The anticonvulsant effects of Ducrosia anethifolia (Boiss) essential oil are produced by its main component alpha-pinene in rats

ABSTRACT Ducrosia anethifolia has been recommended as a remedy for neurological disorders. However, the anticonvulsant effects of D. anethifolia essential oil (DAEO) and its major constituent α-pinene have not yet been clarified. Methods: A rat model of pentylenetetrazole (PTZ)-induced convulsions was used. Oxidant and antioxidant parameters were assayed in the temporal lobe. Results: The data showed that DAEO (50, 100 and 200 mg/kg, i.p.) and α-pinene (0.2 and 0.4 mg/kg i.p.) delayed the initiation time, and reduced the duration of myoclonic and tonic-clonic seizures following PTZ injection. The PTZ produced oxidative stress so that malondialdehyde and hydrogen peroxide levels were increased and catalase and peroxidase activity decreased. Pretreatment with DAEO and α-pinene significantly inhibited the above-mentioned enzymatic changes in PTZ-treated animals. Conclusion: The results suggest that α-pinene, at teast in part, was responsible for the induction of the anticonvulsant and antioxidant effects of DAEO in rats.

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.

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. 2) Duration: the time interval from the onset to termination of seizures or death of the animal.
3) Percent of death: the number of rats that died after PTZ injection among the rats of a particular group. 4) 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 −1 cm −1 and expressed in units (U). One 'U' is defined as µmol of MDA formed min −1 mg −1 protein.

Hydrogen peroxide
Hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 within 30 seconds at 240 nm. The assay solution contained 50 mM potassium phosphate buffer (pH 7.0) and 15 mM H 2 O 2 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 -1 cm -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.

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.

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).

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).

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).

H 2 O 2 levels
The PTZ-treated rats showed a significant increase in H 2 O 2 levels in the temporal lobe. Alternatively, DAEO, α-pinene and diazepam significantly decreased PTZ-induced H 2 O 2 production (Figure 4).

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).

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.   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

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 GABA A 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, 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. 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.   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 H 2 O 2 , 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 GABA A receptors. Our experiment contributes to our knowledge of the pharmacology of D. anethifolia (Boiss).