Hello Everyone,
I am planning to use ferrozine assay to measure the chelation ability of plant extracts (containing polyphenols) by reacting FeCl2 (ferrous) with the extract of interest then add ferrozine to react with the remaining Fe+2 in the solution giving a complex that can be measured spectrophotometrically. I found another paper used ferric-ferrozine to measure the antioxidant power (similar to FRAP assay) by reacting ferrozine with FeCl3 (ferric) to form ferric-ferrozine complex then adding the extract to measure its capability to reduce Fe+3 into Fe+2 and ferrous-ferrozine complex can be measured the same way as the first method.
I am confused between the two methods; can anyone guide me through choosing the best one (I want to measure the chelation ability)?
Thank you,
Fatma
Use Fe2+. Fe(phen3)3+ is a strong oxidant and will be reduced by polyphenols.
Chelation ability is best showing using Ferrous-ion chelating activity (FICA) assay. I used it during my PhD to test the ferrous chelating activity of seaweed extracts.
I used the method described by Wang et al. (2009). In a 96-well plate 100 µl of each sample extract (1 mg/ml) were mixed with 150 µl distilled water and 5 µl FeCl2 (2 mM). The reaction was initiated by the addition of 5 µl ferrozine (5 mM). Following 10 mins incubation at room temperature the absorbance was measured at 562 nm using a plate reader . The assay positive control contained distilled water and all assay reagents. The colour blank (containing sample extracts and all reagents apart from ferrozine) was used to correct the colour of the seaweed extracts. The % ferrous ion-chelating activity (FICA) was calculated as follows: FICA, % = (1 - ((Abs562nm Seaweed extract - Abs562nm Colour blank) /Abs562nm Control)) × 100
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