Abstract: This study evaluated the central action of Waltheria indica extract. Aqueous ethanolic extract of the plant showed bioactivity in acetic-acid induced stretches in animal model. The central effects of the most biologically active fraction (ethyl acetate) of extract of Waltheria indica was evaluated in mice using the elevated plus maze paradigm and the strychnine and leptazol-induced convulsions. Sedative effect was studied using the amylobarbitone-induced sleeping time. The extract fraction significantly (p< 0.05) increased the amylobarbitone sleeping time and protected (100%) mice from death due to pentylenetetrazole convulsion. The extract failed to protect mice against strychnine convulsion, even though it delayed the time of onset of death. The exploratory activity was also significantly (p< 0.05) decreased in the extract treated mice. The extract blocked leptazole-induced convulsion, potentiated amylobarbitone sleeping time and decreased exploratory activity, indicating anticonvulsant and sedative actions.
INTRODUCTION
Waltheria indica (synonym Waltheria americana) is a plant widespread throughout the tropics (Trease and Evans, 1996; Burkill, 2000). It belongs to the family Sterculiaceae and is commonly called sleepy morning. In Nigeria, the plant is widely used to treat many diseases such as giddiness and diarrhoea (Heinrich et al., 1992), cough, headache and fever (Gills, 1992). Aerial part of the plant is boiled and cooled and the resulting aqueous solution is used traditionally to treat convulsion in children (Mallam Bashir, personal communication). The present study was based on the hypothesis that Waltheria indica contains bioactive substances that have great potentials for treatment of various illnesses. Though, there are ample scientific and empirical evidences supporting the use of plant-derived formulations for treatment of diseases, medical professionals require scientific evidence of efficacy and safety of these herbal medicines (Gamaniel and Wambabe, 1996; Bizimenyera et al., 2006). Hence there is the need for the scientific validation of Waltheria indica before it can gain wider acceptance and use in the orthodox medicine. The chemical characterization of the Waltheria indica leaf extract showed the presence of mucilages, tannins, reducing sugars, saponins, alkaloids, steroid derivatives and flavonoids (Harboune and Baxtler, 1993; Burkill, 2000). Manske (1975) has reported that the extract of Waltheria indica is physiologically active and scientific elucidation of its diverse bioactivities is required. As some of the diseases traditionally treated with Waltheria indica (headaches and convulsion) are of neural origin, this present study was carried out using standard methods to evaluate some effects of extract fraction of Waltheria indica on the Central Nervous System (CNS).
MATERIALS AND METHODS
Plant Material
Fresh Waltheria indica aerial parts (stem, leaves and flowers) were
collected from fields around Samaru-Zaria, Kaduna State, Nigeria in August 2006
for this study. Taxonomic identification was confirmed by Dr. M.S. Abubakar
of the Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical
Sciences, Ahmadu Bello University, Zaria, Nigeria. A voucher specimen (NPR/2006)
has been deposited at the Departmental herbarium.
Preparation for Extraction
The plant material was washed, air-dried and oven-dried for 2 h in the Department
of Pharmacognocy and Drug Development, Ahmadu Bello University, Zaria. It was
then pounded into powder and sieved.
Extraction
Five hundred grams of the powdered material was soxhlet extracted with aqueous
ethanol (60% v/v). The aqueous ethanol extract, upon concentration, yielded
a yellow greenish residue hitherto called the extract (15.3% w/w). The extract
was suspended in water and defated with petroleum ether. It was then successively
partitioned with N-butanol, acetone and ethyl acetate and the respective fractions
were concentrated over water-bath. The fractions were tested for bioactivity
against acetic acid-induced writhes (Koster et al., 1959) and tail immersion
tests in mice (Turner, 1965).
Animals
Adult albino mice weighing 21.5-27 g were purchased from the animal house
of the Department of Pharmacy, Ahmadu Bello University, Zaria-Nigeria. The animals
were maintained under standard nutritional and environmental conditions, having
access to water and food ad libitum. Feeding was withdrawn 12 h before
experimentation.
Acute Toxicity Test
The acute toxicity (oral LD50) of the extract of Watheria
indica aerial portion was estimated in 25 albino mice using the standard
method of Lorke (1983).
Exploratory Behavioural Studies
The elevated plus maze test was used to determine the effects of extracts
on exploratory activities in mice (Lister, 1987; Espejo, 1997; Brown et al.,
1999). The elevated plus maze was built according to the description of Lister
(1987) with two open arms (30x5x15 cm) and two closed arms (30x5x15 cm) extending
from a central, open square (5x5 cm). The floor of the maze was painted black
which was used to measure locomotion. A black lip (4 mm high) was attached to
the floor of the open arms to prevent the mice from falling off (Trullas and
Skolnick, 1993). The maze was elevated on a pedestal to a height of 45 cm above
the floor. All the experiments were carried out under white light according
to Lee and Rodgers (1990).
Twenty four mice were used for the study. They were divided into 3 groups, 1, 2 and 3 each comprising 8 mice. Mice in groups 1 and 2 were administered the extracts at a doses of 100 and 200 mg kg-1, respectively. Each mice was placed in the central square of the maze facing an open arm and its behaviour (head dips and stretch attend postures) observed. The number of head dips and stretch attend postures were observed for 10 min each and recorded. The tests were carried out 30 min after pretreatment of the animals with doses of extract (100 and 200 mg kg-1), groups 1 and 2, respectively. Standard drug, chlorpromazine (1 mg kg-1) was given to the third group.
Effects of Extract on Amylobarbitone-Sodium Sleeping Time
Twenty mice of both sexes weighing 20.8-25.5 g were randomly divided into
4 groups of 5 mice each. They were treated with amylobarbitone sodium 30 min
after varying doses of extract of Waltheria indica were administered.
Mice in group 1 received only amylobarbitone sodium (20 mg kg-1)
and they served as control. Mice in group 2 were administered 50 mg kg-1
of extract 30 min before the administration of amylobarbitone sodium (20 mg
kg-1), while those in group 3 were given first 100 mg kg-1
of extract and 30 min later they were administered 20 mg kg-1 amylobarbitone
sodium. Group 4 mice were administered first 200 mg kg-1 of extract
and 20 mg kg-1 of amylobarbitone sodium 30 min later. All injections
were given intraperitoneally and the duration of sleep was defined as the time
between losing and regaining the righting reflex (Carlini and Burgos, 1979).
Effects of Extracts on Pentylenetetrazole (Leptazol)-Induced Convulsion
Twenty adult albino mice of both sexes, weight 22.6-27.1 g were randomly
divided into 4 groups of 5 mice each. They were housed in plastic cages and
were given access to food and water ad libitum.
Group 1 which serve as control was given only the vehicle, 20 mL kg-1 normal saline. Group 2 received a convulsive dose of 100 mg kg-1 of leptazol only and served as reference. Group 3 received 100 mg kg-1 of leptazole 30 min after administration of 100 mg kg-1 extract, while group 4 was given 100 mg kg-1 of leptazole 30 min after treatment with 200 mg kg-1 extract. Leptazole was administered and all animals received 30 mg kg-1 pentazocine intraperitoneally before treatment with strychnine and pentylenetetrozol to alleviate pains associated with convulsion.
Effects of Extracts on Strychnine-Induced Convulsion
Mice were randomly divided into 4 groups of 5 mice each. Group 1 received
vehicle (20 mL kg-1 normal saline) to serve as control. Group 2 receive
2 mg kg-1 strychnine alone and served as reference. Group 3 was administered
2 mg kg-1 strychnine 30 min after extract treatment of 100 mg kg-1,
while animals in group 4 received 2 mg kg-1 strychnine 30 min after
extract treatment of 200 mg kg-1. Strychnine was administered intraperitoneally.
The latency for convulsive episode in each mice was evaluated in minutes.
Statistical Analysis
Results were expressed as mean±SEM and analyzed using the Students’
t-test and analysis of variance (ANOVA). Values of p<0.05 were considered
statistically significant.
RESULTS
The aqueous ethanolic extract of Waltheria indica was yellow greenish in colour. The acute toxicity study showed low toxicity of the extract (LD50, 875 mg kg-1) in mice when orally administered. The results of screening of the extract fraction for bioactivity showed that the ethyl acetate fraction conferred highest protection (69%) against the acetic acid induced-writhes compared to N-butanol (54%), acetone (50%) and aqueous residue (38%), respectively. The ethyl acetate extract fraction of Waltheria indica also significantly (p<0.05) increased the sleeping time of amylobarbitone sodium dose-dependently in mice. The sleeping time in mice treated with 200 mg kg-1 of the extract was significantly (p<0.05) higher than in those administered 100 mg kg-1 of the extract; 80.0±1.02 and 69.6±0.9 min, respectively. The sleeping time obtained in control mice was 55.6±0.6 min and the value was significantly lower (p<0.05) than those obtained in groups 3 and 4 mice. The result also indicated significant (p<0.05) decrease in exploratory activities (head dip and stretch attend postures) in mice administered 200 mg kg-1 of the extract fraction (Table 1).
| Table 1: | Effects of ethyl acetate aerial parts extract of Waltheria indica on exploratory behaviour in mice |
| N = 10; Statistical analysis was evaluated using students t-test vs control; *: p<0.05 | |
| Table 2: | Effects of ethyl acetate fraction of aerial parts extract of Waltheria indica on pentylenetetrazol-induced convulsion in mice |
| *: p<0.05 when compared with control group; Statistically analysis evaluation by students t-test vs control | |
| Table 3: | Effects of ethyl acetate fraction of extract aerial parts of Waltheria indica on pentylenetetraol-induced convulsion in mice (N = 5) |
| *: p<0.05 when compared with control group; Statistically analysis evaluation by students t-test vs control | |
The effects of ethyl acetate fraction of Waltheria indica on leptazol-and strychnine-induced convulsion in mice respectively (Table 2, 3).
The extract at doses, 100 and 200 mg kg-1 protected the mice from death due to pentylenetetrazole seizures. However, the extract at similar doses could not protect the mice from death due to strychinine seizures even though it significantly (p<0.05) reduced the number of convulsions in mice treated with 200 mg kg-1.
DISCUSSION
The ethyl acetate fraction of the aqueous ethanolic extract of aerial portion of Waltheria indica showed central nervous system depressant activity. This action was demonstrated by its effects on amylobartitone sodium sleeping time, pentylenetetrazole and strychnine-induced convulsions and exploratory activities. Marked decrease in exploratory activities in laboratory animals is a measure of depressant action (Lister, 1990; Harvey, 1991; Brown et al., 1999). The extract treated mice in this study showed decreased gross behaviour, suggesting sedative effects of the extract. This result corroborates the findings of Ming-chin (1998) and that of Wakeel et al. (2004) in which decreased exploratory activity and potentiation of hexabarbitone-induced sleeping time was produced, following the administration of Cistanche deserticola and Ficus platyhylla stem bark respectively, in mice.
Decreased exploratory behaviour in rats treated with extracts of Rauwolfia vomitoria comparable to standard sedative drug, chlorpromazine 1 mg kg-1 was similarly reported by Bisong and Osim (2006). The results obtained in this study also showed significantly (p<0.05) increased amylobarbitone sleeping time, corroborating the findings of Fujimori (1995) and Sandabe et al. (2003) that enhancement of amylobarbitone sodium sleeping time indicates central nervous system depression.
The extract was found to completely prevent death of animal resulting from pentylenetetrazole convulsions, a finding which did not agree with the assertion that most drugs with anticonvulsant actions do not counteract pentylenetretrazole convulsions, but retard them (Loscher et al., 1991). However, the extract failed to protect mice from strychnine convulsions, suggesting differences in mechanisms of action of these convulsive drugs.
In conclusion, extract of Waltheria indica has low toxicity (LD50 870 mg kg-1). It also caused a reduction in locomotor activity and acted apparently synergistically with amylobarbitone to increase the duration of sleep. The findings of this study has established Sedative and anticonvultant action of the plant. The data obtain will add to the large body of evidence collected scientifically to show the immense potentials of medicinal plants use in various traditional system. Further study is required in order to establish the mechanism of action of the extract on the central nervous system.