1. Introduction

In Africa, up to 80% of the population uses traditional medicine, while in industrialized countries the market for herbal medicine is growing steadily ^[1]. Medicinal plants have been used in the treatment and prevention of some infectious diseases such as cancer, malaria, and HIV/AIDS in various parts of Nigeria. Malaria, for instance is endemic throughout the country. World Health Organization (WHO) estimated malaria mortality rate for children under five in Nigeria at 729 per 100,000 ^[2]. The Ministry of Health reported that malaria is responsible for one out of ten deaths in pregnant women and has caused the Federal Government millions of US Dollars annually ^[3].

In Kano and Jigawa States of northern Nigeria, the majority of population relies heavily on traditional practitioners and medicinal plants to meet primary health care obligations. Although orthodox medicine may be available in these areas, herbal medicines are affordable and have often maintained popularity for historical and cultural reasons. Investigations into the chemical and biological activities of plants during the past two centuries have led to the discovery of novel and more effective therapecitic agents ^[4].

Fatope, 1995 ^[5] described a number of reliable and very sensitive bioassay techniques which are indicative of toxicity. These are the one-day duckling bioassay, the chick embryo test, the zebra fish test , test on insect or insect Larvae , the rat multipurpose screen and others. Most of these bioassay techniques cannot be used as rapid, general screening procedure for the detection of toxic secondary metabolites because of costs, specifity, sophistication or objections by animal right actives. As such McLaughlin and co-workers, 1991 ^[6] reported three simple bioassay procedures by his laboratory and successfully used them to direct the isolation of bioactive compounds from plant sources. Brine shrimp test (BST) is the simplest bioassay among the three. The assay was initiated by Meyer et al., 1982 and performed with minor modification in 1991 as a simple, rapid, in-house, bench-top and low cost prescreen for cytotoxicity, insecticidal and anti-malaria activities ^[6].

A positive correlation was successfully established between brine shrimp toxicity and 9KB (human epidermoid carcinoma of nasopharynx) cytotoxicity (p=0.036 and Kappa=0.56) respectively. Furthermore, BST was confirmed to be useful as a prescreen test for antitumour activity in a blind comparison with in vitro cytotoxicity and 3PS (in vitro P388 murine leukemia) activity (p=0.033-0.0331) ^{[8, 9, 10, 11]}. In recent years scientists have been discovering various bioactive compounds with diverse chemical structures, guided by BST a simple and quick bioassay which can be carried out in a chemical laboratory.

In this study the brine shrimp, Artemia cyst was used, a simple zoologic organism (an arthropod). The assay was carried out to investigate the cyclotoxicity of extracts of some medicinal plants useful for malaria therapy in Kano and Jigawa States, northern Nigeria.

2. Materials and Methods

The plant materials (Table 1) for this research work were collected between 7'^h and 23^rd June, 2008 from Kano and Jigawa states of Nigeria. They were authenticated by Prof. Bala Sidi and Baba Ali Garko of Bayero University Herbarium. The voucher numbers of the plants were compared with the ones that were already available at the Herbarium. The plant materials were air - dried and milled.

Table 1. Brine Shrimp Test Activity of Targeted Plant Extracts

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Two hundred grams (200g) of each plant (Table 1) were separately extracted by percolation at room temperature with 1 litre of ethanol for 2 weeks. The percolates were then filtered and the solvent evaporated using the rotary evaporator at 40°C ^[12] to give a residue, Fl. Fl (4 – 4.5g), was partitioned between water and chloroform (300ml, 1:1) in a separatory funnel. After removal of the chloroform layer, the water layer was further washed 3 times with ethyl acetate (200ml) and then separated. The chloroform and ethyl acetate layers were concentrated under vacuum below 40°C to give the chloroform F2 and ethyl acetate, F4 soluble residues. The water soluble layer was concentrated to give F3 residue. F2 was further partitioned between 90% aqueous methanol (200ml) and petroleum ether (200ml). The two layers were separately evaporated under vacuum to dryness to give the petroleum ether soluble residue (F5) and the aqueous methanol soluble reside (F6). The residues were weighed and screened for activity in the brine shrimp lethality test (BST) (Table 1).

The eggs (Premium Grade) of Artemia cysts were purchased from M & M suppliers. Bothell USA. 50mg of eggs were added to a hatching chamber containing Ocean/Sea Water (75ml). The hatching chamber was kept under inflorescent bulb for 48h for the eggs to hatch into shrimp larvae. 20mg of test fractions Fl, F2, F3, F4, F5 and F6 of the various plant species were separately dissolved in 2ml of methanol, from this, 500, 50, and 5µl of each solution was transferred using micro liter syringe (Hamilton Bonaduz AG – Switzerland), into vials corresponding to 1000, 100, and 10µg/ml, respectively. Each dosage was tested in triplicate. The vials (9 per test fraction) and one control containing 500µl of solvent only were allowed to evaporate to dryness in about 48h at room temperature. 4.5ml of Ocean/Sea Water was added to each vial, and 10 larvae of A. cysts (taken 48 - 72h after the initiation of hatching) were added using a Pasteur pipette to each vial. The final volume of solution in each vial was adjusted to 5ml with Ocean/Sea Water immediately after adding the shrimps. One drop of dimethyl sulphoxide (DMSO) was added to the test and control vials before adding the shrimps to enhance the solubility of test materials. LC₅₀ values were determined at 95% confidence intervals by analyzing the data on a computer loaded with a Finney Programme ^[13]. The LC₅₀ values of the brine shrimps obtained for extracts of the plants studied were recorded (Table 1).

3. Results and Discussion

The cytotoxic effects of plant extracts is reported as LC₅₀ values in μg/ml with 95% confidence intervals as determined by a Finney PC computer programme (Table 1). Plant extracts exhibited LC₅₀ values > 100 μg/ml are considered inactive while those with < 200 μg/ml demonstrate very high cytotoxicity.

The strong toxic effect on A. cysts was observed for all ethanol crude (F1) extracts of the targeted plants except A. seyal and D. mespiliformis which were found inactive (LC₅₀ > 100 μg/ml). Their LC₅₀ values varied between 3.5 μg/ml to 246 μg/ml. This is in accordance with the popular use of the plants in the treatment of malaria fever in northern Nigeria.

Highly significant cytotoxicity was recorded on all the fractions of S.kunthiamum, C. kerstingi, J. curcas, C. singueana, E. senegalensis, P. stagninum and S. longepedunculata. The LC₅₀ values in case of C. singueana varied between 3.4 μg/ml to 11 μg/ml. Whereas LC₅₀ of C. kerstingi varied between 5.8 μg/ml to 23 μg/ml. Therefore, it seems that the data on C. singueana agrees with the positive findings of the studies carried out against rodent plasmodia infection, nociception, pyroxia and inflammation in mice and rats ^[14]. Less than 5 μg/ml of lethal concentrations was recorded by the ethanol (F1) leaf extract of J. curcas, ethanol (F1) and ethyl acetate (F4) extracts of C. singueana, methanol fraction (F6) of M. balsamina and petroleum ether (F5) fraction of E. senegalensis bark. All the fractions of D. mespiliformis and A. seyal did not show any activity in brine shrimp test (LC₅₀ > 1000 μg/ml).

In conclusion, over 90% of the plant species used in this study showed significant cytotoxicity in brine shrimp test (BST) at very low LC₅₀ values. The results could lend very strong scientific backing to the claims of the traditional medical practitioners in northern Nigeria, who use the plants to treat malaria and other pathogenic diseases. The results could serve also as a basis for pharmacological and phytochemical research, towards discovering novel bioactive compounds ^[15]. Furthermore, specific bioassay techniques are necessary in order to confirm these findings.

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