Protective Effects of Biarum carduchrum Ethyl Acetate Extract on Seizure Severity,
and Learning in Pentylenetetrazole-Induced Kindling Rats
Youness Teymourivand1, Zahra Hooshmandi1,*, Mahbubeh Setorki2 andSabrieh
- Department of Biology, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran
- Department of Biology, Izeh Branch, Islamic Azad University, Izeh, Iran
* Corresponding author: Zahra Hooshmandi, Department of Biology,
Branch, Islamic Azad University, Sanandaj, Iran. Email: [email protected]
Received 2020 April 23; Revised 2020 July 01; Accepted 2020
Background: Based on traditional beliefs, Biarum carduchrum (family
strengthen the nervous system and prevent from sleep disorder and agitation.
Objectives: This study was performed to investigate the protective effects of the ethyl acetate
Biarum carduchrum leaves against pentylenetetrazol (PTZ)-induced seizure in rats.
Methods: The present experimental research was conducted on 50 male Wistar rats in Iran during
animals were examined in five groups of control, PTZ, intervention (PTZ with 100 and 200 mg/kg ethyl acetate extract
for 10 days),
diazepam (PTZ, 200 mg/kg extract, and diazepam). The research groups were compared in terms of behaviors.
Furthermore, total phenol
and flavonoid levels in the extract were determined using high-performance liquid chromatography.
Results: According to the results, the intervention and diazepam groups had a
lower number of the whole body, tonic, head, and upper organ seizures (P<0.05), and jumps
compared to the PTZ group. Both doses of the extract also significantly decreased immobility time in tail suspension
latency time and significantly enhanced spatial memory in Morris water maze test (P<0.05). This
significantly reduced the levels of nitric oxide and malondialdehyde in the brain and serum (P<0.05) and
antioxidant activity (IC50=200 μg/ml). The extract contained 42.63±0.7494 and
phenol and flavonoid, respectively. The HPLC analysis also revealed the presence of quercetin (30 μg/g) in the
Conclusion: Based on the results, Biarum carduchrum extract can be
depression control and improvement of learning and memory impairments in seizure patients after complementary
Keywords:Biarum carduchrum, Ethyl acetate extract, Learning, PTZ-kindled rats, Seizure
Epilepsy is the most prevalent chronic and debilitating neurological disease that affects 0.5-1% of people
the available treatments, 30% of patients still suffer from epileptic seizures. Although this disease might be
about 60-70% of patients via antiepileptic drugs, the treated patients may suffer from the unwanted side effects of
(1). The hippocampal system is
one of the major areas of the brain involved in epilepsy. Moreover, the hippocampus plays an important role in
and memory, particularly in spatial memory, as lesions in the
CA1 region of the hippocampus cause memory impairments (2). The frequent epileptic
seizures markedly undermine memory and learning (3). It has been found that
electrical and chemical kindling following the application of PTZ can cause learning impairment in laboratory
The hippocampus is involved not only in memory and learning but also in seizure onset, progression, and
neuronal degeneration, especially in the hippocampus CA1 area, alters the function of variable synapses that reserve
information, thereby inducing learning impairment as observed in folding the kindling (7). The PTZ
effects on neuronal membrane characteristics since it blockades chloride channels by coupling with a
A (GABAA) receptor complex and reduces the neurotransmitter-induced chloride induction.
This drug also
affects potassium and calcium channels and releases intracellular calcium ion reserves. It is also applied as a
inducer that is used for the evaluation of antiepileptic drugs in animal models (8). According to a
the concentration of intracellular calcium ion through increasing N-methyl-D-aspartate receptors. The increased
calcium in the cell
protects against the inhibitory effects caused by GABA (9).
Oxidative stress is a condition in which the balance between antioxidants and oxidants is interrupted as the
inconsonance between the production and exhaustion of reactive oxygen and nitrogen species (ROS and RNS,
process is accompanied by the elevation of ROS and RNS levels and the reduction of biological nitric oxide (NO). The
nitrosylation stress pathways lead to inflammatory responses and metabolic mitochondrial process, as well as the
activation of free
radical production (10). The ROS and RNS molecules (e.g.,
dismutase anion, hydroxyl radical, and proxy nitrites) play roles in the immune system and physiological conditions
of the body.
However, they react with fatty acids, proteins, and DNA in the pathologic conditions (accompanied by an increase in
and cause cell damage. Oxidative stress and nitrosylation are involved especially in the diseases of the nervous
system, such as
ischemia, Alzheimer’s, and epilepsy (11, 12).
Biarum carduchrum, belonging to Arecaceae family, is considered one of the most valuable herbs. This
wild growing plant
is found in the Zagros Mountains located in Fars and Kohgiluyeh-Boyer-Ahmad provinces, Iran, as well as in some
areas of Iraq,
Syria, and Turkey (13). The presence of flavonoids and
anthocyanins in Araceae was first reported by Williams et al. (1981). Alkaloids, amines, saponins, cyanotic acids,
are also found in the members of this family (14).
The current research was aimed to determine the protective effects of the ethyl acetate extract of Biarum
leaves on PTZ-induced seizures in rats.
This experimental study was conducted in Kurdestan Province, Sanandaj, located in Iran, in 2019, using Wistar
rats. In order to
establish a chronic epileptic rat model, the animals were induced with epileptic kindling using PTZ (intraperitoneal
of 35 mg/kg/day; CAS:54-95-5; Sigma, St. Louis, MO, USA) or diazepam (Caspian Tamin, Iran). Thiobarbituric acid
(TBA) and sodium
dodecyl sulfate (SDS) were kindly provided by the Saba Co., Ltd. (Sigma, USA). Before the experiments, PTZ and
dissolved in physiological saline. Drug administration was performed between 08:30 and 10:30 a.m. to mitigate the
circadian rhythms; in addition,all equipment was calibrated in this study.
3.2. Extract preparation
Biarum carduchrum was obtained from the local market of Izeh and confirmed by an herbalist (Dr.
reference sample was restored in the herbarium site of the Islamic Azad University of Izeh under the voucher No.
80145. For the
purpose of the study, the leaves were washed and dried at 40°C under vacuum and then milled and sieved for
extraction. In the
next stage, they were added with ethyl acetate and restored for 72 h at room temperature. The extracts were then
filtration and rotation under vacuum at 40°C. Finally, they were dried at the same temperature in an oven under
3.3. Measurement of total phenol compounds
The assessment of phenolic compounds was accomplished using the Folin Ciocalteu method. To this end, 0.5 mL of
before condensation and drying was diluted 10 times with 2.5 mL Folin Ciocalteu reagent and then well mixed by
distilled water and
2 mL sodium carbonate solution (7.5%). The mixture was then inserted in a hot bath (45°C) for 15 min.
absorbance was read by a spectrophotometer at a wavelength of 760 nm. Methanol solution (80%), in addition to
reagents without the
extract, was used as the control sample. The calculated total phenol content (mg in each gram of the dried extract)
compared with a standard calibration curve established based on various concentrations of gallic acid (16).
3.4. Measurement of flavonoids compounds
In brief, 0.5 mL of the extract solution (0.01 g of dried extract per 10 mL of methanol 60%) was mixed with 0.5
mL of 2%
aluminum chloride and 3 ml of 5% potassium acetate. After storing at room temperature for 40 min, the absorption of
the mixture was
determined as compared to that of distilled water at a wavelength of 415 nm. The absorbance value of the mixture was
the standard curve that was prepared based on the absorbance values of different concentrations of quercetin. In
level of flavonoids in the extract was calculated as flavonoids content (mg) in each gram of dried extract (16).
3.5. Diphenyl-β-picrylhydrazyl radical scavenging activity
After the preparation of the extract at various concentrations in distilled water, 1 mL of each concentration was
added at the
equal amount of DPPH solution (0.1 mM, in 95% ethanol). The mixture was then stored for 15 min at room temperature.
In the next
stage, the mixture absorbance was determined at a wavelength of 517 nm using a spectrophotometer. In the control
water (1 mL) was used instead of the extract. The scavenging activity (%) of diphenyl-β-picrylhydrazyl (DPPH)
calculated using the following formula:
DPPH radical scavenging activity (%)=[(control absorbance – sample absorbance)/control
Furthermore, IC50 amount was assessed by plotting the graph of concentration (X-axis) versus the
percentage (Y-axis) (16).
3.6. High-performance liquid chromatography analysis
The HPLC analysis was conducted using the chromatography apparatus, including Waters 2695 separations module and
detector 996 (USA). The injection was performed by the autosampler injector equipment. A 150×4.6-mm column was
chromatographic assay (Eurospher 100-5 C18 analytical column). In addition, elution was carried out using a gradient
methanol (organic phase; solvent A) and distilled water (solvent B) at a flow rate of 1 mL/min. The peaks were
monitored at the
wavelengths ranging from 195 to 400 nm. The volume of 20 µL was injected, and the temperature was adjusted to
data were analyzed using Millennium software (version 32).
3.7. Laboratory animals and grouping
The experiments were conducted at the Islamic Azad University of Sanandaj Kurdestan, Iran. Prior to the study,
was obtained from the Ethics Committee of the university. The animals investigated in this study included 50 male
within a weight range of 150-200 g, supplied from the Center of Animal Breeding Facility in Pasteur Institute,
Karaj, Iran. The
animals were kept under appropriate temperature (21±2°C) and 12 h light/dark cycle with water and food ad
the purpose of the study, the animals were allocated into five groups, each containing 10 cases. In this regard, the
negative control groups were respectively subjected to IP injection of normal saline and PTZ every 48 h for 10 days.
the positive control group was injected with PTZ every 48 h for 10 days and Biarum carduchrum ethyl acetate
mg/kg, daily), followed by diazepam (2 mg/kg) on the 10th day, about 30 min before PTZ injection. The
was also administered with the IP injection of Biarum carduchrum ethyl acetate extract at the doses of 100
and 200 mg/kg
on a daily basis, 30 min before PTZ injection performed every 48 h for 10 days. In order to establish the epilepsy
model, PTZ (35
mg/kg) was administered for 9 days intraperitoneally every 48 h. On day 10, PTZ (60 mg/kg) was injected, and the
degree of seizure were recorded for 30 min. Finally, after obtaining blood samples from the rats under deep
brains were removed and restored at -80°C for the following biochemical tests.
3.8. Passive avoidance memory testing by shuttle box
The measurement of passive avoidance memory was accomplished using the shuttle box. This apparatus contains an
shocker, two chambers (one dark and one bright, connected by a guillotine door), and a 15-watt bulb. The floor of
this device is
composed of a conductive metal grid. The animals were subjected to this fear-based experiment over 4 consecutive
performed trials on the first 2 days were targeted toward training the rats and their acclimatization to the
instrument. The first
day of the experimentation was initiated by leaving the rat in the bright chamber for 5 min. After opening the door
light and dark chambers, the animal voluntarily departed to the dark side and left the place after 5 min. On day 3
experiment, the barrier between the two chambers was removed 20 sec later than the time the rat had previously left
chamber. At this stage, the latency of entering the dark chamber was calculated and regarded as initial latency
(t1). After the rat
entrance to the dark chamber, it was subjected to an electrical shock (1 mA/s); as a result, it left the device
paddling. On the 4th day, the rat remained in the first chamber. After turning on the lamp and opening
the door, the
interval time between the entrance to the bright chamber and dark chamber (up to 300 sec) was calculated and
recorded as delay
through passing (t2) (17).
3.9. Tail suspension test
The tail suspension test was performed using metal bars with a height of 70 cm connecting through a 50-cm rope
longitudinally. The rat was hanged up from its tail, tied by a rope. The test was first begun by jerking the rat.
experiment time duration was 6 min with the first 2 min being used for animal adaptation to the device. The
immobility time (sec)
was evaluated in the next 4 min by means of a chronometer. All measurements were investigated for each rat
3.10. Spatial memory testing
The evaluation of learning, memory, and motor function of the rats was performed using the Moriss water maze. The
included a water pond (136-cm in diameter and 60 cm in height), which was filled up with water (20±1°C)
up to a height
of 25 cm. A 10-cm diameter Plexiglass platform was inserted in the center of the southwest quadrant, about 1 cm
below the water
surface. Each rat was trained four times a day for 4 days. On the 5th day, the test was performed one
time without a
3.11. Measurement of serum nitrite and nitrate
Nitrite and nitrate measurements were performed based on the rate of nitrate to nitrite reduction by cadmium and
the Griess1 and
Griess2 reagents (4).
3.12. Measurement of lipid peroxide levels in the serum and brain
In order to measure lipid peroxide level, 1.5 ml acetic acid (20%), 1.5 ml TBA (0.8%), and 200 μl SDS (8.1%)
were mixed with
200 μl tissue homogenate or serum. The mixture volume was adjusted to 4 mL by distilled water and warmed 60 min
water. After cooling, the reaction mixtures were added with 5 mL n-butanol/pyridine and 1 mL distilled water and
then subjected to
vigorous shaking. The obtained solution was centrifuged for 10 min at 4,000 rpm, and the optical density of the
detected at a wavelength of 532 nm. A comparison was made between the estimated lipid peroxide levels and standard
curve (presented in µmol of MDA) (17).
3.13. Serum and brain total anti-oxidant capacity assay
After the completion of the behavioral tests, heart blood samples were obtained from anesthetized rats before the
their brain. Ferric reducing capacity was detected based on the serum ability to restore ferric ions in the presence
tripyridyl-s-triazine using the colorimetric method (17).
3.14. Statistical analysis
Data analysis was performed in SPSS software (version 20). The normality of data was tested using the
In addition, the comparison of the groups was established based on the ANOVA and Tukey's test. All data were
mean±SD, and the significance of differences was calculated at 95% confidence level.Given the function of
(measuring inter-sample variability), such a value is required for quantitative variables. The calculation
of the sample
size was performed using the freely downloadable G Power software (Faul, Erdfelder, Lang and Buchner, 2007). Based
software, the sample size was determined as 10 animals per group. Considering 20% attrition, this size was divided
by 0.8 as
presented in the following formula:
Corrected sample size=sample size/(1−[% attrition/100])
4.1. Phytochemical analysis of Biarum carduchrum ethyl acetate extract
The total levels of phenolic compounds and flavonoids in the dried extract were estimated at 42.63±0.7494
85.16±6.499 mg/g, respectively. The results of HPLC analysis revealed the presence of quercetin in Biarum
carduchrum ethyl acetate extract at a concentration of 30 μg/g (Figure 1). Furthermore, the
IC50 level of the extract was obtained as 200 μg/mL based on the results of the DPPH inhibitory
activity (Figure 1).
Figure 1. Results of high-performance liquid chromatography analysis and
Biarum carduchrum extract
4.2. Behavioral tests
The frequencies of death rate, tonic, whole body, head, and upper organ seizures, head tick, and jumping and
rotation in the
research groups are depicted in Figure 2 (A-F). The treatment of rats
with Biarum carduchrum ethyl acetate extract at the doses of 100 and 200 mg/kg led to a significant
decrease in the rate
of tonic, total body, head, and upper organ seizures, as well as the number of rotations and jumps, compared to
those in the PTZ
group. However, the numbers of tonic seizures, total body seizures, and rotation and jumping were significantly
lower in the group
receiving diazepam than in the PTZ group.
Based on the results, the number of head ticks underwent a significant decrease in the group injected with 100
carduchrum ethyl acetate extract as compared with that in the PTZ group. According to the results of the tail
Figure 2. Comparison of the frequency of tonic seizures (A), total body
seizures (B), seizures
of the head and upper limbs (C), head tick (D), frequent rotation and jumping (E), and death (F) among the research
groups (* shows
a significant difference with the group administered with PTZ [*: P<0.05, **: P<0.01, and ***: P<0.001])
BB-EA=Biarum carduchrum-ethyl acetate
Figure 3. Comparison of research groups in terms of immobility time based on
suspension test (* shows a significant difference with the PTZ group [***: P<0.001]; # shows a significant
the control group [###: P<0.001])
BB-EA=Biarum carduchrum-ethyl acetate
test (Figure 3), the duration of immobility
in the PTZ-receiving rats showed a significant increase, compared to that in the control group. Furthermore, the
treatment of the
rats with Biarum carduchrum ethyl acetate extract at the doses of 100 and 200 mg/kg resulted in a
significant decrease in
the duration of immobility, compared to the PTZ treatment.
Based on the results, the sequential injection of PTZ resulted in the significant inhibition of the secondary
delay (t2) in the
PTZ group in comparison to that in the control group. However, the group administered with the two doses (100 and
200 mg/kg) of
Biarum carduchrum ethyl acetate extract showed a significant increase in secondary delay,
the PTZ group (Figure 4 A-B).
As the results indicated, the delay time in reaching the platform in the Morris water maze test was significantly
the 3rd and 4th days in the PTZ group than in the control group (Figure 5 A,
B). On the first day,
this delay time was significantly lower in the group injected with 200 mg/kg Biarum carduchrum extract than
in the PTZ
group. According to the results, the delay in reaching the platform significantly decreased in the groups receiving
the doses of
100 and 200 mg/kg of Biarum carduchrum extract during the 2nd, 3rd, and
4th days in
comparison to that in the PTZ group. Furthermore, during the 1st, 2nd, and 4th
days, the frequency
of swimming in the target quadrant was significantly higher in the group administered with 200 mg/kg dose of
carduchrum extract than in the PTZ group.
According to the results of the Morris water maze test (Figure 6 A, B), on the examination
day, the control group had a significantly longer swimming duration and higher swimming frequency in the target
quartile than the
As illustrated in Figure 7 (A-D), the sequential
infusion of PTZ into rats was associated with a significant elevation in the MDA level and a significant decrease in
the brain and
serum antioxidant capacity. However, the treatment of rats by 200 mg/kg of Biarum carduchrum extract
resulted in a
significant increase in the serum antioxidant capacity, compared to PTZ treatment. Moreover, the groups receiving
the doses of 100
and 200 mg/kg of the extract showed a significant elevation in the antioxidant activity of the brain tissue and a
decrease in the MDA levels of the serum and brain tissue.
Figure 4. Comparison of secondary delay among the research groups based on the
test (* shows a significant difference with the PTZ group [**: P<0.01 and ***: P<0.001]).
BB-EA=Biarum carduchrum-ethyl acetate
Figure 5 (A, B). Comparison of delay in reaching the hidden platform (A) and
frequency in the target quartile (B) during the test days among the research groups (* indicates significant
difference with the
PTZ group [*: P<0.05, **: P<0.01, ***: P<0.001])
BB-EA=Biarum carduchrum-ethyl acetate
Figure 6 (A, B). Comparison of swimming time (A) and swimming frequency in the
(B) during the examination day among the research groups (* shows a significant difference with the PTZ group [***:
shows a significant difference with the control group [###: P<0.001])
BB-EA=Biarum carduchrum-ethyl acetate
The results revealed no significant changes in the serum NO levels among the research groups (Figure 8
A, B). According to the
results of this study, the PTZ groups had a significantly higher level of NO level in the brain tissue than the
control group. In
addition, the groups receiving 100 and 200 mg/kg of the extract showed significantly lower levels of NO in the brain
compared to the PTZ group.
The present research evaluated the protective impacts of Biarum carduchrum ethylacetate extract against
PTZ-induced seizures in rats. The results revealed that the rats treated with 100 and 200 mg/kg doses of ethyl
acetate extract had a significant decrease in the number of tonic seizure, total body seizures, seizures of the head
and upper organs, and rotation and jumping. The highest anticonvulsant activity was observed in the seizure-induced
rats administered with extract (200 mg/kg) and diazepam as a GABA agonist. It is proposed that the GABAergic system
has an important role in the epilepsy process. The GABA is a main inhibitor neurotransmitter of the central nervous
system, and more than 25% of the inhibitory neurons are GABAergic. The GABA plays a key role in the modulation of
neuronal activity. This neurotransmitter applies its effects through various receptors. There are two types of GABA
receptors, namely GABAA and GABAB. The GABAA receptors are
Figure 7. Comparison of antioxidant activity in the serum (A) and brain (B) and
malondialdehyde levels of the serum (C) and brain (D) among the research groups (* shows a significant
difference with the PTZ group (**: P<0.01 and ***: P<0.001)
BB-EA=Biarum carduchrum-ethyl acetate
Figure 8. Comparison of nitric oxide levels in the serum (A) and brain (B)
among the research
groups (* indicates a significant difference with the PTZ group (**: P<0.01 and ***: P<0.05)
BB-EA=Biarum carduchrum-ethyl acetate
ionotropic and embed chlorine flow; on the other hand, the GABAB receptors change the function of the
neurons by binding to and activating G proteins and the intracellular messenger system.
According to the literature, the drugs increasing the synaptic rates of GABA by blocking GABA catabolism or
elevating GABA re-absorption are categorized among effective antiepileptics. Benzodiazepines are among these drugs
that elevate the binding rate of GABA to its receptors, and result in the increase of the permeability of chloride
channels. The results of HPLC analysis of Biarum carduchrum ethyl acetate extract were indicative of the
high levels of quercetin flavonoid in the extract. Quercetin is a flavonoid that is found in different fruits and
vegetables, including apple, citrus fruits, berries, onion, cereals, legumes, and tea. Several pharmacological
properties, including antioxidant, anti-inflammatory, and hepatic protective effects, have been reported for
In a study performed by Nassiri-Asl et al. (2013), quercetin at a dose of 50 mg/kg mitigated seizure severity and
enhanced avoidance memory. In addition, pre-treatment with quercetin at the doses of 50 and 100 mg/kg inhibited the
increasing amount of mRNA transcription for the production of GABAA receptor β1 and β3 subunits, 2 h after
kainic acid injection (19). In
terms of innovation, very few studies have investigated this plant and its anti-epileptic effects. One of the
weaknesses of our research was the non-investigation of the molecular mechanism of this plant.
In the current research, diazepam strengthened the anticonvulsant effects of Biarum carduchrum extract,
which could be ascribed to the positive effects of diazepam on the GABAergic system.
Recent studies indicated that oxidative stress and mitochondrial dysfunction can make the brain susceptible to
epileptic seizures. On the other hand, seizure attacks lead to free radicals production and oxidative damage to the
proteins, fats, and nucleic acids in the cells. Therefore, oxidative stress and the production of free radicals are
currently known as the outcomes of seizure attacks (20).
Reactive oxygen species, like superoxide radical, hydrogen peroxide, hydroxyl radical, and oxygen radical, are
generated during normal cell metabolism. The physiological levels of ROS can be neutralized by enzymes, including
superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and peroxiredoxin, or reduced by
non-enzymatic molecules (e.g., vitamins C and E) and the antioxidant defense system (GSH).
However, excessive ROS production reduces the ability of the antioxidant defense system and leads to oxidative
stress. Furthermore, additional ROS reacts with NO and results in the production of RNS, such as peroxynitrite (4).
Seizure induction with PTZ can result in a significant decrease in the levels of GSH, GSSG, cysteine, and thiol
proteins. In addition, there is a report regarding the elevation of carbonyl and D-sulfide proteins in the cerebral
cortex of the mice (6). The
seizure caused by acute PTZ significantly increased NO (five times) in the cerebral cortex (4). The
adjustment of oxidative stress using herbal and chemical drugs is seen as a tool to reduce the damages caused by
epilepsy and the onset of seizure.
In the present study, the results revealed a significant elevation in the peroxidation of lipids and NO
production in the brain and a significant decrease in antioxidant capacity in the rats receiving PTZ injections.
Moreover, the treatment of the seizure-induced rats with Biarum carduchrum ethyl acetate extract was found
to enhance the brain antioxidant capacity and decrease the levels of NO and MDA in the brain. Regarding this, the
Biarum carduchrum extract seems to show its protective effects against the damage caused by PTZ through
dealing with oxidative damage to the nerve cells and strengthening the antioxidant defense.
Based on our results, Biarum carduchrum ethyl acetate extract had a high ability to neutralize DPPH
radicals, indicating the powerful antioxidant activity of the extract. In addition, the evaluation of the total
phenol and flavonoid levels of the Biarum carduchrum extract revealed high levels of these compounds. The
results of Hosseini et al. (2014) showed that Biarum carduchrum extract had a higher capacity of inhibiting
DPPH and reducing its activity than BHT and alpha-tocopherol, respectively (15).
Moreover, the results of the mentioned study were suggestive of the increase of antioxidant activity
with the elevation of the hydromethanol extract concentration, which is due to the entry of more phenolic
compounds into the reaction environment. The increased phenolic compound concentration and hydroxyl group number in
the reaction environment increase the possibility of free radical hydrogenation, thereby leading to the enhancement
of antioxidant activity (15).
Cognitive disorders are mainly seen in patients with epilepsy, which itself can disrupt the cognitive processes.
The common antiepileptic drugs also cause disruptions in the cognitive processes, which is associated with a
significant decline in the quality of life. Epileptic seizures are accompanied by the destruction of nerve cells in
the limbic areas, including CA3, CAL, hippocampus dentate gyrus, amygdala, and entorhinal cortex. The damage to the
neural cells in the hippocampus can lead to memory and learning impairments. The hippocampus acts as a significant
structure in memory processes, and damages to this area of the brain could lead to severe amnesia (21).
According to the reports, the induction of epilepsy in rats by PTZ causes disruption in passive avoidance memory and
reduces the secondary latency time of entering the dark room in the shuttle box test, which is consistent with our
In our study, Biarum carduchrum extract significantly improved spatial memory and passive avoidance
memory in the rats receiving PTZ. It can be concluded that Biarum carduchrum extract prevents memory
destruction by preventing damage to the tissues involved in the memory and learning processes. Quercetin as the
major flavonoid component of Biarum carduchrum ethyl acetate extract can improve learning and spatial
memory by reducing oxidative stress and increasing GSH in the mice (22). In
addition, a single dose of quercetin injected 1 h before receiving scopolamine was reported to improve memory and
learning in zebrafish (23).
Based on the evidence, patients with epilepsy have a significantly higher prevalence of depression than the
general population. Accordingly, depression is one of the serious medical and social problems in epileptic patients,
which affects the quality of life in these patients. In a study, depression was reported to affect almost half of
the patients treated in the epilepsy centers (24).
The administration of Biarum carduchrum extract significantly decreased the immobilization time in the
tail suspension test. Pre-treatment with quercetin as a bioflavonoid has increased the time of social interaction
and reduced the immobility time in mice, indicating its anti-anxiety and anti-depressant effects (25).
Quercetin also dose-dependently shortened the duration of immobilization in the forced swim test in
diabetic mice (26).
According to these studies, the antidepressant effects of Biarum carduchrum could be due to its high
The results of this study revealed that the administration of Biarum carduchrum extract to PTZ-treated
rats significantly decreases the frequency of seizures in the entire body, as well as repeated rotation and
jumping. Additionally, the results of the shuttle box test indicated a significant increase in passive
avoidance memory and spatial memory in the treated rats. The results of the tail suspension test were also
indicative of the shortening of the immobilization time in this group. Therefore, it can be stated that the
protective effects of Biarum carduchrum extract against PTZ-induced seizure are probably obtained by the
modulation of GABA receptors and reduction of the brain oxidative stress markers.
Herein, the authors of the current study extend their gratitude to the staff of the Laboratory of Islamic
Azad University of Sanandaj, Iran, for their technical support. The present research was derived from a thesis
submitted in partial fulfillment of the requirement for a Ph.D. degree.
Authors’ Contribution: Youness Teymorivand conceived and extracted the data, Zahra
Hooshmandi performed the study design, data analysis, and manuscript drafting. Mahbubeh Setorki carried out the
paper revision with complete access to all research data, and Sabrieh Amini checked the integrity of the data
and the accuracy of data analysis, therefore acting as a guarantor.
Conflict of Interests: The authors declare that there is no conflict of interest.
Ethical Approval: The research protocol was approved in 2019 by the Ethics Committee of Sanandaj
Branch, Islamic Azad University, Sanandaj, Iran (Code number: (110483732395782162273123).
Funding/Support: The authors have received no financial support from any agency for this
Funding/Support: Informed consent: Not applicable.
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