Antioxidant and free radical scavenging activity of iron chelators

Abstract

Inside the human body, reactive derivatives of oxygen, known as reactive oxygen species (ROS) such as the superoxide radical (O₂•), hydroxyl radical (•OH) and hydrogen peroxide (H₂O₂), are constantly generated. The ROS easily cause oxidative damage to various biomolecules such as proteins, lipids and DNA leading to various disease conditions. Iron chelators function as antioxidants by scavenging ROS and also reduce the amount of available iron thereby decreasing the quantity of •OH generated by Fenton reactions. In this study, the antioxidant activity of the iron chelators: caffeic acid (CA), 2,3-dihydroxybenzoic acid (DHBA), desferroxamine B (FOB) and benzohydroxamic acid (BHA) were determined using five different in vitro antioxidant assays. The antioxidant assays used were: iron binding ability, reducing ability using the potassium ferricyanide reduction method, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, H₂O₂ scavenging activity and •OH scavenging activity. The standard used for the iron binding ability was Na₂EDTA whereas vitamin C was used as a standard for the remaining assays. The iron chelators showed a concentration dependent increase in their radical scavenging activities as well as their reducing ability. At the concentration of 1 mM, FOB had the highest iron binding ability of 93.7% whereas DHBA had the lowest iron binding ability of 5.0% compared to the standard Na₂EDTA which had 94.8%. The iron chelators, with the exception of BHA, showed good reducing ability than vitamin C. Caffeic acid showed significant DPPH, hydrogen peroxide and hydroxyl radical scavenging activities of 84.7%, 99.8% and 14.5%, respectively. All the iron chelators were observed to show significant activities in all five antioxidant assays.

Keywords: Antioxidant activity; Caffeic acid; Iron binding ability; Radical scavenging activity; Reactive oxygen species; Reducing ability.

Fig. 1.1

Structures of iron chelators used in this study.

Fig. 3.1

Iron binding ability of chelators. Solutions of iron chelators and Na₂EDTA were prepared at 1 mM and 2 mM concentrations and 940 μl aliquot of each solution was used for the iron binding ability in a total volume of 1 ml reaction mixture. Each value is the average of two experiments with error bars indicating standard error of mean (SEM). Na₂EDTA was used as a positive control.

Fig. 3.2

Reducing ability of iron chelators. Solutions of iron chelators and vitamin C at 1 mM and 2 mM concentrations were prepared and 2.5 ml aliquot of each solution was used for the reducing ability assay in a total volume of 7.5 ml reaction mixture. Each value is the average of two experiments with error bars indicating SEM. Vitamin C was used as a positive control.

Fig. 3.3

DPPH radical scavenging activity of iron chelators. Solutions of iron chelators and vitamin C at 1 mM and 2 mM concentrations were prepared and 300 μL aliquot of each solution was used for the DPPH scavenging activity in a total volume of 1.3 ml reaction mixture. Each value is the average of two experiments with error bars indicating SEM. Vitamin C was used as a positive control.

Fig. 3.4

Hydrogen peroxide scavenging activity of iron chelators. (a) and (b) represents H₂O₂ scavenging activity for the iron chelators at 1 mM and 2 mM concentrations respectively. Solutions of iron chelators and vitamin C at 1 mM and 2 mM concentrations were prepared and 500 μL aliquots of each solution was used for the H₂O₂ scavenging activity in a total volume of 1.5 ml reaction mixture. Each bar represents the mean ± SEM; all determinations were performed in duplicate. Vitamin C was used as a positive control.

Fig. 3.5

Hydroxyl radical scavenging activity of iron chelators. Solutions of 1 mM and 2 mM concentrations of iron chelators were prepared and 200 μL aliquots of each solution was used for the •OH scavenging activity in a total volume of 2.2 ml reaction mixture. Each value is the average of two experiments with error bars indicating SEM. Vitamin C was used as a positive control.