Structure-antioxidant activity relationships of flavonoids: a re-examination

The antioxidant and prooxidant activities of flavonoids belonging to several classes were studied to establish their structure-activity relationships against different oxidants. Special attention was paid to the flavonoids quercetin (flavone), taxifolin (flavanone) and catechin (flavanol), which possess different basic structures but the same hydroxylation pattern (3,5,7,3',4'-OH). It was found that these three flavonoids exhibited comparable antioxidant activities against different oxidants leading to the conclusion that the presence of ortho -catechol group (3',4'-OH) in the B-ring is determinant for a high antioxidant capacity. The flavone kaempferol (3,5,7,4'-OH), however, in spite of bearing no catechol group, also presents a high antioxidant activity against some oxidants. This fact can be attributed to the presence of both 2,3-double bond and the 3-hydroxyl group, meaning that the basic structure of flavonoids becomes important when the antioxidant activity of B-ring is small.     

Structure-antioxidant Activity Relationships of Flavonoids: A Re-examination

The antioxidant and prooxidant activities of flavonoids belonging to several classes were studied to establish their structure-activity relationships against different oxidants. Special attention was paid to the flavonoids quercetin (flavone), taxifolin (flavanone) and catechin (flavanol), which possess different basic structures but the same hydroxylation pattern (3,5,7,3',4'-OH). It was found that these three flavonoids exhibited comparable antioxidant activities against different oxidants leading to the conclusion that the presence of ortho -catechol group (3',4'-OH) in the B-ring is determinant for a high antioxidant capacity. The flavone kaempferol (3,5,7,4'-OH), however, in spite of bearing no catechol group, also presents a high antioxidant activity against some oxidants. This fact can be attributed to the presence of both 2,3-double bond and the 3-hydroxyl group, meaning that the basic structure of flavonoids becomes important when the antioxidant activity of B-ring is small.     

Antioxidant protective effect of flavonoids on linoleic acid peroxidation induced by copper(II)/ascorbic acid system

Antioxidants are compounds that can delay or inhibit lipid oxidation. The peroxidation of linoleic acid (LA) in the absence and presence of Cu(II) ion–ascorbate combinations was investigated in aerated and incubated emulsions at 37 °C and pH 7. LA peroxidation induced by copper(II)–ascorbic acid system followed first order kinetics with respect to hydroperoxides concentration. The extent of copper-initiated peroxide production in a LA system assayed by ferric thiocyanate method was used to determine possible antioxidant and prooxidant activities of the added flavonoids. The effects of three different flavonoids of similar structure, i.e. quercetin (QR), morin (MR) and catechin (CT), as potential antioxidant protectors were studied in the selected peroxidation system. The inhibitive order of flavonoids in the protection of LA peroxidation was: morin > catechin ≥ quercetin, i.e. agreeing with that of formal reduction potentials versus NHE at pH 7, i.e. 0.60, 0.57 and 0.33 V for MR, CT, and QR, respectively. Morin showed antioxidant effect at all concentrations whereas catechin and quercetin showed both antioxidant and prooxidant effects depending on their concentrations. The structural requirements for antioxidant activity in flavonoids interestingly coincide with those for Cu(II)-induced prooxidant activity, because as the reducing power of a flavonoid increases, Cu(II)–Cu(I) reduction is facilitated that may end up with the production of reactive species. The findings of this study were evaluated in the light of structure–activity relationships of flavonoids, and the results are believed to be useful to better understand the actual conditions where flavonoids may act as prooxidants in the preservation of heterogeneous food samples containing traces of transition metal ions.     

The Prooxidant Action of Dietary Antioxidants Leading to Cellular DNA Breakage and Anticancer Effects: Implications for Chemotherapeutic Action Against Cancer

Plant-derived dietary antioxidants have attracted considerable interest in recent past for their ability to induce apoptosis and regression of tumors in animal models. While it is believed that the antioxidant properties of these agents may contribute to lowering the risk of cancer induction by impeding oxidative injury to DNA, it could not account for apoptosis induction and chemotherapeutic observations. In this article, we show that dietary antioxidants can alternatively switch to a prooxidant action in the presence of transition metals such as copper. Such a prooxidant action leads to strand breaks in cellular DNA and growth inhibition in cancer cells. Further, the cellular DNA breakage and anticancer effects were found to be significantly enhanced in the presence of copper ions. Moreover, inhibition of antioxidant-induced DNA strand breaks and oxidative stress by Cu(I)-specific chelators bathocuproine and neocuproine demonstrated the role of endogenous copper in the induction of the prooxidant mechanism. Since it is well established that tissue, cellular, and serum copper levels are considerably elevated in various malignancies, such a prooxidant cytotoxic mechanism better explains the anticancer activity of dietary antioxidants against cancer cells.     

Lysophosphatidic acid and sphingosine 1-phosphate: two lipid villains provoking cardiovascular diseases?

Several plant-derived polyphenolic compounds are considered to possess anticancer and apoptosis-inducing properties in cancer cells. Such compounds are recognized as naturally occurring antioxidants but also exhibit prooxidant properties under appropriate conditions. Evidence in the literature suggests that the antioxidant properties of polyphenolics such as gallotannins, curcumin, and resveratrol may not fully account for their chemopreventive effects. We propose a mechanism for the cytotoxic action of these compounds against cancer cells that involves mobilization of endogenous copper and the consequent prooxidant action.     

Flavonoids and urate antioxidant interplay in plasma oxidative stress

Flavonoids are naturally occurring plant compounds with antioxidant properties. Their consumption has been associated with the protective effects of certain diets against some of the complications of atherosclerosis. Lowdensity lipoprotein (LDL) oxidative modification is currently thought to be a significant event in the atherogenic process. Most of the experiments concerning the inhibition of LDL oxidation used isolated LDL. We used diluted human whole plasma to study the influence of flavonoids on lipid peroxidation (LPO) promoted by copper, and their interaction with uric acid, one of the most important plasma antioxidants. Lipid peroxidation was evaluated by the formation of thiobarbituric acid reactive substances (TBARS) and of free malondialdehyde (MDA). The comparative capability of the assayed flavonoids on copper (II) reduction was tested using the neocuproine colorimetric test. In our assay system, urate disappears and free MDA and TBARS formation increase during the incubation of plasma with copper. Most of the tested flavonoids inhibited copperinduced LPO. The inhibition of LPO by flavonoids correlated positively with their capability to reduce copper (II). The urate consumption during the incubation of plasma with copper was inhibited by myricetin, quercetin and kaempferol. The inhibition of urate degradation by flavonoids correlated positively with the inhibition of LPO. Urate inhibited the copperinduced LPO in a concentrationdependent mode. Luteolin, rutin, catechin, quercetin had an antioxidant synergy with urate. Our results show that some flavonoids could protect endogenous urate from oxidative degradation, and demonstrate an antioxidant synergy between urate and some of the flavonoids.     

Inhibitions of the autoxidation of linoleic acid by flavonoids in micelles.

The activities of five flavonoids as chain-breaking antioxidants have been studied for the autoxidation of linoleic acid in cetyl trimethylammonium bromide (CTAB) micelles at 37 degrees C. Flavonols such as quercetin, rutin and morin exhibited antioxidant activities, while two flavanones, naringin and hesperidin, did not suppress the oxidation appreciably. The ratio of rate constants for inhibition and propagation kinh/kp and stoichiometric factor n were determined.     

Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity

The metal chelating properties of flavonoids suggest that they may play a role in metal-overload diseases and in all oxidative stress conditions involving a transition metal ion. A detailed study has been made of the ability of flavonoids to chelate iron (including Fe 3+ ) and copper ions and its dependence of structure and pH. The acid medium may be important in some pathological conditions. In addition, the ability of flavonoids to reduce iron and copper ions and their activity-structure relationships were also investigated. To fulfil these objectives, flavones (apigenin, luteolin, kaempferol, quercetin, myricetin and rutin), isoflavones (daidzein and genistein), flavanones (taxifolin, naringenin and naringin) and a flavanol (catechin) were investigated. All flavonoids studied show higher reducing capacity for copper ions than for iron ions. The flavonoids with better Fe 3+ reducing activity are those with a 2,3-double bond and possessing both the catechol group in the B-ring and the 3-hydroxyl group. The copper reducing activity seems to depend largely on the number of hydroxyl groups. The chelation studies were carried out by means of ultraviolet spectroscopy and electrospray ionisation mass spectrometry. Only flavones and the flavanol catechin interact with metal ions. At pH 7.4 and pH 5.5 all flavones studied appear to chelate Cu 2+ at the same site, probably between the 5-hydroxyl and the 4-oxo groups. Myricetin and quercetin, however, at pH 7.4, appear to chelate Cu 2+ additionally at the ortho -catechol group, the chelating site for catechin with Cu 2+ at pH 7.4. Chelation studies of Fe 3+ to flavonoids were investigated only at pH 5.5. Only myricetin and quercetin interact strongly with Fe 3+ , complexation probably occurring again between the 5-hydroxyl and the 4-oxo groups. Their behaviour can be explained by their ability to reduce Fe 3+ at pH 5.5, suggesting that flavonoids reduce Fe 3+ to Fe 2+ before association.     

Interactions of Flavonoids with Iron and Copper Ions: A Mechanism for their Antioxidant Activity

The metal chelating properties of flavonoids suggest that they may play a role in metal-overload diseases and in all oxidative stress conditions involving a transition metal ion. A detailed study has been made of the ability of flavonoids to chelate iron (including Fe 3+ ) and copper ions and its dependence of structure and pH. The acid medium may be important in some pathological conditions. In addition, the ability of flavonoids to reduce iron and copper ions and their activity-structure relationships were also investigated. To fulfil these objectives, flavones (apigenin, luteolin, kaempferol, quercetin, myricetin and rutin), isoflavones (daidzein and genistein), flavanones (taxifolin, naringenin and naringin) and a flavanol (catechin) were investigated. All flavonoids studied show higher reducing capacity for copper ions than for iron ions. The flavonoids with better Fe 3+ reducing activity are those with a 2,3-double bond and possessing both the catechol group in the B-ring and the 3-hydroxyl group. The copper reducing activity seems to depend largely on the number of hydroxyl groups. The chelation studies were carried out by means of ultraviolet spectroscopy and electrospray ionisation mass spectrometry. Only flavones and the flavanol catechin interact with metal ions. At pH 7.4 and pH 5.5 all flavones studied appear to chelate Cu 2+ at the same site, probably between the 5-hydroxyl and the 4-oxo groups. Myricetin and quercetin, however, at pH 7.4, appear to chelate Cu 2+ additionally at the ortho -catechol group, the chelating site for catechin with Cu 2+ at pH 7.4. Chelation studies of Fe 3+ to flavonoids were investigated only at pH 5.5. Only myricetin and quercetin interact strongly with Fe 3+ , complexation probably occurring again between the 5-hydroxyl and the 4-oxo groups. Their behaviour can be explained by their ability to reduce Fe 3+ at pH 5.5, suggesting that flavonoids reduce Fe 3+ to Fe 2+ before association.     

Antioxidant properties of complexes of flavonoids with metal ions

The formation of complexes of metal ions with the flavonoids quercetin (L1), rutin (L2), galangin (L3) and catechin (L4) has been investigated by UV-visible spectroscopy. The antioxidant activities of the compounds were evaluated by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicalscavenging method. In this work, we have shown that the complexed flavonoids are much more effective free radical scavengers than the free flavonoids. We suggest that the higher antioxidant activity of the complexes is due to the acquisition of additional superoxide dismutating centers. Radical scavenging activities of the compounds were also investigated from an electrochemical point of view. There is a relationship between the logarithm of the antioxidant activity (represented by EC50) and the oxidation potential. The synergic effect of the complexes and ascorbic acid were studied by [13C]-NMR analyses. The results show that ascorbic acid can protect flavonoids from oxidative degradation, and reveal antioxidant synergies between ascorbic acid and the compounds.     

Isolation and characterization of a novel flavonoid possessing a 4,2'-glycosidic linkage from green mature acerola (Malpighia emarginata DC.) fruit

The novel flavonoid, leucocyanidin-3-O-beta-D-glucoside, possessing a 4,2'-glycosidic linkage was isolated from green mature acerola (Malpighia emarginata DC.) puree and given the trivial name "aceronidin." To examine the functions of aceronidin, its antioxidative activity and both its alpha-glucosidase and alpha-amylase inhibition activities, as a potential inhibitor of the sugar catabolic enzyme, were evaluated against those of taxifolin, catechin, isoquercitrin and quercitrin which each have a similar structure. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical quenching activity of aceronidin was stronger than that of alpha-tocopherol and comparable to that of flavonoids. In the yeast alpha-glucosidase inhibitory assay, aceronidin showed significantly greater inhibition than the other flavonoids tested. In the human salivary alpha-amylase inhibitory assay, aceronidin showed inhibition activity. Taken together, these results indicate aceronidin to be a potent antioxidant that may be valuable as an inhibitor of sugar catabolic enzymes.     

Fibrinogen is a co-antioxidant that supplements the vitamin E analog trolox in a model system

Objective: It appears that the atherosclerotic plaque is a prooxidant environment where some molecules that are normally antioxidants, including vitamins C and E, may act as prooxidants that contribute to atherosclerosis by oxidizing LDL. Some molecules can act as co-antioxidants to eliminate this prooxidant effect by recycling or other mechanisms of supplementation. Fibrinogen and other acute phase proteins found in the plaque are antioxidants. We hypothesized that fibrinogen can act as a co-antioxidant to supplement vitamin E thereby eliminating its oxidative effect under prooxidant conditions. We tested a model system for this hypothesis using the vitamin E analogue Trolox in a cell free system.

Methods: LDL was oxidized using 5 umol/l copper. Antioxidant conditions were achieved by adding the antioxidants immediately with LDL, while prooxidant conditions were created by adding antioxidants after a 40 min delay. Oxidation was monitored as the lag phase at 234 nm.

Results: Under antioxidant conditions, the protective effect of fibrinogen and Trolox combined together were about equal to the sum of the anitioxidant effects of each alone (additive), while under prooxidant conditions the combined protection was 54-200% greater (synergistic). These effects were different than those of vitamin C with Trolox in that under antioxidant conditions fibrinogen and Trolox were additive while vitamin C and Trolox showed strong synergistic effects, and in that unlike vitamin C and Trolox fibrinogen showed no prooxidant tendencies under prooxidant reaction conditions.

Conclusions: The data indicated that fibrinogen did act as a co-antioxidant to supplement Trolox and eliminate its prooxidant effect, most probably, by directly quenching the phenoxyl radical, because unlike vitamin C, fibrinogen did not appear to recycle vitamin E. But fibrinogen may act as a universal antioxidant, since unlike Trolox and vitamin C, it showed little tendency toward becoming a prooxidant.     

Effect of catechin O-methylated metabolites and analogues on human LDL oxidation

The effects of catechin metabolites and methylated analogues on LDL oxidation were studied in vitro using either a water-soluble initiator or copper ions to induce lipid peroxidation. Direct addition of catechin O-methylated analogues to the oxidation mixture led to a clear protective effect during lag phase and for the metabolites during both lag and propagation phases. The structure-activity relationships obtained with these selectively O-methylated compounds allowed determination of catechin active moietie: the catechol B-ring. Based on physical chemical studies, these results suggest that the mechanism implied in the scavenging properties of flavan-3-ols is not only hydrogen transfer, as generally described, but mainly an electronic transfer from the phenolate, and that 3'- and 4'-O-methylcatechin seem, moreover, to act as amphiphilic chain-breaking antioxidants. However, the plasma concentration of flavan-3-ols necessary to protect LDL is far greater than those usually found in human plasma. Therefore, the data do not support a direct physiological relevance of flavan-3-ols as antioxidants in lipid processes. Future research should focus on other effects besides simple antioxidant ones.     

The interaction of flavonoids with mitochondria: effects on energetic processes

The study addressed aspects of energetics of isolated rat liver mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, taking into account influences of the 2,3 double bond/3-OH group and 4-oxo function on the C-ring, and o-di-OH on the B-ring of their structures, as well as mitochondrial mechanisms potentially involved in cell necrosis and apoptosis. The major findings/hypothesis, were: The 2,3 double bond/3-OH group in conjugation with the 4-oxo function on the C-ring in the flavonoid structure seems favour the interaction of these compounds with the mitochondrial membrane, decreasing its fluidity either inhibiting the respiratory chain of mitochondria or causing uncoupling; while the o-di-OH on the B-ring seems favour the respiratory chain inhibition, the absence of this structure seems favour the uncoupling activity. The flavonoids not affecting the respiration of mitochondria, induced MPT. The ability of flavonoids to induce the release of mitochondria-accumulated Ca(2+) correlated well with their ability to affect mitochondrial respiration on the one hand, and their inability to induce MPT, on the other. The flavonoids causing substantial respiratory chain inhibition or mitochondrial uncoupling, quercetin and galangin, respectively, also decreased the mitochondrial ATP levels, thus suggesting an apparent higher potential for necrosis induction in relation to the flavonoids inducing MPT, taxifolin and cathechin, which did not decrease significantly the ATP levels, rather suggesting an apparent higher potential for apoptosis induction.     

Comparison of Antioxidant Activity Between Aromatic Indolinonic Nitroxides and Natural and Synthetic Antioxidants

In view of the possible employment of nitroxide compounds in various fields, it is important to know how they compare with other synthetic antioxidant compounds currently used in several industries and with naturally occurring antioxidants. To address this issue, the antioxidant activity of two aromatic indolinonic nitroxides synthesized by us was compared with both commercial phenolic antioxidants (BHT and BHA) and with natural phenolic antioxidants (α-hydroxytyrosol, tyrosol, caffeic acid, α-tocopherol). DPPH radical scavenging ability and the inhibition of both lipid and protein oxidation induced by the peroxyl-radical generator, AAPH, were evaluated. The results obtained show that overall: (i) the reduced forms of the nitroxide compounds are better scavengers of DPPH radical than butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) but less efficient than the natural compounds; (ii) the nitroxides inhibit both linolenic acid micelles and bovine serum albumin (BSA) oxidation to similar extents as most of the other compounds in a concentration-dependent fashion. Since the aromatic nitroxides tested in this study are less toxic than BHT, these compounds may be regarded as potential, alternative sources for several applications. The mechanisms underlying the antioxidant activity of nitroxides were further confirmed by UV–Vis absorption spectroscopy experiments and macroscale reactions in the presence of radicals generated by thermolabile azo-compounds. Distribution coefficients in octanol/buffer of the nitroxides and the other compounds were also determined as a measure of lipophilicity.     

Comparison of antioxidant activity between aromatic indolinonic nitroxides and natural and synthetic antioxidants

In view of the possible employment of nitroxide compounds in various fields, it is important to know how they compare with other synthetic antioxidant compounds currently used in several industries and with naturally occurring antioxidants. To address this issue, the antioxidant activity of two aromatic indolinonic nitroxides synthesized by us was compared with both commercial phenolic antioxidants (BHT and BHA) and with natural phenolic antioxidants (alpha-hydroxytyrosol, tyrosol, caffeic acid, alpha-tocopherol). DPPH radical scavenging ability and the inhibition of both lipid and protein oxidation induced by the peroxyl-radical generator, AAPH, were evaluated. The results obtained show that overall: (i) the reduced forms of the nitroxide compounds are better scavengers of DPPH radical than butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BLT) but less efficient than the natural compounds; (ii) the nitroxides inhibit both linolenic acid micelles and bovine serum albumin (BSA) oxidation to similar extents as most of the other compounds in a concentration-dependent fashion. Since the aromatic nitroxides tested in this study are less toxic than BHT, these compounds may be regarded as potential, alternative sources for several applications. The mechanisms underlying the antioxidant activity of nitroxides were further confirmed by UV-Vis absorption spectroscopy experiments and macroscale reactions in the presence of radicals generated by thermolabile azo-compounds. Distribution coefficients in octanol/buffer of the nitroxides and the other compounds were also determined as a measure of lipophilicity.     

Experimental evidence that flavonoid metal complexes may act as mimics of superoxide dismutase

Radical scavenging activities of flavonoids rutin, taxifolin, (-)-epicatechin, luteolin, and their complexes with transition metal (Fe2+, Fe3+, and Cu2+) towards superoxide were determined using illumination of riboflavin as source and NBT as detector of O*2-. The scavenger potencies of flavonoid metal complexes were significantly higher than those of the parent flavonoids. To elucidate the mechanism of this phenomenon, the rates of superoxide-dependent oxidation of flavonoids and their metal complexes in photochemical system with riboflavin were examined. It was found for the first time that flavonoids bound to metal ions were much less subjected to oxidation compared with those of free compounds. The findings directly demonstrate superoxide scavenging activity of metal ions in complexes with flavonoids and support earlier suggestions that flavonoid metal complexes may exhibit superoxide dismuting activity.     

Measurement of relative antioxidant activity of compounds: a methodological note

The relative activities of some hydrogen-donating antioxidants were assessed by comparing their activities with that of Trolox (Trolox equivalent antioxidant capacity, TEAC) for scavenging the ABTS radical cation (ABTS.+) generated in the aqueous phase. We have verified, however, that TEAC values may change with the concentration of compounds and with the measuring times used. Not withstanding, TEAC values do not differ significantly if the compounds have kinetic curves of ABTS.+ formation similar to that of Trolox. This is the case with ascorbic acid, whose TEAC values, determined by using five concentrations at three different measuring times, are very close. For the flavonoids studied (catechin, rutin, naringenin and silibinin) which have kinetic curves of ABTS.+ formation different from that of Trolox, the TEAC values decrease with increasing concentrations of the compounds for each measuring time, and increase with increasing measuring times for each concentration. In the present study, we conclude that, in order to evaluate relative antioxidant activities of compounds by the ABTS assay, it is essential to perform kinetic studies to assess scavenging of ABTS.+ by these compounds. Therefore, when the TEAC values of compounds are determined for more than one measuring time, we may be sure that all the antioxidant potential of compounds is being considered and whether or not it is possible to establish a hierarchy for their antioxidant activities.     

Action of quinolinic and indolinonic aminoxyls as radical-scavenging antioxidants.

The kinetic studies on the actions of quinolinic and indolinonic aminoxyls in the oxidation of lipid peroxidation induced by free radicals were carried out to evaluate their antioxidant activity. These aminoxyls showed a similar reactivity toward peroxyl radical with alpha-tocopherol. The antioxidant efficacies of aminoxyls against oxidation of methyl linoleate in homogeneous solution were smaller than that of alpha-tocopherol. Hydroxylamine, a reduced form of aminoxyl, possessed a comparative antioxidant efficacy with alpha-tocopherol and was capable of suppressing the consumption of alpha-tocopherol. Aminoxyls showed more potent antioxidant activity than alpha-tocopherol against the oxidation of methyl linoleate micelles induced by peroxyl radical or by a combination of copper ion and hydrogen peroxide. These results suggest that quinolinic and indolinonic aminoxyls may act as potent antioxidants against lipid peroxidation, especially in the presence of a good reductant which reduces aminoxyl radicals to hydroxylamines.     

Dietary flavonoids fail to suppress F2-isoprostane formation in vivo

Dietary antioxidants, including alpha-tocopherol (alpha-TOH) and polyphenolic flavonoid compounds, have been the subject of much research interest, but few studies have investigated interactions between these two antioxidants in vivo. We have conducted a feeding study to determine if supplementation with dietary flavonoids or polyphenol-containing compounds will provide antioxidant protection in tocopherol-deficient animals or exceed the antioxidant protection provided by alpha-TOH alone, using the sensitive and specific measure of lipid peroxidation, F2-isoprostanes. Seventy-two male Sprague Dawley rats were divided into 12 treatment groups to receive either alpha-TOH-sufficient or -deficient AIN93-G diet supplemented with one of five compounds: 0.5% quercetin, catechin, or epicatechin; or 1% cocoa powder or lignin. The fat source was polyunsaturated oil, increased from 7 to 11.05% (w/w with diet) to maximize lipid peroxidation while staying within a physiological range. After 7 weeks of treatment, animals were sacrificed with plasma and hearts analyzed to determine differences in F2-isoprostane levels. None of the treatment compounds significantly decreased plasma or heart F2-isoprostanes compared to the control beyond the significant protection displayed by alpha-tocopherol. We conclude that under these experimental conditions, quercetin, catechin, and epicatechin do not suppress lipid peroxidation in vivo.