Keywords seizure -
Anchusa italica Retz. extract - oxidative stress - mice
Introduction
Epilepsy is one of the most common chronic neurological disorders that affect ∼1%
of the world population.[1 ]
[2 ] Varied range of etiologies such as central nervous system infections, brain injuries,
brain tumors, and cerebrovascular disorders is involved in the development of seizure.[3 ] While the causes of seizures are widely classified, the full mechanisms involved
in the disease are not fully understood. Commonly prescribed drugs are associated
with various side effects, and, in addition, some patients are resistant to these
drugs.[4 ]
[5 ] Therefore, the introduction of new drugs with fewer side effects and more effectiveness
is strongly felt.
Nitric oxide (NO) plays a key role in various pathophysiological and physiological
processes in the nervous system and its related disorders.[6 ]
[7 ] Recently, NO has been shown to be involved in stimulating and exacerbating seizures
in rodents.[8 ] Researchers have shown that the increased levels of NO is associated with decrease
in seizure threshold in the pentylenetetrazole (PTZ) model of seizure in rodents.[9 ]
Besides, several experimental models of seizures have demonstrated an increase in
the oxidative stress markers, such as malondialdehyde (MDA), as the end-product of
lipid peroxidation, in the brain.[10 ] High oxidative stress state and free radical generation are possible causes of seizure-induced
neuronal apoptosis and death.[11 ] Previously, it has been determined that seizures are associated with decrease in
total antioxidant capacity (TAC) in the brain and serum samples.[12 ]
[13 ] Therefore, agents that can reduce the level of oxidative stress markers and increase
antioxidant capacity may potentially have anticonvulsant effects.
Using plant- or herbal-derived compounds for treatment of diseases has currently been
specially considered. Hence, researchers are trying to introduce herbal medicines
for management of diseases.[14 ] Medicinal plants have long attracted much attention and have been used extensively
in traditional medicine. Today, the effects of a large number of these plants in seizures
have been studied and their anticonvulsant effects have been confirmed.[14 ]
[15 ]
[16 ] Recent studies showed that consumption of medicinal plant or natural products as
antioxidants is an approach for management of neurological diseases. Anchusa italica Retz. belongs to the Boraginaceae family, mostly found in temperate regions, especially
in the Mediterranean and tropical regions. A. italica Retz. has many pharmacological properties, including anticancer, antiviral, cardioprotective,
and antioxidants effects.[17 ]
[18 ]
Considering all the information mentioned above, in the current study, using PTZ,
a seizure-provoking agent in rodents, anticonvulsant effects of hydro-alcoholic extract
of the A. italica was investigated, focusing on its possible antioxidative stress properties.
Materials and Methods
Plant Material and Preparation of Hydro-Alcoholic Extract of A. italica
The plant was collected from the margins of Saman city in Chaharmahal va Bakhtiari
province. A. italica flowers were gathered and botanically confirmed by a botanist (Shirmardi, Hamzeh
Ali, PhD, Research Center of Agriculture and Natural Resources, P.O. Box 415, Shahrekord,
Iran) and deposited as specimens in Herbarium of Medical Plants Research Center in
Shahrekord University of Medical Sciences, Shahrekord, Iran (SKUMS-504). In the next
step, by maceration method, 1,000 g of it was macerated in 3,000 mL of 70% ethanol
and kept on a shaker at room temperature for 48 hours. The resulting mixture was filtered
and the solvent was evaporated by a rotary evaporator at 38°C. The extract was completely
dried in the incubator under 40°C.
Standardization of Extract
The total phenolic content of A . italica extract was calculated using Folin–Ciocalteu method. To this end, 1 mg/mL extract
achieved a volume of 3 mL by adding up distilled water and then mixed with 0.5 mL
Folin–Ciocalteu reagent for 10 minutes. After that, 4 mL of sodium carbonate was added
to the consequential mixture. The obtained mixture was kept in the dark for half an
hour and the optical absorbance was read at 765 nm wavelength. The experiments were
conducted in triplicate. The total phenolic content was measured by using a calibration
curve, and the results were expressed as mg of Gallic acid equivalent/g dried extract.[19 ]
Ethical Approval
The present study was conducted following the guidelines for the care and use of laboratory
animals as adopted by the committee on the care and use of laboratory animals of the
Shahrekord University of Medical Science (IR.SKUMS.REC.1396.103).
Experimental Animals
The male NMRI mice, weighing 25 to 30 g, were used in the present study. Animals were
kept under standard laboratory conditions, including 24°C temperature and a 12-hour
darkness cycle with free access to water and food. They were randomly divided into
six groups (n = 10). Animals in group 1 (positive control) received phenobarbital at a dose of
4 mg/kg (i.p.) as a standard antiseizure drug, whereas those in group 2 (negative
control group) received normal saline at a dose of 90 mg/kg (i.p.). Animals in groups
3, 4, and 5 (therapeutic groups) received A. italica extract at the doses of 50, 100, and 200 mg/kg (i.p.). All agents in groups 1 to
5 were administrated 30 minutes before PTZ (i.v.). Animals in group 6 were healthy
controls who received normal saline (i.p.) without PTZ. The dose and time of administrations
were chosen based on our pilot study.
Evaluation of Seizure Threshold
To induce a seizure, PTZ (10 mg/mL) was infused into the tail vein of freely moving
mice at a constant rate (0.3 mL/min) using a 30-gauge needle. After fixing the mice's
tail, the PTZ was injected by a seizure pump. The injection was stopped as soon as
the clonus of the anterior limb was seen. The minimum dose of PTZ for the seizure
was considered as the dose of seizure threshold. In this method, the seizure threshold
was dependent on the PTZ dose and time.[20 ]
Measuring Serum and Prefrontal Cortex Antioxidant Capacity
In the next step, the animals were sacrificed, and their prefrontal cortex and serum
were isolated and transferred for biochemical measurements of the tissue or serum.
Three solutions were used to measure the antioxidant capacity of samples including
solution 1 (1.55 mL of sodium acetate, 8 mL of acetic acid, and 500 mL of distilled
water), solution 2 (270 mg iron chloride, which dissolved in 250 mL of distilled water),
and solution 3 (prepared by dissolving of 47 mg treeazin in 40 mL of hydrochloric
acid [40 mM]). The work-up solution was prepared from 10 mL of solution 1, 1 mL of
solution 2, and 1 mL of solution 3. Also, 25 μL of the serum or homogeneous prefrontal
cortex samples was added to 1.5 mL of the work-up solution and placed at 37°C for
10 minutes, and then the optical absorption at the wavelength of 593 nm was recorded.[21 ]
Measurement of Serum Malondialdehyde
In summary, 0.5 g of thiobarbituric acid was mixed with 80 mL of acetic acid (20%),
adjusted to pH 5/3 by sodium hydroxide, and the volume was diluted with 20% acetic
acid per 100 mL. Next, 100 μL of the serum sample was mixed with 100 μL of sodium
dodecyl sulfate solution (1.8%) and 2.5 mL of the work-up solution. The samples were
placed in boiling water for 1 hour, then cooled and centrifuged at 4,000 rpm. The
optical absorbance of the supernatant was recorded at 523 nm.[22 ]
Measurement of Prefrontal Cortex Malondialdehyde
One gram of the prefrontal cortex tissue was homogenized in 2.5% potassium chloride
(10%) and incubated at 37°C ± 1 in a metabolic shaker for 60 minutes. After 1 hour
of incubation, 1 mL of trichloroacetic acid (5%) plus 1 mL of 67% thiobarbituric acid
was added and mixed well after each stage. The combination of each vial was transferred
to a centrifuge tube and centrifuged at 2,000 g for 15 minutes. Afterwards, the supernatant
was transferred to another tube and placed in a boiling water bath. Ten minutes later,
the test tubes were cooled and the absorbance of each portion was measured at 535 nm.[22 ]
The Nitrite Assay
The Griess reaction was adapted to measure nitrite as depicted. Briefly, the standard
curves for nitrite were prepared, and the samples (50 µL of serum and 100 µL of tissue
suspensions) were added to the Griess reagent. Proteins were precipitated by adding
50 µL of 10% trichloroacetic acid (Sigma-Aldrich). The contents were centrifuged,
and the supernatants were transferred to a 96-well flat-bottomed microplate. Absorbance
was read at 520 nm using a microplate reader, and the final values were calculated
from the standard calibration plots.[23 ]
Statistical Analysis
Statistical analysis was performed using SPSS18 software. One-way analysis of variance
followed by Tukey's post-hoc test was used for data analysis. Values are expressed
as mean ± standard error, and p < 0.05 was considered statistically significant.
Results
Standardization of Extract
Based on Folin–Ciocalteu method, the total phenol content of A . italica extract was determined: 72.2 ± 0.6 mg of Gallic acid equivalent/g of the dried extract.
Seizure Threshold
The results of the mean delay in the onset of seizure (seizure threshold) are presented
in [Fig. 1 ]. Findings showed that the mean delay significantly increased in the extract-treated
group (200 mg/kg; p = 0.0007) as well as phenobarbital group (p = 0.0003), compared with the PTZ group. Furthermore, results showed that seizure
threshold in the group that received phenobarbital was significantly higher than groups
treated with extract at doses of 50 (p = 0.0002), 100 (p = 0.0005), and 200 mg /kg (p = 0.0397).
Fig. 1 The seizure threshold (seconds) in the experimental groups. Data are presented as
mean ± standard error from 10 mice and were analyzed using one-way analysis of variance
followed by Tukey's post-hoc test. ***p < 0.001 compared with the PTZ group, &p < 0.05, and &&&p < 0.001 compared with the PHEN group. EXT, Anchusa italica Retz. hydro-alcoholic extract; PHEN, phenobarbital; PTZ, pentylenetetrazole.
Serum Total Antioxidant Capacity
[Fig. 2 ] indicates that the TAC in the PTZ group significantly decreased, compared with the
control group (p = 0.0452). We observed that all three doses of extract significantly increased the
TAC in comparison with the PTZ group (p = 0.0004). Furthermore, results showed that serum TAC in groups treated with extract
at doses of 50 (p = 0.0003), 100 (p = 0.004), and 200 mg/kg (p = 0.0006) was significantly higher than the group receiving phenobarbital.
Fig. 2 Serum antioxidant capacity (micro mol Fe+2 /mg protein) in the experimental groups. Data are presented as mean ± standard error
from 10 mice and were analyzed using one-way analysis of variance followed by Tukey's
post-hoc test. *p < 0.05 compared with the control group, ###p < 0.001 compared with the PTZ group, and &&p < 0.01 and &&&p < 0.001 compared with the PHEN group. EXT, Anchusa italica Retz. hydro-alcoholic extract; PHEN, phenobarbital; PTZ, pentylenetetrazole; TAC,
total antioxidant capacity.
Serum Malondialdehyde (MDA) Level
According to [Fig. 3 ], serum MDA level in the PTZ group is significantly higher than in the control group
(p = 0.008). Also, A. italica extract at doses of 50, 100, and 200 mg/kg as well as phenobarbital significantly
reduced MDA in the serum samples in comparison with the PTZ group (p = 0.0007). In addition, finding showed that serum MDA level in group treated with
extract at dose of 50 mg/kg was significantly higher than group receiving phenobarbital
(p = 0.0216).
Fig. 3 Serum malondialdehyde levels (micro mol/L) in the experimental groups. Data are presented
as mean ± standard error from 10 mice and were analyzed using one-way analysis of
variance followed by Tukey's post-hoc test. **p < 0.01 compared with the control group, ###p < 0.001 compared with the PTZ group, and &p < 0.05 compared with the PHEN group. EXT, Anchusa italica Retz. hydro-alcoholic extract; MDA, malondialdehyde; PHEN, phenobarbital; PTZ, pentylenetetrazole.
Serum Nitrite Level
Results showed that nitrite level in serum samples in the PTZ groups is significantly
higher than in the control group (p = 0.0002; [Fig. 4 ]). We found that A. italica extract at doses of 100 (p = 0.035) and 200 mg/kg (p = 0.0008) as well as phenobarbital (p = 0.003) significantly reduced the nitrite level in comparison with the PTZ group.
In addition, finding showed that serum nitrite level in group treated with extract
at dose of 200 mg/kg was significantly lower than group receiving phenobarbital (p = 0.0251).
Fig. 4 Serum nitrite levels (micro mol/L) in the experimental groups. Data are presented
as mean ± standard error from 10 mice and were analyzed using one-way analysis of
variance followed by Tukey's post-hoc test. ***p < 0.001 compared with the control group, #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the PTZ group, and &p < 0.05 compared with the PHEN group. EXT, Anchusa italica Retz. hydro-alcoholic extract; PHEN, phenobarbital; PTZ, pentylenetetrazole.
Prefrontal Cortex TAC
As [Fig. 5 ] presents, the TAC in the prefrontal cortex in the PTZ group is significantly lower
than in the control group (p = 0.0004). Results showed that A. italica extract at doses of 50, 100, and 200 mg/kg significantly increased the TAC in the
prefrontal cortex in comparison with the PTZ group (p = 0.0002). Moreover, results showed that prefrontal cortex TAC in groups treated
with extract at doses of 50, 100, and 200 mg/kg was significantly higher than group
receiving phenobarbital (p = 0.0007).
Fig. 5 Antioxidant capacity of prefrontal cortex (micro mol Fe+2 /mg protein) in the experimental groups. Data are presented as mean ± standard error
from 10 mice and were analyzed using one-way analysis of variance followed by Tukey's
post-hoc test. ***p < 0.001 compared with the control group, ###p < 0.001 compared with the PTZ group, and &&&p < 0.001 compared with the PHEN group. EXT, Anchusa italica Retz. hydro-alcoholic extract; PHEN, phenobarbital; PTZ, pentylenetetrazole; TAC,
total antioxidant capacity.
Prefrontal Cortex MDA Level
Based on [Fig. 6 ], the MDA in the prefrontal cortex in the PTZ group is significantly higher than
in the control group (p = 0.0005). We showed that A. italica extract at doses of 50, 100, and 200 mg/kg as well as phenobarbital significantly
decreased the MDA in the prefrontal cortex in comparison with the PTZ group (p = 0.0003). In addition, finding showed that prefrontal cortex MDA level in group
treated with extract at dose of 200 mg/kg was significantly lower than group receiving
phenobarbital (p = 0.0274).
Fig. 6 Level of malondialdehyde (nano mol/mg protein) of prefrontal cortex in the experimental
groups. Data are presented as mean ± standard error from 10 mice and were analyzed
using one-way analysis of variance followed by Tukey's post-hoc test. ***p < 0.001 compared with the control group, ###p < 0.001 compared with the PTZ group, and &p < 0.05 compared with the PHEN group. EXT, Anchusa italica Retz. hydro-alcoholic extract; MDA, malondialdehyde; PHEN, phenobarbital; PTZ, pentylenetetrazole.
Prefrontal Cortex Nitrite Level
As [Fig. 7 ] shows, the level of nitrite in the prefrontal cortex of the PTZ group is significantly
higher than in the control group (p = 0.0004). Results showed that A. italica extract at doses of 50 (p = 0.0475), 100 (p = 0.0042), and 200 mg/kg (p = 0.0006) as well as phenobarbital (p = 0.0002) significantly reduced the nitrite level in the prefrontal cortex in comparison
with the PTZ group. In addition, finding showed that prefrontal cortex nitrite level
in group treated with extract at dose of 200 mg/kg was significantly lower than group
receiving phenobarbital (p = 0.0482) while prefrontal cortex nitrite level in group treated with extract at
dose of 50 mg/kg was significantly higher than group receiving phenobarbital (p = 0.0214).
Fig. 7 Level of nitrite (nano mol/mg protein) of prefrontal cortex in the experimental groups.
Data are presented as mean ± standard error from 10 mice and were analyzed using one-way
analysis of variance followed by Tukey's post-hoc test. ***p < 0.001 compared with the control group, #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the PTZ group, and &p < 0.05 compared with the PHEN group. EXT, Anchusa italica Retz. hydro-alcoholic extract; PHEN, phenobarbital; PTZ, pentylenetetrazole.
Discussion
The findings of the present study showed that hydro-alcoholic extract of A. italica Retz. increased the seizure threshold in PTZ-induced seizure in mice. Results showed
that hydro-alcoholic extract of A. italica Retz. increased the antioxidant capacity as well as decreased the MDA and nitrite
levels in the prefrontal cortex and serum samples ([Fig. 8 ]).
Fig. 8 Graphical abstract of the study.
Given the increasing rate of epilepsy worldwide and the low efficacy and various side
effects of many synthetic drugs, finding new effective agents with low side effects
seem to be necessary.[24 ] Researchers are considering herbal medicines as potential therapies in treatment
of diseases.[25 ]
[26 ] Previous studies have demonstrated that the hydro-alcoholic extract of some medicinal
plants like Viola tricolor significantly enhanced the seizure threshold.[27 ] However, some herbal medicine has not been effective in epilepsy.[28 ] Previous studies have demonstrated that A. italica extract possessed various pharmacological properties in animal model of diseases.[19 ]
[29 ] It has been determined that A. italica has antioxidant properties.[30 ] Also, Torki et al (2018) revealed that A. italica through attenuation of oxidative stress state exerted neuroprotective effects in
brain ischemia model in rats.[19 ] The results of present study showed that A. italica Retz. extract, partially at least, through attenuation of oxidative stress state
increased the seizure threshold. We observed that A. italica Retz. extract at dose of 200 mg/kg had high anticonvulsant efficacy compared with
doses of 50 and 100 mg/kg in PTZ-induced seizure in mice.
The role of oxidative stress in the pathophysiology of seizure has been determined
in several studies.[3 ]
[31 ] In this regard, it has been demonstrated that biomarkers of oxidative stress in
patients with epilepsy are significantly higher than healthy subjects.[32 ] Animal studies indicated that the induction of seizures by PTZ increased production
of free radicals and led to oxidative damage to cells.[33 ] Imbalance in oxidative/antioxidative systems may play a role in oxidative damage
to neurons following seizure attacks.[31 ] In the current study, hydro-alcoholic extract of A. italica Retz. displayed acceptable antioxidant activity in inhibition of 2,2-diphenyl-1-picrylhydrazyl
radicals. Therefore, this ability appears to be related to the antioxidant property
of phenolic compounds present in A. italica Retz. extract. In this study, we found that hydro-alcoholic extract of A. italica Retz. contains high amounts of phenolic contents. The anticonvulsant effects of A. italica Retz. extract seem to be because of the presence of these compounds. Baradaran et
al (2013) revealed that antioxidant activities of medicinal plants are due to secondary
metabolites such as polyphenols.[34 ]
Prefrontal cortex is a part of the brain that is involved in the pathophysiology of
seizures.[35 ] In the present study, the antioxidant capacity of prefrontal cortex and serum samples
in the PTZ group was lower than the control group. Besides, MDA levels in the prefrontal
cortex and serum samples in the PTZ group were significantly higher than in the control
group. In line with previous studies, we found that decrease in antioxidant capacity
as well as increase in lipid peroxidation are associated with seizures.[12 ] Our results showed that A. italica Retz. extract significantly increased the antioxidant capacity in serum and prefrontal
cortex samples. Furthermore, A. italica extract significantly decreased the MDA levels in serum and prefrontal cortex samples.
In general, it can be concluded that A. italica Retz. extract possessed antioxidant effects in PTZ model of seizure in mice.
Previous studies indicated that NO has an excitatory role in the pathogenesis of seizures.[36 ] In this regard, it has been shown that inhibition of NO synthase led to anticonvulsant
effects in different known models of seizure and attenuated seizure development.[37 ] Moreover, it has also been reported that the increase in NO production led to mitochondrial
dysfunction of neuronal cells leading to oxidative stress state and finally increased
the incidence of seizures.[38 ] In this study, nitrite levels in the serum and prefrontal cortex samples increased
in the PTZ groups. We showed that A. italica Retz. extract significantly decreased nitrite levels in the serum and prefrontal
cortex samples.
Overall, our finding demonstrated that A. italica Retz. extract, partially at least, via reducing nitrite and MDA levels as well as
increasing antioxidant capacity in the prefrontal cortex possessed anticonvulsant
effects in PTZ-induced seizure in mice.
Conclusion
According to the present study results, administration of the hydro-alcoholic extract
of A. italica Retz. increased the seizure threshold in PTZ-induced seizure in mice. We found that
anticonvulsant effects of hydro-alcoholic extract of A. italica Retz., partially at least, mediated through its antioxidant properties.
