Plant Materials
Plant Achillea wilhelmsii was purchased from the local market of Nasir Bagh, Board district Peshawar, KPK. Teucrium stocksianum was collected from nearby hills of University of Malakand in the month June - July 2009. The plants were authenticated by Professor Dr. Jehandar Shah, vice chancellor Shaheed Benazir Bhuto University, Sheringal Dir Upper, KPK. Voucher specimens, respectively, AW-2009 for Achillea wilhelmsii and T-01-2009 for Teucrium stocksianum were deposited in the herbarium of Department of Botany, University of Malakand. Ethical Committee of the department of pharmacy approved the experimental protocols as per animal byelaws 2008 of the University of Malakand "Scientific Procedures Issue I".
Preparation of extract and the crude saponins
170 grams of powdered materials of aerial parts of Achillea wilhelmsii and Teucrium stocksianum were extracted with petroleum ether by successive extraction in Soxhlet apparatus followed by extraction with commercial grade methanol. The solvents were subjected to rotary evaporation under vacuum to obtain dry semi solid extracts. The methanol extracts of both plants were further fractionated with n-butanol and water, in equal proportion, to get the n-butanol fraction. The crude saponins were precipitated with ether yielding 12.35 g of crude saponins of Achillea wilhelmsii (CSA) and 9.80 g of crude saponins of Teucrium stocksianum (CST) [18].
Drugs and chemicals
All the reagents used were of analytical grade (E. Merck). Piperazine citrate (GSK) and albendazole (GSK) were used as standard reference drugs in the experiments at concentration 10 mg/ml.
Statistical analysis and calculations
Statistical analysis was performed at 95% confidence interval. P value equal to or less than 0.05 was considered as significant. Microsoft XL sheet and Graph Pad prism were used to calculate mean, SEM and draw the curves for EC50 and LC50.
Brine Shrimp Cytotoxicity
Brine shrimp cytotoxic activity of crude saponins was determined as described by Meyer et al. (1982) with some modifications. Briefly describing, brine shrimp eggs (Artemia salina) were placed on one side of a small tank which was filled with sea water, covered with aluminum foil, and fully aerated. After 48 h incubation at room temperature and under illumination, the resulting nauplii (larvae) were attracted to the other side of the tank with a light source. The nauplii were collected with the help of a dropper.
Stock solutions (10 mg/ml) of CSA and CST were prepared by dissolving 20 mg of each sample in 2 ml of methanol. From the stock solution, 1000 μg/ml, 100 μg/ml, 10 μg/ml, 7.5 μg/ml, 5.0 μg/ml, 2.5 μg/ml and 1.25 μg/ml were prepared by taking 500 μl, 50 μl, 5 μl, 3.75 μl, 2.5 μl, 1.25 μl and 0.625 μl, respectively. The solvents were evaporated from the vials by exposing to evaporation for 24 hours. 2 ml of sea water was added and ten brine shrimps nauplii were transferred with the help of a dropper to each sample vial and the volume of sea water was adjusted to 5 ml. Negative control experiments contained 5 ml of sea water and ten brine shrimps. Survivors were counted after 24 h [19]. Percentage mortality of test concentrations and control was determined using the equation: % mortality = (no. of dead nauplii/initial no. of live nauplii) × 100. Experiments for each concentration were performed in triplicate.
LC50 values less than 100 ppm (100 μg/mL) were considered significant.
Anthelmintic activity
Adult roundworms (Ascaridia galli), tapeworms (Raillietina spiralis) and earthworms (Pheretima posthuma L. Vaill) were used to evaluate in vitro anthelmintic activity. The earth worms were collected from swampy water near the new boys' hostel, University of Malakand, Dir, KPK, Pakistan. Roundworms and tapeworms were obtained from the intestines of freshly slaughtered fowls. Their intestines were treated with normal saline solution to remove all the fecal matters. The worms were collected after dissection of intestines and maintained in normal saline solution, having an average size of roundworms, tapeworms and earthworms as 5-7 cm, 6-7 cm and 7-8 cm, respectively. The assay was performed by using adult earthworms in vitro because the earthworm has high resemblance, both anatomically and physiologically, with the intestinal roundworm parasite Ascaris lumbricoides of human beings. The prelabeled extracted saponins from both plants were prepared in distilled water at concentrations of 10, 20 and 40 mg/ml. Six worms, approximately of equal size, each of Pheretima posthuma, Raillietina spiralis and Ascaridia galli, were placed in petri dishes. Each petri dish contained 25 ml of test solution of the extracts. For reference standards, Albendazole and Piperazine citrate (10 mg/ml each) were used as positive controls, and distilled water was used as the negative control.
The experiments were run in triplicate. Before starting the experiments, standard drugs and test solutions were freshly prepared. Time for paralysis was recorded when no movement was observed except when shaken vigorously, whereas time of death was recorded when the worms did not show any movement by vigorous shaking nor when dipped in warm water (50°C) [20].