CHAPTER THREE 3.0       MATERIALS AND METHODS 3.1       NON-DISPOSABLES Extraction tank, beakers, measuring cylinder, water bath, tests tubes rack, test tubes, sample pipette, calibration pipette, Bunsen burner, matchet, diesel bunder, separating funnels, spatula, cork borer, refrigerator, meter rule, pencil distiller, eccentric oven, incubator, autoclave, clamp stand, inoculating hoop, spirit lamp, masking tape. 3.1.1   Disposables: Whitman Filter paper, petridishes, face mask, cotton wool, Aluminum foil, filter paper, masking tape, pen, permanent marker, hand gloves, hand towel, injection bottles, stock bottles. 3.1.2   Reagents: 1% Aqueous hydrochloric acid 10% Fenic chloride Acetic anhydride Dragendorff reagent Concentrated sulphuric acid Sodium bicarbonate Concentrated hydrochloride acide Bromine water Magnesium metal Ammonium hydroxide solution Fehlin chloride A and B Benzene solution Chloroform solution Methanol (redistilled) Glacial acetic acid 3.1.3   Solvents: Sterile distilled water Ethanol Chloroform Methanol Ethlacetate N-hexane 3.1.4   Media – Nutrient broth 3.1.5   Organisms:                                      Codes Staplylococcus aurous              LIO Escherichia coli                         LIO LIO – locally isolated organism

3.1.6 Drugs: 40 grams of Mangifera indica extract 40 grams of Carica papaya extract 1 gram of methanol, ethylaceta, chloroform and N-hexane fractions of Maifera indica and Carica papaya respectively. Genterincin injection (80mg/2ml) (control). 3.2       Methods: 3.2.1   Collection of Mangifera indica (mango) and Carica papaya The fresh leaves of mango and pawpaw were collected in February, 2014 at Uyo, in Uyo Local Government Area of Ikwa Ibom State of Nigeria. Identification Number for Mangifera indica: UUH, No: 3C, Carica papaya: UUH, No: 18A. 3.2.2   Processing of the leaves of Mangifera indica and Carica papaya The leaves were reduced to smaller sizes using knife and then dried under the sun several days separately. These leaves were further comminuted. The fragments were then stored in a polythene bags free of moisture until required. 3.2.3   Extractions 600g of the comminuted leaves of Mangifera indica and Carica papaya respectively were macerated separately in methanol (redistilled) 1.5L respectively for 72 hours with intermittent shaking, all in separate tank extract was filtered using cotton wool and the filtrate further filtered using whatma no.1 filter paper to have the methanolic filtrate respectively. The extract were then concentrated to a constant weight. The concentrated extracts were weight separately and stored at 4oC in well closed beakers for further analysis.       3.2.4   Phytochemical screening: Phytochemical screening for secondary metabolites in the plant were performed using standard procedures as described by (Sofowora 1993, Trease and Evans, 2006) and Harborne 1998 as follows: 3.2.4.1            Test for alkaloids: about 0.5g each of the leave extract was boiled in 5ml of 1% aqueous hydrochloric acid over a steam bath; the clear filtrate was treated with few drops of dragendoff’s reagents. Turbidity or a pink or red precipitate was taken as preliminary evidence for the presence of alkaloids in the extract (Harborne, 1973). 3.2.4.2            Test for tannins: about 0.5g of each leaves extract was strived with 5ml of distilled water, 5% ferric chloride was added to the filtrates separately. A blue black, green or blue green precipitate was taken as evidence for the presence of tannins (Trease and Evans, 1989).

1. 1. 1. 1. Test for anthraquinones: About 0.5g of each of the leaves extract was boiled with 5ml of 10% sulfuric acid and filtered while hot. The filtrates were each shaken with 2.5ml benzene, the benzene layers were separately and half it own volume of 10% ammonia solution added

A pink, red or violet coloration in the ammonia phase (lower phrase) indicated the presence of anthraquinone derivative in the extract (Trease et al, 1989). 3.2.4.4            Test for flavonoids: Fe pieces of magnesium metal was added to 5ml of the leave extract plus conc Hcl. The precipitated was taken as evidence of preliminary presence of flavonoids (Trease et al, 1989). 3.2.4.5            Test for saponins: About 0.5g of each plant leaves was shaken with water in a test tube frothing which persisted on warning was taken as a preliminary evidence for the presence of saponins (Wall et al, 1952). About 0.5g of each extract was added to about 5ml mixture of 5% sodium bicarbonate and Fehling’s solution and boiled. Presence of the brown precipitated was taken as evidence for the presence of saponin. 3.2.4.6            Test for cardiac glycosides: Lieberman’s test: 0.5g of plant leaves extracts were dissolved in 2ml of acetic anhydride and cooled well in ice. Sulphuric acid was then careful added. A color change from violet to blue green indicated the presence of a steroidal nucleus Shoppee, 1964). Keller –Killiani test: About 0.5g leaves extract was dissolved in 2ml of glacial acetic acid containing one drop of ferric chloride solution. This was then underplayed with 1ml concentrated sulfuric acid. A brown ring at interface indicated the presence of deoxy sugar of cardenolide. 3.2.4.7            Test for terpenoids: A little of each portion was dissolved in ethanol. To it 1ml of acetic anhydride was added to each followed by the addition of conc. H2SO4,  a change in colour from pink to violet showed the presence of terpenoids (Sofowora, 1993).

3.2.5   Partitioning fro both Mangifera indica and Carica papaya: About 40g of Mangifera indica and Carica papaya crude extract was dissolved separately in 300ml of methanol and 100ml of water was then added to the crude extract. The methanolic aqueous extract was them introduced into the separating funnel in the clamp; the following solvents were added separately in decreasing order of polarity; ethyl acetate, chloroform, and n-hexane to have the different fractions. The solvent portions were carefully collected into a beaker and concentrated to dryness to yield the various fraction extracts. The procedures were repeated and each extract fractions stored in clean and well closed beaker at 4oc in a refrigerator for further evaluation.  3.2.6   Preparation of culture medium