Josiane Cillard

Hydroxyl radical scavenging activity of flavonoids

Abstract The flavonoids scavenge hydroxyl ( . OH) radicals generated by UV photolysis of hydrogen peroxide. Free . OH radicals were spin-trapped by 5,5-dimethyl-1-pyrroline N -oxide and the adduct was detected by high pressure liquid chromatography coupled with an electrochemical detector. The scavenging activity of flavonoids decreases in the order: myricetin > quercetin > rhamnetin > morin > diosmetin > naringenin > apigenin > catechin >5,7- dihydroxy -3′,4′,5′-trimethoxyflavone > robinin > kaempferol > flavone. The activity increases with the number of hydroxyl groups substituted in the aromatic B-ring. The presence of a hydroxyl at C-3 and its glycosylation does not further increase scavenging efficiency. It is suggested that the overall antioxidant effect of flavonoids on lipid peroxidation may be due to their . OH and O· − 2 scavenging properties and the reaction with peroxy radicals.

Antioxidant and iron-chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures

The cytoprotective effect of three flavonoids, catechin, quercetin and diosmetin, was investigated on iron-loaded hepatocyte cultures, considering two parameters: the prevention of iron-increased lipid peroxidation and the inhibition of intracellular enzyme release. These two criteria of cytoprotection allowed the calculation of mean inhibitory concentrations (IC50) which revealed that the effectiveness of these flavonoids could be classified as follows: catechin > quercetin > diosmetin. These IC50 values have been related to structural characteristics of the flavonoids tested. Moreover, the investigation of the capacity of these flavonoids to remove iron from iron-loaded hepatocytes revealed a good relationship between this iron-chelating ability and the cytoprotective effect. The cytoprotective activity of catechin, quercetin and diosmetin could thus be ascribed to their widely known antiradical property but also to their iron-chelating effectiveness. These findings increase further the prospects for the development and clinical application of these potent antioxidants.

Antioxidant activity of flavonoids and reactivity with peroxy radical

The autoxidation of linoleic acid and methyl linolenate is inhibited by flavonoids. The antioxidant efficiency of these flavonoids increases with their concentration and in the order fustin < catechin < quercetin < rutin = luteolin < kaempferol < morin for linoleic acid and rutin < catechin < morin = kaempferol for methyl linolenate. Flavonoids are more effective on linoleic acid than on methyl linolenate. The antioxidant activity offlavonoids is related to an inhibition of the formation of trans,trans hydroperoxide isomers of linoleic acid. This inhibition exhibited the great H-atom donating ability of flavonoids to peroxy radical, thus terminating the chain radical reaction.

ROLE OF FLAVONOIDS AND IRON CHELATION IN ANTIOXIDANT ACTION

Publisher Summary This chapter discusses the role of flavonoids and iron chelation in antioxidant action. The potential of flavonoids to inhibit lipid peroxidation in biological models is supposed to reside mainly in their free radical scavenging capacity rather than in their iron chelating activity. This property is considered as a minor mechanism in the antioxidant action, because it has not been clearly established in biological systems. The assessment of a relationship between the antioxidant effect and the iron chelating capacity of flavonoids is subsequently of interest. For this purpose, rat is used hepatocyte cultures as a biological model where lipid peroxidation is induced by iron [Fe(III)] in its complexed form with nitrilotriacetic acid (NTA). The Fe-NTA complex is known to induce a rapid accumulation of iron inside the cells. Nitrilotriacetic acid (NTA) is used to maintain ferric iron in a soluble state; it is a low-affinity iron chelator.

Antioxidants modulate acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line

Exposure of the human skin to ultraviolet radiation (UVR) leads to depletion of cutaneous antioxidants, regulation of gene expression and ultimately to the development of skin diseases. Although exogenous supplementation of antioxidants prevents UVR-induced photooxidative damage, their effects on components of cell signalling pathways leading to gene expression has not been clearly established. In the present study, the effects of the antioxidants alpha-lipoic acid, N-acetyl-L-cysteine (NAC) and the flavonoid extract silymarin were investigated for their ability to modulate the activation of the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) in HaCaT keratinocytes after exposure to a solar UV simulator. The activation of NF-kappaB and AP-1 showed a similar temporal pattern: activation was detected 2 h after UV exposure and maintained for up to 8 h. To determine the capacity of activated NF-kappaB to stimulate transcription, NF-kappaB-dependent gene expression was measured using a reporter gene assay. The effects of the antioxidants on NF-kappaB and AP-1 activation were evaluated 3 h after exposure. While a high concentration of NAC could achieve a complete inhibition, low concentrations of alpha-lipoic acid and silymarin were shown to significantly inhibit NF-kappaB activation. In contrast, AP-1 activation was only partially inhibited by NAC, and not at all by alpha-lipoic acid or silymarin. These results indicate that antioxidants such as alpha-lipoic acid and silymarin can efficiently modulate the cellular response to UVR through their selective action on NF-kappaB activation.

Kinetic evaluation of free malondialdehyde and enzyme leakage as indices of iron damage in rat hepatocyte cultures: Involvement of free radicals

The present study relates to the effect of ferric iron supplementation on lipid peroxidation of adult rat hepatocyte pure cultures. Lipid peroxidation was evaluated by free malondialdehyde (MDA) using size exclusion chromatography (HPLC) as a specific and sensitive method. The ferric iron used under its complexed form with nitrilotriacetic acid (NTA) exhibited a prooxidant activity corresponding to an increase of free MDA recovery in the cells and in the culture medium. This enhancement of lipid peroxidation in the hepatocyte cultures supplemented with ferric iron was correlated with an intracellular enzyme leakage (lactate dehydrogenase and transaminase), suggesting that lipid peroxidation and enzyme release represented good parameters for cytotoxicity evaluation. The toxic effect of Fe-NTA on hepatocyte cultures was a function of the incubation time (from 0 to 48 hr) and of the concentration of ferric iron loading (i.e. 5, 20 and 100 microM). The mechanism by which Fe-NTA induced cellular damage involved free radical production, as increasing amounts of free radical scavengers corresponded to diminishing rates of both total free MDA and enzyme release. However, this reducing capacity varied from one scavenger to another, where they exhibited preferentially a decrease in lipid peroxidation or in enzyme leakage. This suggested a dissociation between the two parameters of cytotoxicity considered. Lipid peroxidation corresponding to alterations of both inner membranes and the plasma membrane, whereas enzyme release mainly corresponded to the damage of plasma membrane. Subsequently, some scavengers (superoxide dismutase, mannitol, alpha tocopherol, beta carotene) presented an intracellular activity, as they reduced mostly lipid peroxidation. Other ones (catalase, dimethylpyrroline N-oxide, thiourea) seemed essentially efficient in protecting the external plasma membrane, as shown an important decrease in enzyme leakage.

Repair of iron-induced DNA oxidation by the flavonoid myricetin in primary rat hepatocyte cultures

Oxidative DNA damage and its repair in primary rat hepatocyte cultures was investigated following 4 h of incubation with the toxic iron chelate, ferric nitrilotriacetate (Fe-NTA), in the presence or absence of the potent protective flavonoid myricetin (25-50-100 microM). Seven DNA base oxidation products were quantified in DNA extracts by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring mode. Concomitantly, DNA repair capacity of hepatocytes was estimated by the release of oxidized-base products into culture media, using the same GC-MS method. A genotoxic effect of Fe-NTA (100 microM) in hepatocytes was evidenced by a severe increase in DNA oxidation over basal levels, with accumulation in cellular DNA of five oxidation products derived from both purines and pyrimidines. This prooxidant effect of iron was also noted by an induction of lipid peroxidation, estimated by free malondialdehyde production. Addition of increasing concentrations of myricetin (25-50-100 microM) simultaneously with iron prevented both lipid peroxidation and accumulation of oxidation products in DNA. Moreover, as an activation of DNA repair pathways, myricetin stimulated the release of DNA oxidation bases into culture media, especially of purine-derived oxidation products. This removal of highly mutagenic oxidation products from DNA of hepatocytes might correspond to an activation of DNA excision-repair enzymes by myricetin. This was verified by RNA blot analysis of DNA polymerase beta gene expression which was induced by myricetin in a dose-dependent manner. This represented a novel and original mechanism of cytoprotection by myricetin against iron-induced genotoxicity via stimulation of DNA repair processes. Since iron-induced DNA damage and inefficient repair in hepatocytes could be related to genotoxicity and most probably to hepatocarcinogenesis, modulation of these processes in vitro by myricetin might be relevant in further prevention of liver cancer derived from iron overload pathologies.

Selective inhibition of NF-κB activation by the flavonoid hepatoprotector silymarin in HepG2: Evidence for different activating pathways

The bioflavonoid silymarin is found to potently suppress both nuclear factor kappa‐B (NF‐κB)‐DNA binding activity and its dependent gene expression induced by okadaic acid in the hepatoma cell line HepG2. Surprisingly, tumor necrosis factor‐α‐induced NF‐κB activation was not affected by silymarin, thus demonstrating a pathway‐dependent inhibition by silymarin. Many genes encoding the proteins of the hepatic acute phase response are under the control of the transcription factor NF‐κB, a key regulator in the inflammatory and immune reactions. Thus, the inhibitory effect of silymarin on NF‐κB activation could be involved in its hepatoprotective property.

Autoxidation of linoleic acid and behavior of its hydroperoxides with and without tocopherols

The autoxidation of linoleic acid dispersed in an aqueous media and the effect of α-, γ- and δ-tocopherols were studied. The quantitative analysis of the hydroperoxide isomers (13-cis,trans; 13-trans,trans; 9-trans,cis; 9-trans,trans) by direct high-performance liquid chromatography exhibited a prooxidant activity of α-tocopherol at high concentration (3.8% by weight of linoleic acid). On the other hand, α-tocopherol at lower concentrations (0.38 and 0.038%) and γ- and δ-tocopherols at high concentration (3.8%) were antioxidant. Furthermore, the addition of tocopherols modified the distribution of the geometrical isomers. The formation of thetrans,trans hydroperoxide isomers was completely inhibited by the highest concentration of the three tocopherols independently of their antioxidant or prooxidant activity and only delayed by the lower concentrations of α-tocopherol. The addition of tocopherols to hydroperoxide isomers reduced the decomposition rate of these isomers in the order α-tocopherol < γ-tocopherol < δ-tocopherol for thecis,trans hydroperoxide isomer and α-tocopherol ≪ γ-tocopherol ⋍ δ-tocopherol for thetrans,trans hydroperoxide isomer. With these hydroperoxides, as during linoleic acid autoxidation, α-tocopherol was completely oxidized whatever its initial concentration, while γ-tocopherol underwent partial oxidation and δ-tocopherol was practically unchanged.

Antioxidant and free radical scavenging activities of the iron chelators pyoverdin and hydroxypyrid-4-ones in iron-loaded hepatocyte cultures: Comparison of their mechanism of protection with that of desferrioxamine

The protective effect on iron-supplemented hepatocyte cultures of three iron chelators, pyoverdin Pa and hydroxypyrid-4-one derivatives CP20 and CP22, was compared to that of the widely known desferrioxamine B (Desferal:DFO), on the basis of two criteria: (a) their effectiveness in inhibiting free malondialdehyde (MDA) production as an index of iron-induced lipid peroxidation; and (b) their ability to reduce intracellular enzyme leakage. In view of these two markers of iron toxicity, the protective effect of these chelators was classified as follows: DFO > CP20 > or = CP22 > Pa. The mechanism of cellular protection was elucidated by investigating both the iron-chelating activity and the free radical scavenging property of these agents. As concerns the iron chelation, DFO and Pa exerted the same rank order as for cytoprotection (DFO > Pa). The free radical scavenging property toward hydroxyl radical .OH and peroxyl radical ROO. was investigated in a cell-free experimental model. The two siderophores, DFO and Pa, appeared to have a lower antiradical activity toward .OH than hydroxypyrid-4-one CP22. This .OH scavenging activity was classified as follows: CP22 >> Pa > DFO. Moreover, the chelators exhibited for the quenching of ROO. the same order of effectiveness as that observed for cellular protection: DFO > CP20 > or = CP22 > Pa. These data indicate that, in addition to the iron-chelating activity which represents the most important property for determining the protection capacity of these iron chelators, their free radical scavenging ability also must be taken into account. This direct demonstration of a strong association between the free radical scavenging activity and the protective effect of iron chelators further increases the prospects for the development and clinical applications of new oral chelating drugs.