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evaporator and later left to dry in the fume extraction hood. Aqueous mixture was evaporated to dryness using the lyophilizer.
3.2.4 Phytochemical Analysis
Quantitative measurement of polyphenols, flavonols, flavanols, alkaloids and carotenoids were performed using spectrophotometry and colorimetry techniques.
3.2.4.1 Determination of Total Polyphenols
Total polyphenols were determined by adapting the method of Waterhouse55. This involved the use of Folin-Ciocalteu reagent and sodium carbonate. 25µl of samples followed by 125µl of 200mM Folin reagent was dispensed into each well. After 5 minutes, 100µl of 7.5% aqueous sodium carbonate was added. A blue colour complex was formed from the reaction between phenolic compounds and Folin’s reagent. The plates were incubated at room temperature for 2 hours. The absorbance was measured at 765 nm.
The intensity of this colour-complex measured is directly proportional to the concentration of phenolic compounds present in the sample. Gallic acid (0, 20, 50, 100, 250 and 500mg/L) in 10% ethanol was used to prepare the standard curve. Results were expressed as mg gallic acid equivalents per gram dry mass of the plant (mgGAE/g DM).
3.2.4.2 Determination of Flavonol Content
Flavonol content was determined using quercetin (0, 5, 10, 20, 40, and 80mg/L) in distilled water as a standard. 50mg of the extracts were weighed and dissolved in water (for the aqueous extract) or 95% ethanol (for ethanolic and ethyl acetate extracts). 12.5µl of samples were pipetted into the 96-well plates. 12.5µl of 0.1% HCl was added to each well, followed by 225µl of 2% HCl. This was left to incubate for 30 minutes at room temperature56. Readings were taken at 360nm and results expressed as mg quercetin equivalent per gram dry mass of the plant (mgQE/g DM).
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3.2.4.3 Determination of Flavanol Content
Spectrophotometric determination of flavanols was done using 4-(dimethylamino)- cinnamaldehyde (DMACA) reagent. DMACA reacts with flavanols to form a characteristic light blue colour. 1mM Catechin hydrate was used to prepare caliberation curve at the following concentrations; 0, 5, 10, 25, 50, 100µM. DMACA reagent was prepared by dissolving DMACA in a solvent-mixture of methanol-HCl (in the ratio 3:1 respectively) to a final concentration of 10µg/ml. 25µl of sample was dispensed into each well, 275µl of DMACA reagent was added to each well. Plates were incubated for 30 minutes at room temperature. Readings were taken at 640 nm. The intensity of the coloration is directly proportional to the concentration of flavonols in the sample. Results were expressed as mg catechin equivalent per gram dry mass of the plant (mgCatechin/g DM).
3.2.4.4 Determination of Total Alkaloids
Total alkaloids was determined using spectrophotometric method described by Fadhil, et al57. The assay is based on the reaction of Bromocresol green (BCG) with alkaloids which produces a yellow-coloured complex. 5mls of 2M sodium phosphate buffer (pH 4.7) was added to 500µl of AD extracts, followed by 5mls of BCG solution. The mixture was vortexed, and the yellow complex formed was extracted with 12mls of chloroform and strongly vortexed. Two layers were formed; the lower layer which contains the alkaloid (yellow complex in chloroform) is pipetted (300µl) into 96 wells-plate, absorbance read at 470nm against blank. 0.1mg/ml Atropine of varying concentrations (8, 12, 16, 20, and 24µg/ml) in 2M sodium phosphate buffer (pH 4.7) was used as a standard reference.
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3.2.5 Antioxidant capacities
3.2.5.1 ORAC Assay
The ORAC assay kinetically measured the peroxyl-radical absorbing potential of the antioxidants present in the plant extracts according to the method of Ou et al58. Trolox;
a water-soluble analogue of vitamin E, was used as the antioxidant standard reference.
The change in fluorescence of the reaction mixture was monitored over a period of 2 hours and recorded every minute (excitation = 485 nm and emission = 535 nm). Results were determined with a regression equation, that relates trolox concentrations with the net area under the kinetic fluorescein decay curve (y = ax2 + bx+ c). The ORAC values were expressed in micromoles of trolox equivalents per gram of sample (μmol TE/g sample).
Working solutions were 1.2mM fluorescein solution, 75mM phosphate buffer (pH 7.4) and 2,2’-Azobis (2-methylpropionamidine) dihydrocholide (AAPH) solution. Samples were diluted (x10) using phosphate buffer. 12µl of samples were pipetted into the wells, followed by 138µl of fluorescein solution. 50µl of AAPH was added and readings were taken. 12µl of samples, followed by 138µl of fluorescein solution and 50µl of AAPH were pipetted into the wells, readings were taken.
3.2.5.2 FRAP Assay
The ferric-reducing power of the plant extracts was determined using spectrophotometry according to the method of Benzie and Strain59. The stock solution (FRAP reagent) was prepared with acetate buffer (300mM, pH 3.6), TPTZ solution, Iron (III) chloride hexahydrate solution and distilled water. The solution mix was in a ratio of 10:1:1:2 respectively. 10µl of sample followed by 300µl of FRAP reagent was pipetted into the microplate. After 30 min of incubation at room temperature, the absorbance was determined at a wavelength of 593 nm. Ascorbic acid was used as the reference
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standard and results expressed as milligram of ascorbic acid per gram of extract (mgAAE/g sample).
3.2.5.3 TEAC Assay
TEAC was used to measure the antioxidant capacity of the plant samples using ABTS (2,2’-azino-bis (3-ethylbenz-thiazoline-6-sulphonic acid)) decolorization assay. This was carried out by assessing the ability of the sample to scavenge/reduce ABTS radical, converting the solution (blue-green) to a colourless product. The extent of decolorization by the sample is proportional to the concentration of the antioxidant present and it is compared to that of trolox; thereby giving TEAC value. The TEAC value was expressed as milligram of trolox equivalent per gram of sample (mgTE/mg sample). 25μl of sample was pipetted into the 96-well plate, and 275μl of ABTS reagent (prepared with 150mM potassium persulfate (K2S2O8) and ABTS in distilled water and placed in the dark room overnight) was added. This was incubated for 30 minutes at room temperature and the readings were taken at 734nm.
3.2.6 Identification of active compounds
Bioactive compounds preseent in the leaves and rhizome were determined using liquid chromatographic methods, as discussed below.
3.2.6.1 High Performance Liquid Chromatography (HPLC)
HPLC analysis was performed on the six extracts. Water (solvent A) and methanol (solvent B) were used for the mobile phase. The gradient program ran from 100% water at 0 minute to 100% methanol at 35 minutes for each sample, after which the column was washed and reconditioned. The sample injection volume was 20 µl, a flow rate of 1 ml/min and a temperature of 25oC was maintained throughout the analytical run. The diode array detector was controlled at a wavelength between 200nm and 700nm. The
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detection wavelengths were 260, 320, 360 and 520nm. Individual compounds were identified by comparing their retention time and UV spectrum with those obtained from their respective standards injected under the same HPLC conditions. Concentration of each compound was calculated using the area of standard, area of sample, injection volume, weight of sample (mg) and extraction volume. Results were expressed as µg/mg of sample.
3.2.6.2 Ultra-Performance Liquid Chromatography and Mass Spectrophotometry
Ultra-performance liquid chromatography and mass spectrophotometry (UPLC-MS) was carried out by connecting a Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS) to a Waters Acquity ultra performance liquid chromatograph to achieve high resolution. Electrospray ionization was used in negative mode with a cone voltage of 15 V, desolvation gas at 650 L/hr and desolvation temperature of 275°C. The rest of the MS settings were optimized for best resolution and sensitivity. Scanning from m/z 150 to 1500 in resolution mode as well as in MSE mode was done for data acquisition. The data acquired in the MSE mode were in two channels, one at a low collision energy (4 V) and the second using a collision energy ramp (40 to 100 V) to obtain fragmentation data. Sodium forbate was used to calibrate the instrument and leucine enkaphalin was used as a lock mass (reference mass) for accurate mass determination. The mobile phase consisted of 0.1% formic acid (Solvent A) and acetonitrile containing 0.1 % formic acid as solvent B. The gradient started at 100%
solvent A for 1 minute and changed to 28 % B over 22 minutes in a linear way. This further changed to 40% B over 50 seconds, a wash step of 1.5 minutes at 100% B followed and re-equilibration to initial conditions for 4 minutes. An injection volume of 2 µL was used and a flow rate of 0.3 mL/min. The column (Waters HSS T3, 2.1 x 100 mm, 1.7 μm) temperature was maintained at 55 ºC.
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3.2.7 Data Analysis
All data were expressed as mean ± standard deviation (SD) of triplicate determinations.
The data were analyzed by ANOVA using GraphPad Prism 5 software and SPSS.
Differences were considered significant at 5% level of significance. Multiple correlation analysis was done on secondary plant compounds vis a vis their antioxidant capacity using bivariate correlation to estimate the Pearson correlation coefficient.