Phytochemical Methodology Extraction of phytochemicals 0.2g of extract was weighed and transferred in a test tube and 15ml ethanol and 10ml of 50%m/v potassium hydroxide was added. The test tube was allowed to react in a water bath at 600c for 3hrsmins. After the reaction time, the reaction product contained in the test tube was transferred to a separatory funnel. The tube was washed successfully with 20ml of ethanol, 10ml of cold water, 10ml of hot water and 3ml of hexane, which was all transferred to the funnel. This extracts were combined and washed three times with 10ml of 10%v/v ethanol aqueous solution. The ethanol solvent was evaporated. The sample was solubilized in 1000ul of pyridine of which 200ul was transferred to a vial for analysis. Quantification by GC-FID The analysis of phytochemical was performed on a BUCK M910 Gas chromatography equipped with a flame ionization detector. A RESTEK 15 meter MXT-1 column (15m x 250um x 0.15um) was used. The injector temperature was 280oC with splitless injection of 2ul of sample and a linear velocity of 30cms-1, Helium 5.0pa.s was the carrier gas with a flow rate of 40 mlmin-1. The oven operated initially at 2000c, it was heated to 3300c at a rate of 30c min-1 and was kept at this temperature for 5min. the detector operated at a temperature of 3200c. Phytochemicals were determined by the ratio between the area and mass of internal standard and the area of the identified phytochemicals. The concentration of the different phytochemicalss express in ug/g. A.D. Buss, M.S. Butler (Eds.), Natural product chemistry for drug discovery, The Royal Society of Chemistry, Cambridge (2010), p. 153 Kelly da, Nelson R. (2014) characterization and quantification by gas chromatography of Phytochemicals. J. -Braz. Chem.. Soc. Vol 25. Qualitative phytochemical Phytochemical screening Qualitative phytochemical analysis The phytochemical analysis of the leaves of Fluted pumkin, Aloe vera and bitter leaf leaves, and black pepper willbecarried out according to the method of Harborne (1998) and Trease and Evans (1983). The methods are shown below: Test for Alkaloids Reagent: 2% HCl Procedure for processing/preparation - Pipette 1.0ml of extract in a test tube - Pipette 5.0ml of 2%HCl in a test tube - Heat the test tube in a water bath (Memmert) for 10minutes - Filter using Whatmann No 1 filter paper - Use the filtrate for the following tests Wagner’s reagent test Principle: Alkaloids under acidic condition and at room temperature reacts with iodine and potassium iodide to give reddish brown precipitate. Reagent: Wagner’s reagent: 2g of iodide and 3g of potassium iodide are weighed, mixed and dissolved in 30ml distilled water and made up to 100ml with distilled water. Procedure - Pipette 1.0ml of filtrate in a test tube - Pipette 1.0ml of Wagner’s reagent in the test tube - Mix properly and observe for colour change A reddish brown precipitate indicates presence of alkaloids. Meyer’s reagent test Principle: Alkaloids under acidic condition and at room temperature reacts with mercuric chloride and potassium iodide to give a cream colour or precipitate. Reagents: Dissolve Meyer’s reagent: 1.4 of mercuric chloride in 60ml distilled water and 4.5g of potassium iodide in 20ml distilled water. The two solutions are mixed and diluted to a 100ml with distilled water. Procedure - Pipette 1.0ml of filtrate in a test tube - Pipette 1.0ml of Meyer’s reagent in the same test tube - Mix properly and observe for colour change. Cream colour precipitate indicates presence of alkaloids (Trease and Evans (1989). Test For Flavonoids: FERRIC CHLORIDE TEST FOR PHENOLIC NUCLEUS: Principle: Phenolic nucleus reacts with ferric chloride at room temperature to give greenish brown or black colour or precipitate. Reagent: 10% Fecl2 (weigh 10.0g Fecl2 and dissolve in 100ml of distilled water). Procedure: - Pipette 1.0ml extract into a test tube. - Pipette 1.0ml 10% ferric chloride in the test tube - Mix and observe any change in color. Greenish brown or black colour/precipitate is an indication of presence of phenolic nucleus. LEAD ACETATE TEST Principle: Flavonoids at room temperature reacts with lead acetate to give a yellow color or precipitate. Reagent: 10% lead acetate(weigh 10.0g lead acetate and dissolve in 100ml distilled water). Procedure: - Pipette 1.0ml extract into a test tube - Pipette 1.0ml of 10% lead acetate solution in the same test tube - Mix properly and observe colour change or precipitate which indicates presence of flavonoids. SODIUM HYDROXIDE TEST Principle: Flavonoids at room temperature and under alkaline pH forms observable precipitate. Reagent: Dilute NaOH: (weigh 40g of NaOH and dissolve in 1 litre of distilled water). Procedure: - Pipette 1.0ml of extract into a test tube. - Pipette 1.0ml of dilute NaOH solution the same test tube - Mix and observe any change in color. - Formation of precipitate indicates presence of flavonoids. (Trease and Evans (1989) Test for Tannins Acid Test Principle: phlobotannins under acidic condition reacts with dilute Hcl to give a red colour or precipitate. Reagent: 1% Hcl, pipette 1.0ml concentrated Hcl and made up to 100ml with distilled water. Procedure: - Add 3.0ml of extract to 2.0 of 1% Hcl Observe presence of red colour or precipitate This indicates the presence of phlobotannins Lead Acetate Test Principle: phlobotannins reacts at room temperature with lead acetate to give dark-blue to black precipitate. Reagent: 5% lead acetate: weigh 5.0g lead acetate and dissolve in 100ml distilled water Procedure: - Pipette into a test tube, 2ml of extract Add 3 drops of 5% lead acetate solution to the extract A dark blue to black precipitate indicates presence of phlebotomy’s. Test for Resins About 0.2g of the powdered materials were extracted with 15ml of 90% ethanol. The alcoholic extract was then poured into 20ml distilled water in a beaker. A precipitate occurring indicates the presence of resins. Test for Saponins About 2g of the powdered sample was boiled in 20ml of distilled water in a water bath and filtered. 10ml of the filterate was mixed with 5ml of distilled water and shaken vigorously for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously, then observed for the formation of emulsion. Test for Cardiac Glycosides One(1ml) of the extract was added 10cm3 of 50% H2So4 and was heated in boiling H2O for 5 minutes. 10cm3 of fehlings solution (5cm3 of each solution A and B) was added and boiled. A brick red precipate indicating presence of glycosides was observed. Test for Terpenoids Five (5ml) of each extract was mixed in 2ml of chloroform (Numex, India), and concentrated H2So4 (3ml) was carefully added to form a layer. A reddish brown coloration of the inner face was formed to show positive results for the presence of terpenoids. DPPH SCAVENGING EFFECTS The ability of the leaf samples to scavenge the DPPH radical was tested in a rapid dot-plot screening and quantified using a spectrophotometric assay. PRINCIPLE DPPH radical reacts with an antioxidant compound that can donate hydrogen, and gets reduced. DPPH, when acted upon by an antioxidant, is converted into diphenylpicryl hydrazine. This can be identified by the conversion of purple to light yellow colour. DOT-PLOT RAPID ASSAY The rapid screening assay was performed by the method proposed by Soler-Rivas et al. (2000). REAGENTS 1. TLC plates (silica gel 60 F254-Merck) 2. DPPH (0.4mM) in methanol PROCEDURE Aliquots of sample samples (3 l) were spotted carefully on TLC plates and dried for 3 minutes. The sheets bearing the dry spots were placed upside down for 10 seconds in a 0.4mM DPPH solution and the layer was dried. The stained silica layer revealed a purple background with yellow spots, which showed radical scavenging capacity. DPPH SPECTROPHOTOMETRIC ASSAY The scavenging ability of the natural antioxidants of the leaves towards the stable free radical DPPH was measured by the method of Mensor et al. (2001). REAGENTS 1. DPPH – 2,2-diphenyl-2-picryl hydrazyl hydrate (0.3mM in methanol) 2. Methanol PROCEDURE The leaf samples (20μl) were added to 0.5ml of 0.1mM methanolic solution of DPPH and 0.48ml of methanol. The mixture was allowed to react at room temperature for 30 minutes. Methanol served as the blank and DPPH in methanol, without the leaf samples, served as the positive control while butylated hydroxytoluene (BHT) served as reference. After 30 minutes of incubation, the discolouration of the purple colour was measured at 518nm in a spectrophotometer (Genesys 10-S, USA). The radical scavenging activity was calculated as follows: Scavenging activity % = 100- A518 (sample) – A518 (blank)× 100 A518 (blank)
