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Anticonvulsant and depressant effects of aqueous extracts of Carum copticum seeds in male rats
Epilepsy & Behavior 22 (2011) 220–225
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Epilepsy & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ye b e h
Anticonvulsant and depressant effects of aqueous extracts of Carum copticum seeds in male rats Mohammad Ebrahim Rezvani a,⁎, Ali Roohbakhsh b, Mohammad Hossein Mosaddegh c, Mansour Esmailidehaj a, Fatemeh Khaloobagheri b, Hossein Esmaeili b a b c
Department of Physiology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Physiology and Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Department of Pharmacology, School of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
a r t i c l e
i n f o
Article history: Received 2 March 2011 Revised 12 July 2011 Accepted 15 July 2011 Available online 16 August 2011 Keywords: Carum copticum Seizure Locomotor activity Anxiety Kindling Folk medicine Herbal medicine
a b s t r a c t In this study, the effects of aqueous extracts of Carum copticum seeds (CCS) were evaluated in kindling models of epilepsy. Additionally, the sedative and anxiolytic effects of the extract were assessed. For pentylenetetrazole (PTZ) kindling, rats received a subconvulsant dose of PTZ (40 mg/kg, ip) every second day and seizure stages were recorded. CCS aqueous extract (200, 400, or 600 mg/kg, ip) was injected 30 minutes prior to each PTZ injection. In electrical kindling, bipolar stimulating and monopolar recording electrodes were implanted stereotaxically in the right basolateral amygdala of male Sprague–Dawley rats. After kindling, the effect of aqueous extracts of CCS (200, 400, or 600 mg/kg, ip) on afterdischarge duration, duration of rearing, forelimb clonus, and loss of equilibrium (stage 5 seizure), and latency to the onset of bilateral forelimb clonus were measured. The sedative and the anxiolytic effects of CCS extracts were evaluated in an open-field apparatus and elevated plus maze, respectively. The results indicate that aqueous extracts of CCS have a significant anticonvulsant effect. Different doses of extract significantly delayed the incidence of every seizure stage in the PTZ model of kindling. Moreover, CCS extract (400 and 600 mg/kg, ip) suppressed afterdischarge duration, latency to the onset of bilateral forelimb clonus, and stage 5 seizure in the electrical kindling model. These results suggest that CCS extract has remarkable antiepileptic and central depressant effects. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Epilepsy is a common neurological disorder that causes physical, psychological, and social abnormalities in patients. One percent of the world's population has epilepsy and approximately 30% of patients are considered to be pharmacoresistant. Kindling models have often been used to study seizure propagation [1] and to perform preclinical evaluation of antiepileptic drugs. Electrical kindling is defined as progressive development of electrographic and motor seizures after repeated daily stimulation of particular brain sites [2]. Pentylenetetrazole (PTZ) kindling is produced by daily injection of subconvulsive doses of PTZ [3]. In both methods, subconvulsive electrical or chemical stimulation finally leads to fully epileptic behaviors or generalized seizures. Plant extracts can be an important source for the development of alternative and complementary treatment of epilepsy. Several plants reputed to possess antiepileptic properties in different cultures have been found to exhibit anticonvulsant activity in different animal models [4]. Carum copticum (family: Apiaceae) is a grassy, annual plant
⁎ Corresponding author. Fax: + 98 351 8203414. E-mail address: [email protected] (M.E. Rezvani). 1525-5050/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2011.07.017
with small and aromatic seeds that grows in Iran, India, and Egypt. In Iranian ancient medicine, the seeds of this plant were used for rheumatoid arthritis, stomach pain, and anxiety [5]. Experimental studies have provided evidence demonstrating the cholinergic and anticholinergic [6–8], bronchodilating [9], analgesic [10], and antihypertensive [8] effects of this plant. In Iran, there are folkloric uses and social beliefs regarding the central depressant, sedative, and antiepileptic effects of Carum copticum seeds (CCS). In addition, during a pilot study, it was observed that CCS-treated rats exhibited depressant and sedative behaviors. On the basis of the above-mentioned evidence, the present study was designed to examine the effects of aqueous extracts of CCS on PTZ- and amygdala-kindled seizures in male rats. Moreover, sedative and anxiolytic effects of the plant seeds were evaluated. 2. Materials and methods 2.1. Plant material and extracting method Dried seeds of Carum copticum were purchased from an herbal pharmacy in Rafsanjan City and a voucher specimen of the plant (RJ 32–534) has been deposited in the herbarium of the School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
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One kilogram of CCS was washed with distilled water and coarsely ground with a blender; the powder obtained was macerated in warm distilled water (50–60 °C) for 48 hours and extracted twice. The resulting extract was concentrated under low pressure in a rotary evaporator and then freeze-dried. For administration, the dried extract was dissolved freshly in 0.9% sterile saline.
CCS aqueous extract or saline 15, 30, or 90 minutes prior to electrical stimulation of the amygdala. In this experiment, afterdischarge duration (ADD), stage 4 latency (S4L), stage 5 duration (S5D), and convulsive behaviors were recorded. In addition, the number of amygdala stimulations required for occurrence of each seizure stage was recorded.
2.2. Chemicals
2.4.3. Experiment 3: Sedative and anxiety tests
Pentylenetetrazole was purchased from Sigma Chemical Company (Poole, UK). PTZ was dissolved in sterile saline (0.9%). Ketamine hydrochloride and xylazine were purchased from Alfasan (Woerden, The Netherlands). All drugs were prepared freshly and injected intraperitoneally.
2.4.3.1. Spontaneous locomotor activity. An open-field test was used to evaluate the sedative effect of the extract. The open-field apparatus (Borj Sanat Co., Iran) consisted of a Plexiglas box (40 cm× 40 cm× 40 cm) equipped with 16 infrared photocells located every 2.5 cm to measure horizontal movements. Thirty minutes after injection of extract or saline, rats were individually placed in the center of the box floor and monitored for 20 minutes. Rats in groups 1–4 received a single dose of saline or three different doses of CCS extract. Other groups were treated with repeated doses of saline or different doses of CCS extract every second day up to day 35, but locomotor activity was recorded only on the last day (18th injection) of treatment. Spontaneous locomotion was recorded as the number of cuts of the beam by the photocell monitoring system [14].
2.3. Animals Male Wistar rats from the Razi Institute (Iran), weighing 200–250 g at the beginning of the study, were used. Animals were housed in standard cages under a 12-hour-light/12-hour-dark cycle (lights on at 07:00) and had free access to food and water. Procedures involving animals and their care were conducted in accordance with the Guide to the Care and Use of Experimental Animals [11]. Approval from the local ethics committee was also obtained. All experiments were done at the same time in the morning (10–12 AM) to avoid a circadian rhythm bias. To reduce the number of kindled animals and their suffering, all distorting factors were eliminated. 2.4. Experimental design To determine the effects of the extract on the development of PTZand amygdala-kindled seizures, the following experiments were designed: 2.4.1. Experiment 1: Pentylenetetrazole kindling (groups 1–4) For PTZ kindling, rats received a subconvulsant dose of PTZ (40 mg/ kg) intraperitoneally every second day. PTZ injections were continued until at least three consecutive stage 5 seizures were elicited [12]. In different groups, rats were injected with 200, 400, or 600 mg/kg CCS aqueous extract or saline 30 minutes prior to administration of PTZ. In these groups, rats were observed for 30 minutes after each PTZ injection and convulsive behaviors were scored as follows: no response, stage 0; motor arrest, stage 1; facial or jaw movements, stage 2; addition of head nodding, stage 3; unilateral forelimb clonus, stage 4; and rearing with bilateral forelimb clonus, stage 5.
2.4.3.2. Elevated plus maze. The elevated plus maze was used to measure anxiety-like behavior of the rats. The maze consisted of two opposite open arms (50 × 10 cm), two opposite closed arms (50 × 10 × 40 cm), and a central platform (10 × 10 cm). The arms were located 50 cm above the floor on the base. The floors of the arms and the walls of closed arms were constructed from black Plexiglas. Thirty minutes after injection of extract or saline, rats were individually placed on the central platform facing an open arm and away from a blinded observer. For 5 minutes, the number of entries into the open and closed arms was recorded. Time spent in the open and closed arms was also measured. The anxiety index was calculated by dividing the number of entries into the open arm by the number of entries into the open and closed arms. 2.5. Assessment of acute toxicity Rats fasted for 12 hours were allocated to five groups of 10 rats per group. Lethality was evaluated after intraperitoneal administration of 750, 1200, 2000, or 3000 mg/kg CCS aqueous extract. Percentage mortality of rats was recorded after 24 hours, and LD50 values were determined graphically [15]. 2.6. Histology
2.4.2. Experiment 2: Amygdala electrical kindling (groups 5–8) For electrical kindling, under ketamine (100 mg/kg, ip) and xylazine (10 mg/kg, ip) anesthesia, animals were stereotaxically implanted with bipolar stimulating and monopolar recording electrodes (twisted into tripolar configuration) terminating in the basolateral amygdala of the right hemisphere (coordinates: A, −2.5 mm; L, 4.8 mm; 7.5 mm below dura). The electrodes (stainless steel, Teflon-coated, 127 μm in diameter, A.M. System Inc., USA) were insulated except at their tips. Two single electrodes were connected to the skull screws, fixed on the cortical surface as earth and differential electrodes. One week after surgery, afterdischarge threshold was determined by amygdala stimulation (60-Hz monophasic square wave, 1-ms duration per wave, for 2 seconds). Stimulation and isolation were provided by a WSI stimulator (WSI Co., Iran). Stimuli were initially delivered at 10 μA and then at 5minute intervals, increasing stimulus intensity in increments of 10 μA until at least 5 seconds of afterdischarges were recorded as previously described [13]. The following experimental groups were considered: First, animals in groups 5 to 8 (n = 6–8 per group) were fully kindled by daily electrical stimulation of the amygdala before any treatment. Then, 24 hours later, the animals were injected with 200, 400, or 600 mg/kg of
At the end of the study, all rats were sacrificed and their brains were removed and fixed in formalin 10% for 1 week. Then, each brain was sectioned and electrode tip placements were determined under a microscope. In the case of abnormality in electrode positions or the presence of tissue damage, the data from that particular animal were not included in the results. 2.7. Phytochemical analysis Quantitative phytochemical analysis of the powdered seeds was conducted to determine the amounts of different compounds including tannins, flavonoids, saponins, alkaloids, and anthraquinones [16]. 2.8. Statistics All data are expressed as means ± SEM. One way ANOVA was performed to compare ADD, S4L, and S5D in different treatments and was followed by Dunnett's test for post hoc analysis. Behavior stages
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were analyzed with the Kruskal–Wallis ANOVA. The Wilcoxon or Mann–Whitney U test was used for comparing data within or between groups, respectively, where data were not normally distributed. In all experiments, P values less than 0.05 were considered statistically significant. 3. Results The LD50 value of the extract was 1737.8 ± 227 mg/kg. This LD50 value suggests that the CCS aqueous extract is relatively safe in rats. Quantitative phytochemical analysis showed that CCS contain tannin 1.83% ± 0.2, flavonoids 9.33% ± 0.11, saponins 0.64% ± 0.15, alkaloids 5.41% ± 0.17, and trace amounts of anthraquinones. 3.1. Effect of CCS extract on kindling rate in the pentylenetetrazole model Fig. 1 illustrates that the aqueous extract of CCS exhibits antiepileptic properties in the PTZ model of kindling. From PTZ injections 1 to 7, there are no interpretable changes (although there are statistically significant differences at a few points) between rats that received saline and those that received different doses of CCS extract. CCS extract 200 or 400 mg/kg (P b 0.01, injections 7–18) and CCS extract 600 mg/kg (P b 0.001, injections 8–18) attenuated seizure severity and delayed all seizure stages. In the saline group, subsequent injection of the same dose of PTZ induced epileptic behaviors in an ascending order. In the CCS groups, none of the rats manifested stage 4 and 5 seizures. By the 11th injection, one of the rats, and by the 14th injection, two of the rats, in the saline group died. However, these results indicate that consecutive injections of CCS extract significantly delay kindling rate and decrease seizure severity. 3.2. Effect of CCS extract on seizure parameters in the amygdala kindling model Fig. 2 illustrates the behavioral and electrophysiological data of different groups of kindled rats. The results show that intraperitoneal administration of aqueous extracts of CCS led to a significant decrease in ADD at doses of 400 and 600 mg/kg (P b 0.01) and a significant increase in S4L at doses of 200, 400, and 600 mg/kg (P b 0.01). Because in each CCS-treated group only two or three rats exhibited stage 5 seizures, the data related to S5D could not be statistically analyzed. However, as shown in Fig. 3, the numbers of amygdala stimulations
Fig. 2. Effect of aqueous extract of Carum copticum (CCS) at the doses of 200, 400, and 600 mg/kg on afterdischarge duration, stage 5 duration and step 4 latency after 15, 30, and 90 min of intraperitoneal injections in amygdala-kindled rats. The rats received daily amygdala stimulation (n = 7–10 rats). Values are means ± SEM; *p b 0.05, **p b 0.01 and ***p b 0.01 compared with saline treated rats.
required for first generation of stage 3, 4, or 5 seizures were greater in the rats treated with CCS extract than in the saline-treated group (P b 0.01). These results indicate an obvious anticonvulsive effect for aqueous extracts of CCS. 3.3. Locomotion and rearing As shown in Table 1, CCS extract at different doses produced a significant decrease in spontaneous locomotor activity and number of rears in rats after the 1st and 18th injections (P b 0.05). In addition, statistical analysis revealed that the rats repeatedly administered (18th injection) CCS extract at doses of 200 and 600 mg/kg (P b 0.05) had a greater decrease in locomotor activity compared with rats that received one injection.
Fig. 1. Effect of the CCS extract of Carum copticum (CCS) on seizures induced by PTZ. The extract was injected prior to PTZ that was injected on every second day. (n = 7–10 rats). *P b 0.05 compared with corresponding points in saline treated group. Values are means ± SEM.
3.4. Elevated plus maze Administration of CCS extract at 400 and 600 mg/kg reduced the number of entries into the open and closed arms, but was unable to
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Administration of aqueous extracts of CCS significantly reduced seizure activity and kindling development rate. The PTZ kindling test is used as a valid model of partial seizures with secondary generalization [17]. Repeated injections of PTZ led to convulsions and epilepsy-like behavior, and finally rats exhibited class 4 (generalization) and class 5 (generalized tonic–clonic) seizures. Attenuation of PTZ-induced convulsions suggests that the antiepileptic effect of CCS extract may be partly mediated by activation of the brain GABAA receptor complex. One well-accepted mechanism by which PTZ induces convulsions is by antagonizing the picrotoxin-sensitive site of the GABAA receptor complex [18,19]. PTZ is a selective blocker of chloride channels in the GABAA receptor complex and repeated exposure of the brain to subconvulsive doses of PTZ leads to suppression of GABA neurotransmission as evidenced by lesser binding of [ 3H]GABA [20] and a decrease in chloride uptake rate induced by GABA [21]. As the results of our study indicate CCS extract has inhibitory effects in the PTZ model and considering that PTZ-induced convulsions are related to suppression of GABA neurotransmission, it is likely that aqueous extract of CCS attenuate epileptic convulsions by enhancing GABAergic neurotransmission in the brain. Kindling of seizures has been used to model the progressive pathogenesis of refractory epilepsy in human [1]. In this experiment, intraperitoneal injection of CCS extract significantly reduced the duration of afterdischarges recorded from the amygdala. It was clearly shown that repeated administration of CCS reduces neuronal activity in the amygdala and possibly other regions of the brain. Because the seizure-protective effect is accompanied by a decrease in S5D and an increase in S4L, it may be concluded that both the partial (1–3) and generalized (4 and 5) seizure stages may be affected by CCS extract. S4L is an index of seizure generalization and its increase implies that CCS extract delays the seizure generalization in rats. Absence of stage 5 or its shortening is also another effect of the CCS extract. In contrast, with PTZ kindling, after amygdala electrical kindling, the function of GABAergic neurotransmission in the hippocampus is intensified [22,23] and the extract might potentiate GABA neurotransmission.
Fig. 3. Progressive development of behavioral manifestation of seizures induced by electrical stimulation of amygdala. Prior to the each stimulation, rats received aqueous extract of Carum copticum (CCS) at the doses of 200, 400 and 600 mg/kg. Results are expressed as the number of stimulations required for the first occurrence of each stage (n = 7–10). Data are the means ± S.E.M. *P b 0.05, **P b 0.01 and ***P b 0.001 from the corresponding value for animals pretreated with saline.
increase the time spent in the open arm. The anxiety index of the control and CCS treated groups remained unchanged. Among the data obtained, the significant reduction in the number of entries into the closed arms compared with the control group was unpredictable (Table 2). 4. Discussion In Iranian folk medicine, Carum copticum seeds have been used as antiepileptic, sedative, and anxiolytic remedies. In the present study, the antiepileptic effect of CCS in the PTZ and amygdala kindling models was evaluated. The aqueous extract of CCS was shown to have anticonvulsant effects in the PTZ and amygdala kindling models.
Table 1 Effects of aqueous extracts of Carum copticum seeds (CCS) on spontaneous locomotor activity and rearing after injections 1 and 18 using the open-field test. Treatment (ip)
n
Saline (control) CCS 200 mg/kg CCS 400 mg/kg CCS 600 mg/kg
10 9 11 10
Locomotor activity (count)
Rearing (count)
After injection 1
After injection 18
After injection 1
After injection 18
642.44 ± 93.85 595.70 ± 51.32 371.55 ± 88.06a 244.04 ± 57.12b
671.25 ± 59.68 402.76 ± 53.24a,c 308.41 ± 71.72a 109.22 ± 61.25b,d
29.55 ± 4.21 20.96 ± 3.56 17.67 ± 2.18a 9.55 ± 3.44b
35.19 ± 5.89 11.56 ± 3.54a 10.49 ± 5.81b 8.22 ± 3.74b
Note. Data are means ± SEM. n = number of rats in each group. a P b 0.05, as compared with corresponding saline-treated group. b P b 0.01, as compared with corresponding saline-treated group. c P b 0.05, as compared with CCS-treated groups with the same dose in injection 1. d P b 0.01, as compared with CCS-treated groups with the same dose in injection 1.
Table 2 Effects of aqueous extract of Carum copticum seeds (CCS) on time spent in and entries into the open and closed arms of the elevated plus maze and calculated anxiety index in independent groups. Treatment (ip)
Saline (control) CCS 200 mg/kg CCS 400 mg/kg CCS 600 mg/kg
n
11 14 10 10
Time (s) spent in
Entries into
Anxiety index
Open arms
Closed arms
Open arms
Closed arms
88.24 ± 10.33 91.11 ± 8.29 40.35 ± 9.20a 35.07 ± 9.27a
207.44 ± 14.53 200.71 ± 22.18 247.54 ± 7.26a 260.11 ± 6.35b
10.21 ± 0.57 8.78 ± 0.48 2.05 ± 0.38b 1.24 ± 0.21b
10.14 ± 0.99 9 .44 ± 0.67 2.65 ± 0.40b 12.01 ± 0.32b
Note. Data are expressed as means ± SEM. n = number of rats in each group. a P b 0.05, as compared with corresponding saline-treated group. b P b 0.01, as compared with corresponding saline-treated group.
0.50 ± 0.06 0.48 ± 0.04 0.43 ± 0.03 0.38 ± 0.04
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Carum seeds have multiple constituents including steroptin, thymine, cumin, amino acids like lysine and threonine, tannins, and dietary fibers [24]. In previous studies, involvement of lysine in the kindling model of epilepsy was observed. L-Lysine can enhance benzodiazepine receptor binding affinity [25,26] and subsequently shows an antiepileptic effect in experimental epilepsy [27,28]. This evidence suggests that the antiepileptic effect of the extract is partly mediated by L-lysine. Carum copticum seeds also demonstrated relaxant and spasmolytic effects on duodenum of laboratory animals [8]. The relaxant and spasmolytic effects of the plant seeds are partly mediated through calcium channel blockade. In addition, there is evidence that calcium channel blockers obviously have anticonvulsive roles in different animal models of epilepsy [29–31]. Thus, it is possible that blockade of Ca 2+ channels is another mechanism by which this extract induces antiepileptic effects. The histamine H1 receptor antagonistic property of CCS [9] cannot be an antiepileptic mechanism for the CCS extract because substantial evidence indicates that histamine, through H1 receptors, plays an important role in suppressing kindled seizures [32–34]. However, the seizure-suppressing effect of selective antagonists of H3 receptors in laboratory animals has been documented in a few studies [35,36]. Therefore, the antiepileptic effect of CCS extract may be due to inhibition of H3 receptors in the brain. The phytochemical screening test showed that flavonoids are the major components in CCS. Some flavonoids have been shown to potentiate brain GABAA receptors [37] and increased GABA-induced currents in rat cortical neurons [38]. Moreover, flavonoids may also be capable of modulating glutamate excitotoxicity via direct scavenging of ROS or by the attenuation of calcium influx [39]. On the basis of this evidence, it is possible that the antiepileptic effects of CCS extract are exerted via potentiation of GABA neurotransmission and/or suppression of glutamate receptors in the brain. The sedative effect of CCS extract was evaluated using the openfield test. Our results imply a depressant effect of CCS at doses that are antiepileptic. Reduced spontaneous locomotor activity is generally attributed to neuronal inhibition. The brain GABAergic system is responsible for sedation and depressive behaviors [40] and the findings also support the possible GABA-mediated property of CCS extract. These results show that CCS induce sedation and central depression. In the elevated plus maze test, the number of entries into the open and closed arms was reduced in CCS-treated animals as compared with controls. The elevated plus maze test is an accepted method for evaluating anxiety-like behavior in laboratory rodents [41,42]. In this maze, decreases in the number of entries into and the time spent in the open arms are indicators of anxiety. Different doses of CCS extract did not increase these indices of anxiety, meaning that the CCS extract does not have anxiolytic-like properties. Furthermore, a significant reduction was observed in the number of entries into the open and closed arms when compared with the control group. It has been demonstrated that evaluation of anxiety in the elevated plus maze depends on exploratory behavior [43,44] and may also be confounded by changes in locomotor activity [36]. In addition, other studies have shown that closed arm entries are a more reliable measure of locomotor activity than total arm entries [43,45]. Thus, the data obtained with the elevated plus maze might represent the suppressive effect of CCS extract on locomotor activity. However, using other animal models of anxiety may provide a more rational deduction about the possible anxiolytic or anxiogenic-like effects of CCS extract. In conclusion, our data indicate that CCS aqueous extract has antiepileptic and sedative effects. This study provides scientific rationale for the use of this aqueous extract of plant seeds for the amelioration of epilepsy observed in traditional medicine in Iran. However, CCS contains multiple components such as steroptin, thymene, tannin, alkaloids, and cumin [24]. Its essential oil also contains thymol, p-cymene, and γ-
cymene. Thus, more studies are necessary to clarify the antiepileptic components and the mechanisms underlying the properties.
Acknowledgment This work was supported by the research deputy of Rafsanjan University of Medical Sciences and Yazd Shahid Sadoughi University of Medical Sciences through a research project. The authors appreciate Ahmadreza Sayyadi and Ali Khodadadi for their assistance during the present study.
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Epilepsy & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ye b e h
Anticonvulsant and depressant effects of aqueous extracts of Carum copticum seeds in male rats Mohammad Ebrahim Rezvani a,⁎, Ali Roohbakhsh b, Mohammad Hossein Mosaddegh c, Mansour Esmailidehaj a, Fatemeh Khaloobagheri b, Hossein Esmaeili b a b c
Department of Physiology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Physiology and Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran Department of Pharmacology, School of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
a r t i c l e
i n f o
Article history: Received 2 March 2011 Revised 12 July 2011 Accepted 15 July 2011 Available online 16 August 2011 Keywords: Carum copticum Seizure Locomotor activity Anxiety Kindling Folk medicine Herbal medicine
a b s t r a c t In this study, the effects of aqueous extracts of Carum copticum seeds (CCS) were evaluated in kindling models of epilepsy. Additionally, the sedative and anxiolytic effects of the extract were assessed. For pentylenetetrazole (PTZ) kindling, rats received a subconvulsant dose of PTZ (40 mg/kg, ip) every second day and seizure stages were recorded. CCS aqueous extract (200, 400, or 600 mg/kg, ip) was injected 30 minutes prior to each PTZ injection. In electrical kindling, bipolar stimulating and monopolar recording electrodes were implanted stereotaxically in the right basolateral amygdala of male Sprague–Dawley rats. After kindling, the effect of aqueous extracts of CCS (200, 400, or 600 mg/kg, ip) on afterdischarge duration, duration of rearing, forelimb clonus, and loss of equilibrium (stage 5 seizure), and latency to the onset of bilateral forelimb clonus were measured. The sedative and the anxiolytic effects of CCS extracts were evaluated in an open-field apparatus and elevated plus maze, respectively. The results indicate that aqueous extracts of CCS have a significant anticonvulsant effect. Different doses of extract significantly delayed the incidence of every seizure stage in the PTZ model of kindling. Moreover, CCS extract (400 and 600 mg/kg, ip) suppressed afterdischarge duration, latency to the onset of bilateral forelimb clonus, and stage 5 seizure in the electrical kindling model. These results suggest that CCS extract has remarkable antiepileptic and central depressant effects. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Epilepsy is a common neurological disorder that causes physical, psychological, and social abnormalities in patients. One percent of the world's population has epilepsy and approximately 30% of patients are considered to be pharmacoresistant. Kindling models have often been used to study seizure propagation [1] and to perform preclinical evaluation of antiepileptic drugs. Electrical kindling is defined as progressive development of electrographic and motor seizures after repeated daily stimulation of particular brain sites [2]. Pentylenetetrazole (PTZ) kindling is produced by daily injection of subconvulsive doses of PTZ [3]. In both methods, subconvulsive electrical or chemical stimulation finally leads to fully epileptic behaviors or generalized seizures. Plant extracts can be an important source for the development of alternative and complementary treatment of epilepsy. Several plants reputed to possess antiepileptic properties in different cultures have been found to exhibit anticonvulsant activity in different animal models [4]. Carum copticum (family: Apiaceae) is a grassy, annual plant
⁎ Corresponding author. Fax: + 98 351 8203414. E-mail address: [email protected] (M.E. Rezvani). 1525-5050/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2011.07.017
with small and aromatic seeds that grows in Iran, India, and Egypt. In Iranian ancient medicine, the seeds of this plant were used for rheumatoid arthritis, stomach pain, and anxiety [5]. Experimental studies have provided evidence demonstrating the cholinergic and anticholinergic [6–8], bronchodilating [9], analgesic [10], and antihypertensive [8] effects of this plant. In Iran, there are folkloric uses and social beliefs regarding the central depressant, sedative, and antiepileptic effects of Carum copticum seeds (CCS). In addition, during a pilot study, it was observed that CCS-treated rats exhibited depressant and sedative behaviors. On the basis of the above-mentioned evidence, the present study was designed to examine the effects of aqueous extracts of CCS on PTZ- and amygdala-kindled seizures in male rats. Moreover, sedative and anxiolytic effects of the plant seeds were evaluated. 2. Materials and methods 2.1. Plant material and extracting method Dried seeds of Carum copticum were purchased from an herbal pharmacy in Rafsanjan City and a voucher specimen of the plant (RJ 32–534) has been deposited in the herbarium of the School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
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One kilogram of CCS was washed with distilled water and coarsely ground with a blender; the powder obtained was macerated in warm distilled water (50–60 °C) for 48 hours and extracted twice. The resulting extract was concentrated under low pressure in a rotary evaporator and then freeze-dried. For administration, the dried extract was dissolved freshly in 0.9% sterile saline.
CCS aqueous extract or saline 15, 30, or 90 minutes prior to electrical stimulation of the amygdala. In this experiment, afterdischarge duration (ADD), stage 4 latency (S4L), stage 5 duration (S5D), and convulsive behaviors were recorded. In addition, the number of amygdala stimulations required for occurrence of each seizure stage was recorded.
2.2. Chemicals
2.4.3. Experiment 3: Sedative and anxiety tests
Pentylenetetrazole was purchased from Sigma Chemical Company (Poole, UK). PTZ was dissolved in sterile saline (0.9%). Ketamine hydrochloride and xylazine were purchased from Alfasan (Woerden, The Netherlands). All drugs were prepared freshly and injected intraperitoneally.
2.4.3.1. Spontaneous locomotor activity. An open-field test was used to evaluate the sedative effect of the extract. The open-field apparatus (Borj Sanat Co., Iran) consisted of a Plexiglas box (40 cm× 40 cm× 40 cm) equipped with 16 infrared photocells located every 2.5 cm to measure horizontal movements. Thirty minutes after injection of extract or saline, rats were individually placed in the center of the box floor and monitored for 20 minutes. Rats in groups 1–4 received a single dose of saline or three different doses of CCS extract. Other groups were treated with repeated doses of saline or different doses of CCS extract every second day up to day 35, but locomotor activity was recorded only on the last day (18th injection) of treatment. Spontaneous locomotion was recorded as the number of cuts of the beam by the photocell monitoring system [14].
2.3. Animals Male Wistar rats from the Razi Institute (Iran), weighing 200–250 g at the beginning of the study, were used. Animals were housed in standard cages under a 12-hour-light/12-hour-dark cycle (lights on at 07:00) and had free access to food and water. Procedures involving animals and their care were conducted in accordance with the Guide to the Care and Use of Experimental Animals [11]. Approval from the local ethics committee was also obtained. All experiments were done at the same time in the morning (10–12 AM) to avoid a circadian rhythm bias. To reduce the number of kindled animals and their suffering, all distorting factors were eliminated. 2.4. Experimental design To determine the effects of the extract on the development of PTZand amygdala-kindled seizures, the following experiments were designed: 2.4.1. Experiment 1: Pentylenetetrazole kindling (groups 1–4) For PTZ kindling, rats received a subconvulsant dose of PTZ (40 mg/ kg) intraperitoneally every second day. PTZ injections were continued until at least three consecutive stage 5 seizures were elicited [12]. In different groups, rats were injected with 200, 400, or 600 mg/kg CCS aqueous extract or saline 30 minutes prior to administration of PTZ. In these groups, rats were observed for 30 minutes after each PTZ injection and convulsive behaviors were scored as follows: no response, stage 0; motor arrest, stage 1; facial or jaw movements, stage 2; addition of head nodding, stage 3; unilateral forelimb clonus, stage 4; and rearing with bilateral forelimb clonus, stage 5.
2.4.3.2. Elevated plus maze. The elevated plus maze was used to measure anxiety-like behavior of the rats. The maze consisted of two opposite open arms (50 × 10 cm), two opposite closed arms (50 × 10 × 40 cm), and a central platform (10 × 10 cm). The arms were located 50 cm above the floor on the base. The floors of the arms and the walls of closed arms were constructed from black Plexiglas. Thirty minutes after injection of extract or saline, rats were individually placed on the central platform facing an open arm and away from a blinded observer. For 5 minutes, the number of entries into the open and closed arms was recorded. Time spent in the open and closed arms was also measured. The anxiety index was calculated by dividing the number of entries into the open arm by the number of entries into the open and closed arms. 2.5. Assessment of acute toxicity Rats fasted for 12 hours were allocated to five groups of 10 rats per group. Lethality was evaluated after intraperitoneal administration of 750, 1200, 2000, or 3000 mg/kg CCS aqueous extract. Percentage mortality of rats was recorded after 24 hours, and LD50 values were determined graphically [15]. 2.6. Histology
2.4.2. Experiment 2: Amygdala electrical kindling (groups 5–8) For electrical kindling, under ketamine (100 mg/kg, ip) and xylazine (10 mg/kg, ip) anesthesia, animals were stereotaxically implanted with bipolar stimulating and monopolar recording electrodes (twisted into tripolar configuration) terminating in the basolateral amygdala of the right hemisphere (coordinates: A, −2.5 mm; L, 4.8 mm; 7.5 mm below dura). The electrodes (stainless steel, Teflon-coated, 127 μm in diameter, A.M. System Inc., USA) were insulated except at their tips. Two single electrodes were connected to the skull screws, fixed on the cortical surface as earth and differential electrodes. One week after surgery, afterdischarge threshold was determined by amygdala stimulation (60-Hz monophasic square wave, 1-ms duration per wave, for 2 seconds). Stimulation and isolation were provided by a WSI stimulator (WSI Co., Iran). Stimuli were initially delivered at 10 μA and then at 5minute intervals, increasing stimulus intensity in increments of 10 μA until at least 5 seconds of afterdischarges were recorded as previously described [13]. The following experimental groups were considered: First, animals in groups 5 to 8 (n = 6–8 per group) were fully kindled by daily electrical stimulation of the amygdala before any treatment. Then, 24 hours later, the animals were injected with 200, 400, or 600 mg/kg of
At the end of the study, all rats were sacrificed and their brains were removed and fixed in formalin 10% for 1 week. Then, each brain was sectioned and electrode tip placements were determined under a microscope. In the case of abnormality in electrode positions or the presence of tissue damage, the data from that particular animal were not included in the results. 2.7. Phytochemical analysis Quantitative phytochemical analysis of the powdered seeds was conducted to determine the amounts of different compounds including tannins, flavonoids, saponins, alkaloids, and anthraquinones [16]. 2.8. Statistics All data are expressed as means ± SEM. One way ANOVA was performed to compare ADD, S4L, and S5D in different treatments and was followed by Dunnett's test for post hoc analysis. Behavior stages
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were analyzed with the Kruskal–Wallis ANOVA. The Wilcoxon or Mann–Whitney U test was used for comparing data within or between groups, respectively, where data were not normally distributed. In all experiments, P values less than 0.05 were considered statistically significant. 3. Results The LD50 value of the extract was 1737.8 ± 227 mg/kg. This LD50 value suggests that the CCS aqueous extract is relatively safe in rats. Quantitative phytochemical analysis showed that CCS contain tannin 1.83% ± 0.2, flavonoids 9.33% ± 0.11, saponins 0.64% ± 0.15, alkaloids 5.41% ± 0.17, and trace amounts of anthraquinones. 3.1. Effect of CCS extract on kindling rate in the pentylenetetrazole model Fig. 1 illustrates that the aqueous extract of CCS exhibits antiepileptic properties in the PTZ model of kindling. From PTZ injections 1 to 7, there are no interpretable changes (although there are statistically significant differences at a few points) between rats that received saline and those that received different doses of CCS extract. CCS extract 200 or 400 mg/kg (P b 0.01, injections 7–18) and CCS extract 600 mg/kg (P b 0.001, injections 8–18) attenuated seizure severity and delayed all seizure stages. In the saline group, subsequent injection of the same dose of PTZ induced epileptic behaviors in an ascending order. In the CCS groups, none of the rats manifested stage 4 and 5 seizures. By the 11th injection, one of the rats, and by the 14th injection, two of the rats, in the saline group died. However, these results indicate that consecutive injections of CCS extract significantly delay kindling rate and decrease seizure severity. 3.2. Effect of CCS extract on seizure parameters in the amygdala kindling model Fig. 2 illustrates the behavioral and electrophysiological data of different groups of kindled rats. The results show that intraperitoneal administration of aqueous extracts of CCS led to a significant decrease in ADD at doses of 400 and 600 mg/kg (P b 0.01) and a significant increase in S4L at doses of 200, 400, and 600 mg/kg (P b 0.01). Because in each CCS-treated group only two or three rats exhibited stage 5 seizures, the data related to S5D could not be statistically analyzed. However, as shown in Fig. 3, the numbers of amygdala stimulations
Fig. 2. Effect of aqueous extract of Carum copticum (CCS) at the doses of 200, 400, and 600 mg/kg on afterdischarge duration, stage 5 duration and step 4 latency after 15, 30, and 90 min of intraperitoneal injections in amygdala-kindled rats. The rats received daily amygdala stimulation (n = 7–10 rats). Values are means ± SEM; *p b 0.05, **p b 0.01 and ***p b 0.01 compared with saline treated rats.
required for first generation of stage 3, 4, or 5 seizures were greater in the rats treated with CCS extract than in the saline-treated group (P b 0.01). These results indicate an obvious anticonvulsive effect for aqueous extracts of CCS. 3.3. Locomotion and rearing As shown in Table 1, CCS extract at different doses produced a significant decrease in spontaneous locomotor activity and number of rears in rats after the 1st and 18th injections (P b 0.05). In addition, statistical analysis revealed that the rats repeatedly administered (18th injection) CCS extract at doses of 200 and 600 mg/kg (P b 0.05) had a greater decrease in locomotor activity compared with rats that received one injection.
Fig. 1. Effect of the CCS extract of Carum copticum (CCS) on seizures induced by PTZ. The extract was injected prior to PTZ that was injected on every second day. (n = 7–10 rats). *P b 0.05 compared with corresponding points in saline treated group. Values are means ± SEM.
3.4. Elevated plus maze Administration of CCS extract at 400 and 600 mg/kg reduced the number of entries into the open and closed arms, but was unable to
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Administration of aqueous extracts of CCS significantly reduced seizure activity and kindling development rate. The PTZ kindling test is used as a valid model of partial seizures with secondary generalization [17]. Repeated injections of PTZ led to convulsions and epilepsy-like behavior, and finally rats exhibited class 4 (generalization) and class 5 (generalized tonic–clonic) seizures. Attenuation of PTZ-induced convulsions suggests that the antiepileptic effect of CCS extract may be partly mediated by activation of the brain GABAA receptor complex. One well-accepted mechanism by which PTZ induces convulsions is by antagonizing the picrotoxin-sensitive site of the GABAA receptor complex [18,19]. PTZ is a selective blocker of chloride channels in the GABAA receptor complex and repeated exposure of the brain to subconvulsive doses of PTZ leads to suppression of GABA neurotransmission as evidenced by lesser binding of [ 3H]GABA [20] and a decrease in chloride uptake rate induced by GABA [21]. As the results of our study indicate CCS extract has inhibitory effects in the PTZ model and considering that PTZ-induced convulsions are related to suppression of GABA neurotransmission, it is likely that aqueous extract of CCS attenuate epileptic convulsions by enhancing GABAergic neurotransmission in the brain. Kindling of seizures has been used to model the progressive pathogenesis of refractory epilepsy in human [1]. In this experiment, intraperitoneal injection of CCS extract significantly reduced the duration of afterdischarges recorded from the amygdala. It was clearly shown that repeated administration of CCS reduces neuronal activity in the amygdala and possibly other regions of the brain. Because the seizure-protective effect is accompanied by a decrease in S5D and an increase in S4L, it may be concluded that both the partial (1–3) and generalized (4 and 5) seizure stages may be affected by CCS extract. S4L is an index of seizure generalization and its increase implies that CCS extract delays the seizure generalization in rats. Absence of stage 5 or its shortening is also another effect of the CCS extract. In contrast, with PTZ kindling, after amygdala electrical kindling, the function of GABAergic neurotransmission in the hippocampus is intensified [22,23] and the extract might potentiate GABA neurotransmission.
Fig. 3. Progressive development of behavioral manifestation of seizures induced by electrical stimulation of amygdala. Prior to the each stimulation, rats received aqueous extract of Carum copticum (CCS) at the doses of 200, 400 and 600 mg/kg. Results are expressed as the number of stimulations required for the first occurrence of each stage (n = 7–10). Data are the means ± S.E.M. *P b 0.05, **P b 0.01 and ***P b 0.001 from the corresponding value for animals pretreated with saline.
increase the time spent in the open arm. The anxiety index of the control and CCS treated groups remained unchanged. Among the data obtained, the significant reduction in the number of entries into the closed arms compared with the control group was unpredictable (Table 2). 4. Discussion In Iranian folk medicine, Carum copticum seeds have been used as antiepileptic, sedative, and anxiolytic remedies. In the present study, the antiepileptic effect of CCS in the PTZ and amygdala kindling models was evaluated. The aqueous extract of CCS was shown to have anticonvulsant effects in the PTZ and amygdala kindling models.
Table 1 Effects of aqueous extracts of Carum copticum seeds (CCS) on spontaneous locomotor activity and rearing after injections 1 and 18 using the open-field test. Treatment (ip)
n
Saline (control) CCS 200 mg/kg CCS 400 mg/kg CCS 600 mg/kg
10 9 11 10
Locomotor activity (count)
Rearing (count)
After injection 1
After injection 18
After injection 1
After injection 18
642.44 ± 93.85 595.70 ± 51.32 371.55 ± 88.06a 244.04 ± 57.12b
671.25 ± 59.68 402.76 ± 53.24a,c 308.41 ± 71.72a 109.22 ± 61.25b,d
29.55 ± 4.21 20.96 ± 3.56 17.67 ± 2.18a 9.55 ± 3.44b
35.19 ± 5.89 11.56 ± 3.54a 10.49 ± 5.81b 8.22 ± 3.74b
Note. Data are means ± SEM. n = number of rats in each group. a P b 0.05, as compared with corresponding saline-treated group. b P b 0.01, as compared with corresponding saline-treated group. c P b 0.05, as compared with CCS-treated groups with the same dose in injection 1. d P b 0.01, as compared with CCS-treated groups with the same dose in injection 1.
Table 2 Effects of aqueous extract of Carum copticum seeds (CCS) on time spent in and entries into the open and closed arms of the elevated plus maze and calculated anxiety index in independent groups. Treatment (ip)
Saline (control) CCS 200 mg/kg CCS 400 mg/kg CCS 600 mg/kg
n
11 14 10 10
Time (s) spent in
Entries into
Anxiety index
Open arms
Closed arms
Open arms
Closed arms
88.24 ± 10.33 91.11 ± 8.29 40.35 ± 9.20a 35.07 ± 9.27a
207.44 ± 14.53 200.71 ± 22.18 247.54 ± 7.26a 260.11 ± 6.35b
10.21 ± 0.57 8.78 ± 0.48 2.05 ± 0.38b 1.24 ± 0.21b
10.14 ± 0.99 9 .44 ± 0.67 2.65 ± 0.40b 12.01 ± 0.32b
Note. Data are expressed as means ± SEM. n = number of rats in each group. a P b 0.05, as compared with corresponding saline-treated group. b P b 0.01, as compared with corresponding saline-treated group.
0.50 ± 0.06 0.48 ± 0.04 0.43 ± 0.03 0.38 ± 0.04
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Carum seeds have multiple constituents including steroptin, thymine, cumin, amino acids like lysine and threonine, tannins, and dietary fibers [24]. In previous studies, involvement of lysine in the kindling model of epilepsy was observed. L-Lysine can enhance benzodiazepine receptor binding affinity [25,26] and subsequently shows an antiepileptic effect in experimental epilepsy [27,28]. This evidence suggests that the antiepileptic effect of the extract is partly mediated by L-lysine. Carum copticum seeds also demonstrated relaxant and spasmolytic effects on duodenum of laboratory animals [8]. The relaxant and spasmolytic effects of the plant seeds are partly mediated through calcium channel blockade. In addition, there is evidence that calcium channel blockers obviously have anticonvulsive roles in different animal models of epilepsy [29–31]. Thus, it is possible that blockade of Ca 2+ channels is another mechanism by which this extract induces antiepileptic effects. The histamine H1 receptor antagonistic property of CCS [9] cannot be an antiepileptic mechanism for the CCS extract because substantial evidence indicates that histamine, through H1 receptors, plays an important role in suppressing kindled seizures [32–34]. However, the seizure-suppressing effect of selective antagonists of H3 receptors in laboratory animals has been documented in a few studies [35,36]. Therefore, the antiepileptic effect of CCS extract may be due to inhibition of H3 receptors in the brain. The phytochemical screening test showed that flavonoids are the major components in CCS. Some flavonoids have been shown to potentiate brain GABAA receptors [37] and increased GABA-induced currents in rat cortical neurons [38]. Moreover, flavonoids may also be capable of modulating glutamate excitotoxicity via direct scavenging of ROS or by the attenuation of calcium influx [39]. On the basis of this evidence, it is possible that the antiepileptic effects of CCS extract are exerted via potentiation of GABA neurotransmission and/or suppression of glutamate receptors in the brain. The sedative effect of CCS extract was evaluated using the openfield test. Our results imply a depressant effect of CCS at doses that are antiepileptic. Reduced spontaneous locomotor activity is generally attributed to neuronal inhibition. The brain GABAergic system is responsible for sedation and depressive behaviors [40] and the findings also support the possible GABA-mediated property of CCS extract. These results show that CCS induce sedation and central depression. In the elevated plus maze test, the number of entries into the open and closed arms was reduced in CCS-treated animals as compared with controls. The elevated plus maze test is an accepted method for evaluating anxiety-like behavior in laboratory rodents [41,42]. In this maze, decreases in the number of entries into and the time spent in the open arms are indicators of anxiety. Different doses of CCS extract did not increase these indices of anxiety, meaning that the CCS extract does not have anxiolytic-like properties. Furthermore, a significant reduction was observed in the number of entries into the open and closed arms when compared with the control group. It has been demonstrated that evaluation of anxiety in the elevated plus maze depends on exploratory behavior [43,44] and may also be confounded by changes in locomotor activity [36]. In addition, other studies have shown that closed arm entries are a more reliable measure of locomotor activity than total arm entries [43,45]. Thus, the data obtained with the elevated plus maze might represent the suppressive effect of CCS extract on locomotor activity. However, using other animal models of anxiety may provide a more rational deduction about the possible anxiolytic or anxiogenic-like effects of CCS extract. In conclusion, our data indicate that CCS aqueous extract has antiepileptic and sedative effects. This study provides scientific rationale for the use of this aqueous extract of plant seeds for the amelioration of epilepsy observed in traditional medicine in Iran. However, CCS contains multiple components such as steroptin, thymene, tannin, alkaloids, and cumin [24]. Its essential oil also contains thymol, p-cymene, and γ-
cymene. Thus, more studies are necessary to clarify the antiepileptic components and the mechanisms underlying the properties.
Acknowledgment This work was supported by the research deputy of Rafsanjan University of Medical Sciences and Yazd Shahid Sadoughi University of Medical Sciences through a research project. The authors appreciate Ahmadreza Sayyadi and Ali Khodadadi for their assistance during the present study.
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