Constructing the interview schedules

2.2.3.0 Determination of Total Alkaloid

5g powdered plant sample was extracted with l00 cm³ solution of methanol:

water (1;1:v:v) it was then filtered and evaporated. The resultant residue was mixed with 20ml of 0.0025M H2SO4 and partitioned with ether to remove unwanted materials. The aqueous fraction was basified with NH4OH solution and was then extracted with excess chloroform to obtain the alkaloid fraction and separated. The chloroform extraction was repeated several times and the bulk extract was concentrated to dryness. The Alkaloid was weighed and the percentage was calculated with reference to the initial weight of the powder.(Trease and Evans,1989 and Nuhu et al.,2000).

43 2.2.3.1 Determination of Total Tannins

Procedure: Powdered sample (0.1g) was put into a 100cm3 conical flask and 50 cm³ of distilled water was added. The flask was gently heated to boiling for 1 hour, filtered hot and the fitrate collected in a 50 cm³ volumetric flask. The residue was washed several times with distilled water and the combined solution made to the volume with distilled water. To 0, 1, 2, 3, 4, and 5cm³of the standard tannic acid and 10 cm³ of the sample solution in a 50 cm³ volumetric flask, 2.5 cm³ Folin-Dense reagent and 10 cm³ Na²CO³ solution were added and made to volume with distilled water. The flasks were allowed to stand for 20 minutes after which Absorbance was measured at 760nm using Spectrophotometer. The calibration curve was plotted from which the concentration of the tannic acid (x) in the sample was extrapolated and tannin content in the sample was calculated using equation.(Allen et al.,1974).

2.2.3.2 Determination of Total phenolics

Total phenol contents in the extracts were determined by the modified folin-ciocalteu method (Wolfe et al., 2003). An aliquot of the plant extract was mixed with 5ml Folin-ciocalteu reagent (previousely diluted with water 1:10, ^v/v) and 4ml (75g/l) of sodium carbonate. The tubes were vortexed for 15s and allowed to stand for 30 min for a colour develop. Absorbance was then measured at 765nm

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using spectrophotometer. Total phenolic contents ware expressed as gallic acid equivalent based on calibration curve.

2.2.3.3 Determination of Total flavonoid

Total flavonoid were estimated using the method of (ordon ez et al., 2006). To 0.5ml of sample extract 0.5ml of 2% AlCl3 ethanol solution was added. After 60 min at room temperature, the absorbance was measured at 420 nm. A yellow colour indicates the presense of flavonoid. Total flavonoid content was expressed as gallic acid equivalent based on calibration curve.

2.2.3.4 Determination of Total proanthocyanidins

Determination of proanthocyanidins was based on the procedure reported by (Sun et al., 1998). A volume of 0.5ml of sample extract solution was mixed with 3ml of 4% vanillin methanol solution and 1.5ml HCl. the mixture was allowed to stand for 15min.The absorbance was then measured at 500nm using spectrophotometer. Total proanthocynidins content was expressed as gallic acid equivalent based on calibration curve.

2.2.4 Hydrogen peroxide radical scarvenging (H2O2) assay

The ability of plant extracts to scavenge hydrogen peroxide is determined according to the method of (Ruch et al.,1989). A Solution of hydrogen proxide (40mM) was prepared in phosphate buffer (50mM, Ph 7.4).The concentration of hydrogen peroxide was determined by absorption at 230nm using a spectrophotometer.1cm³ of Extract 1mg/ml was added to 0.6cm³ hydrogen peroxide solution in buffer and absorbance at 230nm was determined against a

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blank solution containing phosphate buffer without hydrogen peroxide. The percentage of hydrogen peroxide scavenging was calculated as follows.

Where; A0 is the absorbance of control A1 is the absorbance of the test.

2.2.5 Minerals Analysis

Procedure for Mineral Analysis

The mineral elements were analysed using Walinga et al. (1989). The mineral elements in the samples were brought into solution by wet digestion techniques. One gram of the dried samples was put into Kjeldahl digestion flasks to which 24cm³ of a mixture of concentrated; HNO³, H²SO⁴, and 60% HClO⁴ (9:2:1 v/v) was added. The mixture was then allowed to stand overnight to prevent excessive foaming. The flask was put on a heating block and digested to a clear solution, cooled and the content filtered into 50cm³ volumetric flasks through Whatman filter paper. 4cm³ of 5% aqueous lanthanum chloride heptahydrate (LaCl37H2O) was then added as releasing agents to overcome phosphate interference. The flask was then made up to the volume with distilled water. Blank was prepared in similar manner without samples being added.

2.2.5.0 Analysis of Metals using AAS.

In metal analyses (Cu, Mg, Mn, Zn, and Fe) atomic absorption spectrometry (AAS) was used. The principle of the method is based on nebulizing a sample solution into an air-acetylene flame where it vaporized.

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Elemental ions are then atomized and the atoms formed absorb radiation of the characteristic wavelength from the hollow cathode lamp. The absorbance measured, is proportional to the amount of analyte in the sample solution. The concentration of the analyte was calculated using equation.

2.2.5.1 Analysis of Sodium and Potassium using AES

Using flame photometer (corning 400 UK), the galvanometer reading was adjusted to zero with distilled water and then to full scale with standard solution of 25ppm. The other standard solutions were subsequently aspirated along with the sample and blank solution. Calibration curve was plotted from which the concentration of sodium and potassium were determined using equation.

2.2.5.2 Determination of Phosphorus

The phosphorus content of the sample was determined colorimetrically using the vanadate-molybdate blue method (james, 1995). The principle of the method is based on the reaction between phosphorus and ammonium molybdate to form a heteropolyphosphomolybdate complex. The complex was then reduced by tin (II) chloride solution to form a blue-puple complex (molybdenum blue), the intensity of which was measured at 710nm wavelength.

The absorbance measured is directly proportional to the concentration of phosphorus in the sample.

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To a series of 50cm³ volumetric flasks, 0,1,2,3,4, and 5cm³ of the 50ppm standard phosphorus were added. To each flask a drop of phenolphthalein was added and the solution neutralized with ammonia solution (1:4, v/v) and made up to about 40cm³ with distilled water. Furthermore, to each flask, 10cm³ of vanadate-molybdate and 0.7cm³ of 2% tin (II) chloride solutions were added, shaken, made up to volume and set aside in a cupboard for 10 minutes for a colour develop. Similarly, 0.5cm³ each of the digested samples was pipette into a 50cm³ flask and treated as for the standard solutions. The absorbances of both the standards, blank and samples were measured with spectrophotometer 6100 at wavelength of 710nm. The calibration curve was plotted from which the concentration of phosphorus in the sample was extrapolated using equation.

2.2.7 Extraction and chemical Analysis of essential oils

The fresh plant material 100g of each species was subjected to hydro-distillation using a Clevenger type apparatus for 3 hours.The resulting essential oil was kept in refrigerator till analysis, Chemical analysis of the oils were achieved by GC-MS. Gas Chromatograpy coupled with Mass Spectrometry. Components of the essential oils were identified.

Analysis of the essential oil: The GC-MS analysis was performed with a quadruple GC-MS system, Agilent GC model 6890N and Agilent mass selective detector, 5973 series; capillary column (30 m x 0.25 mm; 0.25 μm film thickness). The carrier gas was helium and column head pressure of 15 psi yielding a linear flow rate of 0.8 m/min. The split ratio was 1: 10 and the initial

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column temperature was held at 200oC for 15 min and then raised at 10oC/min and maintained at 260oC until all components had eluted. The components were identified by matching their mass spectra in the Wiley 275 library and their

retention indices were compared with literature values. .