This study investigated the anticonvulsant effects of Epilobium hirsutum extract in a pentylenetetrazole (PTZ)-induced seizure model in mice. Mice were pretreated with 100 or 200 mg/kg of E. hirsutum extract or valproate before being injected with PTZ. The extract increased seizure onset time and decreased duration compared to the PTZ group. Neurological tests and biochemical assays also showed the extract reduced oxidative stress and improved motor function versus PTZ. The results suggest E. hirsutum has anticonvulsant properties potentially due to its antioxidant compounds like flavonoids and phenolic acids.

1. 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 AT E K 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 AT E
2 7 / 9 / 2 0 1 7

2. 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.

3. 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.

4. 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.

6. 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

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. MATERIALS AND METHODS
Materials
- 40 Swiss albino male mice
- PTZ 65 mgkg
- Valproate 100 mgkg
- Extracted plants of 100 and 200 mgkg
- Open field
- Rota rod
- Biochemical assay

13. 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.

14. MATERIALS AND METHODS
METHODS
40 mice were divided in 5 groups/ (n= 8)
• The controlled group
• PTZ group
• Positive group (valproate 100 mgkg and PTZ 65 mgkg)
• Tested group of EH two doses (100 and 200 mgkg)
• On the seventh day, challenge dose of (65mgkg) 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 mgkg and 200
mgkg). 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).

15. 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.

16. 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.

17. 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.

18. MATERIALS AND METHODS
Sample prepares for biochemical assay
- Glutathione peroxidase (GSH-Px) evaluation
- SOD evaluation
- Catalase activity determination
- Glutathione evaluation

19. Figure 1. Effect of valproate (100 mg/kg) and PTZ (65mgkg) 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.

20. Figure 2 . Effect of valproate (100 mg/kg) PTZ (65mgkg) 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.

21. 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

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

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

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

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

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

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

28. 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).

29. DISSOCIATION
• Chemical components of Epilobium hirsutum flavonoids, phenolic acids, steroids, and
tannins, which showed to have antioxidant effect that could suppress the damage
caused by PTZ.
• PTZ kindling model blockers chloride channel of GABA A receptor, increase of the
hyperactivity of glutamic receptors (NMDA) and calcium ions for entering into the
nerve cells and liberation of free radicals that cause local injury of brain tissue by
repeating of sub-convoluted dose or single dose of PTZ administration.
• Valproate, which is one of AEDs, known to suppress seizure provoke by binding
GABA receptor and enhance the GABA ergic inhibitory neurotransmission. It is wide
spectrum mechanism against different type of partial and general seizures.

30. • Theoretically, Epilobium hirsutum at doses of both (100 and 200 mgkg),
significantly increase in onset of convulsion and reduction in the duration of
convulsion with no mortality reported when it was compared with valproate group,
which showed weak effect against the latent dose of PTZ ( 65mgkg) and weaker
comparing it with Epilobium hirsutum, which showed completely seizure
attenuation.
• Biochemically, Epilobium hirsutum at doses of both (100 and 200 mgkg),
significant decreased in the level of MDA (an indication of protein oxidation
damage by PTZ or seizure ) when it was compared with PTZ group.
• while the antioxidant enzymes of (SOD, GSH-Px, CAT, and GSH) showed
significant increase when they were compared with PTZ group.

31. • In our presented experiment, we have undergone mice after seizure provoke for
testing their ability of exploration, and motor coordination and by using open field;
however, all the mice who expressed seizure showed no movement, while the EH
of ( 100 mgkg) that showed attenuated in seizure provoke showed less movement
performance than the controlled group. This was indication of
neuropharmacological continents of the extracted plant in controlling seizure and
their related psychotic disorders.
• Rota rod test used to test animals skeleton muscles relaxation and their balance
performance on Rota rod stand; however, in our present study there was no
significant change in all the groups, which was indication to tell us that the
anticonvulsive effect of the extract plant was not due to muscle relaxation and was
not influenced by the extracted plant components

32. 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 mgkg mg and 200 mgkg
using PTZ kindling model. However, EH 100 mgkg 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.

33. FARTHER RESEARCHES
• From our present research, farther research ought to be done, using sub-
convulsion doses of PTZ (35mgkg) with diazepam.
• We have used two doses (100 and 200 mgkg), different doses might give that
more convincing results.

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

35. 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.

36. 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.

37. 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.