Lurdes Mira

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.

Scavenging of reactive oxygen species by silibinin dihemisuccinate.

Silibinin dihemisuccinate (SDH) is a flavonoid of plant origin with hepatoprotective effects which have been partially attributed to its ability to scavenge oxygen free radicals. In the present paper the antioxidant properties of SDH were evaluated by studying the ability of this drug to react with relevant biological oxidants such as superoxide anion radical (O2-), hydrogen peroxide (H2O2), hydroxyl radical (HO.) and hypochlorous acid (HOCl). In addition, its effect on lipid peroxidation was investigated. SDH is not a good scavenger of O2- and no reaction with H2O2 was detected within the sensitivity limit of our assay. However, it reacts rapidly with HO. radicals in free solution at approximately diffusion-controlled rate (K = (1.0-1.2) x 10(10)/M/sec) and appears to be a weak iron ion chelator. SDH at concentrations in the micromolar range protected alpha 1-antiproteinase against inactivation by HOCl, showing that it is a potent scavenger of this oxidizing species. Luminol-dependent chemiluminescence induced by HOCl was also inhibited by SDH. The reaction of SDH with HOCl was monitored by the modification of the UV-visible spectrum of SDH. The studies on rat liver microsome lipid peroxidation induced by Fe(III)/ascorbate showed that SDH has an inhibitory effect, which is dependent on its concentration and the magnitude of lipid peroxidation. This work supports the reactive oxygen species scavenger action ascribed to SDH.

Plant extracts with anti-inflammatory properties-A new approach for characterization of their bioactive compounds and establishment of structure-antioxidant activity relationships

Geranium robertianum L. (Geraniacea) and Uncaria tomentosa (Willd.) DC. (Rubiaceae) plant extracts, frequently used in traditional medicine for treatment of inflammatory and cancer diseases, were studied to identify potential bioactive compounds that may justify their therapeutic use and their underlying mechanisms of action. Since some of the pharmacological properties of these plant extracts may be linked to their antioxidant potential, the antioxidant activity, in relation to free radical scavenging, was measured by the ABTS/HRP and DPPH() assays, presenting U. tomentosa the higher activity. The antioxidant activity was also evaluated by scavenging of HOCl, the major strong oxidant produced by neutrophils and a potent pro-inflammatory agent. U. tomentosa was found to be a better protector against HOCl, which may justify its effectiveness against inflammatory diseases. SPE/LC-DAD was used for separation/purification purposes and ESI-MS/MS for identification/characterization of the major non-volatile components, mainly flavonoids and phenolic acids. The ESI-MS/MS methodology proposed can be used as a model procedure for identification/characterization of unknowns without the prerequisite for standard compounds analysis. The ESI-MS/MS data obtained were consistent with the antioxidant activity results and structure-activity relationships for the compounds identified were discussed.

Study of the effects of dietary fish intake on serum lipids and lipoproteins in two populations with different dietary habits.

Increased concentrations of n-3 polyunsaturated fatty acids (PUFA), namely eicosapentaenoic acid (20:5; EPA) and docosahexaenoic acid (22:6; DHA), have been shown to be beneficial in coronary artery disease (CAD). In the present study, the relationships between fish intake and concentrations of serum EPA and DHA and the effects of these fatty acids on serum lipids and lipoproteins were investigated. Two groups of men, one living in a fishing village and the other in a farming village, participated in this study. The daily fish consumption was ten times greater in the fishing village group than in the rural village group and the mortality from IHD in the rural village was four times higher. Serum concentrations of EPA and DHA were significantly higher in the fishing village group (P < 0.001). In this group, the serum concentration of arachidonic acid (20:4; AA), was significantly lower (P < 0.001), and the ratio EPA:AA was twice that of the rural village (P < 0.001). Moreover, in the fishing village group, the serum triacylglycerol and total cholesterol levels were significantly lower than those observed in the rural village (P < 0.01 and P < 0.05 respectively). In the fishing village group the serum LDL-cholesterol concentration was also lower, although the difference was not significant. Our results reinforce the hypothesis that a high intake of n-3 PUFA provides protection against CAD.

Influence of the metabolic profile on the in vivo antioxidant activity of quercetin under a low dosage oral regimen in rats

Flavonoids are known to possess a broad set of pharmacological effects, some of which have been attributed to their antioxidant properties and, more recently, to cell signalling modulation. Nevertheless, flavonoids are extensively metabolized and their metabolites are the potential bioactive forms in vivo. Therefore, a first and crucial step to understand the mechanisms underlying potential health benefits of flavonoids is knowledge of their metabolites and their biological activities.

Protection by Flavonoids Against the Peroxynitrite-mediated Oxidation of Dihydrorhodamine

Peroxynitrite anion is a reactive and short-lived species and its formation in vivo has been implicated in several human diseases. In view of the potential usefulness of compounds that can protect against peroxynitrite or their reactive intermediates, a study focused on flavonoid compounds was carried out. Since the reactivity of peroxynitrite may be modified by [Formula: See Text] which is an important plasma buffer, the protection of flavonoids against peroxynitrite was evaluated by their ability to inhibit the peroxynitrite-mediated dihydrorhodamine (DHR123) oxidation with or without physiological concentrations of bicarbonate. Flavonoids from different classes were studied to elucidate which structural features are required for an effective protection. The most efficient flavonoids on protecting DHR123 against oxidation by peroxynitrite have their ability diminished in the presence of bicarbonate, but they maintain the hierarchy established in the absence of bicarbonate. The flavones are the most effective flavonoids and their effects depend mainly on the number of hydroxyl groups. These must include either a catechol group in the B-ring or a hydroxyl group at the 3-position. This work also included some isoflavones, flavanones and a flavanol, which enable us to conclude about the importance of another structural feature: the 2,3-double bond. These results indicate that the ability of flavonoids to protect against peroxynitrite depends on some structural features, also important to scavenge oxygen free radicals and to chelate metal ions. The most efficient flavonoids are effective at low concentrations with IC50 of the same magnitude as Ebselen, a selenocompound that has been reported to be excellent at protecting against peroxynitrite. Their effectiveness at low concentrations is an important aspect to take into account when characterizing a compound as an antioxidant with biological interest.

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.

Antioxidants in medicinal plant extracts. A research study of the antioxidant capacity of Crataegus, Hamamelis and Hydrastis.

The antioxidant capacity of extracts of Crataegus oxyacantha, Hamamelis virginiana, Hydrastis canadensis, plants native to Europe and North America which have long been used in herbal medicine for the treatment of cardiac and circulatory functions, has been investigated. The total antioxidant potential conferred by all hydrogen donating antioxidants present in these extracts has been assessed by the ABTS assay and the relative order of antioxidant potential has been established. Gas chromatography coupled to mass spectrometry (GC‐MS) has been used for the chemical identification of the antioxidant volatile compounds present in the extracts. The GC‐MS data were related to the results obtained using the ABTS assay. Copyright

NADH oxidase activity of rat liver xanthine dehydrogenase and xanthine oxidase—contribution for damage mechanisms

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of and H2O2 during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.

Structure and antioxidant activity of brominated flavonols and flavanones

Hypobromous acid (HOBr) produced by both eosinophil peroxidase (EPO) and myeloperoxidase (MPO) is a stronger oxidant than HOCl, and is also essential for optimal and efficient microbial killing. Considering the potential cytotoxic effect of HOBr, if it is formed outside the phagosome, it should be useful to scavenge it in order to protect the nearby tissues. In this study the ability of selected flavonoids to protect against HOBr mediated oxidation reactions was performed through a competitive reaction, and the resulting products identified by high performance liquid chromatography (HPLC) and electrospray ionization tandem mass spectrometry(ESI-MS/MS). Several structural features were found to be important to confer high antioxidant activity to flavonoids towards HOBr: the C2=C3 double bond and the 3-OH group in the C-ring, and the presence of both 5-OH and 7-OH groups in the A-ring. The MS results showed that flavonoids are dibrominated in the A-ring, suggesting that (except for fisetin) bromination occurs at C6 and C8 positions, through an electrophilic aromatic substitution reaction. The chemical modifications achieved by bromination of flavonoids have changed their biological properties, presenting their brominated derivatives higher antioxidant activity, as radical scavengers, and higher lipophilicity, than the parent flavonoids. Brominated flavonoids may then diffuse easily through membranes increasing the intracellular concentration of the compounds. These locally formed metabolites may also interact with signaling cascades involving cytokines and regulatory transcription factors, thus playing a role in inflammation and in the regulation of immune response.