Anti-convulsant action and amelioration of oxidative stress by Glycyrrhiza glabra root extract in pentylenetetrazole- induced seizure in albino rats.

OBJECTIVES: The aim of the present study was to evaluate the anti-convulsant potential of aqueous and ethanol e xtract of Glycyrrhiza glabra (AEGG and EEGG) and its action on markers of oxidant stress in albino rats.
MATERIALS AND METHODS: The aqueous and ethanol extract of Glycyrrhiza glabra was tested at three doses viz. 100, 200, and 400 mg/kg i.p. for its anti-convulsant activity using pentylenetetrazole (PTZ)-induced seizure in rat. The effect of EEGG (400 mg/kg, i.p.) on oxidative stress markers like malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) of rat brain tissue homogenate was tested.
RESULTS: The onset of seizure was delayed (P < 0.01) by all the three doses of EEGG, but the duration of convulsion was reduced (P < 0.01) only in higher dose level (200 and 400 mg/ kg), whereas AEGG up to 400 mg/kg did not alter any of the parameters significantly. Biochemical analysis of rat brain tissue revealed that MDA was increased (P < 0.01), whereas SOD and CAT were decreased (P < 0.01) in PTZ-induced seizure rat, whereas pre-treatment with EEGG (400 mg/kg) decreased (P < 0.01) the MDA and increased (P < 0.01) both SOD and CAT, indicating attenuation of lipid peroxidation due to increase in antioxidant enzymes.
CONCLUSION: The results demonstrated that EEGG poses anti-convulsant potential and ameliorates ROS induced neuronal damage in PTZ-induced seizure.

Introduction

Over excitation of excitatory amino acids is commonly accepted mechanism for the genesis of epilepsy.[1] Prolonged epileptic discharges could lead to a large number of changes and cascades of events in cellular level, results in mitochondrial dysfunction increased production of reactive oxygen species (ROS) and activation of intrinsic mitochondrial apoptotic pathways, plays an important role in neuronal cell death after seizure.[2] Mitochondria is the primary site of ROS production and making them vulnerable to oxidative stress and damage, which can affect ATP production, antioxidant defense, and synaptic glutamate homeostasis.[3] Oxidative damage to one or more of these cellular targets may affect neuronal excitability and increase seizure susceptibility.[4] Mitochondrial dysfunction has been identified as a potential cause of epileptic seizure and therapy-resistant form of severe epilepsy.[5] Therefore, the main trend of the current investigations is to search for a novel anti-epileptic drugs with antioxidant properties.[6]

The roots of Glycyrrhiza glabra L. (Family: Leguminosae) contains bioactive compounds like glycyrrhizin, glycyrrhetinic acid, liquiritin, liquiritigenin, and glabridin. Most of the pharmacological activities of glycyrrhiza extract are attributed to 18β-glycyrrhetinic acid.[7] It shows various CNS activities like anti-convulsant, memory-enhancing, anti-depressant, and cerebro-protective effect.[8-11] Till now, no study has been reported on anti-oxidative potential of Glycyrrhiza glabra extract after PTZ-induced seizure in rat. Thus, the objective of the present study was to test the anti-convulsant potential of the extract and its action on markers of oxidant stress after PTZ-induced seizure.

Materials and Methods

Collection of Plant and Preparation of Extract

The coarse powder of Glycyrrhiza glabra root was procured from Yucca enterprises, Mumbai, India. The ethanol and aqueous extract of Glycyrrhiza glabra was prepared by soaking 500 gm of powdered plant material with 500 ml of the respective solvent for 24 hrs at room temperature. The macerated powders were then extracted in a soxhlet extractor containing 500 ml of respective solvent for 36 hrs. The crude extracts were dried under reduced pressure using rotary evaporator (Heidolph, Germany) leaving a dark brown residue. The yield for aqueous extract was 15.4% w/w, and ethanol extract was 16% w/w respectively. The resultant extracts were stored in desiccators for further investigation. Fresh solutions of each extracts were prepared by reconstituting with normal saline.

Drugs and Chemicals

Diazepam, thiobarbituric acid, DTNB reagent, sulfosalicylic acid (Himedia Laboratories Ltd., Mumbai, India), pentylenetetrazole (Sigma-Aldrich Chemie, Germany), trichloroacetic acid (BDH Fine Chemical, Mumbai, India), tris buffer, pyrogallol, potassium dihydrogen phosphate, sodium biphosphate (Merck, Mumbai, India), hydrochloric acid, hydrogen peroxide, sodium carbonate, sodium hydroxide (SD Fine Chemicals, Mumbai, India), sodium potassium tartarate, bovine serum albumin (Loba Chemie, Mumbai, India), and copper sulfate (RFCL Limited, New Delhi, India) were used in this study.

Experimental Animals

Adult albino rats (150 to 250 g) of either sex were obtained from the stock of the animal house, Roland institute of pharmaceutical sciences, Berhampur, Odisha (India) (Regd no. 926/ab/06/CPCSEA, dated 22.2.2006). Animals were housed in polypropylene cages maintained at an ambient temperature of 25 ± 1°C on a 12 h light/dark cycle with free access to standard chow pellet and water ad libitum. Animals were acclimatized to laboratory conditions for one week before testing and are kept overnight fasting but not water prior to an experiment. Experimental protocol was approved by Institutional Animal Ethical Committee (IAEC).

Acute Toxicity

Acute toxicity was determined according to the OECD guidelines No.423. Female albino rats (n = 3 per step) were selected by random sampling technique. The rats were kept fasting for overnight providing with water ad libitum. The aqueous and ethanol extract of Glycyrrhiza glabra were administered separately to two set of rats (n = 3 per set) at a dose of 300 mg/kg by intra-gastric tube. Food was withheld for further 3-4 hrs and observed once in every 30 min during the first 24 hrs and daily thereafter, for a period of 14 days for any mortality. If mortality was not observed for any animal, then the procedure was repeated again with higher doses such as 1000 and 2000 mg/kg, and the animals were observed for toxic symptoms as per the above procedure.[12]

Experimental Design

After acclimatization, the animals were randomly divided into five groups of six rats each (n = 6). Group-I: Normal saline, (10 ml/kg, i.p.), group-II: Diazepam (2 mg/kg, i.p), group-III, IV, V: Received three graded doses (100, 200, and 400 mg/kg, i.p.) of AEGG. Same protocol was also followed for studying the effect of EEGG.

PTZ-induced Seizure

The anti-convulsant activity of test drug was determined using PTZ-induced seizure test. The albino rats were selected two weeks prior to conducting the experiment by injecting the pentylenetetrazole (30 mg/kg, s.c.) in the scruff of neck. Only those animals showing clonic convulsions within 30 min were selected for the present study. After one week, all the treatments were administered intraperitonially; after 30 min, PTZ was administered (80 mg/kg, s.c.) in the scruff of neck. Then, convulsive behavior was observed for a period of 30 min. The parameters measured were onset of clonic convulsion and duration of convulsion. The ability of the test compound to prevent this feature or prolong the latency or onset of the clonic convulsions was taken as indication of anti-convulsant activity.[13]

Biochemical Analysis of Oxidant Stress Markers after PTZ- Induced Seizure

The animals were randomly divided into three groups each containing six rats (n = 6). Group-I: Normal control group (Received normal saline), group-II: Seizure control group (Received normal saline + PTZ), and group-III: Test group (Received EEGG 400 mg/kg, i.p. + PTZ). All the treatments were administered for two successive days. On second day, 30 min after the treatment, PTZ (60 mg/kg, i.p.) was administered only to group-II and III to induce seizure and were observed for a period of 30 min to assess mortality, and then rats were sacrificed for biochemical estimation of antioxidant markers.

Brain Sample Preparation and Biochemical Evaluation

All the rats were sacrificed by decapitation, the brains were quickly removed and were washed with cold saline solution, followed by 50 mM Tris-HCl buffer (pH 7.4), blotted dry and weighed. Then, it was placed into a glass bottles, labeled, and stored in a deep freezer (-25°C) until processing (maximum 10 hrs). The tissues were homogenized in four volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.4) using a glass Teflon homogenizer (Elektrocrafts, Mumbai) for 2 min at 5000 rpm after cutting it into small pieces. The homogenate was used for estimation of MDA. The homogenate was then centrifuged (Remi, India) at 5000×g for 60 min to remove the debris. The clear upper supernatant fluid was extracted with an equal volume of ethanol/chloroform (5:3 v/v) mixture and centrifuged at 5000×g for 30 min, the clear upper ethanol layer was taken and used for SOD, CAT and protein assay.[14] All the preparations were performed at 4°C, and then MDA,[15] SOD,[16] CAT,[17] and protein[18] were estimated.

Statistical Analysis

The results were expressed as mean ± SEM. Data were analyzed by one-way ANOVA, followed by Dunnett's multiple comparison tests was performed using Graph Pad Prism version 5.00 for windows, San Diego, California, USA. P < 0.05 was considered as significant.

Results

The ethanol and aqueous extract of Glycyrrhiza glabra did not show any mortality up to 1000 mg/kg. There were slight gross behavioral changes observed in all the doses up to 3 hrs [Table 1].

Table 1

Behavioral assessment of rats pre-treated with aqueous and ethanol extracts of Glycyrrhiza glabra at 2000 mg/kg dose

Effect of Aqueous Extract of Glycyrrhiza glabra on PTZ -Induced Seizure

Subcutaneous administration of PTZ elicited various phases of convulsions such as myoclonic jerking, tonic extension of fore/hind limb, and finally death. AEGG at all the three doses level did not show any significant delay in onset as well as decrease in duration of clonic convulsion compared to control. The onset time of clonic convulsion was delayed by 6% with AEGG (400 mg/kg), in contrast, diazepam (2 mg/kg) showed 100% delay [Table 2].

Table 2

Effect of aqueous extract of Glycyrrhiza glabra (AEGG) on pentylenetetrazole (PTZ)-induced seizure in rat

Effect of Ethanol Extract of Glycyrrhiza glabra on PTZ -Induced Seizure

The EEGG at 100 mg/kg delayed (P < 0.01) only the onset of clonic convulsion, but at 200 and 400 mg/kg delayed (P < 0.01) the onset as well as reduced (P < 0.01) the duration of clonic convulsion compared to control. The onset time of clonic convulsion was delayed by 100% with EEGG (400 mg/kg) and was comparable with diazepam 2 mg/kg [Table 3].

Table 3

Effect of ethanol extract of Glycyrrhiza glabra (EEGG) on pentylenetetrazole (PTZ)-induced seizure in rat

Effect of Ethanol Extract of Glycyrrhiza glabra on Oxidant Stress Markers

The biochemical analysis of MDA, SOD, and CAT of the brain homogenate were performed for antioxidant activity. PTZ-induced seizure increased (P < 0.01) the MDA, whereas decreased (P < 0.01) the CAT and also decreased (P < 0.05) the SOD in seizure control group compared to normal control group. Pre-treatment with EEGG (400 mg/kg) decreased the MDA (P < 0.01) and increased (P < 0.01) both the SOD and CAT compared to seizure control group. The SOD and CAT level of the brain homogenate increased up to the normal control group signifies restoration of enzymes [Table 4].

Table 4

Effect of ethanol extract of Glycyrrhiza glabra (EEGG) on lipid peroxidation and antioxidant enzymes level in rat brain after PTZ-induced seizure

Discussion

In the present study, it was observed that AEGG (400 mg/ kg) did not produce any delay in onset and decrease in duration of seizure compared to control [Table 2]. The observed effect is in agreement with the findings of Nassiri et al., which states that AEGG (300 mg/kg) delays the onset time and decrease the duration of seizure significantly in mice but lower doses (60-200 mg/kg), the duration of seizure was only decreased compared to control.[19] The variation in dose may be use of different species of animal or variation in percentage of active phytoconstituents. The EEGG showed a significant delay in onset and decrease in duration of convulsion compared to control [Table 3]. The observed effect is in agreement with the findings of Ambawade et al., which states that the EEGG significantly and dose-dependently delayed the onset of PTZ-induced seizure.[20]

Epileptic seizures results in free radical production and oxidative damage to cellular proteins, lipids and DNA, resulting neuronal cell death.[2] Enhanced production of ROS excides the antioxidant scavenging capacity of the cell and results in development of oxidative stress. Under normal physiological conditions, tissue injury caused by free radicals is controlled by antioxidant defense system. The most important enzymatic antioxidants are superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), non-enzymatic antioxidants are glutathione and uric acid.[14]

It has been reported that after single dose administration of PTZ, it leads to increase in oxidative stress and lipid peroxidation.[21] Our study supports the above hypothesis that PTZ-induced seizure activity enhanced the MDA level, while the antioxidant enzymes SOD and CAT was reduced in rat brain homogenate of seizure control group compared to normal control group. The elevated level of MDA, decrease in SOD and CAT indicates increased oxidative stress, and attenuation of antioxidant defense leads to ROS-induced damage [Table 4]. It may be supposed that decrease in antioxidant enzymes in the brain of PTZ-induced seizure rat is due to increased formation of oxygen-free radical, which causes lipidperoxidation and increases MDA. The level of MDA in the brain homogenate of EEGG-treated rats was lowered and the level of SOD and CAT was increased compared to seizure control group and was almost equivalent to normal control group, which indicates attenuation of lipidperoxidation and increase in antioxidant defense [Table 4].

The ability of the extract to attenuate the oxidative stress during PTZ-induced seizure and increase in SOD and CAT indicates neuroprotective property of the extract, this observation is in agreement with Muralidharan et al., which states that the level of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase were reduced and lipidperoxidation was increased due to hypoxia and were restored to near normalcy by administration of AEGG. This suggests that cerebro-protective effect in hypoxic rats may be mediated by antioxidant effects of AEGG.[11] Therefore, the present effect against oxidative stress in PTZ-induced seizure may be mediated by antioxidant effects of EEGG.

The anti-convulsant effect of Glycyrrhiza glabra was already reported, but the antioxidant effect during seizure was not reported till now. This was the first study in which EEGG was used to prevent the PTZ-induced neuronal injury via: ROS generation in brain. In conclusion, the result of the present study demonstrated the anti-convulsant and antioxidant properties of the EEGG in the PTZ-induced seizure.