Fresh leaves of A. montanuswith stalks were collected/ harvested in the months of March and April along a stream in northern part of Katsina-Ala township of Katsina-Ala Local Government Area of Benue State. Katsina-Ala town is located on latitude 7o 10ʹ N and longitude 9o 19ʹ E in the middle belt (Guinea Savannah) of Nigeria. A sample of the plant was brought to Zaria and was identified/ authenticated by a plant taxonomist at the Herbarium, Department of Biological Sciences, Ahmadu Bello University, Zaria, where a voucher specimen was deposited and assigned a voucher number 7037.

The harvested leaves were air-dried at room temperature until a constant weight was obtained, then pounded in a wooden mortar with pestle. The pulverized product weighing about 6 kg was stored in an air-tight nylon bag under cool, dark conditions at room temperature until use.

During extraction, 1 kg (250 g per separating funnel) of the pulverized product was soaked in 95% ethanol (EtOH) in the ratio 1:6 w/v in four separating funnels (pre-plugged with cotton wool) for 72 hours. The extracted solution collected in excess of the solvent was transferred in evaporating dishes and concentrated to dryness over water baths at 60oC. The dried extract obtained after concentration was then weighed to determine the percentage yield. Thereafter, 100 g of the mixture was suspended in 300 mL of 17.65% methanol (MeOH) in a large beaker. The solution was then partitioned with chloroform in equal volume in a separating funnel to yield chloroform (CHLO) and aqueous (AQ) portions. Lastly, the aqueous portion was further partitioned with n-butanol in equal volume also, to obtain final n-butanol (BuOH/BUT) and aqueous extracts. In each partitioning step, the mixtures were vigorously shaken to re-suspend the particles. Impurities were pooled together in a separate beaker and discarded. The different portions collected in separate conical flasks were again concentrated to residue over the water bath at 60oC and weighed to determine percentage yield in terms of the mass of the crude ethanol extract (CEE). These different fractions of leaf extracts of A. montanuswere packed in clean air-tight glass bottles and stored at room temperature in the laboratory hood until used.

One gramme of crude ethanol leaf extracts of A. montanuswas dissolved in 100 mL of distilled water in three test tubes and subjected to qualitative phytochemical screening employing standard screening tests 18.

Thin Layer Chromatography (TLC) was carried out on the crude ethanol leaf extracts of A. montanusand the different fractions thereof using the method of Mehta 19. Little quantities of the Crude Ethanol, n-Butanol, Chloroform and Aqueous Extract of A. montanusleaf were fetched with a spatula and dissolved in 5 mL in their respective solvents in well labeled Bijou bottles. These were used to spot 10 cm x 5 cm TLC plates (already coated with 0.25 mm silica gel 60F254 that were marked at about 1.0 cm intervals and 1.5 cm from the bottom) using capillary tubes and were allowed to air dry. The plates were then placed (the spotted portion downwards) in an air-tight TLC tank that was already charged with eluting solvent (chloroform-ethyl acetate-formic acid, 5:4:1). The respective plant constituent(s) were allowed to drag upward along the stationary phase from the spots by capillary action until the solvent front was seen reaching the top. The plates were then removed, marked, air-dried and sprayed with the detecting (spray) reagents (1% FeCl₃, Dragendoff s and Anisaldehyde-H₂SO₄ in 10% ethanol) 20 in the fume hood to develop the chromograms. Retention factor (Rf) value of the compound(s) was calculated by dividing the distance travelled by the compound (solute) by that travelled by the solvent front 1819.

Ninety (5-10 weeks) apparently healthy albino mice, Mus musculus of both sexes weighing between 15 and 25 g were obtained and were housed in well fenestrated plastic cages and allowed free access to standard feed and tap water. Within the two weeks of acclimatization, faecal pellets obtained per rectum from the group set aside for anthelmintic studies were analysed and the mice treated with broad spectrum anthelmintic, albendazole at 10 mg/kg for deparasitization.

Heligmosomoidesbakeriadult were obtained from Helminthology Laboratory of the Department of Veterinary Parasitology and Entomology, University of Nigeria, Nsukka-Nigeria where they were maintained in mice. Both infected mice and infective (L₃) larvae were transported to Ahmadu Bello University, Zaria. They were immediately multiplied and maintained under laboratory conditions.

The modified method of 21 was used to obtain clean infective larvae used for anthelmintic trial. The larval suspension was allowed to stand on the bench for one hour. The supernatant fluid was carefully aspirated using Pasteur pipette. To ensure an even distribution of the larvae, the suspension was mixed by gentle shaking of the tube. Thereafter, an aliquot of 0.1 mL was drawn onto a clean microscope slide and larvae counted under x4 objective. This was repeated four more times, after which mean count obtained was used to determine the number of larvae/mL of the suspension.

Experimental infection of mice was by oral route (gavage). Mice were well restrained at the scruff and 0.1 mL of the larval suspension inoculated right into the oesophagus via a blunted tip slightly curved 18 gauge needle mounted on tuberculin (1 mL) syringe. One hundred and fifty (150) infective larvae (L₃) of H. bakeriwere inoculated into each worm-free mouse.

Seventy (70) mice positive for ova of H. bakeriat 15^th day post infection (PI) were randomly assigned into 14 experimental (treatment) groups of five mice each. Twelve (12) groups were administered with four (4) graded doses of CEE, BUT and AQ fractions. The remaining two (2) groups were controls, {positive control (Albendazole, ABZ 10 mg/kg) and negative control (Distilled water, DW 5 mL/kg)}. Each of the estimated doses of the various fractions of A. montanuswere administered to mice (according to grouping previously described) for five consecutive days; on the 18^th to 22^nd days post infection (PI) 22. Doses were chosen at a common logarithmic interval of 0.08 for all extract-treated groups. All treatments were administered orally.

On the 23^rd day PI, all the mice were deprived of food but not water for 24 hours so as to empty the gastrointestinal tract to make worm counting easier. Mice were euthanized in chloroform chamber and necropsied. Modified method 23 was used for worm count.

Anthelmintic efficacies of the extracts were accessed by counting the worms in the treated animals and comparing with counts from the untreated control mice. The percentage deparasitization (DP), (%DP- reduction in the worm counts) for the various groups was then calculated using the formula:

Where: N= mean worm count in untreated group.

n = mean worm count in treated groups 24.

Seventy percent (70%) reduction in worm count or more was considered significant at p<0.05 8.

The worm count data generated were summarized in tables and expressed in percentages (percentage deparasitization). One-way analysis of variance (ANOVA) and Tukey's Multiple Comparison Post Test (Graphpad Instat) were used for data analysis. The results were expressed as Mean ± SEM. Difference between Means of treated and untreated control groups was considered significant at p<0.05.

Extraction of pulverized leaf of A. montanus showed that 1,000 g yielded 255.84 g of crude ethanol (EtOH) extract. Solvent partitioning of 100 g of crude (EtOH) extract yielded 31.42 g, 11.58 g and 3.00 g of aqueous (AQ), n-butanol (BuOH) and chloroform (CF) fractions (extracts) respectively. The remaining portion was discarded as residue. The colour and percentage yields of the plant material is as shown in Table 1. Generally, partitioning with water resulted in the highest quantity of crude extract, while CF gave the least quantity. Figure 1

Acanthus montanus was selected according to earlier reports of anthelmintic efficacy 17 and folkloric claims. Among the fractions tested, n-butanol extractproduced profound anthelmintic activity. The degree of deparasitization obtained in this study is similar to that obtained from other studies with plants that were found to produce anthelmintic effects 2524. The results of the anthelmintic study indicate that n-butanol portion of A. montanusleaf produced significantly higher deparasitization followed by crude ethanol and then aqueous portions. The efficacy of the n-butanol extract (portion), for instance increased with dose, an indication of graded response of the parasite to the drug 2627. This is probably due to the activity of tannins that was found to be more in the n-butanol extract (fraction). Different classes of anthelmintics are known to show profound effects on the physical activities, generally culminating into loss of mobility and mortality of helminth parasites in a dose-dependent manner 28. The active principle(s) in the A. montanusextracts responsible for this anthelmintic activity might be individual phytocomponents as detected during phytochemical screening or a number of them working in synergy. Plant secondary metabolites found in this study have been reported to occur in other plants with anthelmintic activity 257. In the present study, tannins (condensed tannins) were likely responsible for the observed profound anthelmintic activity 29 due to its abundance in the extract screened phytochemically and confirmed by thin layer chromatography. Chemically, tannins are polyphenolic compounds which are uncouplers of oxidative phosphorylation in helminth parasites 30. Some synthetic phenolic anthelmintics as niclosamide, oxyclozanide and bithionol, are reported to interfere with energy generation in helminth parasites by uncoupling oxidative phosphorylation 31. It is possible that the large amount of tannins detected in the n-butanol leaf extracts of A. montanus produced similar effects. Another possible anthelmintic effect of tannins is that they can bind to free proteins in the gastrointestinal tracts of host animal 32 or glycoprotein on the cuticle of the parasite 33, and may thus cause death.

Polyphenolic compounds which are reported to be present in leaves of mainly dicotyledonous plants are potent anthelmintics. Condensed tannins which are derived from flavonol are soluble in water and are capable of precipitating proteins. They are reported to be found in cell walls or stored in vacuoles, stems, leaves, flowers or seeds 3435. Tannins protect the intestine from reinfection by tanning proteins in the lining of the gut (intestine) 36. Consumption of vegetative portions of such plants is advocated. This may explain why the plant s (A. montanus) aerial parts are used as vermifuge by the Etulo natives of Benue State, Nigeria. Its tannin content may explain its anthelmintic activity.