FRAP assay measures the reducing ability of antioxidants

FRAP assay measures the reducing ability of antioxidants against oxidative effects of reactive oxygen species. The antioxidant potential is estimated by ability of anti-oxidants to reduce TPRZ-Fe (III) complex to TPTZ-Fe (II). Total antioxidant power may be considered analogous to total reducing power. In the current study chloroform and benzene extracts of A. calamus exhibited greater total antioxidant power in FRAP assay compared to crude aqueous extract whereas other extracts did not show anti-oxidant activity. Metal chelating capacity is claimed as one of the antioxidant mechanisms since it reduces the concentration of the catalytic transition metal in lipid peroxidation. The iron chelating capacity test measures the ability of antioxidants to compete with ferrizone in chelating ferrous ions [34]. In the presence of samples possessing chelating activity, the formation of complexes is decreased. Among the eight extracts of A. calamus, cold water, NaOH and crude aqueous extracts showed a higher chelating activity and the lowest activity was found in chloroform extract. The cold water extract showed maximum chelating activity when compared with other extracts. Phenolic compounds may contribute directly to antioxidant action. It is reported that phenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans [35]. Polyphenols are potential protecting agents against lethal effects of oxidative stress and offer protection of DNA by chelating redox-active transition metal ions [36]. Here, the benzene extract of A. calamus had the highest phenolic content compared with other extracts. Similarly, the benzene extract exhibits high free radical scavenging action. Hence high anti-oxidant potential of benzene extract might be due to high free radical scavenging action coupled with high phenol content. Hydroxyl radicals generated by the Fenton reaction cause phospholipase a2 inhibitor induced breaks in DNA strands to yield its open circular or relaxed forms. Exposure of plasmid DNA to Fenton\'s reagent ultimately results in DNA strand breaks, mainly due to the generation of hydroxyl radical and subsequent free radical-induced reaction in plasmid DNA. Hydroxyl radicals react with nitrogenous bases of DNA producing base radicals and sugar radicals. The base radicals in turn react with the sugar moiety causing breakage of sugar phosphate backbone of nucleic acids resulting in strand breaks. Different extracts of A. calamus except the cold water extract showed DNA damage protecting activity in our present study. A higher DNA protection activity was found in the petroleum ether, benzene, chloroform, alcohol and NaOH extracts compared with other extracts. The DNA protective activity of A. calamus may be due to high hydroxyl radical scavenging action of the extracts, as hydroxyl radicals are major DNA damaging radicals. In addition high phenolic content of the extracts might also contribute for DNA protection. It is well documented that mitochondria are the major source of intracellular ROS. The major single-organ oxygen consumers are the liver and brain, consuming 20.4% and 18.4%, respectively. Therefore, the liver mitochondria serve as an optimal sub cellular system to evaluate the efficacy of the antioxidants. In the current study, the impact of antioxidant activity of A. calamus on the biological system was investigated in isolated rat liver mitochondria by creating oxidative stress using FeSO4 and ascorbic acid. We observed a decrease in the mitochondrial ROS production in the presence of benzene extract of A. calamus compared to other extracts. It is to be noted that the benzene extract of A. calamus has most potent antioxidant property as it was effective in all in vitro assays excepting ferrous iron chelating action, whereas other extracts did not exhibit anti-oxidant action on par with this extract. However in vitro studies have to be supported by in vivo observations to determine whether extract has ability for antioxidant action in biological system. Hence, the antioxidant potential has to be tested in vivo by creating oxidative stress. Since benzene extract was more potent, it was tested for anti-oxidant action in vivo.