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African Journal of Biotechnology Vol. x(xx), pp. xxx-xxx, xx xxxxx, 2011
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Volatile constituents and behavioral change induced by
Cymbopogon winterianus leaf essential oil in rodents
Bárbara L. S. Leite1, Thais T. Souza1, Angelo R. Antoniolli1, Adriana G. Guimarães1, Rosana S.
Siqueira1, Jullyana S. S. Quintans1, Leonardo R. Bonjardim1, Péricles B. Alves2, Arie F.
Blank3, Marco Antonio Botelho4, Jackson R. G. S. Almeida5, Julianeli T. Lima5, Adriano A. S.
Araújo1 and Lucindo J. Quintans-Júnior1*
1Departamento de Fisiologia, Universidade Federal de Sergipe. Campus Universitário “Prof. Aloísio de Campos” CEP:
49100-000, São Cristóvão, SE, Brazil.
2Departamento de Química, Universidade Federal de Sergipe. Campus Universitário “Prof. Aloísio de Campos” CEP:
49100-000, São Cristóvão, SE, Brazil.
3Departamento de Agronomia, Universidade Federal de Sergipe. Campus Universitário “Prof. Aloísio de Campos” CEP:
49100-000, São Cristóvão, SE, Brazil.
4nstituto Federal de Educação Ciência e Tecnologia do Ceará, Departamento de Pesquisa. Laboratório de
Biotecnologia. CEP: 62700-000, Canindé, CE, Brazil.
5Colegiado de Ciências Farmacêuticas, Universidade Federal do Vale do São Francisco. CEP 56.306-410, Petrolina,
PE, Brazil.
Accepted 9 June, 2011
Cymbopogon winterianus Jowitt (‘Java citronella’) is an important essential oil yielding aromatic grass
cultivated in India and Brazil and its volatile essential oils extracted from its leaves are used in
perfumery, cosmetics, pharmaceuticals and flavoring industries. However, there is no report on any
psychopharmacological study of C. winterianus leaf essential oil (LEO) available to date. In this study,
the pharmacological effects of the LEO were investigated in animal models and its phytochemical
analyses. GC-MS analysis showed a mixture of monoterpenes, as citronellal (36.19%), geraniol (32.82%)
and citronellol (11.37%). LEO exhibited an inhibitory effect on the locomotor activity of mice, an
antinociceptive effect by increasing the reaction time in the writhing and capsaicin tests. All doses
induced a significant increase in the sleeping time of animals not having modified however, the latency.
The LEO did not alter the remaining time of the animals on the rota-rod apparatus. These results
suggest a possible central effect.
Key words: Cymbopogon winterianus, essential oil, CNS, behavioral effects, analgesic.
INTRODUCTION
Traditional health care is utilized by the majority of the
low income population in Brazilian northeast. This is
especially true of treatment for mental health problems.
*Corresponding author. E-mail: lucindo_jr@yahoo.com.br or
lucindo@pq.cnpq.br. Tel/Fax: 55-79-2105-6645 or +55-79-
2105-6474.
Abbreviation: LEO, Leaf essential oil; EO, essential oil.
Besides, it have been described as a hypothetical
potential to affect chronic conditions such as
anxiety,depression, headaches, pain treatment or
epilepsy, which does not respond well to conventional
treatments (Carlini, 2003). A great number of scientists
and organizations turn their attention to traditional
therapies in order to find and conserve important
resources (Akerele, 1990). However, medicinal plants
have been an important source of new drugs with
biological activity (Quintans-Júnior et al., 2008a, 2011
The genus Cymbopogon Spreng (Poaceae) is
characterized by its species possessing great variability
in morphology and chemotypes. Most species of the
genus are aromatic and yield volatile oils of important
commercial values (Blank et al., 2007). Cymbopogon
winterianus Jowitt (Java citronella) is an important
essential oil yielding aromatic grass cultivated in India
mainly in the lower hills of Assam, Karnataka and
Southern Gujarat. The steam volatile essential oils
extracted from its leaves are used in perfumery,
cosmetics, pharmaceuticals and flavoring industries
(Tanu et al., 2004). The main traditional use is as a
repellent (Tawatsin et al., 2001). Folk medicine
practitioners in northeastern Brazil use the infusion of the
fleshy leaves and unguent for the treatment of anxiety,
sedative and pain disorders (Quintans-Júnior et al.,
2008b). However, there is little published information
about biological effects of this plant. Menezes et al.
(2010) demonstrate that C. winterianus leaf essential oil
(LEO) induces hypotension due to a decrease in
peripheral vascular resistance secondary to
vasodilatation and these effects appear to be mainly
mediated by Ca+2 channel blocking. Additionally,
preliminary study realized in our laboratory with the LEO
showed anticonvulsant and antinociceptive properties in
rodents (Quintans-Júnior et al., 2008b; Leite et al., 2010).
The aim of this study was to perform phytochemical
screening of the LEO and to investigate its central
nervous system (CNS) activity.
MATERIALS AND METHODS
Plant material and essential oil extraction
Leaves were collected in February 2007 from the cultivation of the
C. winterianus genotypes established at the Research Station
"Campus Rural” of the Federal University of Sergipe (10°C 55’ S,
37°C 11’ W), Brazil and a voucher sample was deposited in the
Herbarium of the Department of Biology of the same University.
Plants were cut 20 cm above soil level in Spring at 09:00 h and
dried at 40°C in a forced air oven (Marconi®, Brazil) for 5 days. The
essential oil (EO) of those leaves were extracted by hydrodistillation
for 3 h (Carvalho-Filho et al., 2006), using a Clevenger-type
apparatus (British Pharmacopoeia, 1988). The oils were s eparated
from the aqueous phase and kept in the freezer (-20°C) until further
use. The oil content was estimated based on dry herbage weight
using three samples of 75 g of dry leaves (American Spice Trade
Association, 1985). 3.4% essential oil content was obtained.
Gas chromatography – mass spectrometry
Oil s ample analysis was performed on a Shimadzu QP5050A
(Shimadzu Corporation, Kyoto, Japan) system comprising a AOC
20i autosampler and gas chromatograph interfaced to a mass
spectrometer (GC-MS) instrument employing the following
conditions: column J and W Scientific DB-5MS (Folsom, CA, USA)
fused silica capillary column (30 cm x 0.25 mm i.d, 0.25 µm coating
thickness, composed of 5% phenylmethylpolysiloxane),
helium(99.999% purity) was used as c arrier gas at a constant flow
of 1.2 ml/min and an injection volume of 0.5 µl was employed (split
ratio of 1:83) injector temperature 250°C and ion-source
temperature 280°C. The oven t emperature was programmed fr om
50°C (isothermal for 2 min), with an increase of 4°C/min., to 200°C,
then 10°C/min to 300°C, ending with a 10 min isothermal at 300°C.
The mass spectra were taken at 70 eV with scanning speed of 0.85
scan/s from 40 to 550 Da.
Gas-chromatography (GC-FID)
Quantitative analysis of the chemical c onstituents was performed by
flame ionization gas c hromatography (FID), using a Shimadzu GC-
17A (Shimadzu Corporation, Kyoto, Japan) equipment, under the
following operational conditions: capillary ZB-5MS column (5%
dimethylpolysiloxane) fused silica capillary column (30 m x 0.25 mm
i.d., 0.25 µm coating thickness) from Phenomenex (Torrance, CA,
USA), under the same conditions GC-MS. The essential oil
composition was reported as a relative percentage of the total peak
area.
Identification of essential oil constituents
Identification of individual components of the essential oil was
performed by computerized matching of the acquired mass spectra
with those stored in NIST21 and NIST107 mass spectral library of
the GC-MS data system. Retention indices (RI) for all c ompounds
were determined according to the method of Van den Dool and
Kratz (1963) for each constituent as previously described (Adams,
2007).
Drugs
Polyoxyethylene-sorbitan monolated (Tween 80) and cremophor
was purchased from Sigma (USA) and Diazepam (DZP) was
obtained from Cristália (Brazil). All drugs and the essential oil were
administered orally (per os, p.o.) in volumes of 0.1 ml/10 g (mice).
Animals
Male Swiss mice (28 to 32 g), with 2 to 3 months of age, were used
throughout in this study. The animals were randomly housed in
appropriate cages at 22 ± 1°C on a 12 h light/dark cycle (lights on
06:00 to 18:00) with free access to f ood (Purina) and water. They
were used in groups of 8 animals each. Experimental protocols and
procedures were approved by the Universidade Federal de Sergipe
Animal Care and Use Committee (CEPA/UFS N°010/07).
Acute toxicity (LD50)
This test was performed according to a method described by Lorke
(1983), with modifications, where the acute toxicity of LEO was
assessed by orally route (per os, p.o.). Groups of 10 animals each
were separated and received doses of 500, 750, 1000, 2000 or
3000 mg/kg of LEO. The animals were observed daily for 48 h and
a number of deaths of animals were registered and lethal dose 50%
(LD50) calculated (Litchfield and Wilcoxon, 1949).
Behavioral effects
Behavioral screening of the mice was performed following
parameters described by Almeida et al. ( 1999) and animals (n = 8,
each group) were observed at 0.5, 1.0 and 2.0 h after per os (p.o.)
administration of LEO (25, 50 and 100 mg/kg). Control group
received saline/tween-80 0.2% (vehicle).
Locomotor activity
Mice were divided into four groups of 10 animals each. Vehicle
(saline/tween-80 0.2%) and LEO (25, 50 and 100 mg/kg, p.o.) were
injected. The spontaneous locomotor activity of the animals was
assessed in a cage activity (50 × 50 × 50 cm) in 0.5, 1 and 2 h after
administration (Asakura et al., 1993).
Motor coordination test (rota-rod test)
A rota-rod tread mill device (AVS®, Brazil) was used for the
evaluation of motor coordination. Mice were placed on a horizontal
rotation rod set at a rate of 9 rpm (Perez et al., 1998). Initially, the
mice able to r emain on the rota-rod apparatus longer than 180 s (9
rpm) were selected 24 h before the test. Sixty minutes after the
administration of either vehicle (saline/tween-80 0.2%), LEO ( 25, 50
and 100 mg/kg, p.o.) or diazepam (1.5 mg/kg, i.p.), each animal
was tested on the rota-rod apparatus and the time (s) remained on
the bar for up to 180 s was recorded after 1 h.
Pentobarbital-induced hypnosis
Sodium pentobarbital, at a hypnotic dose of 50 mg/kg (i.p.), was
injected into four groups (n = 10) of the mice 60 min after
pretreatment with s aline/tween-80 0.2% (vehicle) and LEO (25, 50
and 100 mg/kg, p.o.), respectively. The latency (the interval
between the injection of s odium pentobarbital and the loss of the
righting reflex) and duration of sleeping time (the interval between
the loss and recovery of the righting reflex) were recorded
(Elisabetsky et al., 1995).
Acetic acid-induced writhing
This study was performed according to Koster et al. (1959). Mice (n
= 8, per group) were injected intraperitoneally (i.p.) with 0.85%
acetic acid at a dose of 10 ml/kg. LEO (25, 50, and 100 mg/kg,
p.o.). The reference drug, morphine (MOR, 3 mg/kg), was
solubilized in saline + 1 drop of Tween-80 0.2% (vehicle) and was
administered i.p. to different groups of the mice 1 h before the
acetic acid injection. Subsequently, the writhing was counted for 20
min after a latency period of 5 min.
Capsaicin-induced nociception
The method used was s imilar to that described previously
(Sakurada et al., 1992). Mice were individually placed in a
transparent Plexiglas cage (25 × 15 × 15 cm) observation chamber.
Following the adaptation period, 20 µl of capsaicin (1.6 µg/paw
prepared in a phosphate-buffered solution) was inj ected under the
skin of the dorsal surface on the right hind paw. The mice were pre-
treated with LEO (25, 50 and 100 mg/kg, p.o.) 60 min before
injection of the algogen. The control animals received a similar
volume of vehicle. After this process, pairs of mice were placed
individually in different Plexiglas cage for 5 min f ollowing capsaicin
injection. The amount of time spent licking the injected paw was
timed with a chronometer and was considered indicative of
nociception.
Statistical analysis
The data obtained were evaluated by one-way analysis of variance
(ANOVA) followed by Dunnett`s test. Differences were c onsidered
to be statistically significant when p < 0.05.
RESULTS
GC-MS analysis showed a mixture of monoterpenes,
being citronellal (36.19%), geraniol (32.82%) and
citronellol (11.37%) as the main compounds in the EO
(Table 1).
The LD50 calculated to per os (p.o.) administration of
the LEO in mice was 1,953.8 mg/kg with confidence
interval of 1,580.9 to 2,326.7 mg/kg. LEO at doses of 25,
50, 100, 200 and 400 mg/kg (p.o.) showed depressant
activity on CNS based on the following behavioral
alterations in animals after 0.5, 1 and 2 h treatment:
decrease of the spontaneous activity, palpebral ptosis,
ataxia, analgesia and sedation. These effects were dose-
dependent.
The doses of 25, 50 and 100 mg/kg (p.o.) LEO caused
a significant decrease of ambulation (number of squares
crossed) at 0.5, 1 and 2 h after administration (Figure 1).
As shown in Figure 2a, LEO at all doses did not affect the
latency of pentobarbital-induced hypnosis. However, LEO
at 25, 50 and 100 mg/kg (p.o.) significantly increased the
sleeping time compared with the control group animals
(Figure 2b).
In the rota-rod test, LEO-treated mice did not show any
significant motor performance alterations with doses of
25, 50 and 100 mg/kg. As might be expected, the CNS
depressant diazepam (1.5 mg/kg) reduced the time of
treated animals on the rota-rod apparatus (Figure 3).
Figure 4 shows that LEO was significantly (p < 0.001)
reduced, in a dose-dependent manner, the number of
writhing movements induced by the p.o. administration of
the acetic acid solution. In the capsaicin test, LEO
significantly reduced the licking time compared with the
control group (Figure 5) only in higher doses.
DISCUSSION
In this study, the pharmacological effects of the C.
winterianus leaf essential oil (LEO) were investigated in
animal models and it characterized a
psychopharmacological effect of this essential oil on the
CNS. The results obtained and the LD50 values
represent a low toxicity of LEO and they were similar to
the ones observed for other essential oils (Fandohan et
al., 2008). The LEO increases the sleeping time induced
by sodium pentobarbital in a dose-dependent manner,
decrease ambulation without alter motor coordination
Table 1. Chemical composition and retention indices of the constituents of the EO.
RT(min)
a
Compound
b
(%)
c
RI
d
8.452 6-Metil-5-hepten-2-one 0.23 984
8.600 Myrcene 0.16 988
9.975 Limonene 2.37 1028
10.225 β -(z)-Ocimene 0.35 1035
10.600 β - (e)-Ocimene 0.23 1045
11.533 Not identified 0.28 1071
12.558 Linalool 1.39 1099
14.350 Isopulegol 1.11 1148
14.525 Citronellal 36.19 1152
14.725 Iso-isopulegol 0.33 1158
16.508 N-decanal 0.46 1206
17.242 Citronellol 11.37 1226
17.642 Neral (z-citral) 4.53 1237
18.125 Geraniol 32.82 1251
18.717 Geranial (e-citral) 5.84 1267
21.592 Citronellyl acetate 0.75 1348
22.572 Geranyl acetate 1.17 1377
23.958 β-Caryophyllene 0.42 1417
Total 99.62
aRetention time; bcompounds listed in order of elution from an DB-5MS column; cpercentage based on FID peak
area normalization; dcalculated using the equation of Van den Dool and Kratz (1963).
0
10
20
30
40
25
50
100
LEO (mg/kg)
Vehicle
1.5
DZP
Number of squares
crossed
25
50
100
LEO (mg/kg)
Vehicle
1.5
DZP
25
50
100
LEO (mg/kg)
Vehicle
1.5
DZP
0.5
h
1
h
2
h
*
*
*
**
**
**
**
** **
**
Figure 1. Effect of LEO on locomotor activity of mice. The parameters evaluated were the number of squares crossed in
activity cage. Values are the mean ± SEM for 10 mice; statistical differences versus control group were calculated using
ANOVA, followed by Dunnett’s test (n = 10). *p < 0.05 or **p < 0.01.
performance of animals. Additionally, LEO produced
significantly analgesic effect at all doses in the writhing
and capsaicin tests.
A general pharmacological screening with the LEO
demonstrated some behavioral change in mice, as
decrease of the spontaneous activity, palpebral ptosis
ataxia, analgesia and sedation. These signals showed
possible evidence that the effects on CNS are similar to
drugs that reduce the CNS activity (Fernández-Guasti et
al., 2001; Morais et al., 2004).
0
100
200
300
*
**
*
25 50 100
LEO (m g/ kg)
Ve hicle 1.5
DZP
**
Duration of sleeping
(min)
B)
0
1
2
3
4
25 50 100
LEO (mg/kg)
Ve hicle 1.5
DZP
Onset of sleeping (min.)
A)
Figure 2. Effect of LEO on pentobarbital-induced hypnosis in mice. The parameters
evaluated were the onset of sleeping (A) and duration of sleeping (B). Values are mean ±
SEM for 10 mice, *p < 0.01; **p < 0.001, as compared to vehicle
LEO caused a significant reduction of ambulation of
animals in the test of spontaneous movement after 0.5, 1
and 2 h of its administration in the doses of 25, 50 and
100 mg/kg, that corroborates with the hypothesis of the
LEO reduces the CNS activity, it was reported that
reduction of the ambulation of the animals is
characteristic of psychopharmacological drugs
(Fernández-Guasti et al., 2001).
The LEO 25, 50 and 100 mg/kg (p.o.) had an increase
in the total time of sleep of the animals, but did not have
an increase in the latency for the induction of sleep
compared with the control group. It is established that the
potencialization of the time of sleep induced by
pentobarbital must be a sedative or hypnotic action that is
attributed to the involvement of central mechanisms in
the regulation of sleep (N’Gouemo et al., 1994) and
involves the enhancement of the GABAergic system
(Steinbach and Akk, 2001; Sivam et al., 2004).
Previous studies suggested that the CNS depression
and the nonspecific muscle relaxation effect can reduce
the response of motor coordination (Gonçalves et al.,
2008). We did not see any interference with the motor
0
50
100
150
200
*
25 50 100
LEO (mg/kg)
Ve hicle 1.5
DZP
Time (s) on Rota rod
Figure 3. Time (s) on the Rota-rod observed in mice after p.o. treatment with vehicle (control),
LEO (25, 50 and 100 mg/kg) or Diazepam (DZP, 1.5 mg/kg). The motor response was recorded
for the following 180 s after drug treatment. Statistical differences versus control group were
calculated using ANOVA, f ollowed by Dunnett’s test (n = 10) *p < 0.05.
0
10
20
30
**
**
**
Number of writhings
25 50 100
LEO (mg/kg)
Veh icle 3
MOR
*
Figure 4. Antinociceptive effect of LEO in the acetic acid-induced writhing test in mice. Vehicle
(control), LEO (25, 50 and 100 mg/kg, p.o.) or morphine (MOR) were administered 60 min
before acetic acid injection. Values are mean ± SEM for 10 mice, *p < 0.05 or **p < 0.001, when
compared with control, one-way ANOVA.
0
25
50
75
**
**
Licking time (s)
25 50 100
LEO (mg/kg)
Ve hicle 3
MOR
Figure 5. Antinociceptive effect of LEO in the c apsaicin test in mice. Vehicle (control), LEO (25,
50 and 100 mg/kg, p.o.) or morphine (MOR) were administered 60 min before acetic acid
injection. Values are mean ± SEM for 10 mice, *p < 0.01 or **p < 0.001, when compared with
control, one-way ANOVA.
coordination of the animals in the rota-rod test, therefore,
eliminating a nonspecific muscle relaxation effect of LEO
at the doses used.
Acetic acid-induced is a standard, simple and sensitive
test for measuring analgesia induced by both opioids and
peripherally acting analgesics (Hunskaar and Hole,
1987). In this test, pain is elicited by the injection of an
irritant such as acetic acid into the peritoneal cavity which
produces episodes of characteristic stretching (writhing)
movements and inhibition of the number of episodes by
analgesics is easily quantifiable. Oral administration of
LEO produced marked inhibition of the abdominal
constriction. However, although, the writhing response
test is very sensitive, it has a poor specificity as an
analgesic screening test
Sakurada et al. (1992) proposed the capsaicin-induced
pain model for the study of compounds that act on pain of
a neurogenic origin. Studies have shown that capsaicin
evokes the release of neuropeptides, excitatory amino
acids (glutamate and aspartate) nitric oxide and
proinflammatory mediators in the periphery and transmits
nociceptive information to the spinal cord (Sakurada et
al., 2003). The results indicated a significant reduction in
neurogenic nociception caused by the intraplantar
injection of capsaicin, showing that LEO caused
significant effects in this model. LEO may be good
candidates for the treatment of neuropathic conditions, in
which effective treatment is difficult (Akada et al., 2006).
CG-MS analyses showed a mixture of monoterpenes
(main compounds): citronellal (36.19%), geraniol
(32.82%) and citronellol (11.37%). Biological activities
described for eugenol and citronellal include myorelaxant,
anticonvulsant, antinociceptive and sedative (Quintans-
Júnior et al., 2008b; Melo et al., 2010). Quintans-Júnior et
al. (2010a) demonstrated analgesic effect of the
citronellal on orofacial nociception in rodents. Another
article published by our group demonstrated
anticonvulsant activity of many monoterpenes, such as
carvacrol, (−)-borneol and citral (Quintans-Júnior et al.,
2010b). Additionally, De Sousa et al. (2006)
demonstrated that citronellol possesses anticonvulsant
activity due to the reduction of neuronal excitability mainly
through the voltage-dependent Na+ channels and by
facilitation of the inhibitory synaptic input by simply
activating GABAA.
Based on the results obtained, it is possible to suggest
that LEO has CNS activities, as hypnotic, sedative and
antinociceptive, which might involve a central GABAergic
system. Pharmacological, toxicological and chemical
studies are continuing, in order to characterize the
precise mechanism(s) responsible for the CNS action
and also to identify other monoterpenes present in C.
winterianus leaf essential oil. Finally, the CNS action
demonstrated in this study supported at least in part, the
ethnomedical uses of this plant.
ACKNOWLEDGEMENTS
We thank Mr Osvaldo Andrade Santos for the technical
support. We would like to thank the Fundação de Apoio à
Pesquisa e à Inovação Tecnológica do Estado de
Sergipe/FAPITEC-SE for the fellowship support. Author
Bárbara Lima Simeoni Leite has scholarships from Rede
Nordeste de Biotecnologia (RENORBIO).
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