Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 381, Issue 5, pp 415–426

Comparative anticonvulsant activities of the essential oils (EOs) from Cymbopogon winterianus Jowitt and Cymbopogon citratus (DC) Stapf. in mice

Authors

  • Monalisa Ribeiro Silva
    • Department of Physiology and Pharmacology, Faculty of MedicineFederal University of Ceará (UFC)
  • Rafael Matos Ximenes
    • Department of Physiology and Pharmacology, Faculty of MedicineFederal University of Ceará (UFC)
  • José Galberto Martins da Costa
    • Department of Biological and Health SciencesRegional University of Cariri (URCA)
  • L. Kalyne A. M. Leal
    • Department of PharmacyFederal University of Ceará (UFC)
  • Amanda A. de Lopes
    • Department of Physiology and Pharmacology, Faculty of MedicineFederal University of Ceará (UFC)
    • Department of Physiology and Pharmacology, Faculty of MedicineFederal University of Ceará (UFC)
    • Faculty of Medicine of Juazeiro do Norte (FMJ)
ORIGINAL ARTICLE

DOI: 10.1007/s00210-010-0494-9

Cite this article as:
Silva, M.R., Ximenes, R.M., da Costa, J.G.M. et al. Naunyn-Schmied Arch Pharmacol (2010) 381: 415. doi:10.1007/s00210-010-0494-9

Abstract

The fresh leaves of Cymbopogon citratus are a good source of an essential oil (EO) rich in citral, and its tea is largely used in the Brazilian folk medicine as a sedative. A similar source of EO is Cymbopogon winterianus, rich in citronellal. The literature presents more studies on the EO of C. citratus and their isolated bioactive components, but only a few are found on the EO of C. winterianus. The objective of the present study was then to study, in a comparative way, the effects of both EOs on three models of convulsions (pentylenetetrazol, pilocarpine, and strychnine) and on the barbiturate-induced sleeping time on male Swiss mice. The animals (20–30 g) were acutely treated with 50, 100, and 200 mg kg−1, intraperitoneally, of each EO, and 30 min later, the test was initiated. The observed parameters were: latency to the first convulsion and latency to death in seconds. Furthermore, the in vitro effects of the EOs were also studied on myeloperoxidase (MPO; a biomarker for inflammation) and lactate dehydrogenase (LDH; an index of cytotoxicity) releases from human neutrophils. The EOs radical-scavenging activities were also evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The results showed that both EOs were more active on the pentylenetetrazol-induced convulsion model, and C. citratus was even more efficient in increasing latency to the first convulsion and latency to death. Both parameters were potentiated in the presence of a lower dose of diazepam (reference drug) when associated to a lower dose of each EO (25 mg kg−1). Besides, their anticonvulsant effects were blocked by flumazenil, a known benzodiazepine antagonist. This effect was somewhat lower on the pilocarpine-induced convulsion, and better effects were seen only with the EOs’ higher doses (200 mg kg−1). A similar result was observed on the strychnine-induced convulsion model. Both EOs potentiated the barbiturate-induced sleeping time. However, C. citratus was more efficient. Interestingly, both EOs completely blocked the MPO release from human neutrophils and showed no cytotoxic effect on the LDH release from human neutrophils. On the other hand, only a very low or no effect on the DPPH assay was observed with C. winterianus and C. citratus, respectively, indicating that the radical scavenging activity did not play a role on the EOs’ effects. We conclude that the mechanism of action of the anticonvulsant effect of the EOs studied is, at least in part, dependent upon the GABAergic neurotransmission. In addition, their effects on inflammatory biomarkers can also contribute to their central nervous system activity.

Keywords

Anticonvulsant and anti-inflammatory activitiesCymbopogon citratusC. winterianusSedativeAntioxidantEssential oil

Introduction

Numerous herbal medicines are recognized to present bioactive constituents that have a potential to affect chronic conditions, such as anxiety, depression, headaches, or epilepsy. They could be useful for patients not responding well to conventional treatments (Carlini 2003; Blank et al. 2007). A good example is essential oils (EOs) present in aromatic plants that exhibit a variety of biological properties, such as analgesic, anticonvulsant, and anxiolytic ones (Viana et al. 2000; Matos 2007; Quintans-Júnior et al. 2008).

The Cymbopogon genus presents more than 100 species found in tropical countries (Lorenzi and Matos 2003), and from those, 56 are aromatic and then rich in essential oils (concentrated hydrophobic liquid, which carries volatile and aromatic compounds). Furthermore, monoterpenes are chemical constituents found in essential oils of fruits, vegetables, and several herb species. They are naturally occurring hydrocarbons, composed by the condensation of two isoprenes and, although widely distributed in the plant kingdom, are mainly found in essential oils. The ten-carbon isoprenoids are derived from the mevalonate pathway in plants, but are not produced by mammals, fungi, or other species. They function physiologically as chemoattractants or chemorepellents (McGarvey and Croteau 1995) and are largely responsible for the distinctive fragrance of many plants. A number of monoterpenes have antitumor activities and prevent the carcinogenesis process at both its initiation and promotion/progression stages. In addition, monoterpenes are effective in treating early and advanced cancers (Gould 1997; Crowell 1999).

Cymbopogon citratus Stapf. (Poaceae), known worldwide as lemongrass (“capim-santo” in the Brazilian folk medicine), is widely used in tropical countries for the treatment of hypertension, gastrointestinal disturbances, anxiety, epilepsy, and other central nervous system (CNS) disorders (Viana et al. 2000; Singi et al. 2005; Matos 2007; Quintans-Júnior et al. 2008). Cymbopogon winterianus Jowitt (Poaceae), known as java citronella (“capim-citronela” in Brazil), is mainly used as an insect repellent and air freshener (Guenther 1972; Matos 2007). However, healers in Northeast Brazil use the infusion of citronella fresh leaves for the treatment of anxiety and epilepsy (Quintans-Júnior et al. 2008).

The essential oils of these two Cymbopogon species are similar, but differing in major constituents. Thus, C. citratus EO is rich in citral [a compound of neral (37%) and geranial (45%)] and mircene (12%). On the other hand, C. winterianus EO is rich in citronellal (61%) and geraniol (19%) (Craveiro et al. 1981; Matos 2007). It was shown (Blank et al. 2007) that seasonal changes had significant effects on the yield and volatile oil content of C. winterianus, and maximum volatile oil yields were observed at 9:00 a.m. at all seasons. Many pharmacological activities are described for C. citratus as a CNS depressant, spasmolytic, analgesic drug, and an antimicrobial against Helicobacter pylori (Nsour et al. 2000; Viana et al. 2000; Matos 2007). Additionally, there is little information on C. winterianus besides some reports on its pharmacological activities as an antifungal and acaricide (Guenther 1972; Blank et al. 2007; Matos 2007). Preliminary behavioral screening carried out in our laboratory with the EO of C. winterianus leaves showed a depressant activity on the CNS.

The aim of the present work was, therefore, to investigate in a comparative way, the anticonvulsant effects of the EO from C. citratus and C. winterianus leaves in experimental models of convulsion in mice. Besides, the antioxidant potential, as evaluated by their radical scavenging activity, as well as the effects on lactate dehydrogenase (LDH; an indicator of cytotoxicity) and myeloperoxidase (MPO; a biomarker for inflammation) releases from human neutrophils was also assessed.

Materials and methods

Plant material and essential oil extraction

C. winterianus and C. citratus fresh leaves were collected (in the morning) at the Medicinal Plants Garden of the Research Laboratory on Natural Products, of the Regional University of Cariri (URCA), Ceará, Brazil, and authenticated by Prof. Afranio Fernandes of the Department of Biology of the Federal University of Ceará. Voucher specimens of each plant are deposited at the Prisco Bezerra Herbarium (C. citratus, EAC 41,832; C. winterianus, EAC 43,194). The EOs were obtained by steam distillation of fresh leaves (300 g) and maintained protected from light and heat until use. Yields were 0.47% and 0.49% for C. citratus and C. winterianus, respectively. For the pharmacological assays, the EO was suspended in 0.5% Cremophor® (polyethoxylated castor oil), diluted in distilled water up to the desired concentration, and sonicated before use.

Gas chromatography and mass spectrometry analyses

The identification of volatile constituents was performed using a Hewlett-Packard 5890 Series II gas chromatograph, equipped with an HP-5971 mass selective detector and an HP-5 (25 cm × 0.2 mm × 0.33 mm) capillary column. Gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) were performed using split/splitless injections, with the injector set at 220°C, the column at 60°C, with a heating lamp of 3°C min−1, a final temperature of 240°C for 7 min, and the flame ionization detector set at 250°C. Helium was used as a carrier gas at 1 mL min−1. The GC–MS electron ionization system was set at 70 eV. A sample of the EO was solubilized in ethyl acetate for the analyses. Retention indices were determined by co-injection of hydrocarbon standards. The oil components were identified by comparison with data from the literature, the profiles from the Wiley 138 and Nist 98 libraries, and by co-injection of authentic standards, when available. Results from both EOs are shown in Figs. 1 and 2 and Tables 1 and 2.
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Fig. 1

The essential oils (EOs) of C. citratus (Cc; a, c) and C. winterianus (Cw; b, d) significantly increased the latency to the first convulsion and the latency to death in the model of PTZ-induced convulsions. EOs (50, 100, and 200 mg kg−1, i.p.) were acutely administered 30 min before the test initiation. Diazepam (DZP, 1 mg kg−1) was used as the reference drug. a, b, and c vs. controls, at p < 0.05, 0.01, and 0.001, respectively, from 8 to 16 animals per group (ANOVA and Student–Newman–Keuls as the post hoc test)

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Fig. 2

Effect of the combination of the essential oils (EOs) from C. citratus (Cc) and C. winterianus (Cw) plus diazepam (DZP) on the PTZ-induced convulsion. Latency to the first convulsion (a) and latency to death (b) of the EOs from C. citratus and C. winterianus in the model of PTZ-induced convulsions in mice. DZP was used at a lower dose (0.2 mg kg−1) alone or associated to each EO (25 mg kg−1, i.p.). Data from 12 animals per group. a vs. controls; b vs. DZP and c vs. C. citratus or C. winterianus, respectively (a); a vs. controls, b vs. DZP (b) (ANOVA and Student–Newman–Keuls as the test post hoc)

Table 1

Chemical constituents and their respectives percentages of the essential oil from fresh leaves of Cymbopogon citratus

RT (min)a

Constituent

Percentage

4.52

Myrcene

14.4

5.33

Citronellal

0.10

7.15

Linalol

0.24

8.99

β-(E)-ocimene

0.24

9.24

Terpineol-4-ol

1.24

11.20

Neral

37.42

11.88

α-(E)-ocimene

0.32

12.06

Geranial

45.47

Total

 

99.43

aRelative retention time (RT) from the literature (Adams 2001)

Table 2

Chemical constituents and their respectives percentages of the essential oil from fresh leaves of Cymbopogon winterianus

RT (min)a

Constituent

Percent

8.02

Limonene

2.25

10.22

Citronellyl acetate

0.69

11.05

Citronellal

60.96

14.61

Citronellol

11.52

15.40

Geraniol

19.03

18.55

Linalool

0.54

19.48

Geranial

0.87

24.13

Isopulegol

0.57

24.90

Neryl acetate

0.76

Total

 

97.19

aLiterature-relative retention time (RT) (Adams 2001)

Animals

Male and female Swiss mice (weighing 20–30 g; 8 weeks old) were used. The animals were maintained in cages, with free access to food and water and kept in a standard light–dark cycle (12/12 h) and controlled temperature of 22°C. The number of animals per group ranged from 8 to 24, and the doses used ranged from 25 to 200 mg kg−1, intraperitoneally (i.p.), based on pilot experiments and on data from the literature. The experiments were performed according to the Guide for the Care and Use of Laboratory Animals, from the US Department of Health and Human Services, Institute of Laboratory Animal Resources, Washington, DC (1985), submitted and approved by the local Animal Resources Committee.

Drugs and reagents

Pilocarpine, pentylenetetrazol, strychnine, and pentobarbital were purchased from Sigma (MO, USA) and dissolved in distilled water before use. Cremophor, also purchased from Sigma and used as an emulsifying agent for the EO suspension, was suspended in distilled water. Diazepam (Valium, ampoule) was from Roche, Brazil. Valproic acid (Depakene, syrup) was from Abbott, and carbamazepine (Carmazin, pills) was from Lab. Teuto, Brazil. All other reagents were of analytical grade.

Pilocarpine-induced convulsions

In the pilocarpine-induced convulsions, the animals (16 to 24 per group) were previously treated with the EOs of C. citratus or C. winterianus at the doses of 50, 100, and 200 mg kg−1 (i.p.), 30 min before the administration of the convulsant agent (pilocarpine, 350 mg kg−1, i.p.). Negative controls received only the vehicle solution (0.5% Cremophor®, i.p.), and positive controls received carbamazepine or valproic acid intraperitoneally, at the doses of 25 and 50 mg kg−1, respectively, also administered 30 min before pilocarpine. We evaluated the latency to the first seizure, death latency, and the percentage of survival in 24 h. The status epilepticus (SE) was defined as continuous seizures for a period longer than 30 min. The mortality rate was recorded up to 24 h after the pilocarpine administration and expressed as percentage of survival.

Pentylenetetrazol- and strychnine-induced convulsions

For these two experimental models, the animals (8 to 16 per group) were treated with C. citratus and C. winterianus EO at the doses of 50, 100, and 200 mg kg−1 (i.p.). The negative controls were treated with the vehicle solution (0.5% Cremophor®, in distilled water, i.p.) and the positive controls with diazepam (DZP) as the reference drug at the dose of 1 mg kg−1 (i.p.). Thirty minutes after the administration of the EOs (treated groups) or vehicle (controls), the animals received pentylenetetrazol (PTZ) 80 mg kg−1 or strychnine 2 mg kg−1, intraperitoneally. Observed parameters were the latency to the first seizure, latency to death, as well as the percentage of survival in 24 h. Those animals that did not die within a 2-h period were further observed for 24 h, in order to determine the percentage of survival and then were sacrificed by cervical dislocation.

Effect of the EOs from C. citratus and C. winterianus plus diazepam on PTZ-induced convulsion

Since both EOs were more efficacious against PTZ-induced convulsions, we decided to demonstrate any possible DZP effect potentiation in this model. The animals were pretreated with the EOs of C. citratus or C. winterianus (25 mg kg−1) or with diazepam (0.2 mg kg−1). Other groups were treated with the combination of the EO plus diazepam. All animals were administered 30 min later with PTZ (80 mg kg−1). These combination groups were compared to the controls, to groups receiving only EOs of C. citratus or C. winterianus (25 mg kg−1), and to diazepam-treated groups (0.2 mg kg−1). The latency to the first seizure, death latency, and percentage survival in 24 h were evaluated. Those animals that did not die up to a 2-h period were observed afterwards for 24 h to determine the percentage of survival.

Effect of the EOs from C. citratus and C. winterianus plus flumazenil on the PTZ-induced convulsion

In order to evaluate the participation of the gamma aminobutyric acid (GABA)ergic system on the EOs effects, in the PTZ-induced convulsion model, the animals (nine per group) were divided into the following groups: control (distilled water), DZP (1 mg kg−1), C. citratus or C. winterianus (200 mg kg−1 of each), and the combinations of flumazenil (Flu) (5 mg kg−1) plus DZP, Flu plus C. citrates, and Flu plus C. winterianus. The drugs were administered intraperitoneally, and 30 min later, the animals were injected with PTZ (80 mg kg−1). In the case of combinations, the animals were firstly administered with Flu, and 15 min later, with DZP or the EO, followed by PTZ (after 15 min of the drug’s combination). The latency to the first convulsion and latency to death in seconds were the registered parameters.

Pentobarbital-induced sleeping time

Both EOs presented some degree of sedation, which was intensified at higher doses. Thus, we decided to verify any possible potentiation of the EO on the barbiturate-induced sleeping time test in mice. The animals (16 per group) were treated with the EOs at the doses of 50, 100, and 200 mg kg−1 (i.p.). The negative control group was treated by the intraperitoneal route with the vehicle solution, and the positive control group was treated with DZP (2 mg kg−1). Thirty minutes after the EO and control treatments, all animals (16 animals per group) received sodium pentobarbital (40 mg kg−1). The time since the injection up to the loss of the righting reflex was recorded as sleeping latency, and the time elapsed between the loss and the voluntary recovery of the righting reflex was considered as the sleeping time (Vasconcelos et al. 2007).

Determination of 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity in vitro

The method was that described by Saint-Cricq de Gaulejac et al. (1999). Briefly, an aliquot (0.1 mL) of the EO suspension (200 and 400 μg/mL) or α-tocopherol (5 and 50 μg/mL) was mixed with 3.9 mL of 1,1-diphenyl-2-picrylhydrazyl (DPPH) (0.3 mM in a 1:1 methanol/ethanol solution). The mixture was vortexed for 1 min, left standing at room temperature for 30 min, and the absorbance determined at 517 nm. The percentage of inhibition was calculated according to the following equation: percent inhibition = [Ao − (Ac/Ao)] × 100, where Ao was the absorbance of the control (without the EO) and Ac was the absorbance in the presence of EO.

LDH release from human neutrophils

After isolation, the cells (5.0 × 106/ml) in a suspension were incubated with the EOs (1, 10, and 100 µg/mL) or indomethacin (100 µM, as a reference drug), vehicle or Triton X-100 at 0.05% (known to cause cell lysis and used as a positive control) for 15 min at 37°C. Then, the LDH release was determined according to the manufacturer’s instructions (LDH liquiform of Labtest Diagnosis, MG, Brazil). The increasing LDH leakage was expressed by the absorbance decrease determined at 340 nm.

MPO release from human neutrophils

Following Lucisano and Mantovani (1984), 2.5 × 106 human leukocytes were suspended in buffered Hanks balanced solution containing calcium and magnesium. The preparations usually contained predominantly neutrophils (85.0 ± 2.8%), and the cell viability, as determined by the Trypan blue test, was 97.7 ± 0.94%. The cells were incubated with the EOs (0.01, 0.1, and 1 μg/mL) for 15 min at 37°C. Human neutrophils were stimulated by the addition of phorbol myristate acetate (PMA, 0.1 μg/mL) for 15 min at 37°C. The suspension was centrifuged for 10 min at 2,000×g at 4°C. Aliquots (50 μL) of the supernatants were added to phosphate-buffered saline (100 μL), phosphate buffer (50 μL, pH 7.0), and H2O2 (0.012%). After 5 min at 37°C, 3,3′,5,5′-tetramethylbenzidine (TMB; 1.5 mM, 20 μL) was added, and the reaction was stopped by 30 μL of sodium acetate (1.5 M, pH 3.0). The absorbance was determined at 620 nm.

Statistical analysis

The data were expressed as means ± SEM. Analysis of variance (ANOVA) was followed by the Student–Newman–Keuls as a post hoc test. The results were considered statistically significant at p < 0.05.

Results

Chemical compositions of the EOs from C. citratus and C. winterianus

In the chemical composition of the EO of C. citratus, monoterpene compounds, hydrocarbons, ketones, aldehydes, and esters were found. The GC method was used to identify and quantify these compounds. The essential oil is characterized by the presence of neral and geranial (37.42% and 45.47%, respectively, that lead to the formation of citral), mircene (12.44%), terpineol-4-ol (1.14%), β-o-cimene (0.24%), and linalool (0.24%). The major components in the C. winterianus volatile oil were identified as citronellal (60.96%), geraniol (19.03%), citronellol (11.52%), limonene (2.55%), geranial (0.87%), neryl acetate (0.76%), citronellyl acetate (0.69%), isopulegol (0.57%), and linalool (0.54%). The results are shown in Tables 1 (C. citratus) and 2 (C. winterianus).

Effects of EOs from C. citratus and C. winterianus on the PTZ-induced convulsions in mice

In the PTZ-induced convulsion group, the animals treated with the C. citratus EO (50, 100, and 200 mg kg−1) significantly increased in a dose-dependent manner (99% and 4.7 and 5.9 times, respectively) the latency to the first seizure as compared to the control group. A similar pattern was observed for the death latency where increases of 7.8, 10.3, and 15.6 times were seen with the doses of 50, 100, and 200 mg kg−1, respectively. All animals treated with the C. citratus EO presented convulsions, and the percentage survival in 24 h was 31.2, 53.1, and 62.5 for the tested doses. On the other hand, no animals survived in the control group. The animals that received the C. winterianus EO (50, 100, and 200 mg kg−1) were less responsive and showed an increased latency to the first seizure only at the highest dose (3.3 times increase). However, better responses to the death latency were demonstrated with the doses of 50, 100, and 200 mg kg−1, with increases of 7.0, 8.3, and 14.2 times, respectively, as well as in the percentage of survival (43.7, 56.2, and 93.7) (Fig. 1). The association of a low diazepam dose (0.2 mg kg−1, i.p.) with lower doses of C. citratus and C. winterianus EOs (25 mg kg−1) showed a significant increase in all parameters analyzed, as presented in Fig. 2. Furthermore, similar to DZP, the anticonvulsant effects of both EOs were significantly blocked by the pretreatment with flumazenil, a known benzodiazepine antagonist, as shown in Fig. 3.
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Fig. 3

Flumazenil (Flu) antagonizes the anticonvulsant effects of the essential oils (EOs) from C. citratus (Cc) and C. winterianus (Cw) in the model of PTZ-induced convulsion. a Latency to the first convulsion. b Latency to death. EOs (200 mg kg−1, i.p.) were administered 30 min before the test initiation. In the case of drug combination, Flu (5 mg kg-1, i.p.) was administered 15 min before DZP (1 mg kg−1, i.p.) or the EO, followed by PTZ (80 mg kg−1, i.p.) 15 min later. Data from nine animals per group. a vs. control (ANOVA and Student–Newman–Keuls as the post hoc test)

Effects of the EOs from C. citratus and C. winterianus on the strychnine-induced convulsion in mice

In the model of strychnine-induced convulsions, latencies to the first convulsion were unchanged after the administration of C. citratus EO and increased with the highest dose (200 mg) of C. winterianus EO (2.5 times increase) as compared to controls. Similarly, while C. citratus did not change the death latency, C. winterianus-treated animals increased this parameter in 1.3, 1.5, and 2.1 times, as related to controls, at the doses of 50, 100, and 200 mg kg−1, respectively (Fig. 4). The percentage of survival increased only by 12.5 after administration of the EOs from both species.
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Fig. 4

Effects of the EOs from C. citratus (Cc) and C. winterianus (Cw) on the latency to the first convulsion (a, b) and on the latency to death (c, d) in the model of strychnine-induced convulsions in mice. EOs (50, 100, and 200 mg kg−1, i.p.) were acutely administered 30 min before the test initiation. Data from 8 to 16 animals per group. a vs. controls, b vs. DZP (a, b); a vs. controls (c, d) (ANOVA and Student–Newman–Keuls as the post hoc test)

Effects of the EOs from C. citratus and C. winterianus on the pilocarpine-induced convulsions in mice

In the model of convulsions induced by pilocarpine, a significant 2.2 times increase in the latency to the first seizure was observed, only in the group of animals treated with 200 mg kg−1 of the C. citratus EO, while similar increased rates (around 1.4 times) were observed in this parameter after administration of the EO of C. winterianus at the doses of 100 and 200 mg kg−1. As far as the death latency is concerned, while increases of 1.1, 2.2, and 3.9 times were demonstrated after C. citratus at the doses of 50, 100, and 200 mg kg−1, increases ranging from 1.2 to 1.5 were observed after C. winterianus at the same doses (Fig. 5). The rates of survival ranged from 22% to 28% for both species, at the highest doses. All groups showed convulsions.
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Fig. 5

Effects of the EOs from C. citratus (Cc) and C. winterianus (Cw) on the latency to the first convulsion (a, b) and on the latency to death (c, d), in the model of pilocarpine-induced convulsions in mice. EOs (50, 100, and 200 mg kg−1, i.p.) were acutely administered 30 min before the test initiation. Data from 16 to 24 animals per group. a vs. controls, b vs. reference drugs (a, b); a vs. controls (c, d) (ANOVA and Student–Newman–Keuls as the post hoc test)

Effects of the EOs from C. citratus and C. winterianus on the barbiturate-induced sleeping time in mice

The data from the barbiturate-induced sleeping time are shown in Fig. 6. Significant increases of 34% and 41% were observed in the sleep latency after C. citratus administration at the doses of 100 and 200 mg kg−1. DZP, used as a positive control, increased this parameter by 44%. An increase of 2 times was demonstrated in the sleep time duration with the highest dose of C. citrates, while an increase of 3.7 times was observed after the DZP treatment. However, C. winterianus did not alter the sleep latency but increased by 2.1 and 3.3 times in the sleep duration at the doses of 100 and 200 mg kg−1, respectively.
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Fig. 6

The EOs from C. citratus (Cc) and C. winterianus (Cw) potentiated the sleep latency (a, b) and the sleeping time (c, d), in the model of barbiturate-induced sleeping time in mice. EOs (50, 100, and 200 mg kg−1, i.p.) were acutely administered 30 min before the test initiation. Data from 11 to 16 animals per group. a vs. controls (ANOVA and Student–Newman–Keuls as the post hoc test)

Radical scavenging activity of the EOs from C. citratus and C. winterianus as determined by the DPPH assay

This assay is used for the screening of antioxidant activity. Our results did not show any radical scavenging activity with the EO of C. citratus at the concentrations used (200 and 400 µg/mL). However, although also not very efficient, C. winterianus showed some effect especially at the highest concentration (400 µg/mL). On the contrary, vitamin E (50 µg/mL) used as reference drug, presents a radical scavenging activity, significantly decreasing the absorbance as related to controls (Fig. 7).
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Fig. 7

The EOs from C. citratus (Cc) and C. winterianus (Cw) at the concentrations of 200 and 400 µg/mL did not show any radical scavenging activity, as evaluated by the DPPH assay in vitro. Data from three samples. a vs. controls (ANOVA and Student–Newman–Keuls as the post hoc test)

Effects of the EOs from C. citratus and C. winterianus on the releases of MPO and LDH from human neutrophils in vitro

A complete inhibition of the phorbol myristate acetate, PMA-stimulated MPO (a biomarker for inflammation) release from human neutrophils, was observed in the presence of both EOs, at concentrations ranging from 0.01 to 10 µg/mL as compared to dimethyl sulfoxide (DMSO). A significant effect was observed even with the lower concentration (Fig. 8). Similarly, no cytotoxicity was demonstrated with either EOs, as assessed by the LDH assay up to 100 µg/mL concentration (Fig. 9).
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Fig. 8

The EOs of C. citratus (Cc) and C. winterianus (Cw) at the concentrations of 0.01, 0.1, and 1 µg/mL significantly inhibited the myeloperoxidase (MPO) release from phorbol myristate acetate (PMA)-stimulated human neutrophils in vitro. Data from two to seven samples. a vs. Hanks solution (control); b vs. DMSO (ANOVA and Student–Newman–Keuls as the post hoc test)

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Fig. 9

The EOs of C. citratus (Cc) and C. winterianus (Cw) at the concentrations of 1, 10, and 100 µg/mL did not alter the LDH release from human neutrophils in vitro, indicating the absence of cytotoxicity. Data from two to eight samples. a vs. Triton X-100; b vs. Hanks solution (ANOVA and Student–Newman–Keuls as the post hoc test)

Discussion

C. citratus and C. winterianus are popularly used in Brazil, mainly due to their actions on the central nervous system, however, with few controlled studies about this type of activity. The present work provides data on the effects of the EOs from fresh leaves of both species on the mouse central nervous system, focusing mainly on their anticonvulsant and sedative/hypnotic activities. These data are indicative of a depressive activity on the CNS (Almeida et al. 1999).

There are various chronic models though to reflect human epilepsy. Unfortunately, at the moment, it is not possible to judge which model is best suited for developing new strategies in the search for antiepileptic drugs. Rather, a battery of models should be used to avoid false negative or positive predictions (Löscher 2002). In the present work, the anticonvulsant property of the EOs was evaluated by experimental procedures, and one of them, the pilocarpine-induced convulsion, is widely used to investigate antiepileptic drugs, since it is a model of temporal lobe epilepsy. All of these models are of a high predictive value for detection of clinically effective drugs (White 1997). Pentylenetetrazol, pilocarpine, and strychnine are convulsant agents (Nicoll 2001; Rang et al. 2003). We showed the EOs to be able to modify the progress of convulsive episodes induced by both PTZ and pilocarpine, not only the latency to the first convulsion but also the death latency. The mortality rate was significantly reduced mainly in the PTZ-induced convulsions model.

According to a previous work (De Sarro et al. 1999a, b), PTZ may be exerting its convulsant effect by inhibiting the activity of the gamma aminobutyric acid (GABA), at GABAA receptors. PTZ is considered to be a GABAergic antagonist (Riazi et al. 2008) and to interact with the GABAA receptor (Huang et al. 2001). An earlier study (Walsh et al. 1999) showed that an acute injection of PTZ caused transient changes in GABAA receptor mRNA levels, without altering the receptor number, but affecting the coupling mechanism between the GABA and benzodiazepine sites on the GABAA receptor. More recently, it was reported that cyclooxygenase (COX) inhibitors dose-dependently protected against PTZ-induced convulsions and antagonized the effect of flumazenil against PTZ-induced convulsion, further confirming the GABAergic mechanism (Dhir et al. 2006). In the present work, the EOs anticonvulsant effects were also blocked by the pretreatment with flumazenil, a known benzodiazepine antagonist. The gamma aminobutyric acid is the major inhibitory neurotransmitter implicated in epileptic processes. The enhancement or inhibition of the neurotransmission of GABA will attenuate or enhance convulsions, respectively (Meldrum 1981; Gale 1992; Westmoreland et al. 1994).

The PTZ test identifies drugs with efficacy against non-convulsive absence crisis as well as myoclonic seizures. The behavioral results obtained with the EOs from fresh leaves of C. citratus and C. winterianus are in accord with the ethno-pharmacological use of these plants, suggesting anticonvulsant and sedative/hypnotic activities. Although the EOs from both species delayed both parameters (latency to the first convulsion as well as death latency), the effects of the EO of C. citratus on the latency to the first convulsion were more intense. However, both EOs significantly increased the number of survivals. Since the EOs delayed the occurrence of PTZ convulsion, it is probable that they may be interfering with GABAergic mechanisms in order to exert their anticonvulsant effects. This point was corroborated by the potentiation of the DZP anticonvulsant effect on the PTZ-induced convulsions, as observed after its association with a lower dose of each EO.

Recently, it was reported that the EO of C. winterianus presents a CNS depressant activity, significantly reduced PTZ and picrotoxin-induced seizures in rodents, and also increased latencies to clonic seizures induced by strychnine (Quintans-Júnior et al. 2008). However, they used doses higher than ours of up to 400 mg kg−1. Similarly, another work (Blanco et al. 2009) studied the anxiolytic, hypnotic, and anticonvulsant properties of the EO of C. citratus in mice. The authors used even higher doses, 0.5 and 1 g kg−1, and observed that the EO increased the pentobarbital-induced sleeping time, delayed clonic seizures induced by PTZ, and blocked tonic extensions induced by maximal electroshock. According to them, these results indicate that the EO of C. citratus elevates the seizure threshold and/or blockades seizures spread. Although their results agree with ours, the use of higher doses could decrease the selectivity of the EO studied.

When pilocarpine, an agonist for cholinergic receptors, was administered as the convulsing agent, the onset of convulsions was decreased, but the death latency was delayed with higher doses of the EOs. It is worth emphasizing that, in this model, the EO of C. citratus was more effective. Generally, the pilocarpine administration induces cholinergic symptoms in the central (tremor) and peripheral (salivation and diarrhea) nervous systems, as could be noticed in the animals treated with the EOs. Alterations in the extracellular hippocampal glutamate (increased levels) and GABA (decreased levels) have been observed after the intrahippocampal administration of 10 mM of pilocarpine in rats. While pilocarpine seems to be responsible for the seizure onset, those two amino acids are responsible for maintaining the sustained seizure activity (Smolders et al. 1997a, b, c).

Focally evoked pilocarpine-induced seizures were completely prevented by vigabatrin (γ-vinyl-GABA), an antiepileptic drug acting as an irreversible inhibitor of the GABA-metabolizing enzyme. However, we used carbamazepine and valproic acid as standard antiepileptic drugs and noticed that valproic acid offered a better protection against pilocarpine-induced convulsions. It has also been shown that pilocarpine-induced increases in the extracellular levels of glutamate and dopamine in the rat hippocampus are promptly reduced by diazepam (Khan et al. 1999). It has been shown that pilocarpine-induced status epilepticus (SE) can lead to a downregulation of metabotropic glutamate receptors such as mGuR8, suggesting that this condition of SE is associated with a deteriorated autoregulation of glutamate release (Kral et al. 2003).

The oxidative stress has been also implicated in several diseases, including epilepsy. Thus, it was reported that the protective effect of levetiracetam, a new antiepileptic drug against pilocarpine-induced convulsions is mediated, at least in part, by the reduction of lipid peroxidation and hippocampal oxidative stress (Oliveira et al. 2007). Over-excitation of excitatory amino acids has been recognized as an important mechanism in seizure genesis, wherein free radicals have been also suggested to play a critical role (Gupta et al. 2002; Gupta and Briyal 2006). However, in our study, we could not observe any significant radical scavenging effect of the EOs as evaluated by the DPPH assay.

On the other hand, strychnine (STR) causes convulsions by antagonizing glycine receptors, increasing postsynaptic excitability and increasing ongoing activity in dorsal horn neurons (Game and Lodge 1975; McGaraughty and Henry 1998). Strychnine-sensitive glycine receptors are primarily localized in the brain stem and spinal cord, where they are major mediators of postsynaptic inhibition (Kehne et al. 1992). A compound acting like a glycine receptor agonist would be potentially effective as an antiepileptic agent. Our results demonstrate that the acute administration of the EO from C. winterianus, but not that of C. citratus, on STR-induced convulsions significantly increased the latency to the first seizure and death latency only at the highest dose. Therefore, this effect suggests a possible involvement of the glycinergic neurotransmission with the mechanism of action of that EO.

A decrease in sleeping latency and an increase in sleeping time duration induced by pentobarbital, as observed after the treatment with the EOs, are conventional parameters related to a sedative/hypnotic property. Nevertheless, it is a non-specific test, since compounds that decrease the rate of hepatic biotransformation of barbiturates can show the same behavioral effects, as central nervous system depressant drugs (Fujimoto et al. 1960). Although, in the present study, the two EOs increased both parameters, in the barbiturate-induced sleeping time, the EO of C. winterianus was somewhat more efficacious mainly on the sleeping time. Although it has been recently shown (Blanco et al. 2009) that the EO of C. citratus increased the sleeping time, the doses used by these authors were much higher.

In the present study, we also demonstrated that both EOs potently inhibited the MPO (a biomarker for inflammation) release from PMA-stimulated human neutrophils. Besides, neither EO showed any cytotoxicity as evaluated by the LDH assay. Inflammatory processes such as the production of inflammatory cytokines and related molecules have been described in the brain after seizures induced in experimental models and in human epilepsy as well (Vezzani 2005).

Our results suggest that the EO from C. citratus has the potential to alter the course of convulsive episodes, interfering in the seizure threshold and/or blocking the seizure propagation. Since isolated citral or myrcene were not able to protect mice against convulsive episodes Viana et al. 2000 or to show anxiolytic activity (Vale et al. 2002), the results obtained with the EOs are probably due to a synergistic action of other compounds present in small amounts. Nevertheless, our results reinforce the therapeutic potential of the two species’ EOs and point out to the biological and cultural value of studies on traditional folk medicine as a source of new drugs for the treatment of central disturbances.

These data lead us to conclude that both the C. winterianus and C. citratus leaf EOs have a CNS depressant activity and anticonvulsant properties, justifying the use of the plant infusion by traditional medicine practitioners in the treatment of epilepsy. Although the EOs seem to affect preferentially the GABAergic neurotransmission, other pathways such as glycinergic ones are probably also involved and cannot be discarded. Furthermore, the possible EOs’ effects on inflammation, as demonstrated by their activities on the PMA-stimulated MPO release from human neutrophils might also play a role.

Acknowledgments

We are grateful to the Organic Chemistry Department of the Federal University of Piauí (UFPI), Brazil, for the GC–MS analysis of the essentials oils and to Professor M.O.L. Viana for the orthographic revision of the manuscript. This work was supported by fellowships from the Brazilian Coordination for Qualifying University Professors—CAPES.

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