Prooxidant action of knipholone anthrone: Copper dependent reactive oxygen species generation and DNA damage

Knipholone (KP) and knipholone anthrone (KA) are natural 4-phenylanthraquinone structural analogues with established differential biological activities including in vitro antioxidant and cytotoxic properties. By using DNA damage as an experimental model, the comparative Cu(II)-dependent prooxidant action of these two compounds were studied. In the presence of Cu(II) ions, the antioxidant KA (3.1–200 μM) but not KP (6–384 μM) caused a concentration-dependent pBR322 plasmid DNA strand scission. The DNA damage induced by KA could be abolished by reactive oxygen species scavengers, glutathione and catalase as well as EDTA and a specific Cu(I) chelator bathocuproine disulfonic acid. In addition to Cu(II) chelating activity, KA readily reduces Cu(II) to Cu(I). Copper-dependent generation of reactive oxygen species and the subsequent macromolecular damage may be involved in the antimicrobial and cytotoxic activity of KA.     

New advances in molecular mechanisms and the prevention of adriamycin toxicity by antioxidant nutrients

Anthracyclines (doxorubicin, daunorubicin, epirubicin, and idarubicin) are currently the most effective group of anti-neoplastic drugs used in clinical practice. Of these, doxorubicin (also called adriamycin) is a key chemotherapeutic agent in cancer treatment, although its use is limited as a consequence of the chronic and acute toxicity associated with this drug. The molecular mechanisms of doxorubicin account for both the anti-cancer and the toxic side effects. Many antioxidants have been assayed, with positive or negative results, to prevent the toxicity of doxorubicin. The present review has two main goals: (1) to report the latest findings regarding the molecular mechanisms of doxorubicin toxicity; (2) to update our understanding of the role of natural antioxidants in preventive therapy against doxorubicin-induced toxicity. This review provides new evidence for the chemoprevention of doxorubicin toxicity, making use of natural antioxidants – in particular vitamin E, vitamin C, coenzyme Q, carotenoids, vitamin A, flavonoids, polyphenol, resveratrol, antioxidant from virgin olive oil and selenium – and offers new insights into the molecular mechanisms of doxorubicin toxicity with respect to DNA damage, free radicals and other parameters.     

Selective iNOS inhibition reduces renal damage induced by cisplatin

Cisplatin is a chemotherapeutic agent used in the treatment of several cancer tumors; however, nephrotoxicity has restricted its use. Reactive oxygen species and peroxynitrite, which is formed by the reaction between superoxide anion and nitric oxide (NO*), are implicated in cisplatin-induced nephrotoxicity. In contrast, both toxic and beneficial effects of NO* have been suggested in cisplatin-induced nephrotoxicity. Therefore, nowadays the role of NO* in this experimental model remains controversial. The aim of the present work was to elucidate the role of NO* in cisplatin-induced renal damage using N-[3-(aminomethyl)benzyl]acetamidine (1400W), a selective and irreversible inhibitor of iNOS. The mRNA levels of iNOS were increased in cisplatin-treated rats. The administration of 1400W reduced the cisplatin induced histological damage, renal dysfunction (increase in proteinuria and kidney injury molecule expression and decrease in creatinine clearance), tubulointerstitial infiltration, oxidative stress (increase in renal malondialdehyde and inmmunostaining for 4-hydroxy-2-nonenal) and nitrosative stress (immunostaining for 3-nitrotyrosine). In addition, the administration of 1400W was unable to modify systolic blood pressure in control rats. Our data demonstrate that selective iNOS inhibition reduces the cisplatin-induced nephrotoxicity and nitrosative stress which strongly suggest that in this experimental model (1) the NO* production is toxic and (2) iNOS is the main source of NO*.     

Protective effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on copper-induced hydroxyl radical generation in the rat heart

The present study was examined the effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on Cu(II)-induced hydroxyl radical generation (OH) in the extracellular fluid of rat myocardium. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium. When Cu(II) was infused through the microdialysis probe, Cu(II) increased in OH formation trapped as 2,3-DHBA in the dialysate. When fluvastatin (100 microM) was administered to Cu(II) (50 microM)-pretreated animals, the levels of 2,3-DHBA at 300 min after administration of fluvastatin significantly decreased. In cumulative dose dependent experiments, three concentrations of Cu(II), 10, 25 and 50 microM, were infused through the microdialysis probe in the rat myocardium. A positive linear correlation between Cu(II) and the formation of 2,3-DHBA (R(2)=0.980) was observed. However, when corresponding experiments were performed with fluvastatin (100 microM) pretreated animals, the level of 2,3-DHBA decreased. These results suggest that blocking LDL oxidation by fluvastatin may attenuate Cu(II)-induced OH formation in the rat heart.     

Ascorbic acid protects against lipopolysaccharide-induced intra-uterine fetal death and intra-uterine growth retardation in mice

Lipopolysaccharide (LPS) has been associated with adverse developmental outcomes including embryonic resorption, intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR) and preterm labor. Reactive oxygen species (ROS) mediate LPS-induced developmental toxicity. Ascorbic acid is an antioxidant. In the present study, we investigated the effect of ascorbic acid on LPS-induced IUFD and IUGR in mice. All ICR pregnant mice except controls received an intraperitoneal (75 microg/kg, i.p.) injection of LPS daily on gd 15-17. The experiment was carried out in three different modes. In mode A, the pregnant mice were pretreated with a single dose (500 mg/kg, i.p.) of ascorbic acid before LPS. In mode B, the pregnant mice were administered with a single dose (500 mg/kg, i.p.) of ascorbic acid at 3h after LPS. In mode C, the pregnant mice were administered with 500 mg/kg (i.p.) of ascorbic acid at 30 min before LPS, followed by additional dose (500 mg/kg, i.p.) of ascorbic acid at 3h after LPS. The number of live fetuses, dead fetuses and resorption sites was counted on gd 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. Results showed that maternally administered LPS significantly increased fetal mortality, decreased fetal weight and crown-rump and tail lengths of live fetuses, and retarded skeletal ossification in caudal vertebrae, anterior and posterior phalanges, and supraoccipital bone. LPS-induced IUFD and IUGR were associated with lipid peroxidation and GSH depletion in maternal liver, placenta and fetal liver. Pre-treatment with ascorbic acid significantly attenuated LPS-induced lipid peroxidation, decreased fetal mortality, and reversed LPS-induced fetal growth and skeletal development retardation. By contrast to pre-treatment, post-treatment with ascorbic acid had less effect on LPS-induced IUFD, although post-treatment significantly attenuated LPS-induced lipid peroxidation and reversed LPS-induced fetal growth and skeletal development retardation. Furthermore, post-treatment with ascorbic acid reduced the protective effects of pre-treatment on LPS-induced IUFD. All these results suggest that pre-treatment with ascorbic acid protected against LPS-induced fetal death and reversed LPS-induced growth and skeletal development retardation via counteracting LPS-induced oxidative stress, whereas post-treatment had less effect on LPS-induced IUFD.     

Antioxidant properties of Thonningianin A,isolated from the African medicinal herb,Thonningia sanguinea

The antioxidant properties of Thonningianin A (Th A), an ellagitannin, isolated from the methanolic extract of the African medicinal herb, Thonningia sanguinea were studied using the NADPH and Fe2+/ascorbate-induced lipid peroxidation (LPO), electron spin resonance spectrometer and the deoxyribose assay. Th A at 10 microM inhibited both the NADPH and Fe2+/ascorbate-induced LPO in rat liver microsomes by 60% without inhibitory effects on cytochrome P450 activity. Th A was similar to the synthetic antioxidant, tannic acid, as an inhibitor of both the NADPH and Fe2+/ascorbate-induced LPO but potent than gallic acid, vitamin C and vitamin E. While Th A poorly scavenged the hydroxyl radical generated by the Fenton reaction it dose-dependently scavenged 1,1-diphenyl-2-picrylhydrazyl, superoxide anion and peroxyl radicals with IC50 of 7.5, 10 and 30 microM, respectively. Furthermore, Th A showed inhibitory effects on the activity of xanthine oxidase with an IC50 of 30 microM. In the deoxyribose assay both T. sanguinea and its methanolic component Th A showed only site-specific (Fe3+ + H2O2) but not non-site-specific (Fe3+ + EDTA + H2O2) hydroxyl radical scavenging suggesting chelating ability for iron ions. Spectroscopic studies showed that Th A enhanced absorbance in the visible region in the presence of Fe2+ ions. These results indicate that the antioxidant properties of Th A involve radical scavenging, anti-superoxide formation and metal chelation.     

Different antioxidant effects of polyphenols on lipid peroxidation and hydroxyl radicals in the NADPH-, Fe-ascorbate- and Fe-microsomal systems

Antioxidant and pro-oxidant effects of 14 naturally occurring polyphenols (PP) on rat liver microsomal lipid peroxidation (LP) and hydroxyl radical (*OH) production were studied in NADPH-dependent, 50 microM Fe(2+)-500 microM ascorbate (Fe-AA) or 50 microM Fe(2+) system, respectively. LP determined by the thiobarbituric acid method was inhibited in the NADPH system by flavonols and trans-resveratrol that were more effective than other flavonoids and derivatives of benzoic and cinnamic acid and were mostly more efficient than in the Fe-AA system. Inhibition of LP in the Fe system was higher by one order of magnitude than in the Fe-AA system. *OH production in the NADPH system, measured by formaldehyde production, was decreased by myricetin, fisetin and quercetin, but increased by kaempferol, morin and trans-resveratrol, indicating that z.rad;OH played a minor role in LP, which all of these PP inhibited. None of these PP at up to 40 microM concentration quenched *OH in the Fe-AA system. All tested PP, except trans-resveratrol and gentisic acid, spectrally interacted with Fe(2+) or Fe(3+), indicating formation of complexes or oxidation of PP. In contrast to the NADPH system we found no correlation between Fe(2+) chelation and inhibition of Fe-AA- or Fe-dependent LP indicating that iron chelation did not play a significant role in the two latter systems. It is concluded that the inhibition of LP by PP was apparently due to their hydrogen donating properties rather than chelation of iron.     

Antioxidant and antigenotoxic activities in Acacia salicina extracts and its protective role against DNA strand scission induced by hydroxyl radical

The antioxidant potency of Acacia salicina extracts was investigated. Total antioxidant capacity was determined using an ABTS⁺ assay. Superoxide radical scavenging was measured using riboflavin-light-nitro blue tetrazolium (NBT) assay. In addition, the content of phenols, total flavonoids and sterols were measured in the tested extracts. The petroleum ether exhibited a potent scavenging activity toward ABTS radical cations. Whereas, chloroform extract showed the highest activity against superoxides radicals and was also able to protect pKS plasmid DNA against hydroxyl radicals induced DNA damages. The antimutagenicity of these extracts was assayed using the Ames assay against Salmonella typhimurium TA98 and S. typhimurium TA 1535 tester strains at different concentrations. These extracts decreased significantly the mutagenecity induced by sodium azide (SA) and 4-nitro-o-phenylenediamine (NOP). The antioxidant and antimutagenecity activities exhibited by A. salicina depended on the chemical composition of the tested extracts.     

Interactions of peroxynitrite with uric acid in the presence of ascorbate and thiols: implications for uncoupling endothelial nitric oxide synthase

It has been suggested that uric acid acts as a peroxynitrite scavenger although it may also stimulate lipid peroxidation. To gain insight into how uric acid may act as an antioxidant, we used electron spin resonance to study the reaction of uric acid and plasma antioxidants with ONOO-. Peroxynitrite reacted with typical plasma concentrations of urate 16-fold faster than with ascorbate and 3-fold faster than cysteine. Xanthine but not other purine-analogs also reacted with peroxynitrite. The reaction between ONOO- and urate produced a carbon-centered free radical, which was inhibited by either ascorbate or cysteine. Moreover, scavenging of ONOO- by urate was significantly increased in the presence of ascorbate and cysteine. An important effect of ONOO- is oxidation of tetrahydrobiopterin, leading to uncoupling of nitric oxide synthase. The protection of eNOS function by urate, ascorbate and thiols in ONOO(-)-treated bovine aortic endothelial cells (BAECs) was, therefore, investigated by measuring superoxide and NO using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) and the NO-spin trap Fe[DETC]2. Peroxynitrite increased superoxide and decreased NO production by eNOS indicating eNOS uncoupling. Urate partially prevented this effect of ONOO- while treatment of BAECs with the combination of either urate with ascorbate or urate with cysteine completely prevented eNOS uncoupling caused by ONOO-. We conclude that the reducing and acidic properties of urate are important in effective scavenging of peroxynitrite and that cysteine and ascorbate markedly augment urate's antioxidant effect by reducing urate-derived radicals.     

The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: acute studies

Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 microM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 microM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 microM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.     

Antioxidant, prooxidant and cytotoxic activity of hydroxylated resveratrol analogues: structure-activity relationship

Resveratrol (trans-3,4',5-trihydroxystilbene), a naturally occurring hydroxystilbene, is considered an essential antioxidative constituent of red wine possessing chemopreventive properties. However, resveratrol and even more its metabolite piceatannol were reported to have also cytostatic activities. In order to find out whether this is related to antioxidative properties of those compounds, we synthesized five other polyhydroxylated resveratrol analogues and studied structure-activity relationships between pro-/antioxidant properties and cytotoxicity. Radical scavenging experiments with O(2)(*-) (5,5-dimethyl-1-pyrroline-N-oxide/electron spin resonance (DMPO/ESR)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (photometry) revealed that 3,3',4',5-tetrahydroxystilbene (IC(50): 2.69microM; k(9): 443000M(-1)s(-1)), 3,4,4',5-tetrahydroxystilbene (IC(50): 41.5microM; k(9): 882000M(-1)s(-1)) and 3,3',4,4',5,5'-hexahydroxystilbene (IC(50): 5.02microM), exerted a more than 6600-fold higher antiradical activity than resveratrol and its two other analogues. Furthermore, in HL-60 leukemic cells hydroxystilbenes with ortho-hydroxyl groups exhibited a more than three-fold higher cytostatic activity compared to hydroxystilbenes with other substitution patterns. Oxidation of ortho-hydroxystilbenes in a microsomal model system resulted in the existence of ortho-semiquinones, which were observed by ESR spectroscopy. Further experiments revealed that these intermediates undergo redox-cycling thereby consuming additional oxygen and forming cytotoxic oxygen radicals. In contrast to compounds with other substitution patterns hydroxystilbenes with one or two resorcinol groups (compounds 1 and 3) did not show an additional oxygen consumption or semiquinone formation. These findings suggest that the increased cytotoxicity of ortho-hydroxystilbenes is related to the presence of ortho-semiquinones formed during metabolism or autoxidation.     

Investigating the potential neuroprotective effects of statins on DNA damage in mouse striatum

We investigated the protective effect of pravastatin, simvastatin and atorvastatin on striatal DNA damage as a potential method to conserve and protect the nigrostriatal neurons. C57Bl/J6 mice were treated with combinations of a statin and the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). DNA damage, DNA sensitivity to H(2)O(2)in vitro, and DNA repair capacity was measured using the single cell gel electrophoresis (comet) assay. MPTP treatment increased DNA damage in the striatum. Contrary to expectation, the statins studied here did not protect DNA against H(2)O(2) induced damage but did in fact cause DNA damage. Treatment with simvastatin showed a significant increase in levels of DNA damage (p0.0018) and DNA damage induced in vitro with H(2)O(2) was significantly increased by pravastatin (p0.0001). DNA repair and repair capacity was slightly increased by simvastatin, significantly increased by pravastatin (p=0.0093), but slightly decreased by atorvastatin. In the MPTP treated groups, pre-treated with statins, pravastatin (p0.0036) and simvastatin (p0.021), increased the level of DNA damage, while atorvastatin did not exhibit a significant effect. All three statins, administered prior to MPTP, offered slight protection against H(2)O(2) induced DNA damage and simvastatin and atorvastatin decreased the DNA repair capacity of the cells insignificantly. Treatment with statins, under the experimental conditions used, increased baseline levels of DNA damage, but DNA repair processes were left intact because the amount of repair also increased. Oxidative damage, however, largely exceeded the extent of DNA repair of mice treated with both the statins and MPTP.     

Phenolics from monofloral honeys protect human erythrocyte membranes against oxidative damage

The aim of the present work was to analyze the phenolic extracts from two monofloral Cuban honeys for their in vitro total antioxidant capacity, phenolic compounds content and free radical scavenging activity. The phenolic extracts, rich in lipophilic compounds, were tested further for their ability to inhibit AAPH-induced oxidative damage (hemolysis, lipid peroxidation and cytosolic depletion of reduced glutathione and decrease of superoxide dismutase activity) in erythrocytes. Results indicate an important total antioxidant capacity measured by TEAC and ORAC assays, as well as a relevant radical scavenging activity performed by EPR. Moreover, 13 phenolic compounds were identified using HPLC–LC/MS with quercetin as the most abundant flavonoid. The results also show that both extracts were able to inhibit erythrocytes oxidative damage, and that this may likely be due to their incorporation into cell membranes and their ability to cross it and reach the cytosol. In fact, flavonoid uptake by erythrocytes was further confirmed by testing quercetin, which efficiently incorporated into erythrocytes. Overall, this study indicates that honey contains relevant antioxidant compounds responsible, at least in part, for its biological activity and that uptake of its flavonoids may provide defense and promote cell functions in erythrocytes.     

Cardioprotective effects of a proanthocyanidin-rich fraction from Croton celtidifolius Baill: Focus on atherosclerosis

Proanthocyanidins are the most abundant polyphenols in human diets. Epidemiological studies have pointed to proanthocyanidins as promising molecules that could prevent the development of several coronary syndromes by inhibiting the atherogenic process. The present study was designed to investigate the antiatherogenic effects of a proanthocyanidin-rich fraction (PRF) obtained from Croton celtidifolius Baill (Euphorbiaceae) barks. In isolated human LDL, PRF caused a concentration-dependent inhibition of Cu²⁺-induced oxidative modifications, evidenced by the increasing of the lag phase of lipid peroxidation and decreasing in the oxidation rate (V_max), moreover, the protein moieties from LDL were protected against Cu²⁺-induced oxidation. In human umbilical vein endothelial cells (HUVECs), PRF reduced the ROS production stimulated by oxidized LDL. Herein, we demonstrate that oral treatment with PRF improved endothelium-dependent vasorelaxation in hypercholesterolemic LDL receptor knockout mice (LDLr^−/−), however, the fraction did not modify plasma lipids and atherosclerotic lesion size in this experimental model. Finally, our results showed for the first time that PRF prevent isolated LDL oxidation, decrease oxidative stress in endothelial cells and improve endothelial function in mice.     

Antioxidant properties of ursodeoxycholic acid

We have investigated potential antioxidant properties of the clinically relevant bile acid UDCA, which reaches therapeutic concentrations up to 0.09 and 29 mM, respectively, in human plasma and bile. UDCA was an excellent scavenger of OHz.rad; generated by FeCl(3)-EDTA, H(2)O(2) and ascorbate in the deoxyribose oxidation test, showing IC(min) and IC(50) values of 0.02 and 0.2 mM, respectively, and a second-order rate constant for reaction with OHz.rad; of 2+/-0.1 x 10(10)M(-1)s(-1). Notably, the drug could enhance at 1.5 mM concentration the antioxidant capacity of human bile against OHz.rad;-induced deoxyribose oxidation. UDCA also showed antioxidant effects in the deoxyribose test performed with nonchelated iron ions, such as Fe(2+) plus H(2)O(2) (IC(min): 7 mM, IC(50): 20 mM) or Fe(3+) plus H(2)O(2) and ascorbate (IC(min): 0.3 mM, IC(50): 5 mM), and inhibited ferrozine-Fe(2+) and desferrioxamine-Fe(3+) complexes formation with IC(50) values of, respectively, 12 and 0.3 mM, indicating that the drug interacts more with iron(III) than with iron(II). Moreover, UDCA significantly inhibited phospholipid liposome peroxidation induced by the OHz.rad;-generating system FeCl(3)-EDTA, H(2)O(2) and ascorbate (IC(min): 0.75 mM, IC(50): 3 mM), and by peroxyl radicals generated in the aqueous phase by AAPH (IC(min): 8 mM, IC(50): 14 mM). UDCA, even at 25 mM concentration, was ineffective on the lipoperoxidation mediated by Fe(2+) alone, but at the same concentration counteracted significantly that by Fe(3+) plus ascorbate, further pointing to its preferential antioxidant interaction with iron(III).In conclusion, UDCA has direct antioxidant properties, which are especially relevant against Fe(3+)- and OHz.rad;-dependent biomolecular oxidative damage; such properties are evident at therapeutically relevant drug concentrations, suggesting that UDCA could act as an antioxidant in vivo.     

Lipid peroxidation induced by indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacin radicals

Some of the side-effects of using indomethacin (IM) involve damage to the gastric mucosa and liver mitochondria. On the other hand, neutrophils infiltrate inflammatory sites to damage the tissues through the generation of reactive oxygen species by myeloperoxidase. The stomach and intestine have large amounts of peroxidase. These findings suggest that peroxidases are involved in tissue damage induced by IM. To clarify the basis for the tissue damage induced by IM in the presence of horseradish peroxidase (HRP) and H2O2 (HRP-H2O2), lipid peroxidation was investigated. When IM was incubated with liver microsomes in the presence of HRP-H2O2 and ADP-Fe3+, lipid peroxidation was time-dependent. Catalase and desferrioxamine almost completely inhibited lipid peroxidation, indicating that H2O2 and iron are necessary for lipid peroxidation. Of interest, superoxide dismutase strongly inhibited lipid peroxidation, and it also inhibited the formation of bathophenanthroline-Fe2+, indicating that reduction of the ferric ion was due to superoxide (O2-). ESR signals of IM radicals were detected during the interaction of IM with HRP-H2O2. However, the IM radical by itself did not reduce the ferric ion. These results suggest that O2- may be generated during the interaction of IM radicals with H2O2. Ferryl species, which are formed during the reduction of iron by O2-, probably are involved in lipid peroxidation.     

Comparative study on antioxidant activity of different varieties of commonly consumed legumes in India

Legumes are rich source of proteins, dietary fiber, micronutrients and bioactive phytochemicals. Thirty different varieties of commonly consumed legumes in India, were screened for phenolic content and antioxidant activity using, radical scavenging [(1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid, (ABTS⁺)], Ferric Reducing Antioxidant Power (FRAP) and metal ion (Fe²⁺) chelation assays. Legumes varied largely in their antioxidant activity. Horse gram, common beans, cowpea (brown and red) and fenugreek showed high DPPH radical scavenging activity (>400 units/g), while lablab bean (cream and white), chickpea (cream and green), butter bean and pea (white and green) showed low antioxidant activity (<125 units/g). Green gram, black gram, pigeon pea, lentils, cowpea (white) and common bean (maroon) showed intermediate activity. Similar trend was observed when the activity was assessed with ABTS⁺ and FRAP assays. Thus most of the varieties having light color seed coat, except soybean exhibited low antioxidant activity. While legumes having dark color seed coat did not always possessed high antioxidant activity (e.g. moth bean, black pea, black gram, lentils). Antioxidant activity showed positive correlation (r² > 0.95) with phenolic contents, in DPPH, ABTS⁺ and FRAP assays, whereas poor correlation (r² = 0.297) was observed between Fe²⁺ chelating activity of the legumes and phenolic contents.