The ferrous ion-chelating activity was determined following the method described by Engin Celep with slight modifications (Celep et al., 2012) (link). 2 mL of sample solution (1-5 g/L) was mixed with 7.4 mL of ethanol (55%), 0.2 mL of ferrous chloride solution (2 mm) and 0.4 mL of ferrozine solution (5 mm). The mixture was thoroughly mixed and allowed to stand at room temperature for 20 min. The absorbance of the mixture was then measured at 562 nm. Ascorbic acid was used as a positive control. The ferrous ion-chelating activity was calculated using the following equation:
where, D1 was the absorbance of the sample, and D0 was the result of the distilled water.