3. Extraction and preliminary phytochemical analyses 1 Introduction
3.2.2 Extraction procedure
3.2.2.1 Preliminary extraction procedure
The dried fine powder (1 g) of the plant was added in each centrifuge tube and this was followed by 10 mL of different solvents (Hexane, Chloroform, Dichloromethane, Ethyl acetate, Acetone, Ethanol, Methanol, Butanol and Water). The mixtures were shaken separately for 10 minutes at a speed of 200 rpm in a shaking incubator (New Brunswick Scientific Co., INC.). The extracts were filtered using cotton wool and a funnel into labelled pre-weighed glass vials and the solvents were evaporated using a fan. The quantity of the plant extracts was determined by subtracting the mass of the empty pre-weighed glass vials from the mass of the dried crude extracts in the vials.
The extracts were reconstituted in acetone to a final concentration of 10 mg/mL for subsequent assays.
3.2.3 Phytochemical screening on TLC
The phytochemical profiles of the crude extracts were determined on TLC by loading 10 µL of 10 mg/mL solution. The solutions of the extracts were loaded using a micropipette, at the bottom of the TLC plate, just 1 cm away from the base of the TLC plate. The plates were then put inside the saturated TLC tank that contained the following solvent: Ethyl acetate: Methanol: Water (30:5.4:5) [EMW] (polar), Chloroform: Ethyl acetate: Formic acid: 10:8:2 [CEF] (intermediate polarity: acidic), Benzene: Ethanol: Ammonium hydroxide: 18:2:0.2 [BEA] (nonpolar/basic) (Kotze and Eloff, 2002). Following the development, the plates were observed under the UV light (254 and 365 nm) to view separated compounds. The Vanillin-sulphuric acid reagent (0.1 g vanillin: 28 mL methanol: 1 mL sulphuric acid) was sprayed on the chromatograms and heated at 110°C in an oven to observe the colour development and the visualisation of non-fluorescing compounds.
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3.2.4 Preliminary screening for Phyto-constituents 3.2.4.1 Saponins
Exactly 0.5 g of the powdered leaf was suspended in a 5 mL of tap water and thereafter, the mixture was vigorously shaken and heated. The formation of froths indicated the presence of saponins (Odebiyi and Sofowaro, 1978).
3.2.4.2 Terpenes/terpenoids
The powdered leaf (0.5 g) was mixed with 2 mL of chloroform and 3 mL concentrated sulphuric acid. The formation of reddish-brown colour indicated the presence of terpenoids (Odebiyi and Sofowaro, 1978).
3.2.4.3 Phlobatannins
The powdered leaf (0.5 g) was dissolved in 5 mL of distilled water and filtered. The filtrate was boiled with 2 mL of 1% hydrochloric acid (HCL). The formation of a red coloured precipitate indicated the presence of phlobatannins (Borokini and Omotoyo, 2012)
3.2.4.4 Tannins
The powdered leaf (0.5 g) was dissolved in 5 mL of distilled water and then boiled gently and cooled. The solution of 1 mL was added in the test tube as well as 3 drops of 1% ferric chloride solution. The mixture was underplayed with 1 mL of concentrated sulphuric acid (H2SO4). The formation of a blue-black, brown-green, green or blue- green colour indicated the presence of tannin (Borokini and Omotayo, 2012).
3.2.4.5 Cardiac glycosides
The powdered leaf (1 g) was treated with 2 mL of glacial acetic acid containing one drop of 1% ferric chloride solution and the mixture was then underplayed with 1 mL of concentrated sulphuric acid (H2SO4). The sample was observed for colour change to indicate the presence of cardiac glycosides (Odebiyi and Sofowara, 1978).
3.2.4.6 Flavonoids
The diluted ammonia (5 mL) was added to a portion of the aqueous filtrate of the plant extract, followed by the addition of 1 mL of concentrated sulphuric acid (H2SO4). The sample was observed for colour change to indicate the presence of flavonoids (Borokini and Omotayo, 2012).
39 3.2.4.7 Steroids
Acetic anhydride (2 mL) was added to 0.5 g of powdered leaf, followed by the addition of 2mL of sulphuric acid (H2SO4). The sample was observed for colour change to indicate the presence of steroids (Borokini and Omotayo, 2012).
3.2.4.8 Alkaloids
The powdered leaf (0.5 g) was extracted with 95% ethanol in a shaking incubator (200 rpm) for 10 minutes and filtered using cotton wool and funnel. The extract was dried under a fan. The residue was redissolved in 5 mL of 1% of hydrochloric acid (HCL), followed by the addition of 5 drops of Drangendoff’s reagent. The colour change was observed to indicate the presence of alkaloids (Odebiyi and Sofowara, 1978).
3.2.5 Quantification of major phytochemicals 3.2.5.1 Total phenolic content
The quantity of phenolics present in each extract was determined by using the Folin- Ciocaltleu reagent method (Humadi and Istudor, 2008), with minor modifications. The plant material (10 mg/mL) was reduced to a concentration of 0.05 mg/mL in a test tube. Exactly 100 µL of 0.05 mg/mL of each plant extracts was transferred to a test tube and diluted with 900µL of distilled water, followed by an addition of 250 µL of Folin-Ciocaltleu reagent. A volume of 1.25 mL of 7% of Sodium carbonate was added to stop the reaction and the mixture was incubated in the dark at a room temperature for 30 min. A 10S UV-VIS spectrophotometer (Thermo Scientific Genesys) was used to determine the absorbance of the mixtures at 550 nm and the results were recorded.
A blank was prepared similarly except that the plant extracts were replaced by acetone. The results were obtained from the linear regression formula: 𝑦 = 3.025𝑥 − 0.1141. The Gallic acid standard curve was expressed as milligram gallic acid equivalence/ gram of the extract (mg of GAE/ gram extract). The concentrations of the standard used (1.25, 0.63, 0.31, 0.16, 0.08 mg/mL) and the experiment were conducted in triplicates.
3.2.5.2 Total tannin content
The Folin-Ciocalteu method described by Tambe and Bhamber (2014) was used to determine the tannin content in the plant extracts. Briefly, a volume of 50 µL of 7.5 mg /mL of each extract was added to a clean test tube containing 3.8 mL of distilled water.
A volume of 0.25 mL of Folin-Ciocalteu reagent was added to the mixture; the mixture
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was vortexed and then 0.5 mL of a 35% solution of sodium carbonate was added into the mixture. The mixture was transferred to a 10 mL of a volumetric flask and the volume of the mixture was made up to 10 mL by adding distilled water. The mixture was vortexed and incubated at room temperature for 30 minutes in the dark. A blank was prepared similarly as the test solutions without adding any extract but acetone.
The absorbance of the mixture was measured using the A 10S UV-VIS spectrophotometer (Thermo Scientific Genesys) at 725 nm. Gallic acid was used as a standard, and reference standard solutions were prepared, and the absorbance for the solutions was measured against the blank. The Tannin content was expressed as a milligram gallic acid equivalence/ gram of extract (mg GAE/g extract), which was calculated using the Gallic acid standard curve with equation, 𝑦 = 3.878𝑥 − 0.0023.
The concentrations of the standard used (1, 0.5,0.25, 0.125, 0.0625 mg/mL) and the experiment were conducted in triplicates.
3.2.5.3 Total flavonoid content
The flavonoid content was determined by the aluminium chloride colorimetric assay described by Tambe and Bhambar (2014). Briefly, 100 µL of 10 mg/mL was added to 4.9 mL of distilled water in a clean test tube, followed by an addition of 300 µL of 5%
sodium nitrite dissolved into distilled water. The mixture was left at a room temperature for 5 minutes. Thereafter, 300µL of aluminium chloride dissolved in distilled water was added to the mixture. The reaction was incubated at a room temperature for 5 minutes, after 2 mL of sodium hydroxide was added to the solution. The mixture in the test tube was then made up to 10 mL with distilled water. The blank was prepared in the same manner; however, 100 µL of acetone was added instead of the plant extracts. The absorbance of the mixture was determined using the A 10S UV-VIS spectrophotometer (Thermo Scientific Genesys) at a wavelength of 510 nm. The total flavonoid content of the sample was expressed as a milligram quercetin equivalence/gram of extract (mg QE/g extract) using the equation (𝑦 = 0.6522 − 0.0006) obtained from the quercetin standard curve. The concentrations of the standard used (500, 250, 125, 62.5, 31.25 µg/mL) and the experiment were conducted in triplicates.