proinflammatory and anti-inflammatory cytokines in seizures. Adv Exp Med Biol 2004; 548:123–33. [31] Bharti S, Rani N, Krishnamurthy B, Arya DS. Preclinical evidence for the pharmacological actions of naringin: a review. Planta Med 2014;80(6):437–51. [32] Liu Y, Wu H, Nie YC, Chen JL, Su WW, Li PB. Naringin attenuates acute lung injury in LPS-treated mice by inhibiting NF-κB pathway. Int Immunopharmacol 2011;11(10): 1606–12. [33] Moult PR. Neuronal glutamate and GABAA receptor function in health and disease. Biochem Soc Trans 2009;37(Pt 6):1317–22. [34] Gale K. Role of GABA in the genesis of chemoconvulsant seizures. Toxicol Lett 1992; 64–65:417–28. [35] Mortazavi F, Ericson M, Story D, Hulce VD, Dunbar GL. Spatial learning deficits and emotional impairments in pentylenetetrazole-kindled rats. Epilepsy Behav 2005; 7(4):629–38.