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Received 29 July 2000; received in revised form 13 December 2000; accepted 12 January 2001
Abstract
The methanolic extract of rhizomes of Cyperus articulatus, a plant used in traditional medicine in Africa and Latin America for
many diseases, possesses anticonvulsant activity in mice. This extract protected mice against maximal electroshock (MES)- and
pentylenetetrazol (PTZ)-induced seizures. It also delayed the onset of seizures induced by isonicotinic acid hydrazide and strongly
antagonized N-methyl-D-aspartate-induced turning behavior. The ED50 for protection against seizures was 306 (154– 541) mg/kg
intraperitoneally (i.p.) for the PTZ test and 1005 (797–1200) mg/kg i.p. for the MES test. The ED50 of methanolic extract against
N-methyl-D-aspartate-induced turning behavior was 875 (623– 1123) mg/kg i.p. C. articulatus L. methanolic extract protected 54%
of mice from seizures induced by strychnine at the dose of 1000 mg/kg i.p. but had no or a moderate effect only against
picrotoxin- or bicuculline-induced seizures. With these effects, the rhizome of C. articulatus L. possesses anticonvulsant properties
in animals that might explain its use as a traditional medicine for epilepsy in Africa. © 2001 Elsevier Science Ireland Ltd. All
rights reserved.
0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
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146 E.N. Bum et al. / Journal of Ethnopharmacology 76 (2001) 145–150
Table 1
Effect of the methanolic extract on STR-induced tonic seizures in micea
a
Latency in s, means 9 SD (nE10). *PB0.01, **
PB0.05. (Correction for multiple t-test by Bonferroni method).
Table 2
Effect of the methanolic extract on BIC and PIC-induced seizures in micea
Time of onset of seizures (s) Percentage of protected mice Latency of seizures (s)
a
Latency in s, means 9 SD (nE10).
previously (Ngo Bum et al., 1996). In short: the dried ‘positive control group’ receiving 3 mg/kg CGP 37849
rhizomes of C. articulatus were ground. The powder of NMDA antagonist that normally provide 100% protec-
the plant was extracted with ethyl acetate (in order to tion (Schmutz et al., 1990).
study ethyl acetate extract, because ethyl acetate can
extract components that are a little beat less polar than
2.3.2. Pentylenetetrazol test
the methanol ones) and the supernatant filtered. The
The method has been described previously (Schmutz
solid phase was extracted with methanol. The extract,
et al., 1990). In brief, clonic seizures were induced in
filtered and dried, constituted the methanolic extract
male mice by the i.p. injection of 70 mg/kg pentylenete-
(ME) that was tested in some models of epilepsy. The
trazol (PTZ). The protective effect of the ME was
ME was diluted in 40% DMSO (dimethyl sulfoxide) in
recorded. The time of onset of seizures in non-protected
distilled water and administered intraperitoneally (i.p.)
mice was also recorded. There were two control groups,
1 h before the test. The following doses of the ME were
used: 50, 100, 200, 500, 1000 and 2000 mg/kg.
2.2. Animals
one receiving placebo and a positive control group animals without seizures, the latency to the onset of
receiving 0.1 mg/kg clonazepam. seizures and the time of tonic extension of hind extrem-
ities in MES. The ED50 values (dose at which 50% of
2.3.3. Strychnine test the animals are protected) and 95% confidence limits
The method has been described previously (Lehmann were determined by using the SAS probit procedure
et al., 1988). In brief, strychnine (STR) convulsions with Abbot’s correction. For the latency to the onset of
followed by death were induced in male mice by the i.p. seizures and the time of tonic hind limb extension
injection of 2.5 mg/kg STR nitrate. A protective effect seizure in the MES test, the mean values of the control
of the ME given i.p. 1 h prior to STR was recorded and groups were compared to the mean values of the groups
compared to the one of 3 mg/kg clonazepam. The treated with the extracts using the correction for multi-
number of animals, which survived more than 10 min ple t-test by Bonferroni method. The Fisher exact test
served as criterion of protection. The time to onset of (two-tail) was used to compare percentage of protected
death was recorded in non-protected mice. mice in each case. The doses of ME are in mg/kg.
2.5. Chemicals
2.3.4. Isonicotinic acid hydrazide (isoniazid; INH) test
Animals were injected i.p. with INH 250 mg/kg
PTZ, BIC, PIC, NMDA, STR, INH, carbamazepine
(Bernasconi et al., 1988) 1 h after the administration of
are from sigma chemical, USA; CGP 37849 from No6ar-
the ME, and the time to onset of clonic or tonic
tis, Basle, Switzerland.
seizures was recorded. Data of the control group
(treated with placebo) were compared to data of the
group treated with the ME. The positive control group 3. Results
received diazepam, 10 mg/kg orally (per os).
3.1. Effect of the methanolic extract on
NMDA-induced turning beha6ior
2.3.5. Bicuculline test
Animals were injected i.p. with bicuculline (BIC) 2.7
The turning behavior induced by NMDA (75 mg/kg)
mg/kg (Masereel et al., 1998; Palmer et al., 1999) 1 h
was antagonized by the dose of 3 mg/kg i.p. of the
after the administration of the ME. The time to onset
NMDA antagonist CGP 37849 (Schmutz et al., 1990).
of clonic or tonic seizures was recorded in the control
The ME also prevented mice from turning. This effect
group (treated with placebo) and in the group treated
was dose-dependent: 20%, 60% and 90% of the animals
with the ME.
did not show turning behavior at the doses of 500, 1000
and 2000 mg/kg i.p., respectively. At the highest dose,
2.3.6. Picrotoxin test
the protection provided by the extract was better than
The method has been described previously (Lehmann
the one provided by CGP 37849. In non-protected
et al., 1988). In brief, clonic seizures were induced in
animals, the time of the onset of the turning behavior
male mice by the i.p. injection of 7.5 mg/kg picrotoxin
was also delayed by the ME (control group: 10169290
(PIC). A protective effect of the extract against PIC-in-
s; ME 500 mg/kg: 14039283 s). The ED50 in protect-
duced clonic seizures was recorded. A 0.4 mg/kg dose
ing animals against turning behavior was 875 (623–
of clonazepam was used as positive control.
1123) mg/kg i.p. (Fig. 1 a and b).
2.3.7. Maximal electroshock test 3.2. Effect of the methanolic extract on PTZ-induced
The method has been described previously (Schmutz seizures
et al., 1990; Wamil et al., 1994). In brief, tonic convul-
sions of the hind extremities of mice were induced by The ME dose-dependently protected animals against
passing alternating electrical current (50 Hz, 18 mA, 0.2 clonic seizures induced by PTZ but did not delay the
s) through temporal electrodes. For each experiment onset of seizures in non-protected animals. At the dose
one group served as a negative control (placebo) and of 100 mg/kg i.p., the ME protected 40% of mice
one group as a positive control (carbamazepine, 30 against seizures. The dose of 2000 mg/kg provided
mg/kg p.o.). The number of animals protected from protection for 90% of mice. The ED50 was 306 (154–
tonic hind limb extension seizure and the time spent in 541) mg/kg i.p. Fig. 2 shows the dose-response relation-
this position were determined in each dose group. ship of the effect of the ME.
min to 48.89 10.6 min at the dose of 200 mg/kg ip., are of predictive relevance regarding the clinical spec-
and from 38.896.l min to 71.2916.8 min at the dose trum of activity of experimental compounds (Rogawski
of 2000 mg/kg i.p. This is a 183% increase compared to and Porter, 1990; Kupferberg and Schmutz, 1998). Be-
control. At this dose the ME was nearly as efficacious cause the MES and PTZ tests are assumed to identify
as diazepam, 10 mg/kg (Fig. 3). anticonvulsant drugs effective against generalized tonic-
clonici partial seizures and generalized clonic seizures,
3.4. Effect of the methanolic extract on STR-induced respectively (Löscher and Schmidt, 1988; Rogawski and
seizures and exitus Porter, 1990; De Deyn et al., 1992; Kupferberg and
Schmutz, 1998), the effect of the ME in these tests
ME at the dose of 1000 mg/kg i.p. protected 54% of could therefore suggest anticonvulsant efficacy against
mice from tonic seizures and death induced by STR 2.5 the above mentioned seizures types in man. As PTZ
mg/kg, that is 69% of the effect of clonazepam, 3 mg/kg and INH have been shown to interact with the GABA
(78% protection). The same dose only slightly but sig- neurotransmitter and the GABA receptor complex
nificantly delayed the onset of the seizures from 4.8 mm (Doctor et al., 1982; Löscher and Schmidt, 1988; De
to 6.5 mm in non-protected animals (Table 1). Deyn et al., 1992), antagonism of PTZ- and INH-in-
duced seizures suggests that the extract of C. articulatus
3.5. Effect of the methanolic extract on BIC- and L. might have effects on GABA-ergic neurotransmis-
PIC-induced seizures sion. But these effects do not seem to be related to the
GABA or PIC sites of the GABA receptor complex (De
The ME at a dose of 2000 mg/kg did not possess a Deyn et al., 1992) because BIC- and PIC-induced
significant effect against seizures induced by BIC and seizures were not significantly antagonized. It has been
PIC (Table 2). shown previously that an extract of rhizhomes of C.
articulatus selectively antagonized NMDA receptor-me-
3.6. Effects of the methanolic extract on MES-induced diated neurotransmission in rat cortical wedge in vitro
seizures (Ngo Bum et al., 1996). These results are in accordance
with the present in vivo studies since ME dose-depen-
The ME antagonized MES-induced seizures. The dently antagonized NMDA-induced turning behavior
doses of 1000 and 2000 mg/kg i.p. of ME protected in mice. The moderate antagonism of the ME in STR-
55% and 95% of mice, respectively. This effect was induced seizures suggests that additional mechanisms
comparable to that of carbamazepine (30 mg/kg), a might be involved. The multiplicity of putative mecha-
standard antiepileptic drug. The ED50 of the ME was nisms of action and the broad spectrum of anticonvul-
1005 (797–1200) mg/kg i.p. (Fig. 4). sant activity of the ME might be due to the presence of
different active components in the ME interacting
3.7. Unwanted effects simultaneously. These mechanisms and their contribu-
tion to the anticonvulsant properties of the ME will be
Mice treated with 1000 and 2000 mg/kg i.p. of ME better understood once the active components in the
showed ataxia 1 h after its administration. At lower rhizome of C. articulatus are identified. Although, the
doses, no clear unwanted effects were observed. traditional medicine uses the decoction but not the ME to
treat epilepsy, the results of this study confirms the
presence of anticon6ulsant properties in the rhizomes of
4. Discussion and conclusion C. articulatus. Comparison between the ME extract and
de decoction could be made after the study of the effects
The ME of rhizomes of C. articulatus L. contains at of the decoction in the same animal models of epilepsy.
least one component that antagonizes chemically and
electrically induced seizures in mice. The ME of C.
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