Ultrospec 2100

Fe²⁺ chelating activity was measured following a previously reported method using the 2,2′-bipyridyl competing assay.^{45 (link)} Test samples included varying concentrations of GA ranging from 10–100 μM in 1% sodium dodecyl sulfate. Prior to analyses, 0.2 mL of each sample was added into 0.2 mL of FeSO₄ solution (1 mM). The mixture was then mixed with 0.8 mL of 0.1% 2,2′-bipyridyl solution (in 0.2 M HCl), 0.4 mL of 10% (w/v) hydroxylamine HCL and 2.5 mL of ethanol. Absorbance was obtained at wavelength of 524 nm with an UltroSpec 2100 instrument (Biochrom Ltd, Cambridge, UK). Chelating activity was calculated by the following equation: % inhibition = [(ΔA_control − ΔA_treatment)/ΔA_control] × 100%. Each value was expressed as mean ± SD from triplicate experiments.

The ferric reducing antioxidant power (FRAP) of the extracts were measured following published methods (Maksimović et al., 2005 (link); Tsai et al., 2002 ). The FRAP reagent contained 100 mL acetate buffer (3 mM, pH 3.6), 10 mL 2,4,6-tripyridyl-s-triazine (10 mM) in 40 mM hydrochloric acid, and 10 mL FeCl₃·6H2O (20 mM). Briefly, 1.5 mL of freshly prepared FRAP reagent was mixed with 50 μL of each extract (final concentration of 100 μg/mL) and the absorbance was measured at 593 nm after 5 minutes using an UltroSpec 2100 instrument (Biochrom Ltd, Cambridge, UK). L-ascorbic acid (0.1 – 3 mM) was used as the positive control and to generate a calibration curve. The FRAP capacity of each extract was expressed as ascorbic acid equivalents (AAE)/mg where AAE is defined as follows: the reducing power of 1 mg dry plant material is equivalent to the reducing power of 1 nM of L-ascorbic acid (Maksimović et al., 2005 (link)). All data were the average of three individual experiments.