Phytochemical and Anticonvulsant Studies on the Aqueous Ethanol Extract of the Root-Back of Ficus Abutilifolia ( Miq . ) Miq . ( Family : Moracea )

Ficus abutilifolia, belonging to the family Moraceae is a small to medium sized tree that grow mostly in the African continent. It was reported to be used traditionally, in promoting fertility in humans and in the treatment of skin wart and management of epilepsy. Preliminary phytochemical investigation of the powdered root revealed the presences of flavonoids, saponins and tannins among others. The intraperitoneal LD50 of the 70% aqueous ethanol extract was found to be 2154.1 mg/Kg in mice. The anticonvulsant studies of the extract revealed that a single administration (at the dose of 100 – 400 mg/Kg) produced a dose-dependent protection against MEST; however, the extract did not offer significant protection against pentylenetetrazoleand 4amino pyridine–induced seizures. These finding suggest some level of protection by the aqueous ethanol extract against MES induced seizure in chicks, thereby giving support to the traditional claim for the use of the plant in the treatment and/or management of convulsion and epilepsy.


INTRODUCTION
Plants and their products have been in use in the treatment of chronic ailments, pains and infectious diseases before the modern civilization.And to date, they still remain the almost exclusive source of drugs for more than 60% of the world's population, especially in developing countries, and also serve as important source of new drugs, new drug leads, and new chemical entities.It is in record that the potential of plants in general and higher plants in particular as a source of new drugs has not been fully explored.Some individual plant extract may have been subjected to specific pharmacological test (e.g. for cardiac activity only) however, the same extract may be examined for other types of activities such as pain reliving, anti-inflammation, antidiarrhea etc (Balunas and Kinghorn, 2005;Li and Vederas, 2009;Phillipson, 2001).Ficus abutilifolia (Miq.)Miq. is commonly called large-leaved rock fig or rock wild fig and belongs to the family Moraceae.It is a small to medium sized, deciduous to semi-deciduous tree that may grow up to 15 m high (though it seldom exceeds 5 m).The bark of the plant is yellowish-white, smooth and flaking.The trunk is usually twisted.Leaves are broadly ovate and cordate at base, they usually measured 7.5 -20.0 X 6.5 -18.0 cm.Fruits are 1.5 -2.5 cm in diameter usually borne singly or in pairs at the leaf axils, smooth or slightly hairy.
A decoction of the leaf of the plant is reported to be used in promoting fertility in humans and the milky latex is used to remove skin warts.Bark decoction is taken by men as a strengthening tonic (Burring, 2006).
Information made available to us (personal communication, 2004) showed that the root of F. abutilifolia is also used as part of a preparation in the management of epilepsy.We therefore, investigate and report here the preliminary phytochemical screening and for the first time the anticonvulsant properties of the F. abutilifolia root-bark.

Plant Material
The plant specimen was collected in October, 2006 around Basawa-Zaria, Kaduna State Nigeria.It was identified by the herbarium keepers of the Department of Biological Sciences, Ahmadu Bello University, Zaria-Nigeria, where a specimen (voucher specimen number 900, 742) was deposited for future reference.The root of the plant was cleaned, air dried and ground to powder using pestle and mortar.

Extraction of the Plant Material
Powdered plant (150 g) was macerated with 750 ml of 70% aqueous ethanol for four days after which, it was filtered and evaporated to dryness on water bath to give a brownish residue.The residue, subsequently referred to as the extract was stored in an air tight container until required for further use.

Animals
Swiss albino mice of both sexes, weighing 18 -25 g were obtained from the Animal House facility of the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria.One day old Ranger cockerels were obtained from National Animal Production Research Institute (NAPRI) Shika-Zaria, Nigeria.The mice were kept under well-ventilated conditions, 12 hours light/dark cycle, temperature of 25 ± 2 0 C and fed on standard laboratory animal Feeds and had access to water ad-libitum.

Preliminary Phytochemical Screening
The powdered root of F. abutilifolia was subjected to preliminary phytochemical analysis to test for the presence of phytochemical constituents using the following methods: Anthraquinones [Borntrager's test: 100 mg of powdered plant in 5 ml of chloroform, filtered.2 ml filtrate + 2 ml 10% NH 4 OH.A bright pink colour indicates the presence of anthraquinones; Modified Borntrager's test: 200 mg plant material boiled in 5ml 10% HCl, filtered.Filtrate extracted with 5ml benzene, and benzene layer shaken with 5 ml 10% NH 4 OH.A rose pink or cherry red colour indicates the presence of anthraquinone derivatives (Evans, 1996).

Acute Toxicity Study
LD 50 determination was conducted using the method of Lorke (1983).Nine mice were divided into 3 groups of 3 mice each.The first group received the extract (i.p.) at a dose of 1000mg/kg; group 2 received the extract at a dose of 100mg/kg (i.p.), while the last group received the extract at the dose of 10mg/kg body weight.Animals were observed for general signs and symptoms of toxicity including mortality over a period of 24 hours.
In the second phase, 4 mice were divided into 4 groups of one mouse each.The extract was administered at the dose of 600, 1000, 1600, and 2900 mg/Kg (i.p.) to animals respectively, based on the result of the first phase.LD 50 was calculated as the square root of the geometrical mean of highest non lethal dose for which the animal survived and the lowest lethal dose for which the animal died.

Anticonvulsant Activity Maximal Electroshock-Induced Seizures in Chicks (MEST)
The methods previously described by Swinyard and Kupferberg (1985) and Browning, (1992) were employed.Fifty one day old cockerels were randomly divided into 5 groups of 10 chicks per group.The first group received normal saline i.p.; groups 2 -4 received the extracts (100, 200 and 400 mg/kg, i.p. respectively).While the fifth group received phenytoin 20 mg/kg, (i.p.).Thirty minutes later, maximal electroshock was delivered to induce seizures in the chicks using Ugo basile electroconvulsive machine (model 1801) with corneal electrodes placed on the upper eyelid of the chick after dipping them in normal saline.The current, shock duration, frequency and pulse width were set and maintained at 90mA, 1.0 sec, 200 Hz and 1.0 ms -1 respectively.An episode of tonic extension of the hind limbs of the chicks was considered as full convulsions.Lack of tonic extension of the hind limbs was regarded as protection.The recovery time was taken for the unprotected animals.

Pentylenetetrazole-Induced Seizures in Mice (Sc-PTZ)
The method of Swinyard et al. (1952) was employed.Twenty-five mice were randomly divided into 5 groups of five mice per group.The first group which served as negative control was treated with normal saline (i.p.).Groups 2 -4 received different doses of the aqueous ethanol extract reconstituted in water (100, 200 and 400 mg/kg, i.p. respectively).Group 5 which served as positive control was treated with 200 mg/kg i.p. valproic acid.
Thirty minutes later, 85mg/Kg of freshly prepared solution of pentylenetetrazole was administered subcutaneously to all the mice.The mice were observed for 30 minutes for the onset and incidence of seizures.An episode of clonic spasm of at least 5 seconds was considered as seizure.Lack of threshold convulsion during 30 minutes of observation was regarded as protection.The number of mice protected was noted and the anticonvulsant properties of the extract expressed as percentage protection.

4-Amino Pyridine-Induced Seizure in Mice
The method of Yagamuchi and Rogawski, (1992) was adopted.Twenty-five mice were randomly divided into 5 groups of five mice per group.The first group which served as negative control was treated with normal saline.Groups 2 -4 received different doses of the aqueous ethanol extract reconstituted in water (100, 200 and 400 mg/Kg, i.p. respectively).Group 5 which served as positive control was treated with 20 mg/Kg (i.p.) phenobarbitone.
Thirty minutes later, 15mg/Kg of freshly prepared solution of 4-aminopyridine was administered subcutaneously to all the mice.The mice were observed for 30 minutes for characteristic behavioral signs such as hyperactivity, trembling, intermittent forelimb extension, tonic seizure and death.Lack of threshold convulsion during 30 minutes of observation was regarded as protection.The number of mice protected was noted and the anticonvulsant properties of the extract expressed as percentage protection.

Statistics Analysis
Data were expressed as Mean ± Standard Error of Mean.Statistical analysis was carried out using one-way ANOVA, followed by Dunnett's test and Chi-square for percentage protection and P<0.05 was considered significant.

RESULTS AND DISCUSSION
Preliminary phytochemical screening tests for the powdered root of F. abutilifolia (table 1) revealed the presence of saponins, flavonoids, tannins, terpenoids and/or steroids.However, alkaloids were not detected in the root.The presence of the above chemical compounds may alone or in combination contribute to the observed anticonvulsant effect of the root extract.The median lethal dose of the aqueous ethanol extract of F. abutifolia was estimated to be 2, 154.1 mg/Kg body weight.The acute toxicity index (LD 50 ), serve as a means of giving an idea about the toxic effect of any potential drug substances.The result of the present study shows that the 70% aqueous ethanol extract of F. abutilifolia has an LD 50 of over 2,000 mg/Kg body weight in mice when administered through intraperitoneous route.The extract can be regarded as slightly toxic but the risk of acute intoxication is minimal (Lorke, 1983).
The aqueous ethanol root extract of F. abutifolia dose dependently protected the animals against maximal electroshock seizure with the highest protection of 40% produced at the dose of 400 mg/Kg.However, the extract did not offer any significant protection against pentylenetetrazole and 4-aminopyridine induced seizures in mice.The hind limb tonic extension (HLTE) produced electrically as in the maximum electroshock test (MEST), is a common feature in many animal species including humans.And the response of the brains of the animals to anticonvulsant is similar to that of humans.Extracts of F. abutilifolia afforded dose depended protection to the laboratory animals against the HLTE showing the ability of the extract to inhibit or prevent seizure discharge within the brainstem seizure substrate (Browning, 1992).This suggests that the 70% aqueous extract of the plant under study contain some compounds that may be beneficial in the treatment of generalized tonic-clonic and partial seizure.The chemically-induced seizure using PTZ test usually identifies drugs that raise seizure threshold in the brain (White et al, 1998).The standard convulsant PTZ has also been shown to interact with γ-amino butyric acid (GABA) -a neurotransmmiterand the GABA receptor complex (Bum et al, 2001).Drugs that inhibit the PTZ activity such as diazepam and valproic acid exert their effect by enhancing GABA mediated inhibition in the brain.The inability of the aqueous ethanol extract of F. abutilifolia to protect the animals against PTZ-induced seizure suggests that it may not be effective in the treatment of absence and/or myoclonic seizures.The potassium channel blocker -4-aminopyridine -is a convulsant that penetrates the blood-brain barrier (Yagamuchi and Rogawski, 1992), and causes convulsion by enhancing spontaneous and evoked neurotransmitters.The inability of the extract to afford any protection to the laboratory animals against the chemically-induced seizure by 4-aminopyridine indicates that the extract does not produce its activity via potassium channel.

CONCLUSION
F. abutilifolia root extracts afforded protection to laboratory animals against maximum electroshock, indicating that it may be useful in the management of grand mal epilepsy.However, the absence of any significant protection to the animals against the chemically induced seizures of PTZ-and 4AP suggests that, at this dose the extracts may not be beneficial in the management of petit mal epilepsy.

Table . 1
: Preliminary Phytochemical Screening Result of Powdered root-bark of F. abutilifolia.

Table . 2
: Effects of different doses of aqueous ethanol extract of F. abutilifolia root-bark on the convulsive activities of electroshock .

Table . 3
: Effects of aqueous ethanol extract of F. abutilifolia root on pentylenetetrazole-induced seizure in mice.

Table . 4
: Effects of different doses of aqueous ethanol extract of F. abutilifolia root on the convulsive activities of 4-aminopyridine.