T. C.

REPUBLIC OF TURKEY

YUZUNCU YIL UNIVERSITY

INSTITUTE OF HEALTH SCIENCE

INVESTIGATION THE EFFECT OF PENTHYLENTETRAZOLE INDUCING EPILEPSY MODEL USING

Epilobium hirsutum EXTRACTION

Sara Sami DZHAFAR

DEPARTMENT OF BASIC PHARMACEUTICAL SCIENCES

(PHARMACOLOGY)

MASTER THESIS

E X A M I N I N G C O M M I T T E E C H A I R M A N

P R O F. D R . S U A T EK İ N

A S S T . P R O F. D R . O R U C A L L A H V E R D I Y E V P R O F. D R . E R D I N Ç T Ü R K

( S U P E R V I S O R ) M E M B E R M E M B E R

T H E S I S A D M I S S I O N D A T E

2 7 / 9 / 2 0 1 7

INTRODUCTION

Epilepsy

Epilepsy is one of the most complicated hyperactivity neurological disease, which affect

different parts of the brain normal performance like movement, awareness, sensation,

consciousness and behavior. Seizure duration can take a few seconds to a few minutes;

however, backing up to the normal stage was gradually with consciousness and awareness

return, which considered the end stage of epilepsy.

INTRODUCTION

Epidemiology

Globally epilepsy considers one of the chronic burden brain electrical disorder that approached to

reach up to 50 million of the world population. WHO studies have showed the prevalent of

epilepsy is about 80% in low and middle income countries, while annual cases of high income

countries were between 30 and 50 per 100, 000. In Turkey, despite the a few studies done around

the country, the prevalence rate of epilepsy showed to reach (5.3/1,000) separated between 8.8 in

1,000 in rural and 4.5 in 1,000 in urban areas that considered being for developed countries, while

the lowest rate prevalence reported to be in Japan, with 1.5 per 1,000.

INTRODUCTION

The pathologic of epilepsy

Epilepsy can arise either from idiopathic (genetic) or symptomatic (acquire) or even both. Acquire

like stroke, brain tumors, meningitis, or injury of the both brain or spinal cord, oxygen and glucose

impairment. While genetic like malformation of the mitochondria, voltage channel or synapses and

receptors or physiologically like change in cerebral blood flow, distribution in blood brain barrier,

increase intracranial pressure, ischemic hemorrhages.

According to their generation type

The first generation Ethosuximide, phenytoin, phenobarbital,

valproic acid, carbamazepine, fenitoin

The second generation Zonisamide, oxcarbaze, gabapentin,

lamotrigine, levetiracetam, felbamate-

pine, rufinamid, tiagabine, pregabalin

topiramate, vigabatrin, clobazam

The third generation Ezogabine, lacosamide, perampanel

Commonly used newer

antiepileptic drugs

Topiramate, lamotrigine, levetiracetam

ALTERNATIVE TREATMENTS

Supplements like : Pyridoxine, ascorbic acid, selenium, Omega 3, vit E, porpolis, magnesium.

- Physiology treatment: Of relaxation , yoga, sleepiness.

- Diets: Ketogenic diet and the Atkins diet.

- Surgical intervenes: Vagus nerve stimulator (VNS), deep brain stimulation (DBS), removing the

affected part of the anterior temporal lobe along with the hippocampus and amygdala.

- Medicinal plant: Brassica nigra, Nigella sativa, , Ginkgo biloba.

THE CONCEPT OF ETHNOPHARMACOLOGY

Ethnopharmacology science works on testing the hypothesis of observation, description,

and experimental investigation of phytochemical compounds then producing these data for

pharmacology experimental lab to investigate these indigenous components and their rule

in the biological activities.

EPILOBIUM HIRSUTUM

Epilobium hirsutum exist in the most areas of Van in Turkey and was used by folk in

relieving symptoms of seizures by boiling the plant after drying it and drink it as an herbal

tea, which our experiment was based on and depending on folk used of the aerial parts of

the plants.

ADVANTAGE OF USING MEDICAL PLANTS

• The advantage of using herbal plant in controlling seizure is their availability in the wild and

almost all over the world, has less side effects comparing them with drugs and it is accessible to

almost to all average income populations with less or no financial need.

• The disadvantage of some medicinal plants that herbals can be contaminated, if they are not

probably collected and saved.

• Taking them may interrupt the regime of the administrated ADEs from increasing their sedative

effect or simulative effect or even containing epileptogenic compounds or influence their

mechanism of action.

• Moreover, herbals density and the quality of the active ingredients usually not known by the

patient, which may in return interfere with the patient's health condition, in to more badly

condition.

THE AIM OF THE STUDY

Our aim in this study was proving the interest of Ethnopharmacology in pharmacology lab by

exploring the anticonvulsant effect of Epilobium hirsutum using experimental male mice and PTZ

seizure inducer. Moreover, open field, as well as Rota rod tests, supported our research by providing

additional information of animal locomotors behavior and muscular relaxation. We have enhanced

our experiment using biochemical assay to evaluate the brain defend system in the presence of

seizure inducer of PTZ.

MATERIALS AND METHODS

Materials

- 40 Swiss albino male mice

- PTZ 65 mg\kg

- Valproate 100 mg\kg

- Extracted plants of 100 and 200 mg\kg

- Open field

- Rota rod

- Biochemical assay

MATERIALS AND METHODS

METHODS

Preparation of lyophilized extracts

Lyophilized extraction was more likely for our research as using water extract. It provided us

stabile compounds by devoid the product from microbial formation and active enzyme activation

as well as oxidization reaction, and being able to save it for 5 years since it doesn't contain water

or humidity.

MATERIALS AND METHODS

METHODS

40 mice were divided in 5 groups/ (n= 8)

• The controlled group

• PTZ group

• Positive group (valproate 100 mg\kg and PTZ 65 mg\kg)

• Tested group of EH two doses (100 and 200 mg\kg)

• On the seventh day, challenge dose of (65mg\kg) was administered to the all groups except the controlled one.

All the groups undergone seizure provoked except the controlled and both doses of EH (100 mg\kg and 200

mg\kg). Rota rod apparatus were used to evaluate motor coordination, muscle relaxation and open field

apparatus were used to evaluate learning, and exploration behavior.

• The last step was capturing the brain for biochemical assays to evaluate lipid peroxidation enzymes of MDA,

and antioxidant enzyme activities such as (SOD, GSH-Px, GSH, and CAT).

MATERIALS AND METHODS

Pentylenetetrazole-induced convulsions

1. Duration of convulsion (number of mice showing convulsions

2. Onset of convulsions (elapsed time from PTZ injection until convulsion occurred

3. Mortality for the duration of 30 min

seizures intensity was evaluated according to 5-point scale

Stage 1: Ear and facial twitching.Stage 2: Head nodding, head clonus and myoclonic jerks.Stage 3: Unilateral forelimb clonus.Stage 4: Rearing with bilateral forelimb clonus.Stage 5: Generalized Tonic–Clonic seizure (GTCS) with loss of righting reflex.

If no convulsion occurred during the limited time, the animals considered to be protected by the extracted plant.

MATERIALS AND METHODS

Evaluation of neurological deformities

1. Open field test

It used to measure mental activity, anxiety, exploration, depression, and locomotion as well as

seizures psychotic emotion. However, increase in the count of mice movement was regarded

to central nervous stimulation while a decrease in count of mice movement regard to central

nervous depressant activity.

MATERIALS AND METHODS

2. Rota rod test

• Rota-rod test uses to test the skeleton muscles relaxation as it assesses motor coordination,

motor learning, intoxication, sedation, stamina, motor memory (long-term skill or procedural

memory) and balances of the mice. This by testing the ability of mice to remain on a rotating

rod during the 300 seconds. However, the mouse were faild off the rod rotating at different

speeds or under continuous acceleration.

MATERIALS AND METHODS

Sample prepares for biochemical assay

- Glutathione peroxidase (GSH-Px) evaluation

- SOD evaluation

- Catalase activity determination

- Glutathione evaluation

Figure 1. Effect of valproate (100 mg/kg) and PTZ (65mg\kg) and the two doses of pilobium hirsutum (100

and 200 mg/kg) on the latency of arriving to phase 5 of seizure. n = 8 in each group.

Figure 2 . Effect of valproate (100 mg/kg) PTZ (65mg\kg) and the two doses of pilobium hirsutum (100

and 200 mg/Kg) on the time in the phase 5 n = 8 in each group. a: p<0.001, b, b1 p<0.01, c, c1 p<0.05

shows significant difference as compared to PTZ-kindled group.

RESULTS

Groups Onset of clonic

convulsion ((s)

SEM

Duration of

convulsion (s)

SEM

The number of

mortality

Control 0.00±0.00 0.00±0.00 0.00±0.00

PTZ 2,20± 5.28c 4,33 ± 0,33a,b 1/7 (14%)

100 mg EH+PTZ 2,80± 8,70 2,00 ± 0,00b,c 1/7 (14%)

200 mg EH+PTZ 4,50± 6,72c,c1 1,80 ± 0,20a,b1 0/5 (0.0%)

PTZ + VPA 2,04± 1,47c1 3,60 ± 0,25cb1 0/6 (0.0%)

X X

a: p<0.001, b,b1,: p<0.01, c,c1: p<0.05 (Similar letter fields show significance at that letter level).

Table 1. Effect of Epilobium hirsutum (EH) on the pentylenetetrazole-induced convulsion in mice

Table 2. Comparison of parameters in Open-field test among 4 different experimental groups

Figure 3. MDA evaluation level in (nmol/mg pt.) anoug the 5 groups

Figure 4. GS-HPX evaluation level in (IU/mg pt.) among the 5 groups

Figure 7. GSH evaluation level in (µmol/g pt.) among the 5 groups

Figure 5. SOD evaluation level in (IU/mg pt) among the 5 groups

Figure 6. CAT evaluation level in (IU/mg pt.) among the 5 groups

Table 3. Antioxidant enzymes (SOD, GSH-Px and CAT) activities, MDA and GSH in control, PTZ,

100 mg EH+PTZ, 200 mg EH+PTZ and PTZ + VPA groups in brain tissue samples.

a: p<0.001, b,b1,: p<0.01, c,c1: p<0.05 (Similar letter fields show significance at that letter level).

DISSOCIATION

• Chemical components of Epilobium hirsutum flavonoids, phenolic acids, steroids, andtannins, which showed to have antioxidant effect that could suppress the damagecaused by PTZ.

• PTZ kindling model blockers chloride channel of GABA A receptor, increase of thehyperactivity of glutamic receptors (NMDA) and calcium ions for entering into thenerve cells and liberation of free radicals that cause local injury of brain tissue byrepeating of sub-convoluted dose or single dose of PTZ administration.

• Valproate, which is one of AEDs, known to suppress seizure provoke by bindingGABA receptor and enhance the GABA ergic inhibitory neurotransmission. It is widespectrum mechanism against different type of partial and general seizures.

• Theoretically, Epilobium hirsutum at doses of both (100 and 200 mg\kg),significantly increase in onset of convulsion and reduction in the duration ofconvulsion with no mortality reported when it was compared with valproate group,which showed weak effect against the latent dose of PTZ ( 65mg\kg) and weakercomparing it with Epilobium hirsutum, which showed completely seizureattenuation.

• Biochemically, Epilobium hirsutum at doses of both (100 and 200 mg\kg),significant decreased in the level of MDA (an indication of protein oxidationdamage by PTZ or seizure ) when it was compared with PTZ group.

• while the antioxidant enzymes of (SOD, GSH-Px, CAT, and GSH) showedsignificant increase when they were compared with PTZ group.

• In our presented experiment, we have undergone mice after seizure provoke fortesting their ability of exploration, and motor coordination and by using open field;however, all the mice who expressed seizure showed no movement, while the EHof ( 100 mg\kg) that showed attenuated in seizure provoke showed less movementperformance than the controlled group. This was indication ofneuropharmacological continents of the extracted plant in controlling seizure andtheir related psychotic disorders.

• Rota rod test used to test animals skeleton muscles relaxation and their balanceperformance on Rota rod stand; however, in our present study there was nosignificant change in all the groups, which was indication to tell us that theanticonvulsive effect of the extract plant was not due to muscle relaxation and wasnot influenced by the extracted plant components

In the conclusion, investigating the anticonvulative effect of lyophilized extract

of Epilobium hirsutum showed potential anticonvulsant effect and less toxicity

in the experimental male mice at the doses of 100 mg\kg mg and 200 mg\kg

using PTZ kindling model. However, EH 100 mg\kg considered being the

depended dose. In addition, as the experimental epileptic animals expressed

mediation of oxidative stress and free radicals, it suggested that Epilobium

hirsutum prevent seizure provoke by the action of antioxidant mechanism.

However, this suggestion cannot be rely on from a single research.

FARTHER RESEARCHES

• From our present research, farther research ought to be done, using sub-

convulsion doses of PTZ (35mg\kg) with diazepam.

• We have used two doses (100 and 200 mg\kg), different doses might give that

more convincing results.

FARTHER RESEARCHES

• As ours study was carried in vivo, in vitro study is also suggested.

FARTHER RESEARCHES

• Studying the enzymes by taking blood tests might facilitate the process of

the biochemical assay or even gives useful elucidations.

• Studying with bigger animal like rats, rabbits or monkeys may facilitate

biochemical assay and breaking the gatekeeper of most done researches.

FARTHER RESEARCHES

In our experimental we have used lyophilized method in plant

extraction study the plant with different extraction methods may

improve the active components.

FARTHER RESEARCHES

• As we have collected the plant from the coastal line of Van lake; different areas

of different climates may shows more interesting results of different

concentration of different active components of the extracted plant.