Keywords:
Pentytenetetrazote - seizures - oxidative stress
Palavras-chave:
Ducrosia anethifolia - α-pinene - Pentilenotetrazol - Crise - Estresse oxidativo
Epilepsy is one of the oldest conditions known to man and is the third most common
neurological disorder after stroke and Alzheimer's disease. Approximately 1% of the
world's population suffers from epilepsy[1]. Anti-epileptic drugs are usually the first choice of treatment for epilepsy but
approximately one-third of people with epilepsy do not respond to the drugs. Anti-epileptic
drugs do not cure epilepsy, but can prevent seizures from occurring. Recently, it
has been proposed that seizures and status epilepticus may be associated with oxidative
stress[2]. Oxidative stress, due to the increase in the activity of glutamatergic transmitters,
plays a crucial role in the induction of neuronal cell death[3]. Since the brain utilizes the greatest amount of oxygen compared with other organs,
it is particularly at risk of oxidative stress[4].
Experimental models of epilepsy have been developed to find the basic mechanisms of
epileptic seizures and new therapeutic approaches. The chemical kindling induced by
the pentylenetetrazole (PTZ) is one of the most-widely used models for the induction
of convulsions in animals.
Medicinal plants have recently become a major target in the search for new drugs and
have led to compounds to treat epilepsy accompanied by oxidative stress[5],[6]. Ducrosia anethifolia Boiss, known in Persian as Moshgak, Roshgak, and Moshkbu, belongs to the Apiaceae
family. It is one of the three species of Iranian Ducrosia growing wild in southeastern Iran, in the mountainous regions of the Kerman province[7]. In Iranian traditional Medicine, the whole herb—especially its aerial parts—has
been used as an analgesic for headache, backache, as well as for the treatment of
colic, and colds. It is also used to relax the body and mind, allowing a restful sleep[8]. Furthermore, antianxiolytic effects of D. anethifolia essential oil (DAEO) have been reported[9]. The antioxidant, antimicrobial, antimycobacterial, antifungal, and central nervous
system depressant effects of this plant and other species of Ducrosia have been reported in pharmacological and biological studies[10]. Phytochemical studies of DAEO revealed that aliphatic aldehydes and other monoterpene
hydrocarbons such as limonene, citronellal, terpinolene, myrcene, α-pinene, pulegone,
p-cymene and coumarins such as pangelin are the main components of D. anethifolia aerial parts[11]. High performance liquid chromatography (HPLC) analysis of DAEO indicated the presence
of terpenoids such as α-pinene as one of the major components. Terpenes constitute
the major portion of the essential oils and, somehow, are responsible for the medicinal
plant's pharmacological activities such as antinociceptive, anti-inflammatory and
anticonvulsant effects[12].
It has been reported that α-pinene has anticonvulsant and antioxidant properties[13]. However, there is no scientific information to validate the anticonvulsant activity
of this plant in experimental animals. Therefore, the present study was designed to
determine the possible effects of DAEO, and its major component α-pinene, on PTZ-induced
seizure and brain oxidative stress in male rats.
METHODS
Animals
Adult male Wistar rats weighting 200-250g were prepared from the Animal House of Shahid
Bahonar University of Kerman. The animals were housed in a room with photoperiod control
(a 12-hour light/dark cycle) and temperature (22 ± 2°C). Food and water was available
ad libitum. All experimental procedures were approved by the Animal Research Ethics Committee
of the Kerman Neuroscience Research Center, Kerman, Iran (EC/95).
Drugs
Pentylenetetrazole, α-pinene and diazepam were purchased from Sigma-Aldrich Co. The
drugs were dissolved in a saline solution (0.9%) and injected intraperitoneally (i.p.)
in a volume of 1 ml/kg of the rat's body weight.
Plant material
Fresh aerial parts (leaves and flowers) of D. anethifolia were collected, in July, from the Lalehzar mountainous area in Kerman province, Iran,
at an altitude of 2,800 m. The voucher specimens were deposited at the herbarium of
Shahid Bahonar University of Kerman (Code number: 1371). The material was dried at
room temperature and used for distillation. The essential oil was isolated by hydrodistillation
of the fresh aerial parts for 4 hours, and then dried over anhydrous sodium sulfate
14 and stored in a refrigerator (4°C).
Acute toxicity
Seven rats were treated with the DAEO (500 mg/kg, i.p.) and the mortality and morbidity
were determined.
PTZ-induced seizures
Pentylenetetrazole (80 mg/kg, i.p.) was injected to induce convulsions in rats. Diazepam
(2 mg/kg, i.p.) and DAEO (25, 50, 100 and 200 mg/kg, i.p.) and α-pinene (0.2 and 0.4
mg/kg, i.p.) were administered 30 minutes before receiving PTZ. The seizure parameters
were precisely monitored for 40 minutes after each PTZ injection in all groups. The
following parameters were measured using a stopwatch in seconds, and behaviors were
recorded with a CD camera.
The resultant seizures were classified according to the modified Racine scale[14] as follows:
-
Stage 0: no response.
-
Stage 1: ear and facial twitching.
-
Stage 2: myoclonic jerks without rearing.
-
Stage 3: myoclonic jerks, rearing.
-
Stage 4: turning over onto side position, tonic-clonic seizures.
-
Stage 5: turning over onto back position, generalized tonic-clonic seizures.
-
Latency: the time between PTZ injection and the onset of seizures[15].
-
Duration: the time interval from the onset to termination of seizures or death of
the animal.
-
Percent of death: the number of rats that died after PTZ injection among the rats
of a particular group.
-
Protection percentage: the number of rats that responded to the test[16]. P%=1-(nt/Nt) (nc/NC) ×100.
Biochemical measurements
After behavioral assessment, the animals were euthanized under deep anesthesia, and
the temporal lobes of the brains were dissected and stored at −80°C until the day
of assay.
Brain lipid peroxidation
Lipid peroxidation products such as malondialdehyde (MDA) are considered to be reliable
indicators of oxidative damage[17]. Temporal lobe tissue (0.5 g) was homogenized in 10 mg of 0.1% trichloroacetic acid;
the homogenate was centrifuged at 15,000 rpm for 15 minutes to 1.0 mg aliquot of the
supernatant; and 4.0 mg of 0.5% thiobarbituric acid in 20% trichloroacetic acid was
added. The mixture was heated at 95°C for 30 minutes and then cooled in an ice bath.
After centrifugation (10,000 rpm for 10 minutes), the absorbance of the supernatant
was recorded at 532 nm (Biochrom WPA Biowave II UV/Visible Spectrophotometer). The
thiobarbituric acid reactive substances content was calculated according to its extinction
coefficient of 155mM−1cm−1 and expressed in units (U). One ‘U’ is defined as μmol of MDA formed min−1mg−1 protein.
Hydrogen peroxide
Hydrogen peroxide (H2O2) was determined by the method described by Velikova et al., (2000). Temporal lobe
tissue (0.5 g) was finely ground with trichloroacetic acid (5 ml of 0.1 % w/v) and
centrifuged at 10,000 × g for 15 minutes. Phosphate buffer (0.5 ml, pH 7.0) and 1 ml potassium iodide were
added to the 0.5 ml supernatant. Its absorbance was recorded at 390 nm after overtaxing
using a UV visible spectrophotometer.
Total soluble proteins
Total proteins were estimated using the Bradford method and bovine serum albumin was
used as the standard.
Antioxidant enzymes activities
Temporal lobe tissue (0.5 g) was finely ground under chilled conditions in 3 ml of
phosphate buffer (50 mM with pH 7.5) for the extraction of antioxidant enzymes. Centrifugation
of the mixture was performed at 10,000 × g for 10 minutes at 4°C. The supernatant was recentrifuged at 15,000 × g for 10 minutes and the resultant extract stored at −20°C for determination of the
activity of antioxidant enzymes.
Evaluation of catalase activity
The activity of catalase (CAT) was estimated by monitoring the decrease in absorbance
of H2O2 within 30 seconds at 240 nm. The assay solution contained 50 mM potassium phosphate
buffer (pH 7.0) and 15 mM H2O2 and 100 μl enzyme extract[18].
Evaluation of peroxidase activity
Peroxidase (POD) activity was assayed according to the method of Plewa et al.[19], based on the amount of tetraguaiacol absorbed after formation, by oxidation, of
guaiacol catalyzed by this enzyme in 3 minutes at a wavelength of 470 nm using an
extinction coefficient of tetraguaiacol, ε = 26.6 mM−1cm−1.
HPLC analysis
The obtained essential oil was analyzed using HPLC (Agilent Technologies, 1200 Infinity
series, USA) equipped with a 1260 Infinity Quaternary Pump and a 1260 Infinity Variable
Wavelength Detector. An Agilent 1260 Infinity Manual Injector fitted with a 20 μL
sample loop was used to introduce the samples. The analytes were separated on a Restek
Ultra C18 (250 mm × 4.6 mm, 5μm) column (USA). Chromatograms were processed by an
Agilent HPLC Chem Station (Rev. B.04.03).
Statistical analysis
The data are expressed as mean ± SEM. Comparison between groups was made by analysis
of variance followed by the Tukey test. Differences between experimental groups of
each point with p < 0.05 were considered statistically significant.
RESULTS
Acute toxicity
The essential oil of D. anethifolia has shown no mortality up to a dose of 500 mg/kg. However, we used doses of 25, 50,
100 and 200 mg/kg in this study.
Anticonvulsant activity assessment
Effect of DAEO on PTZ-induced seizures
The essential oil showed dose-dependent effects against PTZ-induced seizures. It could
significantly reduce the number of convulsing animals. Pretreatment with DAEO (50,
100 and 200 mg/kg) and α-pinene (0.2 and 0.4 mg/kg) significantly reduced mortality
rate and attenuated PTZ-induced seizures ([Table]).
Table
The effect of Ducrosia anethifolia essential oil and α-pinene on pentylenetetrazole (PTZ) induced seizures in rats.
|
Treatment and doses (mg/kg, i.p)
|
Duration of seizure (sec.)
|
% Mortality
|
% Protection
|
|
Myoclonic
|
Tonic
|
Tonic-clonic
|
|
PTZ
|
34 ± 03
|
31 ± 42
|
165 ± 86
|
100
|
14
|
|
PTZ + Diazepam 2 mg/kg
|
2 ±14***
|
4 ± 85***
|
4 ± 14***
|
0
|
100
|
|
PTZ + D. anethifolia 25 mg/kg
|
20 ± 01
|
40 ± 01
|
162 ± 71
|
71
|
29
|
|
PTZ + D. anethifolia 50 mg/kg
|
10 ± 42**
|
13 ± 57*
|
13 ± 42***
|
0
|
100
|
|
PTZ + D. anethifolia 100 mg/kg
|
13 ± 71**
|
18 ± 14
|
35 ± 57***
|
14
|
86
|
|
PTZ + D. anethifolia 200 mg/kg
|
17 ± 42*
|
19 ± 14
|
27 ±14***
|
14
|
86
|
|
PTZ + α-pinene 0.2 mg/kg
|
16 ± 71*
|
10 ± 57**
|
31 ± 57***
|
42
|
58
|
|
PTZ + α-pinene 0.4 mg/kg
|
13 ± 43**
|
8 ± 42**
|
21 ± 71***
|
28
|
72
|
Data are presented as duration of myoclonic, tonic and tonic-clonic seizures and represent
percentage of the mortality and protection criteria (n = 7).
***p < 0.001,
**p < 0.01,
*p < 0.05, compared with PTZ-treated control rats. PTZ: pentylenetetrazole
Effect of DAEO and α-pinene on the onset of seizure
The DAEO (50, 100 and 200 mg/kg, i.p.) significantly delayed the onset of PTZ-induced
seizures. However, diazepam and α-pinene had no significant effects on the onset of
seizure ([Figure 1]).
Figure 1 The effect of Ducrosia anethifolia essential oil (DAEO) and α-pinene on the onset of seizure of pentylenetetrazole (PTZ)-induced
convulsion in rats.Histograms represent mean ± SEM for seven animals. ***p < 0.001,
**p < 0.01, versus PTZ group by analysis of variance with Tukey's post hoc test.
Effect of DAEO and α-pinene on the duration of seizure
The essential oil at doses of 50, 100 and 200 mg/ kg, α-pinene (0.2 and 0.4 mg/kg)
and diazepam could significantly alter the duration of seizures in PTZ-treated rats.
However, 25 mg/kg of DAEO had no effect on the duration of seizures ([Figure 2]).
Figure 2 The effect of Ducrosia anethifolia essential oil (DAEO) and α-pinene on the duration of PTZ-induced seizure in Rats.Histograms
represent mean ± SEM (n=7). ****p < 0.001 versus PTZ-treated group. The data were
analyzed by one-way analysis of variance with Tukey's post hoc test.
Biochemical measurements
MDA levels
The PTZ injection significantly increased brain temporal lobe MDA levels, which were
significantly attenuated by DAEO (50, 100, 200 mg/kg) and α-pinene (0.2 and 0.4 mg/kg)
([Figure 3]).
Figure 3 The effect of Ducrosia anethifolia essential oil (DAEO) and α-pinene on the temporal lobe MDA levels in the PTZ seizure
models.Data represent means ± SEM (n=7), ***p < 0.001 and *p < 0.05 compared with
nontreated normal rats. +++p < 0.001 versus PTZ-injected group.
H2O2 levels
The PTZ-treated rats showed a significant increase in H2O2 levels in the temporal lobe. Alternatively, DAEO, α-pinene and diazepam significantly
decreased PTZ-induced H2O2 production ([Figure 4]).
Figure 4 The effect of Ducrosia anethifolia essential oil (DAEO) and α-pinene on the temporal lobe H2O2 levels in the rat PTZ seizure models.Data represent means ± SEM (n=7), ***p < 0.001
and **p < 0.01 compared with the control nontreated groups. +++p < 0.001 compared
with PTZ-treated animals.
The effect of DAEO and α-pinene on brain CAT and POD activities in PTZ-treated animals
The brain CAT and POD activities were significantly decreased following PTZ administration.
However, DAEO (50,100 and 200 mg/kg), α-pinene (0.2 and 0.4 mg/kg) and diazepam could
prevent the effect of PTZ on CAT and POD activities ([Figure 5] and [6]).
Figure 5 The effect of Ducrosia anethifolia essential oil (DAEO) and α-pinene on catalase activity in the temporal lobe of the brain in
the PTZ seizure model.Data represent means ± SEM (n=7), ****p < 0.001, **p < 0.01
and *p < 0.05 compared with the control non-treated groups. +++p < 0.001 and ++p <
0.01 compared with PTZ-treated animals.
Figure 6 The effect of Ducrosia anethifolia essential oil (DAEO) and α-pinene on peroxidase activity in the temporal lobe in
the PTZ seizure model.Data represent means ± SEM (n=7), ****p < 0.001, **p < 0.01
compared with the control untreated groups. +++p < 0.001 and ++p < 0.01 compared with
PTZ-treated animals.
HPLC analysis
According to the obtained HPLC spectrum of essential oil of D. anethifolia, there was a major peak following retention times (min): 6.950 ([Figure 7]). The peak for the reference standard, α-pinene, appeared at the retention time
(min) of 6.866.
Figure 7 HPLC chromatogram of Ducrosia anethifolia essential oil (DAEO).
DISCUSSION
In the present work, the effects of DAEO and α-pinene were studied. Ducrosia anethifolia essential oil and α-pinene were initially evaluated in a behavioral study that gave
a good indication of the reduction of seizures. Additionally, the results showed that
DAEO and α-pinene were able to significantly decrease the oxidative stress factors
after seizures induced by PTZ.
The PTZ method is a valid model of convulsion for the study of generalized myoclonic
(absence) seizures[20],[21],[22]. It has been demonstrated that oxidative stress resulting from free radicals plays
a critical role in the genesis of epilepsy and in post-seizure neuronal death. The
brain is particularly susceptible to oxidative stress damage[4],[23],[24]. Traditionally, medicinal plants with antioxidant properties have been candidates
for preventing oxidative damage and epilepsy[25]. The phytochemical and HPLC analysis by Hajhashemi et al.[9] showed that DAEO had a wide spectrum of bioactive compounds, and terpenoids were
its major components[9]. The antinociceptive, anticonvulsant and anti-inflammatory properties of monoterpenes,
such as α-pinene, carvacrol, γ-terpineol, citronellol and linalool have been reported[26],[27]. Pentylenetetrazole induces convulsion by inhibiting GABA receptors-chloride channel
complexes. It appears that the inhibitory effect of DAEO against PTZ-induced seizure
may occur through the rise of the convulsion threshold in the brain via the stimulation
of GABA receptors[28]. The α-pinenes, as major components of DAEO, have a promoting effect on GABAA receptors and increase the postsynaptic GABA-dependent chloride flows, as well as
being a potent inhibitor of acetylcholinesterase[13]. The major inhibitory neurotransmitter in the brain is GABA and the inhibition of
its neurotransmission has been thought to be a critical factor in epilepsy[29]. The standard antiepileptic drugs, phenobarbital and diazepam, can induce their
antiepileptic effects by enhancing GABA neurotransmission. Glutamate and glutamatergic
receptors are located in both central and peripheral nervous systems and may be responsible
for most of the excitatory neurotransmission.
In addition to GABA dysregulation, it has been indicated that excitatory amino acids
are also involved in the initiation and propagation of seizures[30],[31]. Citronellal, citronellol, myrcene and β-pinene, the DAEO monoterpenes, have NMDA
receptor antagonist activities and can protect neurons against overstimulation[30],[32]. Activation of NMDA receptors generally increases intracellular calcium influx,
which raises neuronal excitation and excitability mainly via stimulation of cAMP-dependent
signaling molecules including adenylyl cyclases and protein kinase A[33]. Especially, it has been reported that down-regulation of the cAMP-response element-binding
protein is correlated with the suppression of epileptic seizures[34]. It has been reported that linalool, a DAEO constituent compound, exerts a considerable
anticonvulsant activity in a rat model of PTZ-kindling via modulation of glutamatergic
currents[35]. In addition, linalool inhibits adenylate cyclase in chick retinas[36]. Thus, DAEO anticonvulsant capacity, at least in part, is mediated by modulation
of intracellular second messengers such as calcium and glutamate. However, additional
studies are still required to clarify this important issue in more details.
In the present study, PTZ-induced seizures could increase the levels of oxidative
stress indicators such as MDA and H2O2, and decrease the activities of antioxidant enzymes, CAT and POD[37],[38]. It has been demonstrated that the use of free radical scavengers in the treatment
of epilepsy provides an important perspective that will be the driving force for future
drug design of novel antiepileptics[39]. Pretreatment with DAEO and α-pinene could prevent the seizures and thus decrease
oxidative stress. The data showed a dose-dependent effect of DAEO against seizure-induced
oxidative stress in experimental models of seizures.
Potential antioxidant therapy that includes either natural antioxidants or agents
is capable of augmenting the functions of these enzymes[40]. Earlier reports have shown that the natural drugs like DAEO have antioxidant properties
because of the presence of α-pinene, citronellal, γ-terpinene, myrcene and limonene[41].
Taken together, the data suggest that DAEO and α-pinene have antiepileptic activities.
This effect may be due to their antioxidant properties and possible activation of
GABAA receptors. Our experiment contributes to our knowledge of the pharmacology of D. anethifolia (Boiss).
