Phythochemical screening and anticonvulsant activity of Cymbopogon winterianus Jowitt (Poaceae) leaf essential oil in rodents.
Cymbopogon winterianus (Poaceae) is used for its analgesic, anxiolytic and anticonvulsant properties in Brazilian folk medicine. This report aimed to perform phythochemical screening and to investigate the possible anticonvulsant effects of the essential oil (EO) from fresh leaves of C. winterianus in different models of epilepsy. The phytochemical analysis of EO showed presence of geraniol (40.06%), citronellal (27.44%) and citronellol (10.45%) as the main compounds. A behavioral screening demonstrated that EO (100, 200 and 400mg/kg; ip) caused depressant activity on CNS. When administered concurrently, EO (200 and 400mg/kg, ip) significantly reduced the number of animals that exhibited PTZ-and PIC-induced seizures in 50% of the experimental animals (p < 0.05). Additionally, EO (100, 200 and 400mg/kg, ip) significantly increased (p < 0.05) the latencies of clonic seizures induced by STR. Our results demonstrated a possible activity anticonvulsant of the EO.
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Keywords: Cymbopogon winterianus; CNS depressant; Anticonvulsant activity; Pentylenetetrazol; Picrotoxin; Strychnine
Epilepsy continues to be a neurological disorder awaiting safer drugs with improved anticonvulsant and anti-epileptogenic effectiveness as currently available drugs fail to provide adequate control of epileptic seizures in about one-third of patients and do not prevent progressive epileptogenic changes are not well understood (Cockerell, 1996). This fact has stimulated a considerable number of research of new anti-epileptic drugs. In this regard, the medicinal plants have been an important source to the development of drugs with this biological activity (Carlini, 2003). Additionally, numerous herbal medicines are recognized as active in the central nervous system (CNS), and they have at least a hypothetical potential to affect chronic conditions such as anxiety, depression, headaches or epilepsy that do not respond well to conventional treatments (Carlini, 2003; Quintans-Junior et al., 2007). In addition, the essential oils are natural products that exhibit a variety of biological properties, such as analgesic (Almeida et al., 2001), anticonvulsant (Almeida et al., 2003) and anxiolytic (Umezu et al., 2002; Almeida et al., 2004).
Cymbopogon winterianus Jowitt (Poaceae), popularly known as "citronella" and "java grass", is an important essential oil yielding aromatic grass cultivated in India and Brazil. The steam volatile essential oils extracted from its leaves are used in perfumery, cosmetics, pharmaceuticals, and flavoring industries (Guenther, 1950; Wealth of India, 1985). C. winterianus essential oil is rich in citronellal, geraniol and citronellol (Cassel and Vargas, 2006). However, folk medicine practitioners in the Brazilian Northeast use the infusion of the fleshy leaves for the treatment of epilepsy and anxiety (oral communication). Little information about the plant specie exists. Previous pharmacological study with Cymbopogon citratus has demonstrated the occurrence of CNS effects and anticonvulsant properties (Blanco et al., 2007). The claim of therapeutic success of the plant in the treatment of epilepsy has not been scientifically scrutinized. Preliminary behavioral screening realized in our laboratory of C. winterianus leaf essential oil (EO) showed depressant activity on CNS. The aim of this work was, therefore, to investigate the anticonvulsant effect of EO in rodents.
Materials and methods
Plant material and essential oil (EO) extraction
Leaves were collected from the cultivation of the C. winterianus genotypes established at the Research Station "Campus Rural da UFS" of the Universidade Federal de Sergipe, Brazil, and a voucher sample has been deposited in the Herbarium of the Department of Biology of Universidade Federal de Sergipe. The leaves of C. winterianus were dried in an oven with air renewal and circulation (model MA-037/18) at 40[degrees]C until complete dehydration has been achieved. The essential oil was obtained by hydrodistillation in a Clevenger-type apparatus using l00g of dried leaves. The oil obtained was dried over anhydrous sodium sulphate, producing yields of 2.34% (v/w). GC-MS analysis were performed in a Shimadzu model QP5050A, according to these experimental conditions; silica capillary column: DB-5MS (30m X 0.25mm X 0.25mm), injector temperature: 230[degrees] C, detector temperature: 250 [degrees]C, temperature program: the column was initially 50 [degrees]C, then raised to 200 [degrees] C at a rate of 4 [degrees]C/min, and finally held at 200 [degrees]C for 10 min, electron impact: 70 eV, carrier gas: helium at 1.2ml/min, scanning speed 0.84 scan [s.sup-1] from m/z 40 to 550 Da. The identification of the components was made through comparison of substance mass spectrum with the database of the GC-MS (NIST21 and NIST107), literature and retention index (Adams, 2001).
Diazepam (DZP), pentylenetetrazole (PTZ), picrotoxin (PIC), phenytoin (PHE), strychnine (STR), polyonxyethylene-sorbitan monolated (Tween 80) and cremophor was purchased from Sigma (USA) and diazepam (DZP), was obtained from Roche (Brazil). All drugs and the essential oil were administered intraperitoneally (ip) in volumes of 0.1 ml/10g (mice).
Male Swiss mice (25-30 g), 2-3 months of age, were used throughout this study. The animals were randomly housed in appropriate cages at 22 [+ or -]1 [degrees]C on a 12h light/dark cycle (lights on 06:00-18:00) with free access to food (Purina) and water. They were used in groups of 10 animals each. Experimental protocols and procedures were approved by the Universidade Federal de Sergipe Animal Care and Use Committee (CEPA/UFS No.010/07).
The behavioural screening of the mice was performed following parameters described by Almeida et al. (1999) and animals were observed at 0.5, 1, and 2h after administration of EO (100, 200 and 400 mg/kg, ip).
PTZ-and PIC-induced convulsion
Modified method of Vellucci and Webster (1984) was used to assess the anticonvulsant effect of the EO. Mice were kept individually in transparent mice cages (25 cm X 15 cm X 15 cm) for 30 min to acclimatize to their new environment before the commencement of the experiment. Seizures were induced in mice with PTZ (60 mg/kg, ip) or PIC (8 mg/kg, ip) and the animals were observed for convulsion for a period of 15 or 20 min, respectively. The onset of tonic-clonic convulsion and the number of animals convulsing or not convulsing within the observation period were noted. Experiments were repeated following the pretreatment of animals with either EO, DZP (3 mg/kg, ip) or control vehicle prior to the administration of any of the convulsant agents used. The ability of the EO to prevent or delay the onset of tonic and tonic-clonic convulsion by the animals was taken as an indication of anticonvulsant activity (Vellucci and Webster, 1984; Amabeoku and Chikuni, 1993). All experiments were carried out between 9.00 and 15.00 h in a quiet room with an ambient temperature of 22 [+ or -] 1 [degrees] C.
The method has been described previously (Lehmann et al., 1988). In brief, strychnine (STR) convulsions followed by death were induced in male mice by the i.p. injection of 3 mg/kg of the STR nitrate. The treatment of the EO was similarly a PTZ- and PIC-test. A protective effect of the EO given (ip) 1 h prior to STR was recorded and compared to the one of 25 mg/kg PHE and with control group (vehicle). The number of animals, which survived more than l0 min served as criterion of protection. The time to onset of death was recorded in non-protected mice.
The parameters data were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's t test. The incidence (%) of clonic or tonic-clonic seizures as well as the mortality were evaluated by Fisher's exact test. Two-factor repeated-measures analysis of variance (ANOVA) of seizures development was performed followed by Tukey's post hoc test. Differences were considered to be statistically significant when p < 0.05.
GC-MS analysis showed a mixture of monoterpenes, being geraniol (40.06%), citronellal (27.44%) and citronellol (10.45%) as the main compounds in the EO (Table 1).
Table 1. Chemical compsition and retention indices of the constituents of the EO RT (min) (a) Compounds (b) (%) I[K.sup.c] 9.992 Limonene 1.58 1027 10.267 -(Z)-Ocimene 0.19 1035 10.642 -(E)-Ocimene 0.09 1045 10.833 Bergamal 0.10 1052 11.558 NI 0.25 1070 12.592 Linalool 0.88 1099 14.383 Isopulegol 0.60 1147 14.525 Citronellal 27.44 1151 14.767 iso-Isopulegol 0.20 1158 14.883 NI 0.09 1161 15.567 NI 0.16 1179 16.533 n-Decanal 0.41 1205 17.192 Citronellol 10.45 1226 17.667 Neral (-Citral) 6.02 1237 18.133 Geranial 40.06 1250 18.750 Geranial (-Citral) 8.05 1267 21.625 Citronelly acetate 0.79 1349 22.600 Geranyl acetate 1.77 1377 24.008 -Caryophyllene 0.55 1418 27.058 -Cadinene 0.23 1512 Total identified 99.41 NI = Not identified. (a) Retention time. (b) Compounds listed in order of elution from an DB-5MS column. (c) Kovats indices were calculated against n-alkanes (C9-C18) on a DB-5MS column.
EO at doses of 100, 200 and 400 mg/kg (ip) showed depressant activity on CNS based on the following behavioral alterations in animals 0.5, 1 and 2 h after treatment: decrease of the spontaneous activity, palpebral ptosis, ataxia, analgesia and sedation. These effects were dose dependent.
Effect of EO on PTZ induced convulsions
As shown in Table 2, EO at the doses of 200 and 400 mg/kg (ip) significantly increased the latency period and reduced the frequency of convulsion. The frequency of survival was significantly increased for EO 200 and 400 mg/kg (ip). DZP (3 mg/kg, ip) completely protected the animals against the tonic convulsion elicited by PTZ (60 mg/kg, ip).
Table 2. Effect of EO on convulsion induced by pentylenetetrazol Treatment Dose Latency (s) (a) % Convulsion % Death (mg/kg) Control - 118.7 [+ or -] 14.6 100 80 DZP 3 900.0 [+ or -] 0.0 (b) 0 (c) 0 (c) EO 100 378.2 [+ or -] 165.1 90 90 EO 200 740.2 [+ or -] 115.5 (b) 50 (c) 30 (c) EO 400 649.3 [+ or -] 158.9 (d) 60 (e) 60 (e) n = 10. (a) Values represent mean [+ or -] S.E.M. (b) p < 0.01 (one-way ANOVA and Dunnett's test), significantly different from control. (c) p < 0.01 (Fisher's test), significantly different from control. (d) p < 0.05 (Fisher's test), significantly different from control. (e) p < 0.05 (one-way ANOVA and Dunnett's test), significantly different from control.
Effect of EO on PIC induced convulsions
In the model of convulsion induced by PIC (Table 3), the EO (200 and 400 mg/kg, ip) induced a significant increase in the latency of convulsion in a dose dependant manner. However, an increased frequency of survival was detected with EO at all doses. The DZP group significantly decreased the development of convulsion in comparison with the control group.
Table 3. Effect of EO on convulsion induced by picrotoxin Treatment Dose Latency (s) (a) % Convulsion % Death (mg/kg) Control - 376.2 [+ or -] 7.1 100 90 DZP 3 1.200 [+ or -] 0.0 (b) 0 (c) 0 (c) EO 100 546.3 [+ or -] 75.3 100 70 (d) EO 200 690.1 [+ or -] 80 (d) 80 (d) 50 (c) EO 400 930 3 [+ or -] 91.1 (b) 60 (d) 70 (d) N = 10. (a) Values represent mean [+ or -] S.E.M. (b) p < 0.01 (one-way ANOVA and Dunnett's test), significantly different from control. (c) p < 0.01 (Fisher's test), significantly different from control. (d) p < 0.05 (Fisher's test), significantly different from control. (e) p < 0.05 (one-way ANOVA and Dunnett's test), significantly different from control.
Effect of EO on STR induced convulsions
Table 4 shows in the control group the STR consistently induced convulsion in 100% of 10 mice. On the other hand, the EO (100, 200 and 400 mg/kg, ip) increase the latency of clonic convulsions significantly different from control (p < 0.05), similarly occurs with group received 25 mg/kg (ip) of PHE.
Table 4. Effect of the EO on STR-induced convulsions in mice Treatment Dose Latency (s) (a) % Convulsion % Death (mg/kg) Control - 113.5 [+ or -] 4.4 100 100 DZP 25 592.5 [+ or -] 21.4 (b) 10 (c) 70 (d) EO 100 204 8 [+ or -] 23.5 (e) 100 100 EO 200 173.4 [+ or -] 13.1 (e) 100 100 EO 400 295.2 [+ or -] 19.0 (e) 90 100 n = 10. (a) Values represent mean [+ or -] S.E.M. (b) p < 0.01 (one-way ANOVA and Dunnett's test), significantly different from control. (c) p < 0.01 (Fisher's test), significantly different from control. (d) p < 0.05 (Fisher's test), significantly different from control. (e) p < 0.05 (one-way ANOVA and Dunnett's test), significantly different from control.
In folk medicine of the Brazilian Northeast the Cymbopogon winterianus ("citronella") is used for treatment of epilepsy and anxiety. In pharmacological behavioral screening, the animals treated with C. winterianus leaf essential oil (EO) showed decrease of response to the touch, palpebral ptosis, ataxia, analgesia, sedation and reduction of motor activity. These data are indicative of depressive activity of the CNS according to Almeida et al. (1999).
Pentylenetetrazole, picrotoxin and strychnine are all convulsant agents (Nicoll, 2001; Rang et al., 2003). Data from this study show that the onset of tonic-clonic convulsion produced by PTZ was significantly delayed by EO (as shown in Table 2). The incidence of mortality was significantly reduced (p < 0.05). According to De Sarro et al. (1999), PTZ may be exerting its convulsant effect by inhibiting the activity of gamma aminobutyric acid (GABA) at GAB[A.sub.A] receptors. Gamma aminobutyric acid is the major inhibitory neurotransmitter which is implicated in epilepsy. The enhancement and inhibition of the neurotransmission of GABA will attenuate and enhance convulsion, respectively (Meldrum, 1981; Gale, 1992; Westmoreland et al., 1994). Since the EO delayed the occurrence of PTZ convulsion, it is probable that it may be interfering with gabaergic mechanism(s) to exert its anticonvulsant effect.
Picrotoxin exerts its convulsant effect by blocking the GAB[A.sub.A] receptor-linked chloride ion channel which normally opens to allow increased chloride ion conductance into the brain cells following the activation of GAB[A.sub.A] receptors by GABA (Faingold, 1987; Meldrum and Rogawski, 2007). Data from this study show that picrotoxin induced convulsion in mice and that EO significantly (p < 0.05, Table 3) attenuated the convulsion. It is probable that EO attenuated PIC convulsion by enhancing GABA neurotransmission. This further supports the hypothesis that EO may be affecting gabaergic mechanism(s) to exert its anticonvulsant activity.
On the other hand, STR cause convulsions by antagonized glycine receptor, increase postsynaptic excitability and increase ongoing activity in dorsal horn neurons (Game and Lodge, 1975; McGaraughty and Henry, 1998). Our results demonstrate that acute administration with EO on STR induced convulsions increased the latency of seizures significantly different from control (p < 0.05). Therefore, this effect suggest of the possible involvement on glycinergic neurotransmission.
However, experimental studies showed that monoterpenes (eugenol, geraniol, citronellol) has CNS depressant activity, anesthetic, myorelaxant and anticonvulsant properties (Dallmeier and Carlini, 1981; Freire et al., 2006; Sousa et al., 2006). Freire et al. (2006) showed which Ocimum gratissimum essential oil, rich in geraniol or eugenol, possess anticonvulsant properties. Additionally, citronellol is an acyclic monoterpene alcohol prevalent in essential oils of C. winterianus (Rao et al., 2004) and in other aromatic plant species (Lis-Balchin et al., 1998). Some of the pharmacological actions of citronellol have been studied (Haginiwa et al., 1963; Hierro et al., 2004; Kubo et al., 1993). Citronellol produced also an increasing effect on the response rate during the alarm period in the conflict test suggesting that it possesses an anti-anxiety effect (Umezu et al., 2002). In recent study, citronellol showed to possess anticonvulsant activity due to the reduction of neuronal excitability mainly through the voltage-dependent N[a.sup.+] channels and by facilitation of the inhibitory synaptic input by simply activating GAB[A.sub.A] (Sousa et al., 2006).
These data lead us to conclude both the C. winterianus leaf essential oil (EO) have a CNS depressant activity and anticonvulsant properties. Thus, lend pharmacological justification to the use of the plant infusion by traditional medicine practitioners in the treatment of epilepsy. It may also not be impossible to suggest that the anticonvulsant activity may be exerted via more than one mechanism especially since the EO is thought to affect both gabaergic and glycinergic mechanisms.
We would like to thank the National Council of Technological and Scientific Development (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico/ CNPq/Brazil)) and the Research Supporting Foundation of State of Sergipe (Fundacao de Amparo a Pesquisa do Estado de Sergipe/FAP-SE) for the financial support.
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L.J. Quintans-Junior (a), *, T.T. Souzaa (a), B.S. Leitea (a), N.M.N. Lessaa (a), L.R. Bonjardima, (a) M.R.V. Santosa (a), P.B. Alves (b), A.F. Blank (c), A.R. Antoniolli (a)
(a) Departamento de Fisiologia, Universidade Federal de Sergipe, Campus Universitdrio "Prof. Aloisio de Campos", CEP 49100-000 Sao Cristovao, SE, Brazil
(b)Departamento de Quimica, Universidade Federal de Sergipe, Campus Universitario "Prof. Aloisio de Campos", CEP 49100-000 Sao Cristovao, SE, Brazil
(c)Departamento de Agronomia, Universidade Federal de Sergipe, Campus Universitario "Prof. Aloisio de Campos", CEP 49100-000 Sao Cristovao, SE, Brazil
* Corresponding author. Tel.: + 55 79 2105 6645; fax: +55 79 210 6474
E-mail addresses: email@example.com, firstname.lastname@example.org (L.J. Quintans-Junior).