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Anticonvulsant Activity of Hydroalcoholic Phoenix dactylifera Fruit Extract and Pimpinella anisum Oil in Mice

Corresponding Author: Mario Giorgi Department of Veterinary Sciences, University of Pisa, Pisa, Italy Fax: +39502210182, Tel: +39 (0)5


Introduction
Epilepsy is not a concept of a single disease, but is a common endpoint of many forms of acquired brain pathology (such as tumors, infection, stroke and traumatic brain injury), or mutation of a single gene (genetic epilepsy) and it can be one component of a neurodevelopmental disorder (Mulley and Mefford, 2011).
A seizure occurrence is the main symptom of epilepsy disease that generally characterized by behavioral changes and physical features which are followed by episodes of abnormal brain electrical activity (Fisher et al., 2014).
One of the key molecules that maintain the inhibitory tone and counterbalances neuronal excitation is the neurotransmitter GABA. When this balance is perturbed, seizures may arise (Treiman, 2001).The hyperexcitability of neurons and excessive production of free radicals result in oxidative damage to brain tissue which has been implicated in the pathogenesis of epilepsy (Parfenova et al., 2012). A community-based study in southern France estimated that up to 22.5% of Patients with epilepsy have drug-resistant epilepsy (Picot et al., 2008). Moreover, dose-related neurotoxicity, cognitive impairment and systemic side effects are the major problems caused by Antiepileptic Drugs (AEDs) (Reynolds and Trimble, 1985). Even in drug-responsive cases, AEDs only suppress seizures without interfering with the epileptogenic disease process that converts a healthy brain into an epileptic one (Loeb, 2011). Accordingly, the discovery of a new anticonvulsant with better efficacy and fewer side effects is of paramount importance. Several plant extracts and products may be useful for the treatment of convulsions or seizures giving a promising source of new antiepileptic drugs (Sucher and Carles, 2015).
Medicinal plants are being regarded as an easily available and potent source of antioxidants as they contain a mixture of different compounds that may act individually or synergistically to cure disease and improve health (Miguel, 2010). Phoenix dactylifera L is one of the oldest crops native to the Middle East and North Africa. It is a monocotyledon woody lasting tree belonging to the Arecaceae family which consists of about 3000 species and 200 genera (Hazzouri et al., 2015). Many studies discussed pharmacological properties of Phoenix dactylifera which includes antioxidant activity (Borochov-Neori et al., 2015), antitumor (Al-Sayyed et al., 2014), hzepatoprotective (Mallhi et al., 2014), immunomodulatory (Karasawa et al., 2011), antidiabetic (Khan et al., 2016) and neuroprotective activity (Majid et al., 2008) and anti-inflammatory effect (Boghdadi et al., 2012).
The Antiepileptic Drugs (AEDs) associated with undesirable side effects which often render treatment difficult; in addition to at least 22.5% of patients are drug resistant (Picot et al., 2008). So that searching for new anticonvulsants is vital. Medicinal and aromatic plants have many constituents which have anticonvulsant action and neuroprotective effects without side effects of chemical drugs. Therefore, the current experiments were conducted to investigate the anticonvulsant and neuroprotective effects of Phoenix dactylifera extract and Pimpinella asinum against experimentally induced convulsions in mice.

Materials
Phoenix dactylifera L. dried fruit was collected in October from a local market in Sharkia province, Egypt. The fruit was separated from seeds, cut into small pieces and ground. Hydroalcoholic Phoenix dactylifera (HAPD) extract was prepared by macerating 500 gm. of Phoenix dactylifera fruit in 2 L of ethanol-water (70/30, v/v) for 48hr at room temperature with frequent shaking, then filtered and the extract was concentrated under reduced pressure on Rotary evaporator until a semisolid residue was obtained (Ragab et al., 2013). Sterilized distilled water was used as a solvent to 100 mg/mL concentration.
The plant materials identified and authenticated by the department of pharmacognocy, faculty of pharmacy, Zagazig University.

Drugs and Chemicals
• Pentylenetetrazole (PTZ) was purchased from (Sigma-Aldrich co. R07AB03) was dissolved in sterilized distilled water to 8.5mg/ml • Sodium valproate (Depakine 200 mg®) was purchased from (Sanofi co. N03AG01) dissolved in sterilized distilled water to 20 mg/ml • Phenytoin sodium (Phenytin 100 mg®) was purchased from (Nile co. N03AB02) dissolved in sterilized distilled water to 10 mg/ml All other chemicals were purchased from standard commercial suppliers and were of analytical grade. All solutions were prepared immediately before use.

Experimental Animals
Seventy male albino mice of 20-30 gm average body weight of each were obtained from the faculty of Veterinary Medicine farm; Zagazig University. The animals were randomly housed in appropriate cages at 22±2°C on a 12 h light/dark cycle with adequate food and water ad-labium.
The experimental protocol was carried out according to the guidelines approved with the Ethical Committee of Zagazig University (No. ZU-IACUC/2/S/18/2018)

Experimental Protocols
Anticonvulsant Activity of Phoenix dactylifera and Pimpinella anisum oil against Pentylenetetrazole induced convulsion in mice For pentylenetetrazole (PTZ) induced convulsions all protective treatments given for 7days and at the end of treatment, PTZ (85 mg/kg) (Al-Taher, 2008), injected I/p one shot. 25 mice were divided into 5 equal groups.
The mice were observed for 1 hour after PTZ injection and onset of the seizure (sec) and generalized seizure (sec) were recorded. After 1 hour of PTZ injection, mice were sacrificed by decapitation and the brains were removed and dissected on an ice-cold surface.
Tissue preparation for biochemical measurements: the brain was taken and washed out in Phosphate Buffer Saline (PBS), pH 7.4. One gram of the brain was homogenated in 5-10 ml of cold buffer (50 mM Tris-HCl).
The homogenate was centrifuged at 10000 rpm or 15 min at 4°C. The supernatant was removed for biochemical assay.

Biochemical Measurements
GABA measurement by using a double antibody sandwich ELISA test according to the published report (Ben-Ari et al., 2007). Glutathione Peroxidase (GPX) was assayed as reported (Paglia and Valentine, 1967). In this assay oxidized glutathione (GSSG), produced upon reduction of an organic peroxide by GPx, is recycled to its reduced state by the enzyme Glutathione Reductase (GR). The oxidation of reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) to Nicotinamide Adenine Dinucleotide Phosphate (NADP+) is accompanied by a decrease in absorbance at 340 nm.
Superoxide Dismutase (SOD) was determined according to method (Nishikimi et al., 1972) and the results were expressed as USOD/mg of protein. One unit of SOD is defined as the amount of enzyme that inhibits the rate of adrenochrome formation in 50%. Malondialdehyde (MDA) as lipid peroxidation marker was assessed as reported (Ohkawa et al., 1979), which is based on the reaction with thiobarbituric acid reactive species (TBARS) during an acid-heating reaction. The absorbance of the product is measured at 534 nm.
Histopathological examination: Parts of the brains were kept in 10% formalin for at least 72 h and then processed for histopathological studies.

Anticonvulsant Activity of Phoenix dactylifera Extract and Pimpinella anisum oil against Maximal Electric Shock in Mice
About 20 mice were divided into 4 equal groups.
At the 7th day, mice received an electrical stimulus with intensity (150 mA, 50-60 Hz, 0.2-second duration) after administration of all drugs by 1hour through mouth electrode to induce hind limb tonic extension. Mice that didn't exhibit hind limb tonic extension considered protected (Toman et al., 1946).

Neurotoxicity Tests Application (Rotarod Test, Forced Swim Test, Horizontal Screen Test)
There were 25 mice divided equally into 5groups. Group (1): mice received distilled water 0.2 ml p. o for 7 days. Group (2): mice received Phoenix dactylifera Hydro alcoholic extract1000 mg/kg per os for 7 days. Group (3): mice received Pimpinella anisum oil 4 ml/kg per os. Group (4): received sodium valproate 100 mg/kg per os for 7 days. Group (5): received phenytoin sodium 25 mg /kg per os for 7 days. At the 7th-day neurotoxicity tests applied: • Rotarod test: For the evaluation of motor coordination rotarod test was performed. Mice were walked on accelerating rotarod, which revolves 5-10 rpm for a period of 120 s. The diameter of the rod was 3.2 cm. Normal mice are able to maintain balance for many minutes, but mice with deficits in motor coordination fall more quickly (Dunham and Miya, 1957). • Forced swim test: According to the method of (Porsolt et al., 1977), forced swim test was performed. Mice were placed in a chamber (Diameter: 45 cm: Height: 20 cm) containing water with a height of 15 cm at 25±2°C. The immobility time through 5 min of the test was measured. Immobility was allocated when no additional activity was observed further than that required to keep the mouse's head over the water. • Horizontal screen test: Neural impairment was also assessed using horizontal screen test. A 13×14 cm square wire screens apparatus mounted horizontally on a steel rod was used.
Mice were held individually on the top of wire screen when the screen inverted through an arc of 180°C, mice unable to climb to an upright position within 2 min were rated as failures (Coughenour et al., 1977).

Statistical Analysis
Data analysis was done using SPSS and the data were expressed as mean ± SE. One way ANOVA test was used for comparison between different groups for parametric data. The p-value ≤0.05 was considered statistically significant.

Results
The Effect of the Hydro-Alcoholic Extract of Phoenix dactylifera and Pimpinella anisum Oil on PTZ-Induced Seizures Administration of 85 mg/kg of PTZ induced a tonicclonic seizure in mice. For PTZ group the onset of convulsions was (59.5±0.65 sec). Administration of HAPD extract, PAO and sodium valproate orally significantly delayed the time of onset of convulsions 98±1.1, 122.5±1.04 and 119.5±0.64 sec, respectively compared with 59.5±0.65 sec for PTZ group.

Effect on Gamma-Aminobutyric Acid (GABA) in the Brain
Administration of PTZ showed significant (p<0.05) decrease in GABA level in the brain (172.3±10.71 µg/g) compared with control group (577.28±47.1 µg/g).

Effect on Brain Malondialdehyde (MDA) as a Biomarker for Lipid Peroxidation
Administration of PTZ showed significant (p<0.05) increase in brain MDA activity (39.35±0.22 n mol/g) compared with (6.5±0.36 n mol/g) for the control group.

Histopathological Picture
They were displayed in (Fig. 3A). The seizures induced by injection of PTZ increased the numerical density of dark neurons which were peculiar among the hippocampus cells and represented about 15-20%. Pretreatment with Phoenix dactylifera protected hippocampal cells from any degenerative or necrotic changes could be observed (Fig. 3B).
It was observed that a few numbers of hippocampus cells have degenerated (darkly appeared) 0.1% in Pimpinella anisum oil treated group (Fig. 3C). Differently, sodium valproate group revealed normal hippocampus cells apparently (Fig. 3D).

Anticonvulsant Activity of Phoenix dactylifera Extract and Pimpinella anisum oil against Maximal Electric Shock in Mice
After the administration of hydroalcoholic Phoenix dactylifera extract 1000 mg/kg p. o, or Pimpinella anisum oil 4 ml/kg p. o or phenytoin sodium 25 mg/kg p. o, the maximal electric shock applied to mice. The animals that didn't exhibit hind limb tonic extension were considered protected. The results of HAPD extract was similar to phenytoin sodium in the protection of mice from maximal electric shock by 80% of mice (Fig. 4).
PAO wasn't able to protect all mice from maximal electric shock as phenytoin sodium and protected only 40% ofmice (Fig. 4)
Phenytoin sodium 25mg/kg bwt significantly decreased swimming activity in mice and decreased immobility time of 229.75±2.48 sec compared with 110.25±1.65sec for the control group (Fig. 5a).
Phenytoin sodium 25 mg/kg, revealed its neurotoxic effect on mice and all mice can't pass the horizontal screen test and fallen down compared to other groups (Fig. 5b).

Discussion
Epilepsy is a prevalent and serious neurological disorder which involves the occurrence of at least one or more epileptic seizures. Worldwide, it affects not less than 50 million people (Behr et al., 2016).
The present study showed that PTZ (85mg/kg) induced onset of convulsion within less than 1minute and strong myoclonic convulsions in line with Deyn and Macdonald (1989) PTZ causes neuron depolarization by increase calcium and sodium influx which produces muscle contraction (Papp et al., 1987).
The present study showed that pretreatment with HAPD extract (1000 mg/kg) delayed onset time of convulsion, delayed time of generalized convulsions and decreased the strength of myoclonic convulsions. These effects could be due to the involvement of the GAB Aergic pathways (as mechanism of action of Phoenix dactylifera effect) as anticonvulsant as reported (Al-Taher, 2008).
The present study revealed that administration of PAO (4 ml/kg) significantly increased the delay of the onset of a seizure in mice and prevented tonic convulsions induced by I .p. injection of PTZ to 20% of mice. An earlier study (Pourgholami et al., 1999) found that Pimpinella Anisum oil I/p block tonic convulsions induced by PTZ thanks to the enhancement of the activity of the Na+-K+ AT Pase Pump. The natural defense system provided by several enzymes such as Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPX) play a vital role for detoxification of free radicals. Many studies demonstrated that oxidative stress plays an important role in the pathogenesis of epileptic seizures (Golechha et al., 2010). Many reports showed that animal models of epilepsy have a high level of MDA and low SOD and GPX levels in brain tissues due to lipid peroxidation (Xie et al., 2012). The results of the present study showed that PTZ (85 mg/kg I/p) caused a significant decrease in the activities of brain Glutathione Peroxidase (GPX) and Superoxide Dismutase (SOD) as well as significant increase in the activities of brain MDA as an end product of lipid peroxidation.
Phoenix dactylifera fruit showed high antioxidant activity, reducing power, free radical scavenging activity which was attributed to its phytoconstituents (flavonoids, phenolics, carotenoids, vitamins) . The findings of our study demonstrated that HAPD has antioxidant action significant raised GPX and SOD levels and decreased MDA levels compared with PTZ and sodium valproate group.
Pimpinella anisum oil is strong antioxidant according to study (Singh et al., 2008). The results of this study appeared to increase levels of GPX and SOD and to decrease MDA levels regarding PTZ and sodium valproate groups.
Low GABA level in the brain leads to neurons firing and generation of convulsions (Watanabe et al., 2002). In the present study, GABA levels were significantly inhibited by PTZ which is a non-competitive GABA antagonist of the gamma-aminobutyric acid (GABA) A receptor complex (Ramanjaneyulu and Ticku, 1984).
HAPD 1000 mg/kg for 1week significantly elevated GABA compared with PTZ group, PTZ elicited a significant decrease in GABA brain level compared with the control group. Another study revealed that daily oral administration of pits of date palm evoked a significant decrease in the side effect of methylprednisolone on some neurotransmitter content in the brain and a significant increase in neurotransmitter contents Norepinephrine (NE), Dopamine (DA) and GABA (Bawazir and Saddiq, 2010).
Indeed, results indicated that Pimpinella anisum oil 4 ml/kg administered orally for 1week elicited a significant increase in GABA level.
The same result was obtained with sodium valproate. This finding is in line with data (Sahraei et al., 2002) who reported that anise oil exerts its effect on opioid receptors via activation of GABAA receptors in mice.
It has also been evidenced that recurrent and even a single convulsive seizure induced by such as pentylenetetrazole (gamma-aminobutyric acid inhibitor) lead to neuronal damage and cell death in form of appearance of dark neurons in different areas of the brain including the pyramidal layer of cortex, reticular formation, hippocampus and limbic system (Bideskan et al., 2015).
The present research found that pentylenetetrazole (85 mg/kg I/p) induced dark neuron formations which were peculiar among the hippocampus cells and represented about 15-20%. Protection with HAPD (1000 mg/kg) prevented dark neuron generation in the hippocampus. The neuroprotective effect of Phoenix dactylifera was earlier reported (Majid et al., 2008), it showing its tendency to reduce brain damage and prevent neurological deficits.
Pimpinella anisum oil (4 ml/kg) gave protection for the brain and hippocampal cells against degeneration and formation of dark neurons except for a few numbers of hippocampus cells (0.1%). This finding was supported (Karimzadeh et al., 2012) that PAO prevented the production of dark neurons and acting as a neuroprotective substance. Mice received an electrical stimulus of sufficient intensity to induce maximal seizures of their hind limbs, with tonic extension as the endpoint of the test (Holmes, 2007). Our results clearly demonstrated the ability of HAPD1000mg/kg to prevent hind limb extension due to MES probably by prolonging the inactivation of sodium channels (Holmes, 2007).
Pimpinella anisum oil 4ml/kg was able to prevent hind limb extension for only 40% for mice compared with 80% for Phoenix dactylifera and phenytoin sodium groups. Neurotoxicity from anticonvulsant compounds in mice manifested by signs of neurological deficits such as sedation, hypo locomotion, ataxia, abnormal gait and cognitive deficits (Loscher and Schmidt, 1988).
Phoenix dactylifera hydroalcoholic extract had no neurotoxic effect on mice and didn't induce any disturbance in motor coordination. No changes in swimming behavior were observed in line with a former study  showing that Phoenix dactylifera extract did not demonstrate any effect on muscle coordination.
Pimpinella anisum oil had relaxant muscle effect and might cause motor impairment for mice (Tirapelli et al., 2007), but the dose that used as anticonvulsant didn't cause any motor disturbance has appeared in the present study (Pourgholami et al., 1999). In this study, we successfully identify Phoenix dactylifera and Pimpinella anisum oil has anticonvulsant and antioxidant activities. Both of them had no neurotoxic effect on mice and didn't induce any disturbance in motor coordination. Further studies should be considered to validate this strategy by fractionation of the Phoenix dactylifera and Pimpinella anisum oil to know the active constituents of these herbal extracts for rapid identification of exact mode of action. A combined effect of the Phoenix dactylifera and Pimpinella anisum oil should be included in the further studies to have the exact evidence of the synergism between these herbal extract in treatment of epilepsy

Conclusion
Phoenix dactylifera and Pimpinella anisum oil have anticonvulsant and antioxidant activity. Both of them have neural central properties to increase GABA level as well as neuroprotective effect.