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Anticonvulsant effect of fisetin by modulation of endogenous biomarkers
Biomedicine & Preventive Nutrition 2 (2012) 215–222
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Original article
Anticonvulsant effect of fisetin by modulation of endogenous biomarkers Kiran S. Raygude , Amit D. Kandhare , Pinaki Ghosh , Subhash L. Bodhankar ∗ Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Pune, Maharashtra, 411038, India
a r t i c l e
i n f o
Article history: Received 10 March 2012 Accepted 25 April 2012 Keywords: Anticonvulsant Brain GABA Fisetin Nitric oxide Xanthine oxidase
a b s t r a c t Epilepsy is a group of disorders of the central nervous system (CNS) bearing its pathological origin in paroxysysmal cerebral dysrhythmia, clinically consisting episodes (seizure) of loss of consciousness. Fisetin is a tetrahydroxy flavone that possesses antiyperlipidemic, antioxidant, anti-inflammatory and antidiabetic potential. The aim of present investigation was to investigate the anticonvulsant activity of fisetin (5, 10 and 25 mg/kg) against pentylenetetrazole (PTZ), strychnine (STR), isoniazid (INH) and maximal electroshock (MES) induced convulsions in mice as well as electrical kindling seizures in rats. Diazepam and phenytoin were used as reference anticonvulsant drugs for comparison. Intraperitoneal administration of PTZ (90 mg/kg), strychnine (5 mg/kg) and isoniazid (300 mg/kg) resulted in hind limb, tonic-clonic convulsion along with lethality in mice, whereas twice daily auricular stimulation resulted progressive severity of seizures in rats. It also significantly altered levels of brain gamma amino butyric acid (GABA) along with nitric oxide (NO) and xanthine oxidase (XO) in mice. Treatment with fisetin (10 and 25 mg/kg) delayed onset of convulsion along with duration of tonic-clonic convulsions as well as it significantly reduced PTZ and STR-induced mortality in mice (P < 0.05 – P < 0.001). It also significantly (P < 0.001) reduced severity of electrically kindled seizures in rats and total number of rats seizure per group. Mice treated with fisetin (10 and 25 mg/kg) significantly increased level of brain GABA whereas it significantly decreased elevated level of brain NO and XO. In conclusion, the findings of present study provide pharmacological credence to anticonvulsant profile of fisetin. The protection against the convulsions and restoration of endogenous enzyme level give an innuendo to its probable mechanism of action which may be mediated through the GABAergic pathway and inhibition of oxidative injury. © 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction Epilepsy, an age-old disease, is portrayed by repeated convulsion. Throughout the world, above 60 million people are encumbered with this disease [1]. It is typified by collection of brain disorders whose indications and rationale were varied. Seizure is a consequence of the abnormal neuronal discharge inside the brain, typically at a focal point, which result into the conscription of surrounding brain regions into epileptiform action [2]. It has been well documented that over excitation of excitatory amino acids receptor trigger the formation of reactive oxygen species (ROS), which result in formation of long lasting seizure and if untreated, can lead to neuronal death. Oxidative stress may also play an important role in seizure-induced brain damage [3,4]. The existing clinically accessible antiepileptic drugs are with unwanted side effects as well as persistent toxicities [5]. Hence, there is still need of further research to find out drugs with relatively less side effects.
∗ Corresponding author. Tel.: +91 20 25437237x229; fax: +91 20 25439383. E-mail address: [email protected] (S.L. Bodhankar). 2210-5239/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2012.04.005
The flavonoid fisetin (FST) (3, 3 , 4 , 7-tetrahydroxyflavone) is present in strawberries, apple, grape, onion, persimmon and cucumber [6] (Fig. 1). Fisetin possess antiyperlipidemic, antioxidant property, anti-inflammatory, anticancer, antiallergic and antidiabetic potential [7–9]. It not only inhibits potently lysosomal enzyme secretion, but also release of arachidonic acid in neutrophils of rats [10]. Further, fisetin inhibits mammalian 5-lipoxygenase and cyclo-oxygenase enzymes [11]. Fisetin has also been reported as a potent inhibitor of the human P-form phenolsulfonyl transferase, which reflects its role as chemo-preventive agent in carcinogenesis induced by sulfation [12]. It increases lipolysis in a dose as well as time dependent pattern which is similar to effect of epinephrine on the l-adrenergic receptor [13]. It is also inhibitor of phosphatidylinositol 3-kinase and protein kinase C [14]. Some authors also described fisetin as an inhibitor of tyrosinase and xanthine oxidase (XO), iron chelator and superoxide scavenger [15]. Fisetin promotes the differentiation of nerve cells. The previous studies have shown that the flavonoid fisetin can activate signaling pathways in hippocampal slices which lead to development of longterm memory. The consequence of this activation is the induction of long-term potentiation (LTP) in hippocampal slices and an increase in long-term memory in mice [16].
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administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (diazepam) drug. Immediately after PTZ administration mice were observed for next 30 min for following symptoms: • • • • •
Fig. 1. Structure of the flavonoid fisetin.
The aim of present investigation was to investigate the anticonvulsant activity of fisetin in various models of convulsion by assessing various behavioral and biochemical parameters. 2. Material and method 2.1. Animals Adult male Swiss albino mice (18–22 g) and male Wistar rats (180–200 g) were purchased from National Institute of Biosciences, Pune and kept in quarantine for 1 week in housed at the institute animal house in groups of six animals per cage at standard laboratory conditions at a temperature of 24 ◦ C ± 1 ◦ C, relative humidity of 45–55% and 12:12 h dark and light cycle. The experiments were carried out between 10:00 am to 5:00 pm. Animals had free access to food (standard chaw pellet, Pranav Agro industries Ltd., Sangli, India) and water ad libitum. Experimental protocols and procedures were approved by the Institutional Animal Ethics Committee (IAEC No. CPCSEA/09/2010). Animals were brought to testing laboratory 1 h before the experimentation for adaptation purpose. The experimentation was carried out in noise free area. 2.2. Drugs and solutions Fisetin (Sigma Aldrich, India), pentylenetetrazole (PTZ) (Sigma Aldrich, India), strychnine (STR) (Sigma Aldrich India), phenytoin (PHY) (Eptoin® , Sun Pharma Ltd., India), diazepam (DZP) (Calmpose® , Ranbaxy Ltd., India) and isoniazid (INH) (Solonex® , Macleods, Mumbai, India) were used in present study. All chemicals were dissolved in saline except fisetin was dissolved in 1% DMSO. The doses of fisetin were selected on the basis of previous studies [16]. All other reagents were purchased from S.D. Fine Chemicals, Mumbai, India. 2.3. Assessment of anticonvulsant activity 2.3.1. Pentylenetetrazole (PTZ) induced convulsions The mice were randomly divided into five groups containing six mice in each group as follows: • • • • •
Group I: fisetin (5 mg/kg, intraperitoneal [i.p.]); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: diazepam (5 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
A previously reported protocol was followed to induced convulsion using PTZ [17]. Vehicle, test drug and standard drugs were administered by intraperitoneal (i.p.) route. PTZ (90 mg/kg) was injected intraperitoneally to mice 45 min after intraperitoneal (i.p.)
Onset of convulsion; Duration of clonic convulsion; Duration of tonic convulsion; Incidence (number of mice showing convulsions); Mortality.
2.3.2. Strychnine (STR) induced convulsions The mice were randomly divided into five groups containing six mice in each group as follows: • • • • •
Group I: fisetin (5 mg/kg, i.p.); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: phenytoin (25 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
A previously reported protocol was followed to induced convulsion using STR [18]. Vehicle, test drug and standard drugs were administered by intraperitoneal (i.p.) route. STR (5 mg/kg) was injected intraperitoneally to mice 45 min after intraperitoneal (i.p.) administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (phenytoin) drug. Immediately after STR administration mice were observed for next 60 min for following symptoms: • • • • •
Onset of convulsion; Duration of clonic convulsion; Duration of tonic convulsion; Incidence (number of mice showing convulsions); Mortality.
2.3.3. Isoniazid (INH) induced convulsions The mice were randomly divided into five groups containing six mice in each group as follows: • • • • •
Group I: fisetin (5 mg/kg, i.p.); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: diazepam (5 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
A previously reported protocol was followed to induced convulsion using INH [19]. Vehicle, test drug and standard drugs were administered by intraperitoneal (i.p.) route. INH (300 mg/kg) was injected intraperitoneally to mice 45 min after intraperitoneal (i.p.) administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (diazepam) drug. Immediately after INH administration mice were observed for next 120 min for following symptoms: • • • • •
Onset of convulsion; Duration of clonic convulsion; Duration of tonic convulsion; Incidence (number of mice showing convulsions); Mortality.
2.3.4. Maximal electroshock (MES) induced convulsions The mice were randomly divided into five groups containing six mice in each group as follows:
K.S. Raygude et al. / Biomedicine & Preventive Nutrition 2 (2012) 215–222
• • • • •
Group I: fisetin (5 mg/kg, i.p.); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: phenytoin (50 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
A previously reported protocol was followed to induce convulsion by maximal electroshock (MES) [20]. Convulsions were induced in mice 45 min after intraperitoneal (i.p.) administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (phenytoin) drug by giving a current stimulus (45 mA, 60 Hz for 0.2 sec) by through auricular electrodes by an electroconvulsometer (Dolphin, India). All animals were observed for duration of hind limb tonic extension (HLTE) and percent protection against HLTE. 2.3.5. Electrical kindling convulsions in rats Kindling results from repetitive subconvulsive electrical stimulation of certain areas of the brain. The animals received two subconvulsive electric shocks per day of 21 mA for 0.1 sec, 3 h apart using auricular electrodes, until all animals exhibited grade 4–5 seizure score according to previously described method [21]. All rats were kindled an equal number of times 12 electric shocks i.e. for 6 days. The severity of convulsions was scored as: 0 = normal; 1 = facial movements; 2 = facial movements with head nodding; 3 = facial movements with head nodding and raising of forelimbs with mild forelimb clonus; 4 = marked rearing to a vertical position and moving the head from side to side and forelimb clonus; 5 = symptoms as in step (4) progressing to falling followed by forelimb and hind limb clonic convulsions. On next day, rats were randomly divided into five groups containing six rats in each group as follows: • • • • •
Group I: fisetin (5 mg/kg, i.p.); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: phenytoin (25 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
Kindling stimulus was given 45 min after intraperitoneal (i.p.) administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (diazepam) drug. Convulsion scores of fisetin treated rats were compared with vehicle and diazepam treated animals. 2.4. Biochemical evaluation 2.4.1. Brain GABA estimation 2.4.1.1. Sample preparation. Forty-five min after vehicle (1% DMSO) or fisetin and 30 min after diazepam (5 mg/kg), phenytoin (25 mg/kg) mice were sacrificed. PTZ (90 mg/kg), INH (300 mg/kg) and STR (3 mg/kg) treated animals were sacrificed as soon as onset of convulsions occurs. Animals which received PTZ (90 mg/kg), INH (300 mg/kg) and STR (3 mg/kg) after 45 min of Fisetin (10 and 25 mg/kg) and sacrificed at the exact time of onset of convulsions. Brain was isolated immediately and transferred to homogenization tube containing 5 mL of 0.01 N hydrochloric acid and homogenized. Brain homogenate was transferred to bottle containing 8 mL of icecold absolute alcohol and kept for 1 h at 0 ◦ C. The content was centrifuged for 10 min at 16,000 rpm, supernatant was collected in petridish. Precipitate was washed with 3–5 mL of 75% alcohol for three times and washes were combined with supernatant. Contents in petridish were evaporated to dryness at 70–90 ◦ C on water bath under stream of air. To the dry mass 1 mL water and 2 mL chloroform were added and centrifuged at 2000 rpm. Upper phase
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containing gamma amino butyric acid (GABA) was separated and 10 L of it was applied as spot on Whatman paper (No. 41). 2.4.1.2. Chromatographic conditions. The mobile phase consisted of n-butanol (50 mL) acetic acid (12 mL) and water (60 mL). The chamber was saturated for half hour with mobile phase. The paper chromatogram was developed with ascending technique. The paper was dried in hot air and then spread with 0.5% Ninhydrin solution in 95% ethanol. The paper was dried for 1 h at 90 ◦ C. Blue color spot developed on paper was cut and heated with 2 mL ninhydrin solution on water bath for 5 min water (5 mL) was added to solution and kept for 1 h Supernatant was decanted and absorbance was measured at 570 nm. 2.4.1.3. Standards and calculations. Stock solution of standard GABA, 1 mg/mL was prepared in 0.01 N HCl. Serial dilutions were prepared to get concentrations 1 ng/10 L to 1000 ng/10 L. To obtain a standard concentration curve for GABA same procedure was followed replacing brain homogenate with standard GABA solutions [22]. 2.4.2. Estimation of total protein Protein concentration was estimated according to previously described method [23], using bovine serum albumin (BSA) as a standard. 2.4.3. Estimation of nitrite/nitrate level The NO level was estimated as nitrite by the acidic Griess reaction after reduction of nitrate to nitrite by vanadium trichloride according to previously described method [24]. The Griess reaction relies on a simple colorimetric reaction between nitrite, sulfonamide and n-(1- naphthyl) ethylenediamine to produce a pink azo-product with maximum absorbance at 543 nm. The concentrations were determined using a standard curve of sodium nitrate and the results were expressed as g/mL. 2.4.4. Estimation of xanthine oxidase (XO) level Xanthine oxidase activity was measured spectrophotometrically by the formation of uric acid from xanthine through the increase in absorbency at 293 nm, according to previously described method [25]. The concentrations were determined using a standard curve of XO solutions and results were expressed as units per gram protein in brain homogenate. 2.5. Statistical analysis Data were expressed as mean ± standard error mean (SEM). The data of “brain GABA”, “nitric oxide” and “xanthine oxidase” was analyzed using one-way analysis of variance (ANOVA), Dunnett’s multiple range test was applied for post hoc analysis. Data of “incidence of convulsion” was analyzed by nonparametric Kruskal–Wallis ANOVA. Data of “mortality” was analyzed by Fisher’s exact test. Data of “percentage seizure free rats” was analyzed by using Kaplan–Meier analysis, log rank (Mantel-Cox) test was applied for post hoc analysis. Analysis of all the statistical data was performed using GraphPad Prism 5.0 (GraphPad, San Diego, USA). P < 0.05 was considered as statistically significant. 3. Results 3.1. Effects of fisetin and diazepam on pentylenetetrazole induced convulsions in mice Intraperitoneal administration of PTZ (90 mg/kg) resulted in hind limb, tonic-clonic convulsion along with lethality in mice. Pretreatment with fisetin (5, 10 and 25 mg/kg) significantly and dose
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Table 1 Effects of fisetin and diazepam on pentylenetetrazole induced convulsions in mice. No. convulsed/No. used
Treatment (mg/kg, i.p.) PTZ
DZP
FST
90 90 90 90 90
– 5 – – –
– – 5 10 25
6/6 0/6### 5/6 3/6## 2/6###
% animals protected
0 100 16.66 50 66.67
Onset of convulsion (Sec) (Mean ± S.E.M.)
0.76 29.64 8.76 16.73 22.68
± ± ± ± ±
0.10 0.27*** 0.41* 1.56** 1.33***
Duration of clonic convulsion (Sec) (Mean ± S.E.M.)
78.96 22.84 73.96 68.22 66.22
± ± ± ± ±
3.40 2.76*** 2.80 2.37* 2.59*
Duration of tonic convulsion (Sec) (Mean ± S.E.M.)
58.92 18.72 53.95 38.92 24.18
± ± ± ± ±
2.13 2.06*** 2.32 2.66** 1.52***
Mortality (% Death)
6/6 (100.00) 0/6 (0.00)$$$ 3/6 (50.00)$ 1/6 (16.66)$$ 0/6 (0.00)$$$
Data are expressed as mean ± S.E.M. n = 6 in each group. Comparison was made with vehicle control group for each test. Data of “onset” and “duration” was analyzed by one-way ANOVA followed by Dunnett’s test (* P < 0.05, ** P < 0.01, *** P < 0.001). Data of “incidence of convulsion” was analyzed by Chi2 test (# P < 0.05, ## P < 0.01and ### P < 0.001). Data of “mortality” was analyzed by Fisher’s exact test ($ P < 0.05, $$ P < 0.01, $$$ P < 0.001). FST: fisetin; PTZ: pentylenetetrazole; DZP: diazepam.
dependently (P < 0.05, P < 0.01 and P < 0.001 respectively) delayed onset of convulsion and reduce PTZ-induced mortality in mice as compared to vehicle control mice. Mice received pre-treatment with fisetin (10 and 25 mg/kg) significantly attenuated (P < 0.01 and P < 0.001 respectively) duration of tonic convulsion and significantly reduced duration of clonic convulsions (P < 0.05 and P < 0.01 respectively) as compared to vehicle control mice. It also significantly reduced the total number of animals convulsed per group (P < 0.01 and P < 0.001 respectively) as compared to vehicle control mice. When compared with vehicle control mice diazepam (5 mg/kg) treated mice showed significant delayed (P < 0.001) onset of convulsion and it significantly (P < 0.001) reduced the duration of tonic and clonic convulsion. Mortality induced by PTZ was also significantly attenuated (P < 0.001) by treatment with diazepam (5 mg/kg) (Table 1). 3.2. Effects of fisetin and phenytoin on strychnine-induced convulsions in mice Strychnine (5 mg/kg, i.p.) induced clonic convulsions followed by tonic extension of hind limbs and mortality in mice. When compared to vehicle control mice, fisetin (10 and 25 mg/kg) treated mice showed significant delayed in onset of convulsion as well as significant reduction in the strychnine-induced mortality in the mice (P < 0.01 and P < 0.001 respectively). Total number of the mice that undergo convulsion by treatment of strychnine (5 mg/kg, i.p.) was also significantly attenuated by pre-treatment with fisetin (10 and 25 mg/kg, P < 0.01 and P < 0.001 respectively). Duration of clonic convulsion was significantly reduced by treatment of fisetin (25 mg/kg, P < 0.05) whereas duration of tonic convulsion also significantly attenuated by treatment with fisetin (10 and 25 mg/kg, P < 0.05 and P < 0.01 respectively) as compared to vehicle control mice. Treatment with phenytoin (25 mg/kg) significantly delayed onset of convulsion as well as it significantly reduced duration of tonic-clonic convulsions (P < 0.001). It also significantly attenuated the strychnine-induced mortality in the mice (P < 0.001) (Table 2). 3.3. Effects of fisetin and diazepam on isoniazid induced convulsions in mice Isoniazid (300 mg/kg, i.p.) elicited tonic-clonic convulsions followed by tonic hind limb extension (THLE) and mortality in mice. Treatment with fisetin (25 mg/kg) significantly attenuated (P < 0.05) the INH-induced mortality in the mice and it also significantly delayed (P < 0.05) onset of convulsion as compared to vehicle control mice. Mice treated with fisetin (10 mg/kg) significantly decreased (P < 0.05) duration of clonic convulsion but it failed to produce any significant decrease in duration of tonic convulsion at any dose as compared to vehicle control mice. Treatment with fisetin (10 and 25 mg/kg) significantly reduced the total number
of mice convulsed per group (P < 0.05) as compared to vehicle control mice. As compared to vehicle control mice, diazepam (5 mg/kg) treated mice showed significant protection against INH-induced morality as well as it significantly delayed onset of convulsion and it significantly attenuated duration of tonic-clonic convulsions (P < 0.001) (Table 3). 3.4. Effects of fisetin and phenytoin on MES induced convulsions in mice In MES test, vehicle control mice showed a typical seizure pattern. The tonic flexion of the limbs occurred immediately after the shock, which then progressed into -THLE followed by either stupor and recovery or death. Mice treatment with fisetin (10 and 25 mg/kg) significantly antagonized (P < 0.01, P < 0.001) duration of MES induced THLE as compared to vehicle control mice. When compared to vehicle control mice, treatment with fisetin (5, 10 and 25 mg/kg) showed significant protection (P < 0.05, P < 0.01 and P < 0.001, respectively) against MES induced mortality. Fisetin at a dose of 25 mg/kg showed complete protection against MES induced mortality as well as total number of mice convulsed per group. Treatment with phenytoin (25 mg/kg) showed significant (P < 0.001) protection against MES induced mortality. It also significantly attenuated (P < 0.001) duration of tonic extension of hind limbs in mice as compared to vehicle control mice (Table 4). 3.5. Effects of fisetin and phenytoin on electrical kindling in rats Twice daily auricular stimulation resulted progressive severity of seizures in rats which reached the THLE phase in 6 days, indicating that the rats were fully kindled. Treatment with fisetin (10 and 25 mg/kg) significantly reduced (P < 0.01 and P < 0.001 respectively) severity of electrically kindled seizures (2.8 ± 0.37 and 1.8 ± 0.37) as compared to vehicle control rats (4.6 ± 0.24). Rats treated with phenytoin (25 mg/kg) also significantly (P < 0.001) attenuated severity of electrically kindled seizures (0.20 ± 0.20) when compared to vehicle control rats (Fig. 2). Rats treated with the fisetin (10 and 25 mg/kg) significantly reduced (P < 0.01 and P < 0.001 respectively) total number of rats seizure per group on 6th day as compared to vehicle control rats. However, treatment with phenytoin (25 mg/kg) also significantly attenuated (P < 0.001) total number of rats seizured per group on 6th day when compared to vehicle control rats (Fig. 3). 3.6. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain GABA level Intraperitoneal administration of PTZ (90 mg/kg) and INH (300 mg/kg) resulted in significant decreased (P < 0.001) in brain GABA level (27.22 ± 1.54 and 28.12 ± 1.41 ng/g respectively) as compared to normal mice (50.82 ± 1.32 ng/g). Mice treated with
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Table 2 Effects of fisetin and phenytoin on strychnine-induced convulsions in mice. Treatment (mg/kg, i.p.) STR
PHY
FST
5 5 5 5 5
– 25 – – –
– – 5 10 25
No. convulsed/No. used
% animals protected
Onset of convulsion (Sec) (Mean ± S.E.M.)
6/6 0/6### 6/6 5/6# 3/6##
0 100 0 16.67 66.67
1.68 59.02 7.60 16.98 38.54
± ± ± ± ±
0.13 0.52*** 0.38 0.66** 1.74***
Duration of clonic convulsion (Sec) (Mean ± S.E.M.)
80.82 23.58 71.38 70.30 67.70
± ± ± ± ±
2.33 1.88*** 2.77 1.87 4.28*
Duration of tonic convulsion (Sec) (Mean ± S.E.M.)
61.34 24.98 50.82 46.84 31.66
± ± ± ± ±
2.06 2.05*** 2.33 3.16* 3.02**
Mortality (% Death)
6/6 (100.00) 0/6 (0.00)$$$ 6/6 (100.00) 4/6 (66.67)$ 2/6 (33.33)$$
Data are expressed as mean ± S.E.M. n = 6 in each group. Comparison was made with vehicle control group for each test. Data of “onset” and “duration” was analyzed by one-way ANOVA followed by Dunnett’s test (* P < 0.05, ** P < 0.01, *** P < 0.001). Data of “incidence of convulsion” was analyzed by Chi2 test (# P < 0.05, ## P < 0.01 and ### P < 0.001). Data of “mortality” was analyzed by Fisher’s exact test ($ P < 0.05, $$ P < 0.01, $$$ P < 0.001). PHY: phenytoin; FST: fisetin; STR: strychnine.
Table 3 Effects of fisetin and diazepam on isoniazid induced convulsions in mice. Treatment (mg/kg, i.p.) INH
DZP
FST
300 300 300 300 300
– 5 – – –
– – 5 10 25
No. convulsed/No. used
% animals protected
Onset of convulsion (Sec) (Mean ± S.E.M.)
6/6 0/6### 6/6 5/6# 5/6#
0 100 0 16.67 16.67
51.78 117.7 54.08 66.38 71.56
± ± ± ± ±
2.52 1.22*** 3.72 3.81 6.40*
Duration of clonic convulsion (Sec) (Mean ± S.E.M.)
76.22 21.90 72.52 62.56 67.24
± ± ± ± ±
2.96 2.02*** 2.70 2.63* 2.62
Duration of tonic convulsion (Sec) (Mean ± S.E.M.)
60.17 24.82 57.02 53.70 56.18
± ± ± ± ±
3.03 2.27*** 4.38 4.39 1.94
Mortality (% Death)
6/6 (100.00) 0/6 (0.00)$$$ 6/6 (100.00) 6/6 (100.00) 4/6 (66.67)$
Data are expressed as mean ± S.E.M. n = 6 in each group. Comparison was made with vehicle control group for each test. Data of “onset” and “duration” was analyzed by one-way ANOVA followed by Dunnett’s test (* P < 0.05, ** P < 0.01, *** P < 0.001). Data of “incidence of convulsion” was analyzed by Chi2 test (# P < 0.05, ## P < 0.01 and ### P < 0.001). Data of “mortality” was analyzed by Fisher’s exact test ($ P < 0.05, $$ P < 0.01, $$$ P < 0.001). FST: fisetin; DZP: diazepam; INH: isoniazid.
fisetin (25 mg/kg) significantly decrease brain GABA level in PTZ treated rats (42.70 ± 1.94 ng/g, P < 0.001) as compared to vehicle control mice. Fisetin (10 mg/kg) treated rats significantly attenuated decreased level of brain GABA in PTZ treated rats (36.66 ± 1.47 ng/g, P < 0.01) as compared to vehicle control mice. Mice treated with fisetin (10 and 25 mg/kg) failed to produce any significant change in brain GABA level in INH (300 mg/kg, i.p.) treated mice as compared to vehicle control mice. When compared with vehicle control mice, diazepam (5 mg/kg) and phenytoin (25 mg/kg) treated mice produced significant increase in brain GABA level in PTZ and INH treated mice (48.44 ± 1.89 and 43.02 ±1.82 ng/g respectively, P < 0.001) (Fig. 4). 3.7. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain nitric oxide level Brain NO level in PTZ (90 mg/kg, i.p.), STR (5 mg/kg, i.p.) and INH (300 mg/kg, i.p.) treated mice (0.214 ± 0.0067, 0.216 ± 0.0163 and 0.196 ± 0.0092 mole/g respectively) was significantly increased (P < 0.001) as compared to normal mice (0.124 ± 0.012 mole/g). Treatment with fisetin (25 mg/kg) significantly attenuated this
elevated level of brain NO in PTZ and STR treated rats (0.156 ± 0.0092 and 0.136 ± 0.008 mole/g, P < 0.01 and P < 0.001 respectively) as compared to vehicle control mice. Mice treated with fisetin (10 mg/kg) significantly decreased brain NO level (0.162 ± 0.006 mole/g, P < 0.01) in STR treated mice, however in PTZ treated mice it failed to do so as compared to vehicle control mice. When compared to vehicle control mice, mice treated with fisetin (10 and 25 mg/kg) failed to produce any significant decreased in brain NO level in INH (300 mg/kg, i.p.) treated mice as compared to vehicle control mice. Mice treated with diazepam (5 mg/kg) and phenytoin (25 mg/kg) significantly antagonized elevated level of NO in brain in PTZ, INH and STR treated mice (0.146 ± 0.008, 0.158 ± 0.006 and 0.142 ± 0.011 mole/g respectively, P < 0.001) (Fig. 5). 3.8. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain xanthine oxidase level Mice treated with PTZ (90 mg/kg, i.p.), STR (5 mg/kg, i.p.) and INH (300 mg/kg, i.p.) results significant increase (7.36 ± 0.32, 6.52 ± 0.53 and 6.84 ± 0.41 U/g respectively, P < 0.001) in brain XO
Table 4 Effects of fisetin and phenytoin on maximal electroshock (MES) induced convulsions in mice. Treatment (mg/kg, i.p.) PHY – 25 – – –
No. convulsed/No. used
% animals protected
Duration of THLE (Sec) (Mean ± S.E.M.)
6/6 0/6### 5/6 2/6## 0/6###
0 100 16.67 66.67 100.00
16.10 0.14 11.75 6.86 1.23
Mortality (% Death)
FST – – 5 10 25
± ± ± ± ±
1.24 0.07*** 1.13 0.86** 0.16***
6/6 (100.00) 0/6 (0.00)$$$ 4/6 (66.67)$ 2/6 (33.33)$$ 0/6 (0.00)$$$
Data are expressed as mean ± S.E.M. n = 6 in each group. Comparison was made with vehicle control group for each test. Data of “duration” was analyzed by one-way ANOVA followed by Dunnett’s test (* P < 0.05, ** P < 0.01, *** P < 0.001). Data of “incidence of convulsion” was analyzed by Chi2 test (# P < 0.05, ## P < 0.01and ### P < 0.001). Data of “mortality” was analyzed by Fisher’s exact test ($ P < 0.05, $$ P < 0.01, $$$ P < 0.001). PHY: phenytoin; FST: fisetin; THLE: tonic hind limb extension.
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level as compared to normal mice (3.24 ± 0.35 U/g). Treatment with fisetin (25 mg/kg) resulted significant decrease in brain XO level (4.42 ± 0.38 and 4.14 ± 0.50 U/g, P < 0.01 and P < 0.05 respectively) as compared to PTZ and STR control mice. In PTZ treated mice elevated level of brain XO was significantly antagonized by pre-treatment of fisetin (10 mg/kg) (5.10 ± 0.48 U/g, P < 0.05) as compared to vehicle control animals whereas in STR treated mice it failed to produced any significant effect in level of brain XO. When compared with vehicle control mice, treatment with diazepam (5 mg/kg) and phenytoin (25 mg/kg) significantly attenuated this elevated level of brain XO in PTZ, INH and STR treated mice (3.82 ± 0.38, 3.68 ± 0.38 and 3.84 ± 0.56 U/g respectively, P < 0.001) (Fig. 6).
4. Discussion
Fig. 2. Effects of fisetin and phenytoin on seizure intensity in fully kindled rats. Data are expressed as mean ± S.E.M. (n = 6 in each group). Comparison was made with vehicle control group for each test. Data was analyzed by one-way ANOVA followed by Dunnett’s test * P < 0.05, ** P < 0.01 and *** P < 0.001 as compared to vehicle control rats.
Fig. 3. Kaplan–Meier analysis of the time of spontaneous seizure appearance after subconvulsive electrical stimulation. The log rank test revealed a significant (* P < 0.05, ** P < 0.01, *** P < 0.001) difference between the different group (n = 6).
Fig. 4. Effects of fisetin, diazepam and phenytoin on PTZ- and INH-induced alteration in brain GABA level. Data are expressed as mean ± S.E.M. (n = 6 in each group) and analyzed by one-way ANOVA followed by Dunnett’s test. Comparison was made with vehicle control group for each test. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to PTZ treated mice. @ P < 0.05, @@ P < 0.01, @@@ P < 0.001 as compared to INH treated mice. # P < 0.05, ## P < 0.01, ### P < 0.001 as compared to normal mice and & P < 0.05, && P < 0.01, &&& P < 0.001 as compared to one another.
Epilepsy is a common chronic neurological disorder caused due to tumours, degenerative conditions or cerebrovascular diseases. The imbalance between excitatory and inhibitory neurotransmission in the brain is an important characteristic of experimental and clinical seizure [21]. Nerve cell depolarization occurres due to the predominancy of excitatory postsynaptic potentials (EPSP) over the inhibitory (IPSP) resulting in the generation of the seizures in the brain. Epilepsy is precipitated due to an array of factors including elevated electrolytes (Na+ , K+ , Ca2+ ) levels, excitatory amino acids (glutamic acid), and inhibitory amino acids (GABA), irregular interneuron connections and abnormal afferent connections from subcortical structures which modulate various intertwining biochemical pathways giving rise to discharges of large numbers of neurons resulting in an epileptic seizure [26]. Depending upon their mechanism of action classical anticonvulsant drugs have been broadly classified into various categories and they are effective against partial and tonic-clonic seizures [27]. The potent antiepileptic drugs act by either inhibiting the voltage gated Na+ channels activation resulting in inhibition of firing of neurons or facilitating pre- or postsynaptic GABA-mediated synaptic transmission and inhibition. This cause enhanced membrane polarization by influx of Cl− ions through GABAA receptor. Some of them also act by inhibiting the GABA metabolism [27]. In the present investigation the pharmacological screening models were selected on the basis of mechanisms involved in the anticonvulsant activity of drugs. PTZ-induced convulsion in mice has been used to screen various drugs as it mimics an array of clinicopathobiological feature of human syndrome [28]. It has been reported that PTZ-induced convulsion model is widely used to identify compounds that are effective against absence and myoclonic seizures. In PTZ-induced convulsions model, PTZ imparts convulsions by its inhibitory effect on GABA-mediated Cl− influx through an allosteric interaction in the Cl− channel [29]. Previously it has been shown that diazepam enhances the GABAergic neurotransmission and gives protection against the PTZ-induced convulsion [30]. Fisetin provides protection against the PTZ-induced convulsions suggesting anticonvulsant property by modulation of GABAergic pathways in brain. In the present investigation, it was found that fisetin increases the level of the brain GABA which provides credence to its anticonvulsant activity by GABA level regulation in brain. STR is a selective competitive antagonist of glycine which blocks its inhibitory effect to produces convulsions. Glycine is vital inhibitory neurotransmitter to motor neurons and interneurons in the brain and other areas of the central nervous system (CNS) [31]. Fisetin and phenytoin exerted their protective effect through their action on glycinergic pathway. INH produces convulsion by interfering with GABA synthesis via down regulation of glutamic acid decarboxylase (GAD)
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Fig. 5. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain nitric oxide level. Data are expressed as mean ± S.E.M. (n = 6 in each group) and analyzed by one-way ANOVA followed by Dunnett’s test. Comparison was made with vehicle control group for each test. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to PTZ treated mice. $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 as compared to STR treated mice. @ P < 0.05, @@ P < 0.01, @@@ P < 0.001 as compared to INH treated mice. # P < 0.05, ## P < 0.01, ### P < 0.001 as compared to normal mice and & P < 0.05, && P < 0.01, &&& P < 0.001 as compared to one another.
activity, resulting in decreased level of GABA [32]. Diazepam produces significant protection against INH-induced convulsions, but fisetin failed to protect against INH-induced seizures and mortality indicating its moderate action on the GABA synthesis mechanism. The drugs which produce effect on Na+ channels are widely used to screen against MES induced convulsions. These drugs are proven to be effective against partial and tonic-clonic seizures in humans [33]. Phenytoin and fisetin exhibited anticonvulsant profile effect via inhibition of the Na2+ channels in partial and tonic-clonic seizures. The focal electrical stimulation is widely used model in neurology and neurosurgery of clinical procedure as well as experimental studies of brain functions. The repetitive administration of highfrequency electrical stimulation of the amygdala as well as hippocampus leads to the development of generalized electrographic and behavioral seizures generally referred as kindling [34]. Phenytoin and fisetin significantly decreases seizure intensity in kindled rats induced by electrical stimulation. It has been documented that oxidative stress plays an important role in the central nervous system in various models of experimental epilepsy [3,35]. Recent studies using 3-mercaptoproprionic
acid suggested that oxidative stress is associated with seizures [36]. Induction of the seizure by means of variety of chemicals result in initiation of array of biochemical processes including membrane phospholipases activation, proteases, nucleases and lipoperoxidation in hippocampus. These alterations result in liberation of free fatty acids, diacylglycerols, eicosanoids and free radicals. Xanthine oxidase plays a pivotal role in generation of oxygen free radicals by degradation of adenine nucleotides [37]. The elevated level of oxygen free radicals brings an array of reactions that are harmful to the neuronal tissue. Hence antioxidants play an important role in the protection of cellular components from free fatty acids, diacylglycerols, eicosanoids and free radicals by quenching the superoxide anion, hydroxyl and peroxyl radicals. Fisetin is a potent antioxidant [7]. The mice pre-treated with fisetin showed decreased activity of this xanthine oxidase (XO) enzyme, which suggests that fisetin may have ability to protect the cellular components against the oxidative stress generated during seizures. Nitric oxide (NO) is an endogenously produced intercellular signaling molecule [38] and depending upon the stimulants and NO related chemicals it may act as an anticonvulsant or a proconvulsant [39,40]. Result of the present investigation reveals that by inhibiting
Fig. 6. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain xanthine oxidase level. Data are expressed as mean ± S.E.M. (n = 6 in each group) and analyzed by one-way ANOVA followed by Dunnett’s test. Comparison was made with vehicle control group for each test. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to PTZ treated mice. $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 as compared to STR treated mice. @ P < 0.05, @@ P < 0.01, @@@ P < 0.001 as compared to INH treated mice. # P < 0.05, ## P < 0.01, ### P < 0.001 as compared to normal mice and & P < 0.05, && P < 0.01, &&& P < 0.001 as compared to one another.
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the level of brain GABA, there is an increase glutamate release which may consequently increase NO production in the hippocampus. Our results are in accordance with the previous findings [41]. In the present investigation, treatment with fisetin restored the elevated level of nitric oxide. PTZ produces specific type of convulsion that is generalized by clonic convulsion whereas MES produces characteristic generalized tonic-clonic impartial convulsions [34,42]. Therefore, our results suggest that fisetin may be useful in the above stated seizure types in human beings. STR produces reflex tonic-clonic and symmetrical types of convulsions and stimulates the whole cerebrospinal axis [43]. Hence, the protective action of fisetin against STR-induced convulsions provides evidence to the theory that it may be useful in amelioration of various types of convulsion. The drugs that are effective against tonic hind limb extension induced by electroshock generally have proven to be effective against partial and tonic-clonic seizures and those in PTZ model are effective against absence seizure in human beings [27]. It could be concluded from the present investigation that fisetin protects endogenous enzyme level, inhibits oxidative damage and modulates GABAergic transmission to exhibit anticonvulsant effect. Further studies are needed to unravel its mechanism of action.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements The authors would like to acknowledge Dr. S. S. Kadam, ViceChancellor and Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, India, for providing necessary facilities to carry out the study. We are also thankful to the All India Council of Technical Education (AICTE), India for financial support by awarding GATE Scholarship to one of the author (Mr. Kiran S. Raygude) for the research work.
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Original article
Anticonvulsant effect of fisetin by modulation of endogenous biomarkers Kiran S. Raygude , Amit D. Kandhare , Pinaki Ghosh , Subhash L. Bodhankar ∗ Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Pune, Maharashtra, 411038, India
a r t i c l e
i n f o
Article history: Received 10 March 2012 Accepted 25 April 2012 Keywords: Anticonvulsant Brain GABA Fisetin Nitric oxide Xanthine oxidase
a b s t r a c t Epilepsy is a group of disorders of the central nervous system (CNS) bearing its pathological origin in paroxysysmal cerebral dysrhythmia, clinically consisting episodes (seizure) of loss of consciousness. Fisetin is a tetrahydroxy flavone that possesses antiyperlipidemic, antioxidant, anti-inflammatory and antidiabetic potential. The aim of present investigation was to investigate the anticonvulsant activity of fisetin (5, 10 and 25 mg/kg) against pentylenetetrazole (PTZ), strychnine (STR), isoniazid (INH) and maximal electroshock (MES) induced convulsions in mice as well as electrical kindling seizures in rats. Diazepam and phenytoin were used as reference anticonvulsant drugs for comparison. Intraperitoneal administration of PTZ (90 mg/kg), strychnine (5 mg/kg) and isoniazid (300 mg/kg) resulted in hind limb, tonic-clonic convulsion along with lethality in mice, whereas twice daily auricular stimulation resulted progressive severity of seizures in rats. It also significantly altered levels of brain gamma amino butyric acid (GABA) along with nitric oxide (NO) and xanthine oxidase (XO) in mice. Treatment with fisetin (10 and 25 mg/kg) delayed onset of convulsion along with duration of tonic-clonic convulsions as well as it significantly reduced PTZ and STR-induced mortality in mice (P < 0.05 – P < 0.001). It also significantly (P < 0.001) reduced severity of electrically kindled seizures in rats and total number of rats seizure per group. Mice treated with fisetin (10 and 25 mg/kg) significantly increased level of brain GABA whereas it significantly decreased elevated level of brain NO and XO. In conclusion, the findings of present study provide pharmacological credence to anticonvulsant profile of fisetin. The protection against the convulsions and restoration of endogenous enzyme level give an innuendo to its probable mechanism of action which may be mediated through the GABAergic pathway and inhibition of oxidative injury. © 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction Epilepsy, an age-old disease, is portrayed by repeated convulsion. Throughout the world, above 60 million people are encumbered with this disease [1]. It is typified by collection of brain disorders whose indications and rationale were varied. Seizure is a consequence of the abnormal neuronal discharge inside the brain, typically at a focal point, which result into the conscription of surrounding brain regions into epileptiform action [2]. It has been well documented that over excitation of excitatory amino acids receptor trigger the formation of reactive oxygen species (ROS), which result in formation of long lasting seizure and if untreated, can lead to neuronal death. Oxidative stress may also play an important role in seizure-induced brain damage [3,4]. The existing clinically accessible antiepileptic drugs are with unwanted side effects as well as persistent toxicities [5]. Hence, there is still need of further research to find out drugs with relatively less side effects.
∗ Corresponding author. Tel.: +91 20 25437237x229; fax: +91 20 25439383. E-mail address: [email protected] (S.L. Bodhankar). 2210-5239/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2012.04.005
The flavonoid fisetin (FST) (3, 3 , 4 , 7-tetrahydroxyflavone) is present in strawberries, apple, grape, onion, persimmon and cucumber [6] (Fig. 1). Fisetin possess antiyperlipidemic, antioxidant property, anti-inflammatory, anticancer, antiallergic and antidiabetic potential [7–9]. It not only inhibits potently lysosomal enzyme secretion, but also release of arachidonic acid in neutrophils of rats [10]. Further, fisetin inhibits mammalian 5-lipoxygenase and cyclo-oxygenase enzymes [11]. Fisetin has also been reported as a potent inhibitor of the human P-form phenolsulfonyl transferase, which reflects its role as chemo-preventive agent in carcinogenesis induced by sulfation [12]. It increases lipolysis in a dose as well as time dependent pattern which is similar to effect of epinephrine on the l-adrenergic receptor [13]. It is also inhibitor of phosphatidylinositol 3-kinase and protein kinase C [14]. Some authors also described fisetin as an inhibitor of tyrosinase and xanthine oxidase (XO), iron chelator and superoxide scavenger [15]. Fisetin promotes the differentiation of nerve cells. The previous studies have shown that the flavonoid fisetin can activate signaling pathways in hippocampal slices which lead to development of longterm memory. The consequence of this activation is the induction of long-term potentiation (LTP) in hippocampal slices and an increase in long-term memory in mice [16].
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administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (diazepam) drug. Immediately after PTZ administration mice were observed for next 30 min for following symptoms: • • • • •
Fig. 1. Structure of the flavonoid fisetin.
The aim of present investigation was to investigate the anticonvulsant activity of fisetin in various models of convulsion by assessing various behavioral and biochemical parameters. 2. Material and method 2.1. Animals Adult male Swiss albino mice (18–22 g) and male Wistar rats (180–200 g) were purchased from National Institute of Biosciences, Pune and kept in quarantine for 1 week in housed at the institute animal house in groups of six animals per cage at standard laboratory conditions at a temperature of 24 ◦ C ± 1 ◦ C, relative humidity of 45–55% and 12:12 h dark and light cycle. The experiments were carried out between 10:00 am to 5:00 pm. Animals had free access to food (standard chaw pellet, Pranav Agro industries Ltd., Sangli, India) and water ad libitum. Experimental protocols and procedures were approved by the Institutional Animal Ethics Committee (IAEC No. CPCSEA/09/2010). Animals were brought to testing laboratory 1 h before the experimentation for adaptation purpose. The experimentation was carried out in noise free area. 2.2. Drugs and solutions Fisetin (Sigma Aldrich, India), pentylenetetrazole (PTZ) (Sigma Aldrich, India), strychnine (STR) (Sigma Aldrich India), phenytoin (PHY) (Eptoin® , Sun Pharma Ltd., India), diazepam (DZP) (Calmpose® , Ranbaxy Ltd., India) and isoniazid (INH) (Solonex® , Macleods, Mumbai, India) were used in present study. All chemicals were dissolved in saline except fisetin was dissolved in 1% DMSO. The doses of fisetin were selected on the basis of previous studies [16]. All other reagents were purchased from S.D. Fine Chemicals, Mumbai, India. 2.3. Assessment of anticonvulsant activity 2.3.1. Pentylenetetrazole (PTZ) induced convulsions The mice were randomly divided into five groups containing six mice in each group as follows: • • • • •
Group I: fisetin (5 mg/kg, intraperitoneal [i.p.]); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: diazepam (5 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
A previously reported protocol was followed to induced convulsion using PTZ [17]. Vehicle, test drug and standard drugs were administered by intraperitoneal (i.p.) route. PTZ (90 mg/kg) was injected intraperitoneally to mice 45 min after intraperitoneal (i.p.)
Onset of convulsion; Duration of clonic convulsion; Duration of tonic convulsion; Incidence (number of mice showing convulsions); Mortality.
2.3.2. Strychnine (STR) induced convulsions The mice were randomly divided into five groups containing six mice in each group as follows: • • • • •
Group I: fisetin (5 mg/kg, i.p.); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: phenytoin (25 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
A previously reported protocol was followed to induced convulsion using STR [18]. Vehicle, test drug and standard drugs were administered by intraperitoneal (i.p.) route. STR (5 mg/kg) was injected intraperitoneally to mice 45 min after intraperitoneal (i.p.) administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (phenytoin) drug. Immediately after STR administration mice were observed for next 60 min for following symptoms: • • • • •
Onset of convulsion; Duration of clonic convulsion; Duration of tonic convulsion; Incidence (number of mice showing convulsions); Mortality.
2.3.3. Isoniazid (INH) induced convulsions The mice were randomly divided into five groups containing six mice in each group as follows: • • • • •
Group I: fisetin (5 mg/kg, i.p.); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: diazepam (5 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
A previously reported protocol was followed to induced convulsion using INH [19]. Vehicle, test drug and standard drugs were administered by intraperitoneal (i.p.) route. INH (300 mg/kg) was injected intraperitoneally to mice 45 min after intraperitoneal (i.p.) administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (diazepam) drug. Immediately after INH administration mice were observed for next 120 min for following symptoms: • • • • •
Onset of convulsion; Duration of clonic convulsion; Duration of tonic convulsion; Incidence (number of mice showing convulsions); Mortality.
2.3.4. Maximal electroshock (MES) induced convulsions The mice were randomly divided into five groups containing six mice in each group as follows:
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• • • • •
Group I: fisetin (5 mg/kg, i.p.); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: phenytoin (50 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
A previously reported protocol was followed to induce convulsion by maximal electroshock (MES) [20]. Convulsions were induced in mice 45 min after intraperitoneal (i.p.) administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (phenytoin) drug by giving a current stimulus (45 mA, 60 Hz for 0.2 sec) by through auricular electrodes by an electroconvulsometer (Dolphin, India). All animals were observed for duration of hind limb tonic extension (HLTE) and percent protection against HLTE. 2.3.5. Electrical kindling convulsions in rats Kindling results from repetitive subconvulsive electrical stimulation of certain areas of the brain. The animals received two subconvulsive electric shocks per day of 21 mA for 0.1 sec, 3 h apart using auricular electrodes, until all animals exhibited grade 4–5 seizure score according to previously described method [21]. All rats were kindled an equal number of times 12 electric shocks i.e. for 6 days. The severity of convulsions was scored as: 0 = normal; 1 = facial movements; 2 = facial movements with head nodding; 3 = facial movements with head nodding and raising of forelimbs with mild forelimb clonus; 4 = marked rearing to a vertical position and moving the head from side to side and forelimb clonus; 5 = symptoms as in step (4) progressing to falling followed by forelimb and hind limb clonic convulsions. On next day, rats were randomly divided into five groups containing six rats in each group as follows: • • • • •
Group I: fisetin (5 mg/kg, i.p.); Group II: fisetin (10 mg/kg, i.p.); Group III: fisetin (25 mg/kg, i.p.); Group IV: phenytoin (25 mg/kg, i.p.); Group V: vehicle control (1% DMSO. i.p.).
Kindling stimulus was given 45 min after intraperitoneal (i.p.) administration of vehicle (1% DMSO) or test drug (fisetin) and 30 min after the standard (diazepam) drug. Convulsion scores of fisetin treated rats were compared with vehicle and diazepam treated animals. 2.4. Biochemical evaluation 2.4.1. Brain GABA estimation 2.4.1.1. Sample preparation. Forty-five min after vehicle (1% DMSO) or fisetin and 30 min after diazepam (5 mg/kg), phenytoin (25 mg/kg) mice were sacrificed. PTZ (90 mg/kg), INH (300 mg/kg) and STR (3 mg/kg) treated animals were sacrificed as soon as onset of convulsions occurs. Animals which received PTZ (90 mg/kg), INH (300 mg/kg) and STR (3 mg/kg) after 45 min of Fisetin (10 and 25 mg/kg) and sacrificed at the exact time of onset of convulsions. Brain was isolated immediately and transferred to homogenization tube containing 5 mL of 0.01 N hydrochloric acid and homogenized. Brain homogenate was transferred to bottle containing 8 mL of icecold absolute alcohol and kept for 1 h at 0 ◦ C. The content was centrifuged for 10 min at 16,000 rpm, supernatant was collected in petridish. Precipitate was washed with 3–5 mL of 75% alcohol for three times and washes were combined with supernatant. Contents in petridish were evaporated to dryness at 70–90 ◦ C on water bath under stream of air. To the dry mass 1 mL water and 2 mL chloroform were added and centrifuged at 2000 rpm. Upper phase
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containing gamma amino butyric acid (GABA) was separated and 10 L of it was applied as spot on Whatman paper (No. 41). 2.4.1.2. Chromatographic conditions. The mobile phase consisted of n-butanol (50 mL) acetic acid (12 mL) and water (60 mL). The chamber was saturated for half hour with mobile phase. The paper chromatogram was developed with ascending technique. The paper was dried in hot air and then spread with 0.5% Ninhydrin solution in 95% ethanol. The paper was dried for 1 h at 90 ◦ C. Blue color spot developed on paper was cut and heated with 2 mL ninhydrin solution on water bath for 5 min water (5 mL) was added to solution and kept for 1 h Supernatant was decanted and absorbance was measured at 570 nm. 2.4.1.3. Standards and calculations. Stock solution of standard GABA, 1 mg/mL was prepared in 0.01 N HCl. Serial dilutions were prepared to get concentrations 1 ng/10 L to 1000 ng/10 L. To obtain a standard concentration curve for GABA same procedure was followed replacing brain homogenate with standard GABA solutions [22]. 2.4.2. Estimation of total protein Protein concentration was estimated according to previously described method [23], using bovine serum albumin (BSA) as a standard. 2.4.3. Estimation of nitrite/nitrate level The NO level was estimated as nitrite by the acidic Griess reaction after reduction of nitrate to nitrite by vanadium trichloride according to previously described method [24]. The Griess reaction relies on a simple colorimetric reaction between nitrite, sulfonamide and n-(1- naphthyl) ethylenediamine to produce a pink azo-product with maximum absorbance at 543 nm. The concentrations were determined using a standard curve of sodium nitrate and the results were expressed as g/mL. 2.4.4. Estimation of xanthine oxidase (XO) level Xanthine oxidase activity was measured spectrophotometrically by the formation of uric acid from xanthine through the increase in absorbency at 293 nm, according to previously described method [25]. The concentrations were determined using a standard curve of XO solutions and results were expressed as units per gram protein in brain homogenate. 2.5. Statistical analysis Data were expressed as mean ± standard error mean (SEM). The data of “brain GABA”, “nitric oxide” and “xanthine oxidase” was analyzed using one-way analysis of variance (ANOVA), Dunnett’s multiple range test was applied for post hoc analysis. Data of “incidence of convulsion” was analyzed by nonparametric Kruskal–Wallis ANOVA. Data of “mortality” was analyzed by Fisher’s exact test. Data of “percentage seizure free rats” was analyzed by using Kaplan–Meier analysis, log rank (Mantel-Cox) test was applied for post hoc analysis. Analysis of all the statistical data was performed using GraphPad Prism 5.0 (GraphPad, San Diego, USA). P < 0.05 was considered as statistically significant. 3. Results 3.1. Effects of fisetin and diazepam on pentylenetetrazole induced convulsions in mice Intraperitoneal administration of PTZ (90 mg/kg) resulted in hind limb, tonic-clonic convulsion along with lethality in mice. Pretreatment with fisetin (5, 10 and 25 mg/kg) significantly and dose
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Table 1 Effects of fisetin and diazepam on pentylenetetrazole induced convulsions in mice. No. convulsed/No. used
Treatment (mg/kg, i.p.) PTZ
DZP
FST
90 90 90 90 90
– 5 – – –
– – 5 10 25
6/6 0/6### 5/6 3/6## 2/6###
% animals protected
0 100 16.66 50 66.67
Onset of convulsion (Sec) (Mean ± S.E.M.)
0.76 29.64 8.76 16.73 22.68
± ± ± ± ±
0.10 0.27*** 0.41* 1.56** 1.33***
Duration of clonic convulsion (Sec) (Mean ± S.E.M.)
78.96 22.84 73.96 68.22 66.22
± ± ± ± ±
3.40 2.76*** 2.80 2.37* 2.59*
Duration of tonic convulsion (Sec) (Mean ± S.E.M.)
58.92 18.72 53.95 38.92 24.18
± ± ± ± ±
2.13 2.06*** 2.32 2.66** 1.52***
Mortality (% Death)
6/6 (100.00) 0/6 (0.00)$$$ 3/6 (50.00)$ 1/6 (16.66)$$ 0/6 (0.00)$$$
Data are expressed as mean ± S.E.M. n = 6 in each group. Comparison was made with vehicle control group for each test. Data of “onset” and “duration” was analyzed by one-way ANOVA followed by Dunnett’s test (* P < 0.05, ** P < 0.01, *** P < 0.001). Data of “incidence of convulsion” was analyzed by Chi2 test (# P < 0.05, ## P < 0.01and ### P < 0.001). Data of “mortality” was analyzed by Fisher’s exact test ($ P < 0.05, $$ P < 0.01, $$$ P < 0.001). FST: fisetin; PTZ: pentylenetetrazole; DZP: diazepam.
dependently (P < 0.05, P < 0.01 and P < 0.001 respectively) delayed onset of convulsion and reduce PTZ-induced mortality in mice as compared to vehicle control mice. Mice received pre-treatment with fisetin (10 and 25 mg/kg) significantly attenuated (P < 0.01 and P < 0.001 respectively) duration of tonic convulsion and significantly reduced duration of clonic convulsions (P < 0.05 and P < 0.01 respectively) as compared to vehicle control mice. It also significantly reduced the total number of animals convulsed per group (P < 0.01 and P < 0.001 respectively) as compared to vehicle control mice. When compared with vehicle control mice diazepam (5 mg/kg) treated mice showed significant delayed (P < 0.001) onset of convulsion and it significantly (P < 0.001) reduced the duration of tonic and clonic convulsion. Mortality induced by PTZ was also significantly attenuated (P < 0.001) by treatment with diazepam (5 mg/kg) (Table 1). 3.2. Effects of fisetin and phenytoin on strychnine-induced convulsions in mice Strychnine (5 mg/kg, i.p.) induced clonic convulsions followed by tonic extension of hind limbs and mortality in mice. When compared to vehicle control mice, fisetin (10 and 25 mg/kg) treated mice showed significant delayed in onset of convulsion as well as significant reduction in the strychnine-induced mortality in the mice (P < 0.01 and P < 0.001 respectively). Total number of the mice that undergo convulsion by treatment of strychnine (5 mg/kg, i.p.) was also significantly attenuated by pre-treatment with fisetin (10 and 25 mg/kg, P < 0.01 and P < 0.001 respectively). Duration of clonic convulsion was significantly reduced by treatment of fisetin (25 mg/kg, P < 0.05) whereas duration of tonic convulsion also significantly attenuated by treatment with fisetin (10 and 25 mg/kg, P < 0.05 and P < 0.01 respectively) as compared to vehicle control mice. Treatment with phenytoin (25 mg/kg) significantly delayed onset of convulsion as well as it significantly reduced duration of tonic-clonic convulsions (P < 0.001). It also significantly attenuated the strychnine-induced mortality in the mice (P < 0.001) (Table 2). 3.3. Effects of fisetin and diazepam on isoniazid induced convulsions in mice Isoniazid (300 mg/kg, i.p.) elicited tonic-clonic convulsions followed by tonic hind limb extension (THLE) and mortality in mice. Treatment with fisetin (25 mg/kg) significantly attenuated (P < 0.05) the INH-induced mortality in the mice and it also significantly delayed (P < 0.05) onset of convulsion as compared to vehicle control mice. Mice treated with fisetin (10 mg/kg) significantly decreased (P < 0.05) duration of clonic convulsion but it failed to produce any significant decrease in duration of tonic convulsion at any dose as compared to vehicle control mice. Treatment with fisetin (10 and 25 mg/kg) significantly reduced the total number
of mice convulsed per group (P < 0.05) as compared to vehicle control mice. As compared to vehicle control mice, diazepam (5 mg/kg) treated mice showed significant protection against INH-induced morality as well as it significantly delayed onset of convulsion and it significantly attenuated duration of tonic-clonic convulsions (P < 0.001) (Table 3). 3.4. Effects of fisetin and phenytoin on MES induced convulsions in mice In MES test, vehicle control mice showed a typical seizure pattern. The tonic flexion of the limbs occurred immediately after the shock, which then progressed into -THLE followed by either stupor and recovery or death. Mice treatment with fisetin (10 and 25 mg/kg) significantly antagonized (P < 0.01, P < 0.001) duration of MES induced THLE as compared to vehicle control mice. When compared to vehicle control mice, treatment with fisetin (5, 10 and 25 mg/kg) showed significant protection (P < 0.05, P < 0.01 and P < 0.001, respectively) against MES induced mortality. Fisetin at a dose of 25 mg/kg showed complete protection against MES induced mortality as well as total number of mice convulsed per group. Treatment with phenytoin (25 mg/kg) showed significant (P < 0.001) protection against MES induced mortality. It also significantly attenuated (P < 0.001) duration of tonic extension of hind limbs in mice as compared to vehicle control mice (Table 4). 3.5. Effects of fisetin and phenytoin on electrical kindling in rats Twice daily auricular stimulation resulted progressive severity of seizures in rats which reached the THLE phase in 6 days, indicating that the rats were fully kindled. Treatment with fisetin (10 and 25 mg/kg) significantly reduced (P < 0.01 and P < 0.001 respectively) severity of electrically kindled seizures (2.8 ± 0.37 and 1.8 ± 0.37) as compared to vehicle control rats (4.6 ± 0.24). Rats treated with phenytoin (25 mg/kg) also significantly (P < 0.001) attenuated severity of electrically kindled seizures (0.20 ± 0.20) when compared to vehicle control rats (Fig. 2). Rats treated with the fisetin (10 and 25 mg/kg) significantly reduced (P < 0.01 and P < 0.001 respectively) total number of rats seizure per group on 6th day as compared to vehicle control rats. However, treatment with phenytoin (25 mg/kg) also significantly attenuated (P < 0.001) total number of rats seizured per group on 6th day when compared to vehicle control rats (Fig. 3). 3.6. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain GABA level Intraperitoneal administration of PTZ (90 mg/kg) and INH (300 mg/kg) resulted in significant decreased (P < 0.001) in brain GABA level (27.22 ± 1.54 and 28.12 ± 1.41 ng/g respectively) as compared to normal mice (50.82 ± 1.32 ng/g). Mice treated with
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Table 2 Effects of fisetin and phenytoin on strychnine-induced convulsions in mice. Treatment (mg/kg, i.p.) STR
PHY
FST
5 5 5 5 5
– 25 – – –
– – 5 10 25
No. convulsed/No. used
% animals protected
Onset of convulsion (Sec) (Mean ± S.E.M.)
6/6 0/6### 6/6 5/6# 3/6##
0 100 0 16.67 66.67
1.68 59.02 7.60 16.98 38.54
± ± ± ± ±
0.13 0.52*** 0.38 0.66** 1.74***
Duration of clonic convulsion (Sec) (Mean ± S.E.M.)
80.82 23.58 71.38 70.30 67.70
± ± ± ± ±
2.33 1.88*** 2.77 1.87 4.28*
Duration of tonic convulsion (Sec) (Mean ± S.E.M.)
61.34 24.98 50.82 46.84 31.66
± ± ± ± ±
2.06 2.05*** 2.33 3.16* 3.02**
Mortality (% Death)
6/6 (100.00) 0/6 (0.00)$$$ 6/6 (100.00) 4/6 (66.67)$ 2/6 (33.33)$$
Data are expressed as mean ± S.E.M. n = 6 in each group. Comparison was made with vehicle control group for each test. Data of “onset” and “duration” was analyzed by one-way ANOVA followed by Dunnett’s test (* P < 0.05, ** P < 0.01, *** P < 0.001). Data of “incidence of convulsion” was analyzed by Chi2 test (# P < 0.05, ## P < 0.01 and ### P < 0.001). Data of “mortality” was analyzed by Fisher’s exact test ($ P < 0.05, $$ P < 0.01, $$$ P < 0.001). PHY: phenytoin; FST: fisetin; STR: strychnine.
Table 3 Effects of fisetin and diazepam on isoniazid induced convulsions in mice. Treatment (mg/kg, i.p.) INH
DZP
FST
300 300 300 300 300
– 5 – – –
– – 5 10 25
No. convulsed/No. used
% animals protected
Onset of convulsion (Sec) (Mean ± S.E.M.)
6/6 0/6### 6/6 5/6# 5/6#
0 100 0 16.67 16.67
51.78 117.7 54.08 66.38 71.56
± ± ± ± ±
2.52 1.22*** 3.72 3.81 6.40*
Duration of clonic convulsion (Sec) (Mean ± S.E.M.)
76.22 21.90 72.52 62.56 67.24
± ± ± ± ±
2.96 2.02*** 2.70 2.63* 2.62
Duration of tonic convulsion (Sec) (Mean ± S.E.M.)
60.17 24.82 57.02 53.70 56.18
± ± ± ± ±
3.03 2.27*** 4.38 4.39 1.94
Mortality (% Death)
6/6 (100.00) 0/6 (0.00)$$$ 6/6 (100.00) 6/6 (100.00) 4/6 (66.67)$
Data are expressed as mean ± S.E.M. n = 6 in each group. Comparison was made with vehicle control group for each test. Data of “onset” and “duration” was analyzed by one-way ANOVA followed by Dunnett’s test (* P < 0.05, ** P < 0.01, *** P < 0.001). Data of “incidence of convulsion” was analyzed by Chi2 test (# P < 0.05, ## P < 0.01 and ### P < 0.001). Data of “mortality” was analyzed by Fisher’s exact test ($ P < 0.05, $$ P < 0.01, $$$ P < 0.001). FST: fisetin; DZP: diazepam; INH: isoniazid.
fisetin (25 mg/kg) significantly decrease brain GABA level in PTZ treated rats (42.70 ± 1.94 ng/g, P < 0.001) as compared to vehicle control mice. Fisetin (10 mg/kg) treated rats significantly attenuated decreased level of brain GABA in PTZ treated rats (36.66 ± 1.47 ng/g, P < 0.01) as compared to vehicle control mice. Mice treated with fisetin (10 and 25 mg/kg) failed to produce any significant change in brain GABA level in INH (300 mg/kg, i.p.) treated mice as compared to vehicle control mice. When compared with vehicle control mice, diazepam (5 mg/kg) and phenytoin (25 mg/kg) treated mice produced significant increase in brain GABA level in PTZ and INH treated mice (48.44 ± 1.89 and 43.02 ±1.82 ng/g respectively, P < 0.001) (Fig. 4). 3.7. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain nitric oxide level Brain NO level in PTZ (90 mg/kg, i.p.), STR (5 mg/kg, i.p.) and INH (300 mg/kg, i.p.) treated mice (0.214 ± 0.0067, 0.216 ± 0.0163 and 0.196 ± 0.0092 mole/g respectively) was significantly increased (P < 0.001) as compared to normal mice (0.124 ± 0.012 mole/g). Treatment with fisetin (25 mg/kg) significantly attenuated this
elevated level of brain NO in PTZ and STR treated rats (0.156 ± 0.0092 and 0.136 ± 0.008 mole/g, P < 0.01 and P < 0.001 respectively) as compared to vehicle control mice. Mice treated with fisetin (10 mg/kg) significantly decreased brain NO level (0.162 ± 0.006 mole/g, P < 0.01) in STR treated mice, however in PTZ treated mice it failed to do so as compared to vehicle control mice. When compared to vehicle control mice, mice treated with fisetin (10 and 25 mg/kg) failed to produce any significant decreased in brain NO level in INH (300 mg/kg, i.p.) treated mice as compared to vehicle control mice. Mice treated with diazepam (5 mg/kg) and phenytoin (25 mg/kg) significantly antagonized elevated level of NO in brain in PTZ, INH and STR treated mice (0.146 ± 0.008, 0.158 ± 0.006 and 0.142 ± 0.011 mole/g respectively, P < 0.001) (Fig. 5). 3.8. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain xanthine oxidase level Mice treated with PTZ (90 mg/kg, i.p.), STR (5 mg/kg, i.p.) and INH (300 mg/kg, i.p.) results significant increase (7.36 ± 0.32, 6.52 ± 0.53 and 6.84 ± 0.41 U/g respectively, P < 0.001) in brain XO
Table 4 Effects of fisetin and phenytoin on maximal electroshock (MES) induced convulsions in mice. Treatment (mg/kg, i.p.) PHY – 25 – – –
No. convulsed/No. used
% animals protected
Duration of THLE (Sec) (Mean ± S.E.M.)
6/6 0/6### 5/6 2/6## 0/6###
0 100 16.67 66.67 100.00
16.10 0.14 11.75 6.86 1.23
Mortality (% Death)
FST – – 5 10 25
± ± ± ± ±
1.24 0.07*** 1.13 0.86** 0.16***
6/6 (100.00) 0/6 (0.00)$$$ 4/6 (66.67)$ 2/6 (33.33)$$ 0/6 (0.00)$$$
Data are expressed as mean ± S.E.M. n = 6 in each group. Comparison was made with vehicle control group for each test. Data of “duration” was analyzed by one-way ANOVA followed by Dunnett’s test (* P < 0.05, ** P < 0.01, *** P < 0.001). Data of “incidence of convulsion” was analyzed by Chi2 test (# P < 0.05, ## P < 0.01and ### P < 0.001). Data of “mortality” was analyzed by Fisher’s exact test ($ P < 0.05, $$ P < 0.01, $$$ P < 0.001). PHY: phenytoin; FST: fisetin; THLE: tonic hind limb extension.
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level as compared to normal mice (3.24 ± 0.35 U/g). Treatment with fisetin (25 mg/kg) resulted significant decrease in brain XO level (4.42 ± 0.38 and 4.14 ± 0.50 U/g, P < 0.01 and P < 0.05 respectively) as compared to PTZ and STR control mice. In PTZ treated mice elevated level of brain XO was significantly antagonized by pre-treatment of fisetin (10 mg/kg) (5.10 ± 0.48 U/g, P < 0.05) as compared to vehicle control animals whereas in STR treated mice it failed to produced any significant effect in level of brain XO. When compared with vehicle control mice, treatment with diazepam (5 mg/kg) and phenytoin (25 mg/kg) significantly attenuated this elevated level of brain XO in PTZ, INH and STR treated mice (3.82 ± 0.38, 3.68 ± 0.38 and 3.84 ± 0.56 U/g respectively, P < 0.001) (Fig. 6).
4. Discussion
Fig. 2. Effects of fisetin and phenytoin on seizure intensity in fully kindled rats. Data are expressed as mean ± S.E.M. (n = 6 in each group). Comparison was made with vehicle control group for each test. Data was analyzed by one-way ANOVA followed by Dunnett’s test * P < 0.05, ** P < 0.01 and *** P < 0.001 as compared to vehicle control rats.
Fig. 3. Kaplan–Meier analysis of the time of spontaneous seizure appearance after subconvulsive electrical stimulation. The log rank test revealed a significant (* P < 0.05, ** P < 0.01, *** P < 0.001) difference between the different group (n = 6).
Fig. 4. Effects of fisetin, diazepam and phenytoin on PTZ- and INH-induced alteration in brain GABA level. Data are expressed as mean ± S.E.M. (n = 6 in each group) and analyzed by one-way ANOVA followed by Dunnett’s test. Comparison was made with vehicle control group for each test. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to PTZ treated mice. @ P < 0.05, @@ P < 0.01, @@@ P < 0.001 as compared to INH treated mice. # P < 0.05, ## P < 0.01, ### P < 0.001 as compared to normal mice and & P < 0.05, && P < 0.01, &&& P < 0.001 as compared to one another.
Epilepsy is a common chronic neurological disorder caused due to tumours, degenerative conditions or cerebrovascular diseases. The imbalance between excitatory and inhibitory neurotransmission in the brain is an important characteristic of experimental and clinical seizure [21]. Nerve cell depolarization occurres due to the predominancy of excitatory postsynaptic potentials (EPSP) over the inhibitory (IPSP) resulting in the generation of the seizures in the brain. Epilepsy is precipitated due to an array of factors including elevated electrolytes (Na+ , K+ , Ca2+ ) levels, excitatory amino acids (glutamic acid), and inhibitory amino acids (GABA), irregular interneuron connections and abnormal afferent connections from subcortical structures which modulate various intertwining biochemical pathways giving rise to discharges of large numbers of neurons resulting in an epileptic seizure [26]. Depending upon their mechanism of action classical anticonvulsant drugs have been broadly classified into various categories and they are effective against partial and tonic-clonic seizures [27]. The potent antiepileptic drugs act by either inhibiting the voltage gated Na+ channels activation resulting in inhibition of firing of neurons or facilitating pre- or postsynaptic GABA-mediated synaptic transmission and inhibition. This cause enhanced membrane polarization by influx of Cl− ions through GABAA receptor. Some of them also act by inhibiting the GABA metabolism [27]. In the present investigation the pharmacological screening models were selected on the basis of mechanisms involved in the anticonvulsant activity of drugs. PTZ-induced convulsion in mice has been used to screen various drugs as it mimics an array of clinicopathobiological feature of human syndrome [28]. It has been reported that PTZ-induced convulsion model is widely used to identify compounds that are effective against absence and myoclonic seizures. In PTZ-induced convulsions model, PTZ imparts convulsions by its inhibitory effect on GABA-mediated Cl− influx through an allosteric interaction in the Cl− channel [29]. Previously it has been shown that diazepam enhances the GABAergic neurotransmission and gives protection against the PTZ-induced convulsion [30]. Fisetin provides protection against the PTZ-induced convulsions suggesting anticonvulsant property by modulation of GABAergic pathways in brain. In the present investigation, it was found that fisetin increases the level of the brain GABA which provides credence to its anticonvulsant activity by GABA level regulation in brain. STR is a selective competitive antagonist of glycine which blocks its inhibitory effect to produces convulsions. Glycine is vital inhibitory neurotransmitter to motor neurons and interneurons in the brain and other areas of the central nervous system (CNS) [31]. Fisetin and phenytoin exerted their protective effect through their action on glycinergic pathway. INH produces convulsion by interfering with GABA synthesis via down regulation of glutamic acid decarboxylase (GAD)
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Fig. 5. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain nitric oxide level. Data are expressed as mean ± S.E.M. (n = 6 in each group) and analyzed by one-way ANOVA followed by Dunnett’s test. Comparison was made with vehicle control group for each test. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to PTZ treated mice. $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 as compared to STR treated mice. @ P < 0.05, @@ P < 0.01, @@@ P < 0.001 as compared to INH treated mice. # P < 0.05, ## P < 0.01, ### P < 0.001 as compared to normal mice and & P < 0.05, && P < 0.01, &&& P < 0.001 as compared to one another.
activity, resulting in decreased level of GABA [32]. Diazepam produces significant protection against INH-induced convulsions, but fisetin failed to protect against INH-induced seizures and mortality indicating its moderate action on the GABA synthesis mechanism. The drugs which produce effect on Na+ channels are widely used to screen against MES induced convulsions. These drugs are proven to be effective against partial and tonic-clonic seizures in humans [33]. Phenytoin and fisetin exhibited anticonvulsant profile effect via inhibition of the Na2+ channels in partial and tonic-clonic seizures. The focal electrical stimulation is widely used model in neurology and neurosurgery of clinical procedure as well as experimental studies of brain functions. The repetitive administration of highfrequency electrical stimulation of the amygdala as well as hippocampus leads to the development of generalized electrographic and behavioral seizures generally referred as kindling [34]. Phenytoin and fisetin significantly decreases seizure intensity in kindled rats induced by electrical stimulation. It has been documented that oxidative stress plays an important role in the central nervous system in various models of experimental epilepsy [3,35]. Recent studies using 3-mercaptoproprionic
acid suggested that oxidative stress is associated with seizures [36]. Induction of the seizure by means of variety of chemicals result in initiation of array of biochemical processes including membrane phospholipases activation, proteases, nucleases and lipoperoxidation in hippocampus. These alterations result in liberation of free fatty acids, diacylglycerols, eicosanoids and free radicals. Xanthine oxidase plays a pivotal role in generation of oxygen free radicals by degradation of adenine nucleotides [37]. The elevated level of oxygen free radicals brings an array of reactions that are harmful to the neuronal tissue. Hence antioxidants play an important role in the protection of cellular components from free fatty acids, diacylglycerols, eicosanoids and free radicals by quenching the superoxide anion, hydroxyl and peroxyl radicals. Fisetin is a potent antioxidant [7]. The mice pre-treated with fisetin showed decreased activity of this xanthine oxidase (XO) enzyme, which suggests that fisetin may have ability to protect the cellular components against the oxidative stress generated during seizures. Nitric oxide (NO) is an endogenously produced intercellular signaling molecule [38] and depending upon the stimulants and NO related chemicals it may act as an anticonvulsant or a proconvulsant [39,40]. Result of the present investigation reveals that by inhibiting
Fig. 6. Effects of fisetin, diazepam and phenytoin on PTZ-, STR- and INH-induced alteration in brain xanthine oxidase level. Data are expressed as mean ± S.E.M. (n = 6 in each group) and analyzed by one-way ANOVA followed by Dunnett’s test. Comparison was made with vehicle control group for each test. * P < 0.05, ** P < 0.01, *** P < 0.001 as compared to PTZ treated mice. $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 as compared to STR treated mice. @ P < 0.05, @@ P < 0.01, @@@ P < 0.001 as compared to INH treated mice. # P < 0.05, ## P < 0.01, ### P < 0.001 as compared to normal mice and & P < 0.05, && P < 0.01, &&& P < 0.001 as compared to one another.
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the level of brain GABA, there is an increase glutamate release which may consequently increase NO production in the hippocampus. Our results are in accordance with the previous findings [41]. In the present investigation, treatment with fisetin restored the elevated level of nitric oxide. PTZ produces specific type of convulsion that is generalized by clonic convulsion whereas MES produces characteristic generalized tonic-clonic impartial convulsions [34,42]. Therefore, our results suggest that fisetin may be useful in the above stated seizure types in human beings. STR produces reflex tonic-clonic and symmetrical types of convulsions and stimulates the whole cerebrospinal axis [43]. Hence, the protective action of fisetin against STR-induced convulsions provides evidence to the theory that it may be useful in amelioration of various types of convulsion. The drugs that are effective against tonic hind limb extension induced by electroshock generally have proven to be effective against partial and tonic-clonic seizures and those in PTZ model are effective against absence seizure in human beings [27]. It could be concluded from the present investigation that fisetin protects endogenous enzyme level, inhibits oxidative damage and modulates GABAergic transmission to exhibit anticonvulsant effect. Further studies are needed to unravel its mechanism of action.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements The authors would like to acknowledge Dr. S. S. Kadam, ViceChancellor and Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, India, for providing necessary facilities to carry out the study. We are also thankful to the All India Council of Technical Education (AICTE), India for financial support by awarding GATE Scholarship to one of the author (Mr. Kiran S. Raygude) for the research work.
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