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Phytomedicine:
International Journal of Phytotherapy & Phytopharmacology, April 2002 v9 i3 p217(7)
Anticonvulsant activity of aqueous
extract of Leonotis
leonurus. E. BienVenu;
G.J. Amabeoku; P.K. Eagles; G. Scott; E.P.
Springfield.
Full Text: COPYRIGHT 2002 Urban & Fischer Verlag
Summary
Water extract of Leonotis
leonurus was tested for anticonvulsant activity
against seizures produced in mice by pentylenetetrazole,
picrotoxin, bicuculline and
N-methyl-DL-aspartic acid (intraperitoneal injections).
L. leonurus extract in the doses of 200 and 400 mg/kg
respectively protected 37.5% and 50% of animals used and significantly (p
<0.05; Student's t-test) delayed pentylenetetrazole
(90 mg/kg)-induced tonic seizures. Similarly, the same doses of L. leonurus extract significantly (p < 0.05; Student's
t-test) delayed the onset of tonic seizures produced by picrotoxin
(8 mg/kg) and N-methyl-DL-aspartic acid (400 mg/kg). However, all the doses of
aqueous extract of L leonurus used did not alter the
seizures induced by bicuculline (20 mg/kg) to any
significant extent. The data suggest that the extract of L. leonurus
has anticonvulsant activity and may probably be acting through non-specific
mechanisms, since it affects both gahaergic and glutaminergic systems. High performance liquid
chromatography (HPLC) and phytochemical tests carried
out respectively show a spectrum profile, characteristic of L. leonurus and the presence of alkaloids, saponins
and tannins in the extract.
Key words: Leonotis
leonurus, aqueous extract, anticonvulsant activity,
seizures, gabaergic and glutaminergic
mechanisms, mice
Introduction
The present study has been focused on the crude water extract of Leonotis leonurus (L.) R. BR. of the family, Lamiaceae, which has a wide natural distribution over large parts of South Africa including the Western Cape Province (Hutchings et al., 1996; Van Wyk et al., 2000). It is known in Afrikaans as "wilde dagga", in Zulu as "umunyane", in Sotho as "lebake", in Xhosa as "umfincafincane" and in Shona as "umhlahlampetu" (Hutchings et al., 1996; Van Wyk et al., 2000). The leaves of L. leonurus have traditionally been smoked for the relief of epilepsy. An infusion and a decoction of the leaf and stem have been used internally for coughs, colds, influenza, bronchitis, high blood pressure and headaches. A tincture of the flower has also been used for the same purpose. Externally, decoctions have been applied to treat boils, eczema, skin disease, itching and muscular cramps (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996, Van Wyk et al., 2000). This study, therefore, investigated the effect of the water extract of L. leonurus on chemically induced seizures by pentylenetetrazole, picrotoxin, bicuculline and N-methyl D-L-aspartic acid in mice. The study also established the chromatographic profile using high performance liquid chromatography (HPLC) method, and identified the groups of chemical compounds present in the water extract using standard phytochemical tests.
.
Material and methods
Collection and identification of plant material
The plant material was collected from
Preparation of aqueous extract of Leonotis leonurus
The leaves of plant species collected were washed with distilled water,
dried in a ventilated oven for 72 hours at 35 [degrees]C and afterwards ground
into fine powder (850 [micro]m) using the Waring
Commercial laboratory blender. A known quantity of the powder (20 g) was
refluxed for 5 hours in boiled water (11), allowed to cool for 24 hours and
filtered. The resultant filtrate was freeze-dried (LSL Secfroid
SR, Model 3021,
Material and methods
Collection and identification of plant material
The plant material was collected from
Preparation of aqueous extract of Leonotis leonurus
The leaves of plant species collected were washed with distilled water,
dried in a ventilated oven for 72 hours at 35 [degrees]C and afterwards ground
into fine powder (850 [micro]m) using the Waring
Commercial laboratory blender. A known quantity of the powder (20 g) was
refluxed for 5 hours in boiled water (11), allowed to cool for 24 hours and
filtered. The resultant filtrate was freeze-dried (LSL Secfroid
SR, Model 3021,
Animals
Male albino mice bought from the
Drugs and chemicals
Pentylenetetrazole (PTZ; Sigma), picrotoxin (Sigma), N-methyl-DL-aspartic acid (NMDLA;
Sigma) and phenobarbitone (Gardenal,
Rhone-poulenc
Anticonvulsant properties assessment
The investigation of anticonvulsant
properties of aqueous extracts of L. leonurus was
carried out using the method of Vellucci and Webster
(1984), and modified by Amabeoku and Chikuni (1993). Seizures were induced in animals with convulsant drugs such as pentylenetetrazole,
picrotoxin, bicuculline and
NMDLA. Mice were observed for 30 minutes for tonic convulsion episode. Seizures
were manifested as tonic hind-limb extension. The ability of the plant extract
to prevent this feature or prolong the latency or onset of the tonic hind-limb
extension was taken as an indication of anticonvulsant activity (Navarro-Ruiz
et al., 1995, 1996; Williamson et al., 1996; Amabeoku
et al., 1998). The onset of tonic convulsions and the proportion of mice
presenting convulsions were recorded. Animals that did not convulse during the
period of observation were considered not having convulsed. Experiments were
repeated with mice pretreated for 15 minutes with L. leonurus
extract, the standard antiepileptic drug, phenobarbitone
or diazepam, or the control vehicle, before the administration of the convulsant drug. Control experiments were also carried out
concurrently with the test experiments.
HPLC
The chromatographic system includes Beckman HPLC system consisting of double
pump Programmable Solvent Module model 126; Diode Array detector Module model 168;
Samsung computer 386 with management System Gold (GoldV601) software supplied
by Beckman; Column, C18 Bondapak 5 [micro]m and dimensions (250 x 4.6 mm). The chromatographic
conditions include Mobile phase: solvent A: 1% acetic acid; solvent B: methanol;
Mode: gradient; flow rate, 1 ml/min; injection volume, 10 [micro]l; detector,
UV at 360 nm; reference standard, Rutin (2.5 g
dissolved in 100 ml of methanol). The HPLC operating conditions were programmed
to give the following: at 0 min, solvent B: 20%; 5 min, solvent B: 40%; 15 min,
solvent B: 60%; 20 min, solvent B: 80% and at 27 min, solvent B: 20%. The run
rate was 30 min.
Phytochemical analysis
Using standard chemical tests, the water extract of L. leonurus
was tested for compounds reported to be bioactive (Sofowora,
1982; Harborne, 1984; Moffat et al., 1986). The
compounds and tests used are presented in Table 1.
Statistical analysis
The results on the onset of seizures were analyzed using the paired
Student's t-test while the proportion of animals that exhibited tonic seizures
was analyzed using the chi-squared test (Tallarida et
al., 1981).
Results
Anticonvulsant properties assessment
Effect of aqueous extract of L. leonurus on pentylenetetrazole-induced seizures
Pentylenetetrazole (90 mg/kg) produced tonic
seizures in all the animals used. A dose of 100 mg/kg of L. leonurus
extract protected 25% of animals against pentylenetetrazole-induced
seizures and did not affect the onset of the seizures to any significant
extent. L. leonurus (200 mg/kg) protected 37.5% of
mice against pentylenetetrazole-induced seizures, and
significantly delayed the latency of the seizures. Similarly, 400 mg/kg of L. leonurus significantly prolonged the latency of the
seizures produced by pentylenetetrazole, and
protected 50% of animals against the seizures. The standard antiepileptic
drugs, phenobarbitone (10 mg/kg) and diazepam (0.50
mg/kg) profoundly antagonized the seizures produced by pentylenetetrazole
(Table 2). The control vehicle did not affect the gross behavior of mice or
alter pentylenetetrazole-induced seizures to any
significant extent (results not shown).
Effect of aqueous extract of L. leonurus on picrotoxin-induced seizures
Picrotoxin (8 mg/kg)
produced tonic seizures in all the animals used. L. leonurus
(100 mg/kg) did not affect the incidence nor the
latency of picrotoxin-induced seizures. L. leonurus (200 mg/kg) significantly delayed the latency, but
did not alter the incidence of seizures produced by picrotoxin
to any significantly extent. A dose of 400 mg/kg of L. leonurus
did not affect the incidence, but significantly prolonged the latency of picrotoxin-induced seizures. Similarly, the standard
antiepileptic drug, phenobarbitone (10 mg/kg) did not
alter the incidence, but profoundly delayed the onset of picrotoxin-induced
seizures. Diazepam (0.50 mg/kg) significantly reduced the number of animals
convulsing and significantly prolonged the latency of seizures produced by picrotoxin (Table 3). The control vehicle did not alter the
seizures or affect the gross behavior of mice (results not shown).
Effect of aqueous extract of L. leonurus on bicuculline-induced seizures
Bicuculline (20 mg/kg) elicited tonic seizures in
all the animals used. However, all the doses of L leonurus
used (100, 200 and 400 mg/kg) did not alter the incidence of bicuculline-elicited seizures significantly. Besides, 400
mg/kg of L leonurus significantly shortened the
latency of bicuculline-induced seizures. Both of the
standard antiepileptic drugs, phenobarbitone (10
mg/kg) and diazepam (0.50 mg/kg) profoundly antagonized seizures produced by bicuculline (Table 4). The control vehicle did not affect
the gross behavior of mice or the seizures produced by bicuculline
significantly (results not shown).
Effect of aqueous extract of L. leonurus on
NMDLA-induced seizures
N-methyl-D-L-aspartic acid (NMDLA; 400 mg/kg) elicited tonic seizures in all
the animals used. L. leonurus (100 mg/kg) did not
affect NMDLA-induced seizures significantly. L. leonurus
(200-400 mg/kg) significantly prolonged the latency of the seizures. A dose of
200 mg/kg of L. leonurus did not affect the incidence
of the seizures while 400 mg/kg protected 12.5% of the mice convulsing.
However, the standard antiepileptic drugs, phenobarbitone
(10 mg/kg) and diazepam (0.50 mg/kg) did not affect the seizures produced by
NMDLA to any significant effect (Table 5). The control vehicle did not affect
the gross behavior of the mice or alter the seizures significantly (results not
shown).
HPLC analysis
According to the HPLC spectrum of water extract of L. leonurus
obtained, the major peaks presented the following retention times (mm): 17.69,
19.22 and 20.76 (Figure 1). The peak for the reference standard, Rutin, appeared at the retention time (mm) of 21.47.
Phytochemical analysis
The standard chemical tests used showed the positive reactions for
alkaloids, saponins (of both steroid and/or triterpenoid groups) and tannins, and the negative
reactions for the rest of compounds tested. In addition, the literature reports
the presence of volatile oil, several unusual diterpenoids
(labdane type lactones) in Leonotis species. Marrubiin
has been isolated as the main diterpenoid lactone from L. leonurus, but its
precursor, premarrubiin, was not found. Two labdane terpenoids, compounds X
and Y occur also in L. leonurus (Van Wyk et al.; 2000).
Discussion and conclusion
In the present study, both the antiepileptic activity of L. leonurus and the probable involvement of GABA (gamma amino
butyric acid) and glutamic acid systems in this
activity were investigated. GABA is the major inhibitory neurotransmitter in
the brain whereas glutamic acid is an excitatory
neurotransmitter in the brain. The inhibition of GABA neurotransmission and the
enhancement of the action of glutamic acid have been
shown to be the underlying factors in epilepsy (Westmoreland et al., 1994; Rang
et al., 1999). Our study shows that the water extract of L. leonurus
protected some of the animals against seizures induced by pentylenetetrazole
(90 mg/kg), picrotoxin (8 mg/kg) and NMDLA (400
mg/kg), and also delayed the latency of the seizures. This is in conformity
with the claims by traditional medicine practitioners that the plant is
effective in the treatment of convulsion.
In this study, pentylenetetrazole
was shown to induce seizures in all the mice used. The mechanism of pentylenetetrazole-induced seizures is uncertain (Rang et
al., 1999). However, according to De Sarro et al.
(1999), pentylenetetrazole may be producing seizures
by inhibiting gabaergic mechanisms. Standard
antiepileptic drugs, phenobarbitone and diazepam are
thought to produce their effects by enhancing GABA-mediated inhibition in the
brain (Rang et al., 1999). It is therefore possible that the anticonvulsant
effects shown in this study by the said drugs against seizures produced by pentylenetetrazole might be due to the activation of GABA
neurotransmission. Since L. leonurus extract
similarly antagonized seizures elicited by pentylenetetrazole
in mice, it is probable therefore that it may also be exerting its
anticonvulsant effects by affecting gabaergic
mechanisms.
In the same study, picrotoxin also produced
seizures in all the mice used. Picrotoxin has been
shown to elicit seizures by antagonizing the effect of GABA by blocking the
chloride channels linked to GABAA-receptors. This prevents the conductance of
chloride ions into the brain, thus inhibiting GAB A-mediated inhibition and
GABA neurotransmission (Westmoreland et al., 1994; Rang et al., 1999). In this
study, phenobarbitone and diazepam were shown to
antagonize the effect of picrotoxin and L. leonurus extract was also shown to delay the latency of picrotoxin-induced seizures. This suggests that L. leonurus may be affecting gabaergic
mechanisms, probably by opening the chloride channels associated with
GABAA-receptors.
Bicuculline is a selective antagonist of GABA at
the GABAA-receptors (Rang et al., 1999). The fact that L. leonurus
aqueous extract did not affect the seizures induced by bicuculline,
suggests that its effects on gabaergic mechanisms do
not affect directly GABAA-receptors.
NMDLA was also shown to elicit seizures in all the mice used. NMDLA, a
specific agonist at the NMDA-receptors, mimics the action of glutamic acid and thus induces seizures by enhancing the glutaminergic system (Rang et al., 1999). It is not
surprising that the standard antiepileptic drugs, phenobarbitone
and diazepam did not alter NMDLA-induced seizures to any significant extent. In
this study, L. leonurus extract was shown to delay
the latency of seizures induced by NMDLA, it may probably, therefore, be
exerting its anticonvulsant effect partly by affecting glutaminergic
mechanisms.
The phytochemical and the HPLC analyses showed the
presence of alkaloids, saponins (of steroid and/or triterpenoid groups) and tannins in the leaves of the
plant, and distinct peaks which may characterize L. leonurus
respectively. In the present state of knowledge of the chemical constituents of
the extracts of this plant, it is not possible to attribute with certainty the
anticonvulsant effect to one or several active principles among those detected
in the aqueous extract of L. leonurus. This is
supported by the literature, according to which some of the groups of compounds
found in the aqueous extract of L. leonurus, such as
alkaloids, did not show any anticonvulsant activity in the experimental animal
model of epilepsy (Akah and Nwaiwu,
1988). Moreover, Van Wyk et al. (2000) also reported
that the actual pharmacological effect of marrubiin,
the main diterpenoid lactone
of L. leonurus, is not known. However, Chauhan et al. (1988) reported that some natural plant triterpenoids evaluated for anticonvulsant ac tivities provided 10-40% protection against PTZ-induced
convulsions in mice. This therefore suggests that saponin
triterpenoids detected in the aqueous extract of the
leaves of L. leonurus might be involved in the
anticonvulsant effect shown by this plant.
In conclusion, the data obtained in this
study indicate that L. leonurus may be said to exert
its anticonvulsant effect via non-specific mechanisms, since it has been shown
to delay the latency of seizures produced by agents affecting both gabaergic and glutaminergic
systems. However more extensive pharmacological, toxicological and phytochemical studies of this plant are needed to fully
elucidate the active constituents, mechanism of action and safety of the plant
as medicinal remedy.
Table 1
Phytochemical screening of aqueous extract of L. leonurus.
Compounds Tests/Reagents
Anthraquinones Borntrager's test, BPC
Alkaloids Dragendorif's reagent and
Mayer's reagent
Cardiac glycosides -- Lieberman's test
-- Keller-Killiani test
Reducing sugars Fehlings reagents
Saponins Frothing test
Tannins Ferric chloride reagent
Table 2
Effect of aqueous extract of L. leonurus (LST) on pentylenetetrazole
(PTZ)-induced seizures in mice.
Dose (mg/kg) No. convulsed/ Animals not
No. used convulsed (%)
PTZ LST Pheno- Diazepam
barbitone
90 -- -- -- 8/8 0
90 100 -- -- 6/8 25
90 200 -- -- 5/8 37.5
90 400 -- -- 4/8 50
90 -- 10 -- 0/8 a 100
90 -- -- 0.50 0/8 a 100
Latency of tonic
convulsion (min.)
PTZ Mean [+ or -] S.E.M.
90 6.38 [+ or -] 0.88
90 9.67 [+ or -] 1.81
90 12.20 * [+ or -] 2.73
90 16.00 ** [+ or -] 1.76
90 --
90 --
* p <0.50
** p < 0.025; vs pentylenetetrazole control (90 mg/kg; i.p.); Student's
t-test.
(a)p < 0.001 vs pentylenetetrazole control (90 mg/kg; i.p.); Chi-squared
test.
Table 3
Effect of aqueous extract of Leonotis leonurus (LST) on picrotoxin
(PIRC)-induced seizures in mice.
Dose (mg/kg) No. convulsed/
No. used
PICR LST Phenobarbitone Diazepam
8 -- -- -- 8/8
8 100 -- -- 8/8
8 200 -- -- 8/8
8 400 -- -- 8/8
8 -- 10 -- 8/8
8 -- -- 0.50 2/8 (a)
Animals not Latency of tonic
convulsed (%) convulsion (min.)
PICR Mean [+ or -] S.E.M.
8 0 14.13 [+ or -] 0.40
8 0 15.38 [+ or -] 0.56
8 0 17.00 * [+ or -] 0.87
8 0 19.37 * [+ or -] 1.00
8 0 24.38 ** [+ or -] 1.32
8 75 22.50 ** [+ or -] 1.75
* p < 0.05
** p < 0.025; vs picrotoxin control (8 mg/kg; i.p.), Student's t-test.
(a)p < 0.1 vs pocrotoxin control (8 mg/kg; i.p.); Chi-squared test.
Table 4
Effect of aqueous extract of Lenotis leonurus (LST) on
bicuculline(BIC)-induced seizures in mice.
Dose (mg/kg) No. convulsed/
No. used
BIC LST Phenobarbitone Diazepam
20 -- -- -- 8/8
20 100 -- -- 8/8
20 200 -- -- 8/8
20 400 -- -- 8/8
20 -- 10 -- 0/8 (a)
20 -- -- 0.50 0/8 (a)
Animals not Latency of tonic
convulsed (%) convulsion (min.)
BIC Mean [+ or -] S.E.M.
20 0 10.88 [+ or -] 0.48
20 0 9.88 [+ or -] 0.69
20 0 10.38 [+ or -] 0.75
20 0 6.63 * [+ or -] 0.48
20 100 --
20 100 --
* p < 0.025; vs bicuculline control (20 mg/kg; i.p.), Student's t-test.
(a)p < 0.001 vs bicuculline control (20 mg/kg; i.p.); Chi-squared test.
Table 5
Effect of aqueous extract of Leonotis leonurus (LST) on
N-methyl-DL-aspartic acid (NMDLA)-induced seizures in mice.
Dose (mg/kg) No. convulsed/
No. used
NMDLA LST Phenobarbitone Diazepam
400 -- -- -- 8/8
400 100 -- -- 8/8
400 200 -- -- 8/8
400 400 -- -- 7/8
400 -- 10 -- 8/8
400 -- -- 0.50 8/8
Animals not Latency of tonic
convulsed (%) convulsion (min.)
NMDLA Mean [+ or -] S.E.M.
400 0 3.50 [+ or -] 0.40
400 0 4.38 [+ or -] 0.26
400 0 5.75 * [+ or -] 0.31
400 12.5 8.71 * [+ or -] 1.42
400 0 2.25 [+ or -] 0.31
400 0 4.00 [+ or -] 0.42
* p < 0.05 vs NMDA control (400 mg/ kg; i.p.), Student's t-test. The
proportion of animals convulsing is compared using Chi-squared test.