The Changes in ionic concentrations observed during hyperexcitation—increased extracellular K+or decreased extracellular Ca2+, for example—may be caused by decreases in extracellular size or volume. Failure of Na-K- pumps due to hypoxia or ischemia is known to promote epileptogenesis in animal models, and interference with Cl--K+ transport, which controls intracellular Cland regulates GABA-activated inhibitory Cl currents, may lead to enhanced excitation. Excitability of synaptic terminals depends on the extent of depolarization and the amount of neurotransmitter released. Synchronization following abnormal bursts of spikes in the axonal branching of thalamocortical relay cells plays a key role in epileptogenesis  (Engelborghset al; 2010) Synaptic Mechanisms: Synaptic pathophysiology of epilepsy and epileptic disorders primarily involves reduced GABAergic inhibitionor enhanced glutamatergic excitation. ^2-5            

Gaba

GABA levels have been shown to be reduced in the cerebrospinal fluid (CSF) of patients with certain kinds of epilepsy, such as infantile spasms and untreated generalized tonic-clonic seizures, and in excised epileptic tissue from patients with drug-resistant epilepsy, suggesting that these patients have decreased inhibition.

Dogs with epilepsy have been shown to have low CSF levels of GABA, and mice genetically susceptible to audiogenic seizures have a lower number of GABA receptors than non-seizure.

• MATERIAL AND METHODS:
  1. 1. Animals:

Swiss albino mice weighing 18-22 gm were purchased from Global Bioresearch Solutions Private Limited, H No 251 Nhavi, Tal - Bhor, Dist- Pune, Pune. The animals were housed in polypropylene cages and maintained under the environmental condition of temperature 25±1 ºC and relative humidity of 45-55 % under a 12h light: 12 dark cycles. The animals had free access to food pellets (Nav Maharashtra Chakan oil mills Ltd., Pune) and water ad libitum. The Institutional Animal Ethics Committee (IAEC) of Loknete shri dadapatil Pharate College of pharmacy, Mandavgan pharata approved all the experimental protocols under the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA). The protocol approval number is 2168/PO/Re/S/22/CPCSEA.

Chemicals:

Instruments Used:

1.1.4.Preparation of drug solution, storage, volume, and route of administration:

1.1.4.1.Kaempferol:

?Preparation of test drug solution:

Drug solution of Kaempferol was prepared by using distilled water a vehicle

?Storage of drug solution:

Kaempferol powder was stored in a desiccator. A fresh drug solution was prepared for each day’s work. The solution was kept in airtight amber-colored bottles and stored at room temperature until ready for use.

?The volume of drug administration:

The volume of Kaempferol solution to be administered was calculated based upon the body weight of animals.

?Route of administration:

Kaempferol solution was administered per oral (p.o.) route.

1.1.4.2.Phenytoin:

?Preparation of standard drug solution:

Solution of Phenytoin was prepared with 1% Sodium-carboxy methylcellulose as the vehicle.

?Storage of drug solution:

Phenytoin powder was stored in a refrigerator below 25 ºC. A fresh drug solution was prepared for each day’s work.

?The volume of drug administration:

The volume of Phenytoin solution to be administered was calculated based upon the body weight of animals.

?Route of administration:

Phenytoin solution was administered through per oral (p.o.) route.

 1.1.5.STR-induced convulsions in laboratory animals:

1.1.5.1.Experimental designs:

The animals were divided randomly into groups with six mice per group as follows:

?Group I: Normal group

The mice received only vehicle (Distilled water).

?Group II: STR control

The mice receive STR (5 mg/kg, i.p.) and only vehicle (Distilled water, 10 mg/kg)

?Group III: Phenytoin (25) group

The mice have received STR (5 mg/kg, i.p.). They were pre-treated with Phenytoin at a dose of 25 mg/kg, p.o., for 7 days.

Group IV: Kaempferol (25) group

The mice have received STR (5 mg/kg, i.p.). They were pre-treated with Kaempferol at a low dose of 25 mg/kg, p.o for 7 days.

?Group V: Kaempferol (50) group

The mice have received STR (5 mg/kg, i.p.). They were pre-treated with Kaempferol at a medium dose of 50 mg/kg, p.o for 7 days.

?Group VI: Kaempferol (100) group

The mice have received STR (5 mg/kg, i.p.). They were pre-treated with Kaempferol at a high dose of 100 mg/kg, p.o for 7 days.

1.1.5.2. Induction of STR-induced convulsions:

?Mice were divided into various groups as mentioned above.

?On 0 day all the behavioural parameter were evaluated before drug administration from 1 to 7 day all animal except normal.

?Pre-treatment was given to all the treatment groups with Kaempferol (25, 50, and 100 mg/kg) and Phenytoin (25 mg/kg) daily for 7 days.

?On 7th days convulsions were induced by administration of STR (5 mg/kg, i.p.) 45 min after drug treatment.

?Onset of convulsion, duration of convulsion was observed was observed post STR administration.

?Post behavioral parameter assessment mice were sacrificed and brain was removed immediately for biochemical analysis. 7-9           

 1.1.5.3.Treatment of Kaempferol and Phenytoin:

Kaempferol (25, 50, and 100 mg/kg) and Phenytoin (25 mg/kg) with different calculated doses based on the animal’s body weight were administered per oral for 7 days.

The observations were recorded on various days in the morning, and doses were administrated immediately afterward.

1.2.Parameter for assessment of the effect of Kaempferol on STR-induced convulsions in mice:

1.2.1.In-vivo parameters:

1.2.1.1.Body weight

?Mice were weighed daily using animal weighing balance.

1.2.1.2.Determination of tonic-clonic convulsions

?The convulsive behavior of each mice for onset and duration of clonic and tonic seizures was observed for 30 min for signs of neurological deficits, especially hind- limb tonic seizures or convulsions, and the resultant seizures were scored as follows:

• unresponsiveness = 0
• mild contractions = 1
• clonic seizures = 2
• tonic seizures = 3 (forelimb and then hindlimb rigidly extended to rear)
• death = 4

?Mice experiencing lethal convulsions were excluded from the study. Mice that exhibited at least three consecutive stage 4 or stage 5 seizures were considered convulsed, and used in this study

1.2.1.3.Locomotor activity:

?The animal locomotor behavior was monitored using Actophotometer. Actophotometer provided with a digital counter, photocell and a light source were used to measure locomotor activity (horizontal movement) of animals.

?Each animal was placed in Actophotometer for 5 minutes and basal activity score was recorded for all animals.

 ?Each animal was treated with respective drug and activity score was recorded after 30 min and 1hr.

?Deceased activity score was taken as index of CNS depression. 10-14            

•Results

1. 1. Effect of kaempferol on body weight:

       
           
       
    Fig. No. 7.1. Graphical representation of effect of kaempferol on body weight

       
           
       
Fig. No. 7.2. Graphical representation of effect of kaempferol on onset and duration of convulsion in STR-induced epilepsy.

1. 2. Effect of kaempferol on locomotor activity during STR-induced post-ictal depression:

              
           
       
     Fig. No. 7.3. Graphical representation of effect of kaempferol on locomotor activity during STR-induced post-ictal depression

1.3.Effect of kaempferol on STR-induced alteration in brain noradrenaline levels:

        
           
       
Fig. No. 7.4. Graphical representation of effect of kaempferol on STR-induced alteration in brain NA levels

brain NA levels compared to STR control rats. Phenytoin (25 mg/kg, p.o.) treatment also showed significantly (P < 0>

Effect of kaempferol on STR-induced alteration in brain dopamine levels:

               
           
       
Fig. No. 7.5. Graphical representation of effect of kaempferol on STR-induced alteration in brain DA levels (25 mg/kg, p.o.)

Effect of kaempferol on STR-induced alteration in brain total protein level:

       
           
       
  Fig. No. 7.6. Graphical representation of effect of kaempferol on STR-induced alteration in

1.6.Effect of kaempferol on STR-induced alteration in brain SOD and GSH level:

       
           
       
Data were analyzed by one-way ANOVA followed by Dunnett’s test. ###P < 0>

There was a significant decrease (P < 0>

1.7.              Effect of kaempferol on STR-induced alteration in brain MDA and nitric oxide level:

       
           
       
Fig. No. 7.8. Graphical representation of effect of kaempferol on STR-induced alteration in brain MDA and NO levels

1.8.Effect of kaempferol on STR-induced alteration in brain Na-K-ATPase level:

       
           
       
Fig. 7.9. Effect of kaempferol on STR-induced alteration in brain Na-K-ATPase level