Epilepsy is a major neurological disorder characterized by recurrent seizures with a lifetime prevalence of 5% (Sander and Shorvon, 1996; Raza et al., 2001). Even though, the modern conventional antiepileptic drugs (AEDs) are effective in approximately 50% of patients, many cases still remain resistant to AED treatment (Heinemann et al., 1994; Shorvon, 1996; Raza et al., 2001). These drugs are associated with vast array of side effects including chronic toxicity, teratogenecity, adverse effects on cognition and behaviour among others (Raza et al., 2001). Thus, due to aforementioned reasons and others; it is pertinent to look for affordable and convenient alternative medicine with view to providing a better protection and activities-particularly medicinal plants. Moreover, medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects and have been used in the discovery and development of new drugs (Farnsworth, 1994; Cragg et al., 1997).
The plant Newbouldia laevis (P. Beauv) Seem or boundary tree is locally called as: Aduruku in Hausa, Ogirisi in Igbo and Akoko in Yoruba languages (Hutchinson and Dalziel, 1963) is a medium sized angiosperm which belongs to the Bignoniaceae family. It grows to a height of about 7-8 (up to 15) m, more usually as shrub of 2-3 m, many-stemmed forming clumps of gnarled branches (Arbonnier, 2004; Usman and Osuji, 2007). It is native to tropical Africa and grows on moist and well-drained soils; extends from Guinea Savannahs to the dense Forests zones.
In Nigeria, it has been found useful as a remedy for earache, sore feet, chest pain, epilepsy and childrens convulsion (Akunyili, 2000). The leaf, stem and fruits have also been used for febrifuge; wound dressing and stomach ache (Iwu, 2000). Recent study has shown the leaf to be a good antibacterial source (Usman and Osuji, 2007).
Earlier studies on the leaves and bark of Congolese Newbouldia leavis revealed the absence of flavonoids; saponins, quinones, terpenes or steroids (Oliver-Bever, 1986). Recent phytochemical studies on the root, root bark and stem of this plant revealed the presence of alkaloids, quinoid and phenylpropanoid amongst others (Gafner et al., 1997; Aladesanmi et al., 1998; Germann et al., 2006). There was no extensive report on the isolation of compounds from the leaves (Usman and Osuji, 2007) and flowers of this species.
In this investigation, the ethanolic flower extract of this plant was subjected for the first time to phytochemical and anticonvulsant study, with the view to evaluating its efficacy as a source of potential antiepileptic agents.
MATERIALS AND METHODS
The flowers of Newbouldia laevis for this work were collected from
Kudingi Village-Samaru Zaria, in the month of August 2005. The studies were
carried out in May 2007 at the Research laboratory, Department of Chemistry,
University of Maiduguri, Maiduguri and Department of Pharmacology and Clinical
Pharmacy, ABU Zaria-Nigeria. The plant specimen was identified by Mr. U.A. Gallah
of the Herbarium Unit, Department of Biological Sciences, Ahmadu Bello University,
Zaria - Nigeria, where a voucher specimen was deposited.
Extraction of Plant Material
The flowers of Newbouldia laevis was dried under shade for several
days and then pulverized into fine powder. About 200 g of the powdered form
was extracted exhaustively with 95% (v/v) ethanol in water using continuous
soxhlet apparatus. The extract was concentrated under reduced pressure to yield
a dark green mass which weighed 38.51 g (19.26% w/w). The crude extract was
then coded NFE-Newbouldia leavis flower extract.
A total of 63 adult male Swiss albino mice weighing between 18-28 g were
obtained from Animal house, Department of Pharmacology and Clinical Pharmacy,
ABU Zaria-Nigeria. The animals were kept under well-ventilated conditions with
12 h light/dark cycle (6 am-6 pm) at room temperature, fed on Standard feeds
(Excel feeds Plc. Kaduna, Nigeria) and allowed water ad libitum.
Pentylenetetrazole (PTZ), Phenobarbitone (PHB) (Pfizer, USA) and 4-amino
pyridine (4-AP) BDH reagent (Poole, UK). All drugs/chemicals were freshly prepared
to the desired concentration with distilled water just before use.
The phytochemical analyses of the crude ethanolic extract of Newbouldia
leavis flower was carried out in order to ascertain the presence of its
constituents utilizing standard conventional protocols (Markham, 1982; Harborne,
1993; Sofowora, 1993; Trease and Evans, 2002).
Acute Toxicity Studies (LD50)
LD50 determination was conducted using the method of Lorke (1983).
In the initial phase mice were divided in to 3 groups of three mice each and
treated with ethanol extract at doses of 10, 100 and 1000 mg kg-1
i.p and observed for 24 h. In the final phase, 3 mice were divided in to 3 groups
of one mouse each and ethanol extract administered at doses of 1600, 2900 and
5000 mg kg-1 i.p and the final LD50 value calculated.
Pentylenetetrazole-Induced Seizure in Mice
The method of Swinyard et al. (1989) was employed. Twenty five mice
were divided into five groups of five mice each. The first, second and third
groups received 50, 100 and 200 mg extract kg-1 body weight. The
fourth group served as control and was given normal saline equivalent to vehicle
given with the extract; while the fifth group was given 20 mg phenobarbitone
(PHB) kg-1 body weight. The drug and extract were administered by
intraperitoneal (i.p.) route. Thirty minutes post treatment, mice in all the
groups received 85 mg pentylenetetrazole kg-1 subcutaneously (s.c.).
Mice were then observed over a period of 30 min. Absence of an episode of clonic
spasm of at least 5 sec duration indicated a compounds ability to abolish
the effect of pentylenetetrazole on seizure threshold.
4-Amino Pyridine-Induced Seizure in Mice
The method adopted for this study was as described by Rogawski and Porter
(1990) and Yagamuchi and Rogawski (1992). Male Swiss albino mice were randomly
divided in to 5 groups each containing five mice. They were allowed to acclimatize
with free access to food and water for a 24 h period before testing. The first,
second and third groups received 50, 100 and 200 mg extract kg-1
body weight. The fourth group was given normal saline equivalent to the vehicle
given the extract while the fifth group received 30 mg PHB kg-1 body
weight. The drug and extract were administered by intraperitoneal route. 15
min post treatment; 4-AP was administered at a dose of 15 mg kg-1
body weight to each mouse s.c. The mice were observed for 30 min for characteristic
behavioural signs, such as hyperactivity, trembling, intermittent forelimb extension,
tonic seizures and death. The ability of the extract to protect the mice from
lethality within 30 min observation period was considered as an indication for
anticonvulsant activity (Yagamuchi and Rogawski, 1992).
All values were expressed as mean±SEM. The data of PTZ and 4-AP tests
were analysed statistically by one way ANOVA. The differences between means
were considered significant.
The preliminary phytochemical analysis of the flower extract of Newbouldia
leavis revealed the presence of cardiac and steroidal glycosides, flavonoids,
tannins while other phytochemicals such as alkaloids, saponins was not detected
as presented in Table 1.
Acute Toxicity Studies
The LD50 in mice was found to be 1264.9 mg kg-1 i.p.
Effects of Ethanolic Flower Extract on PTZ-Induced Seizures
The NFE exhibited slight dose-dependent protection on animals against seizures
induced by PTZ (Table 2) but did not delay the onset of seizures
in non-protected animals (Normal saline group).
|| Phytochemical constituents of the crude ethanolic flower
extract of Newbouldia laevis
|+ = Present, - = Absent
|| Effect of ethanolic flower extract of Newbouldia laevis
on PTZ-induced seizure mice
|One way ANOVA df = 3,12; F = 1.985; P = 0.187; n = 5. * No
significant effect on mean onset of convulsion (p<0.05), since the number
of convulsed animals are few
||Effect of ethanolic flower extract of Newbouldia laevis
on 4-AP-induced seizure in mice
|One way ANOVA df = 3, 19; F = 10.273, n = 5. *, **, *** as
p<0.05, p<0.01, p<0.001, respectively
At the dose of 100 mg kg-1 body weight i.p., the NFE protected 40%
of mice against seizures; while 200 mg kg-1 body weight exhibited
60% protection in mice.
Effects of Ethanolic Flower Extract on 4-Ap-induced Seizures
The NFE exhibited significant dose-dependent delay on the onset of seizures
against 4-AP- induced convulsion seizures. The dosages 50 and 200 mg kg-1
body weight significantly delayed the onset of 8.0±0.45 and 11.2±1.31,
respectively. The extract possesses weak anticonvulsant activity against 4-AP,
since it can only delay the onset significantly (Table 3).
The present study shows that the ethanolic flowers extract of Newbouldia laevis have anticonvulsant activity in PTZ and 4-AP-induced seizures in mice. The toxicity index of the extract in mice indicated that it was moderately toxic.
The PTZ is a known convulsant and anticonvulsant activity in PTZ (s.c) test identifies compounds that can raise the seizure threshold in the brain (White et al., 1998; Raza et al., 2001). Agents (such as phenobarbitone, valproic acid, benzodiazepines) affecting the PTZ test can inhibit absence or myoclonic seizures (Vida, 1995; Hosseinzadeh and Khosravan, 2002). Earlier study has shown that the leaf ethanolic extract exhibit protection against PTZ-induced convulsion in mice (Akunyili, 2000). In line with these findings, this study showed that the extract possesses strong anticonvulsant activity against PTZ-induced seizure in mice.
The K+ channel antagonist-4-amino pyridine is a powerful convulsant in animals and man. The drug readily penetrates the blood-brain barrier and it is believed to induce seizure activity by enhancing spontaneous and evoked neurotransmitter. The profile of drugs effective in this seizure model is distinct from other chemoconvulsant models and more similar to those that prevent tonic hind limb extension in maximal electroshock seizure test. The test is useful to differentiate the mode of action of anticonvulsant drugs (Vogel, 1997).
One potential anti-epileptic mechanism that has been exploited is K+ channels, which play a major role in the control of all aspect of neuronal excitability, including resting membrane potential, responsiveness to synaptic inputs, frequency adaptation and neurotransmitter release (Wickenden, 2002). Drugs like phenytoin which block seizure spread are effective antagonist of seizures induced by K+ channel blockade while those with specific actions on other cellular targets may be weak or inactive, presumably because they are unable to attenuate the spread of intense (non-N-methyl-D-aspartate (NMDA) receptor mediated) excitation evoked by 4-amino pyridine (Yagamuchi and Rogawski, 1992). The results of this research indicated that this extract possesses weak anticonvulsant activity against 4-amino pyridine-induced seizure.
This study therefore, suggests that since earlier studies had shown that the leaf and roots extracts contained potential anticonvulsants, it is hence, imperative to say that the extract of the flower of the same plant could be yet another source.
We therefore, conclude that the anticonvulsant potency of ethanolic flower extract from this plant could favourably suggest the presence of bioactive phytochemicals effective in the therapy of absence seizures and thus help in the control of petit mal; more so, the extract could not be useful in the management of grand mal seizures.
Authors wish to acknowledge the efforts of Mal. Ibrahim of the Department of Pharmacognosy and Drug Development, Ahmadu Bello University, Zaria-Nigeria; for the collection of plant material used for this work.