Anti-plasmodial Property Of Moringa Oleifera Seed Extract On Swiss Mice

CHAPTER THREE
MATERIALS AND METHODS
3.0 Collection of Plant
The seed of Moringa oleifera were freshly obtained directly from it tree at botanical garden  
University of Ilorin, Ilorin Kwara state, the seed were authenticated at the Herbarium of
Department of Plant  Biology, university of Ilorin, Ilorin kwara state, Nigeria by Mr. Bolu Ajayi with the voucher number UILH/001/1008.The seed were kept in at room temperature away from sunlight  untill it is dried. The seed of each plant
was pulverised
separately using a local mortar and pestle. The pulverised seed was kept in an air-tight
container. To obtain the crude extract, using soxhlet extraction process. 200 g of  each
powdery seed was weighed into separate containers using a sensitive weighing balance,
then the extraction yield was calculated by dissolving the measured 200 g in each container  
with 500 ml of petroleum ether.
3.1 Control drug sample   
Chloroquine antimalarial drug was used as a control drug in the experimental animal. It was purchased at Fiolu Pharmaceuticals Ilorin, Kwara state. The tablet was crushed into powder using a mortar and pestle. 100 g of each tablet was crushed and dissolved in 1000 ml of distill water. Oral administration of this aqueous solution was based on the calculations made in relation to body weight of the experimental animals.
 Therapeutic dosage(mg)  x body weight of mice÷400 mg/ml
3.2 Experimental Animals   
 Twenty four swiss mice of both sexes with an average weight of 30 ± 3 g were purchased from the Animal Holding Unit of the Department of Vertinary Physiology, Biochemistry and Pharmacology, Faculty of Vertinary Medicine, University of Ibadan, Nigeria. Animals were housed in wire mesh cages under standard conditions, and the study was conducted in accordance with the recommendations from the declaration of Helsinki on guiding principles in the care and use of animals (Washington, 2011).
3.3 Material and Reagents
Chloroquine, Petroleum ether,Digital weighing balance, syringes, needles, micro pipettes, cotton wool, hand gloves, dissecting kits, stop watch and record book, Phosphate buffered saline, other reagents were of analytical grade.   
3.4        Extraction from the plant leaves       
Performing the Soxhlet method of petroleum  ether extraction
After each plant was crushed, using a pestle and mortar, to provide a greater surface area. The       plant material is ensure to be sufficient to fill the porous cellulose thimble. Following this, the solvent (500 ml of petroleum ether) is added to a round bottom flask, which is attached to a Soxhlet extractor and condenser  on an isomantle. The crushed plant leave of ficus thioningii  is loaded into the thimble, which is placed inside the Soxhlet extractor. The side arm is lagged with glass wool. The solvent is heated using the isomantle and will begin to evaporate, moving through the apparatus to the condenser. The condensate then drips into the reservoir containing the thimble. Once the level of solvent reaches the siphon it pours back into the flask and the cycle begins again. The process should run for a total of 16 hours. Once the equipment has set up the extraction can be left to run without direct supervision. It is not advised to leave the equipment completely alone due to the mix of running water and an electrical appliance, so a technician or other lab user should be made aware. The equipment can be turned on and off when overnight running is not permitted, and the time split over a number of days. For good practice, a control should be added. This could be plant material that has no known antimicrobial effect (for example, a carrier oil such as sunflower oil) at the testing stage Once the process has finished, the petroleum ether should be evaporated using a rotary evaporator, Leaving  a small yield of extracted plant material (about 2 to 3 ml) in the glass bottom flask.the same procedure was repeated for the second leave of achranthes aspera.