Protective effects of nicaraven,a new hydroxyl radical scavenger,on the endothelial dysfunction after exposure of pig coronary artery to hydroxyl radicals

Recently, we have reported that a new synthetic compound, 1,2bis(nicotinamido)-propane (nicaraven), improved cardiac function following preservation and reperfusion. In this study, we investigated the efficacy of nicaraven as a radical scavenger by using an in vitro model of oxidative stress, to clarify mechanisms of the protective effect of this new compound on reperfusion injury in rat heart. Ring segments of epicardial right coronary arteries (RCA) of pig were suspended in organ chambers and exposed to hydroxyl radicals (·OH), generated (by two different systems ) by 0.28 mM FeSO₄/0.28 mM H₂O₂ and DHF/Fe³⁺-ADP (2.4 mM, 43 nM, and 1.56 uM, respectively) to the bathing solution for 60 min. Prior exposure of the coronary arteries to ·OH significantly produced right-ward shift of the dose-response curves of the bradykinin-induced endothelium-dependent relaxations (an increase in the ED₅₀ value for bradykinin by 4.37 and 1.98 times than control in two different ·OH generating systems, respectively), but did not affect the maximum relaxation responses. The presence of nicaraven (10^-4 and 10^-5 M) in the ·OH generating system, shifted the dose-response curves to bradykinin to the control level, suggesting a significant hydroxyl radical scavenging effect of the drug. These results indicate that nicaraven, a new hydroxyl radical scavenger, exhibits a protective effect on hydroxyl radicalinduced endothelial dysfunctions of pig coronary artery.     

Evaluation of hydroxyl radical-scavenging property of purpurogallin using high pressure liquid chromatography

Purpurogallin (PPG) has been used as an additive to edible and non-edible oils or fats to retard oxidation. Its antioxidant mechanism is not known. We investigated the ability of PPG to scavenge exogenously generated hydroxyl radicals (·OH) using a sensitive high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles) with UV light and was trapped with salicylic acid (500 nmoles). Salicylic acid is hydroxylated to produce ·OH adduct products 2,3-and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced concentration-dependent ·OH as estimated by generation of 2,3- and 2,5-DHBA. PPG (100, 200, 300, 400, 500 and 600 nmoles) produced concentration-dependent decreases in ·OH adduct products (approximately 70% inhibition with 600 nmoles of PPG). It did not affect the peak of standard 2,3- and 2,5-DHBA indicating that the decrease in the adduct product generated by H₂O₂ is due to scavenging of ·OH. These results indicate that photolysis of H₂O₂ by UV light produces ·OH and that PPG scavenges ·OH.     

Oxidation of hydroxylamine to nitrite as an assay for the combined presence of superoxide anions and hydroxyl radicals.

The pulse-radiolytic oxidation of hydroxylamine by either hydroxyl radicals (OH), superoxide anions (O₂^?), or a combination of both radicals was investigated. It was found that only OH radicals efficiently attack the substrate, while O₂^? is necessary for the subsequent formation of nitrite. Determination of the latter reaction thus allows the detection of the combined presence of both oxygen radical species.     

In vitro studies of interactions of NO· donor drugs with superoxide and hydroxyl radicals

Nitric oxide (NO·) is a free radical characterized by a high spontaneous chemical reactivity with many other molecules including the superoxide radical (O₂·^–). This complex interaction may generate a peroxynitrite anion (ONOO^–), which behaves as an important mediator of oxidative stress in many pathological states. In the present study, in vitro experiments were performed to assess directly the O₂·^– and hydroxyl (·OH) radical scavenging effects of various NO· donor drugs, i.e. sodium nitroprusside (SNP), sodium nitrite (NaNO₂), molsidomine and SIN 1, at pH 7.4, 7 or 6. Concentrations of NO· in the incubation medium containing the different NO· donor drugs were measured by the assay based on the reaction of Fe-N-methyl-D-glucamine dithiocarbamate (MGD) with NO· that yields a stable spin-adduct measured by electron paramagnetic resonance (EPR). O₂·^– and ·OH generation was characterized by EPR spin trapping techniques, using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). These free radicals were generated from the enzymatic system xanthine-xanthine oxidase, in phosphate buffer adjusted at pH 7.4, 7 and 6. Under these experimental conditions, SNP exhibited the strongest superoxide scavenging properties, characterized by IC₅₀ values expressed in the µmolar range, which decreased at low pH. Addition of SNP (800 µM) to solution containing MGD and Fe²⁺ (5:1) at pH 7 4 produced a three line EPR spectrum which is identified to [(MGD)₂-Fe²⁺-NO]. In control experiments no EPR signal was observed. We obtained the same results with NaNO₂ and an augmentation of the spin-adduct level was noted with the prolongation of the incubation period. In return, molsidomine (2 mM) did not produce, in our conditions, a detectable production of NO·. NaNO₂ displayed a significant superoxide scavenging effect only at pH 6, whilst neither molsidomine nor SIN 1 had any effect. Therefore, the superoxide scavenging properties of SNP, NaNO₂, and molsidomine appeared to be closely related to their potential for NO· release, which partially depends on the pH conditions. The behaviour of SIN 1 is more complicated, the speed of oxygen diffusion probably acting as a limiting factor in NO· formation in our conditions. The production of NO· was detected in presence of SIN 1. The intensity of the complex is comparable with the signal founded with NaNO₂. By contrast, all molecules exhibited hydroxyl radical scavenging properties, highlighting the capacity of ·OH to react with a wide range of molecules. In conclusion, considering the poor chemical reactivity of O₂·^–, the NO· donor drugs/O₂·^– interactions suggest a special relationship between these two radical species, which, in certain pathological states, could lead to the generation of cytotoxic end-products with strong oxidizing properties.     

Antioxidant activity of allicin,an active principle in garlic

Garlic has been claimed to be effective against diseases, in the pathophysiology of which oxygen free radicals (OFRs) have been implicated. Effectiveness of garlic could be due to its ability to scavenge OFRs. However, its antioxidant activity is not known. We investigated the ability of allicin (active ingredient of garlic) contained in the commercial preparation Garlicin to scavenge hydroxyl radicals (·OH) using high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce ·OH adduct products 2,3- and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced a concentration-dependent ·OH as estimated by ·OH adduct products 2,3-DHBA and 2,5-DHBA. Allicin equivalent in Garlicin (1.8, 3.6, 7.2, 14.4, 21.6, 28.8 and 36 g) produced concentration-dependent decreases in the formation of 2,3-DHBA and 2,5-DHBA. The inhibition of formation of 2,3-DHBA and 2,5-DHBA with 1.8 g/ml was 32.36% and 43.2% respectively while with 36.0 g/ml the inhibition was approximately 94.0% and 90.0% respectively. The decrease in ·OH adduct products was due to scavenging of ·OH and not by scavenging of formed ·OH adduct products. Allicin prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner. These results suggest that allicin scavenges ·OH and Garlicin has antioxidant activity.     

Modulation of fatty acid-binding capacity of heart fatty acid-binding protein by oxygen — derived free radicals

Summary In this study, we examined the effects of exposure of heart fatty acid-binding protein (h-FABP) to chemically generated O₂ ^– or OH · with respect to its oleate binding and to its electrophoretic properties. Purified rat h-FABP at 40 M scavenged as much as 30% O₂ ^– and 85% OH ·. On the other hand, when 2 nmol (4 M) FABP were exposed to free radicals, the maximum oleate binding capacity as measured by Scatchard analysis was reduced only by 14% and 27% for O₂ ^– and OH ·, respectively. The electrophoretic pattern of free radical-exposed FABP was not markedly different when examined either by the non-denaturing or by denaturing PAGE, suggesting the absence of any degradation or aggregation of FABP by O₂ ^– or OH ·. It is hypothesized that O₂ ^– or OH · in physiological concentration may not alter the function of FABP markedly in the ischemic-reperfused myocardium.Abbreviations h-FABP Heart Fatty Acid Binding Protein - NEFA Non-Esterified Fatty Acids - O₂ ^– Superoxide anions - OH· hydroxyl radicals - OCI hypohalite radicals - H₂O₂ hydrogen peroxide - HPLC High Pressure Liquid Chromatography     

Antioxidant properties of a North American ginseng extract

A North American ginseng extract (NAGE) containing known principle ginsenosides for Panax quinquefolius was assayed for metal chelation, affinity to scavenge DPPH-stable free radical, and peroxyl (LOO·) and hydroxyl (·OH) free radicals for the purpose of characterizing mechanisms of antioxidant activity. Dissociation constants (Kd) for NAGE to bind transition metals were in the order of Fe²⁺ > Cu²⁺ > Fe³⁺ and corresponded to the affinity to inhibit metal induced lipid peroxidation. In a metal-free linoleic acid emulsion, NAGE exhibited a significant (p 0.05) concentration (0.01-10 mg/mL) dependent mitigation of lipid oxidation as assessed by the ammonium thiocyanate method. Similar results were obtained when NAGE was incubated in a methyl linoleate emulsion containing haemoglobin catalyst and assessed by an oxygen electrode. NAGE also showed strong DPPH radical scavenging activity up to a concentration of 1.6 mg/mL (r² = 0.996). Similar results were obtained for scavenging of both site-specific and non site-specific ·OH, using the deoxyribose assay method. Moreover, NAGE effectively inhibited the non site-specific DNA strand breakage caused by Fenton agents, and suppressed the Fenton induced oxidation of a 66 Kd soluble protein obtained from mouse brain over a concentration range of 2-40 mg/mL. These results indicate that NAGE exhibits effective antioxidant activity in both lipid and aqueous mediums by both chelation of metal ions and scavenging of free radicals.     

Chemical Bases of Nonspecific Immunity

Our knowledge on the nature and quantity of reactive O₂forms generated in phagocytes, particularly in neutrophil leucocytes, and their role in nonspecific immunity is reviewed. In thermodynamical terms, oxygen is a very reactive molecule and, hence, can react with most chemical elements and many organic molecules. In kinetic terms, O₂is rather inert. Its reactivity can be increased either by reduction or excitation. After accepting four electrons, O₂is finally reduced to H₂O. Partial reduction resulting in highly reactive intermediates, namely, superoxide anion (O₂ ^·–), hydrogen peroxide (H₂O₂), and hydroxyl radical (^·OH), is possible. Singlet oxygen (¹O₂) is the product of O₂excitation. Phagocytes acting like agents of nonspecific immunity generate such reactive forms of O₂.     

Effect of chlorogenic acid on hydroxyl radical

Chlorogenic acid (CGA) is considered to act as an antioxidant. However, the inhibitory effects of CGA on specific radical species are not well understood. Electron spin resonance (ESR) in combination with spin trapping techniques was utilized to detect free radicals. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was used as a spin trapping reagent while the Fenton reaction was used as a source of hydroxyl radical (·OH). We found that CGA scavenges ·OH in a dose-dependent manner. The kinetic parameters, IC₅₀ and V_max, for CGA scavenging of ·OH were 110 and 1.27 M/sec, respectively. The rate constant for the scavenging of ·OH by CGA was 7.73 × 10⁹ M^–1 sec^–1. Our studies suggest that the antioxidant properties of CGA may involve a direct scavenging effect of CGA on ·OH.     

Titanium dioxide induced cell damage: A proposed role of the carboxyl radical

Titanium dioxide (TiO₂) nanoparticles have been shown to be genotoxic to cells exposed to ultraviolet A (UVA) radiation. Using the technique of electron spin resonance (ESR) spin trapping, we have confirmed that the primary damaging species produced on irradiation of TiO₂ nanoparticles is the hydroxyl (OH) radical. We have applied this technique to TiO₂-treated fish and mammalian cells under in vitro conditions and observed the additional formation of carboxyl radical anions (CO₂^?) and superoxide radical anions (O₂^?). This novel finding suggests a hitherto unreported pathway for damage, involving primary generation of OH radicals in the cytoplasm, which react to give CO₂^? radicals. The latter may then react with cellular oxygen to form O₂^? and genotoxic hydrogen peroxide (H₂O₂).     

Reduced free radical generation during reperfusion of hypothermically arrested hearts

Several studies indicate the presence of hydroxyl radical (OH·) as well as its involvement in the myocardial reperfusion injury. A transition metal-like iron is necessary for the conversion of superoxide anion (O₂ ^–) to a highly reactive and cytotoxic hydroxyl radical (OH·). In the present study, we have examined the generation of OH· and free iron in reperfused hearts following either normothermic (37°C) or hypothermic ischemia (5°C). Employing the Langendorff technique, isolated rat hearts were subjected to global ischemia for 30 min at 37°C or 5°C and were then reperfused for 15 min at 37°C. The results of the study suggest that both the OH· generation in myocardium and free iron release into perfusate were significantly lower in hearts made ischemic at 5°C as compared to 37°C. Release of myoglobin and lactic acid dehydrogenase into perfusate also followed a similar pattern. Furthermore, in in vitro studies, chemically generated O₂ ^– at 5°C caused a significantly lower rate of oxidation of oxymyoglobin as well as generation of OH° and free iron as compared to 37°C. These results suggest that (1) reperfusion of hypothermic ischemic heart is associated with a reduction in the generation of OH· and cellular damage compared to that of normothermic ischemic heart, and (2) myoglobin, an intracellular protein, is a source of free iron and plays a role in the reperfusion injury mediated by free radicals.Abbreviations OH· hydroxyl radical - O₂ ^– superoxide anion - ODFR oxygen-derived free radicals - KHB Krebs-Henseleit buffer - LDH lactate hydrogenase - SOD superoxide dismutase     

Different Inactivation Behaviors of MS-2 Phage and Escherichia coli in TiO2 Photocatalytic Disinfection

Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO₂ photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (·OH), which is generated on the surface of UV-illuminated TiO₂, plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H₂O₂ and O₂·⁻, etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and Escherichia coli as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction, para-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers, tert-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe²⁺, were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that E. coli is inactivated by both the free and the surface-bound hydroxyl radicals. E. coli might also be inactivated by other ROS, such as O₂·⁻ and H₂O₂, according to the present results.     

Pulse-radiolytic investigations of catechols and catecholamines II. Reactions of Tiron with oxygen radical species

Transient spectra and kinetic data of Tiron (1,2-dihydroxybenzene-3,5-disulphonic acid) are reported, obtained after pulse-radiolytic oxidation by hydroxyl radicals (°OH), superoxide anions (O₂^?) or a combination of both oxygen radicals. The rate constant with °OH radicals was determined at 1.0·10⁹ M^?1·s^?1. Contrary to a previous report (Greenstock, C.L. and Miller, R.W. (1975) Biochim. Biophys. Acta 396, 11–16), the rate constant with O₂^? of 1.0·10⁷ M^?1·s^?1 is lower by one order of magnitude; also the semiquinone absorbs at 300 nm rather than at 400 nm. The ratio of the rate constants with °OH and O₂^? of 100 again demonstrates that any oxidation reaction by the latter radical is unspecific due to the more efficient reaction of °OH radicals, leading to the same products with catechol compounds.     

Evaluation of the Antioxidant Actions of Ferulic Acid and Catechins

We have evaluated the abilities of ferulic acid, (±) catechin, (+) catechin and (-) epicatechin to scavenge the reactive oxygen species hydroxyl radical (OH±), hypochlorous acid (HOCl) and peroxyl radicals (RO₂).

Ferulic acid tested at concentrations up to 5 mM inhibited the peroxidation of phospholipid liposomes. Both (±) and (+) catechin and (-) epicatechin were much more effective. All the compounds tested reacted with trichloromethyl peroxyl radical (CCl₃O₂) with rate constants > 1 × 10⁶M^?1s^?1.

A mixture of FeCl₃-EDTA, hydrogen peroxide (H₂O₂) and ascorbic acid at pH 7.4, has often been used to generate hydroxyl radicals (OH.) which are detected by their ability to cause damage to the sugar deoxyribose. Ferulic acid, (+) and (±) catechin and (-) epicatechin inhibited deoxyribose damage by reacting with OH. with rate constants of 4.5 × 10⁹M^?1s^?1, 3.65 × 10⁹M^?1s^?1, 2.36 × 10⁹M^?1s^?1 and 2.84 × 10⁹M^?1s^?1 respectively. (-) Epicatechin, ferulic acid and the (+) and (±) catechins exerted pro-oxidant action, accelerating damage to DNA in the presence of a bleomycin-iron complex. On a molar basis, ferulic acid was less effective in causing damage to DNA compared with the catechins.

A mixture of hypoxanthine and xanthine oxidase generates O₂ which reduces cytochrome c to ferrocytochrome c. (+) Catechin and (-) epicatechin inhibited the reduction of cytochrome c in a concentration dependent manner. Ferulic acid and (±) catechin had only weak effects.

All the compounds tested were able to scavenge hypochlorous acid at a rate sufficient to protect alpha-1-antiproteinase against inactivation. Our results show that catechins and ferulic acid possess antioxidant properties. This may become important given the current search for “natural” replacements for synthetic antioxidant food additives.     

Zingerone [4-(4-hydroxy-3-methoxyphenyl)-2-butanone] Prevents 6-Hydroxydopamine-induced Dopamine Depression in Mouse Striatum and Increases Superoxide Scavenging Activity in Serum

As superoxide (·O₂^–) and hydroxyl radical (·OH) have been implicated in pathogenesis of Parkinsons disease, free radical scavenging, antioxidant, and neuroprotective agents have attracted attention as ways to prevent progression. We examined effects of zingerone, an alkaloid extracted from ginger root, on 6-hydroxydopamine (6-OHDA)-induced dopamine (DA) reduction in mouse striatum. Zingerone administration 1 h before and for 6 more days following one intracerebroventricular 6-OHDA injection prevented reductions of striatal DA and its metabolites, and increased serum ·O₂^– scavenging activity. Zingerone did not change activities of catalase or glutathione peroxidase in striatum or serum, or ·O₂^– scavenging activity in striatum. Treatment with diethyldithiocarbamate, SOD inhibitor, abolished the protective effect of zingerone against 6-OHDA-induced DA reduction. In vitro, zingerone scavenged ·O₂^– and ·OH and suppressed lipid peroxidation only weakly. Thus, direct antioxidant effects may be a minor component of its putative neuroprotective effect; instead, zingerone acted mainly by increasing systemic superoxide dismutase activity. Effects of zingerone treatment in this model suggest possible value in treatment of Parkinsons disease.     

Lidocaine: a hydroxyl radical scavenger and singlet oxygen quencher

Lidocaine, a local anaesthetic, has been shown to reduce ventricular arrhythmias associated with myocardial infarction and ischemic myocardial injury and its protective effects has been attributed to its membrane stabilizing properties. Since oxygen radicals are known to be produced during ischemia induced tissue damage, we have investigated the possible antioxidant properties of lidocaine and found that lidocaine does not scavenge 0₂ ^–· radicals at 1 to 20 mM concentrations. However, lidocaine was found to be a potent scavenger of hydroxyl radicals and singlet oxygen. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic techniques. Lidocaine inhibited DMPO-OH adduct formation in a dose dependent manner. The amount of lidocaine needed to cause 50% inhibition of that rate was found to be approximately 80 M and at 300 M concentration it virtually eliminated the DMPO-OH adduct formation. The production of OH-dependent TBA reactive products of deoxyribose was also inhibited by lidocaine in a dose dependent manner. Lidocaine was also found to inhibit the ¹O₂-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation in a dose dependent manner. ¹O₂ was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers and was detected as TEMP-¹O₂ adduct by EPR spectroscopy. The amount of lidocaine required to cause 50% inhibition of TEMP-¹O₂ adduct formation was found to be 500 M. These results suggest that the protective effect of lidocaine on myocardial injury may, in part, be due to its reactive oxygen scavenging properties. These results may also explain the membrane stabilizing actions of lidocaine by scavenging OH · and ¹O₂ that are implicated in membrane lipid peroxidation.     

Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence

The ability of paraquat radicals (PQ^+.) generated by xanthine oxidase and glutathione reductase to give H₂O₂-dependent hydroxyl radical production was investigated. Under anaerobic conditions, paraquat radicals from each source caused chain oxidation of formate to CO₂, and oxidation of deoxyribose to thiobarbituric acid-reactive products that was inhibited by hydroxyl radical scavengers. This is in accordance with the following mechanism derived for radicals generated by γ-irradiation [H. C. Sutton and C. C. Winterbourn (1984) Arch. Biochem. Biophys.235, 106–115] PQ^+. + Fe³⁺ (chelate) → Fe²⁺ (chelate) + PQ⁺⁺ H₂O₂ + Fe²⁺ (chelate) → Fe³⁺ (chelate) + OH^? + OH.. Iron-(EDTA) and iron-(diethylenetriaminepentaacetic acid) (DTPA) were good catalysts of the reaction; iron complexed with desferrioxamine or transferrin was not. Extremely low concentrations of iron (0.03 μm) gave near-maximum yields of hydroxyl radicals. In the absence of added chelator, no formate oxidation occurred. Paraquat radicals generated from xanthine oxidase (but not by the other methods) caused H₂O₂-dependent deoxyribose oxidation. However, inhibition by scavengers was much less than expected for a reaction of hydroxyl radicals, and this deoxyribose oxidation with xanthine oxidase does not appear to be mediated by free hydroxyl radicals. With O₂ present, no hydroxyl radical production from H₂O₂ and paraquat radicals generated by radiation was detected. However, with paraquat radicals continuously generated by either enzyme, oxidation of both formate and deoxyribose was measured. Product yields decreased with increasing O₂ concentration and increased with increasing iron(DTPA). These results imply a major difference in reactivity between free and enzymatically generated paraquat radicals, and suggest that the latter could react as an enzyme-paraquat radical complex, for which the relative rate of reaction with Fe³⁺ (chelate) compared with O₂ is greater than is the case with free paraquat radicals.     

Generation of hydroxyl radicals by soybean nodule leghaemoglobin

Leghaemoglobin, a protein present in root nodules of soybean (Glycine max (L.) Merr.), generates the highly reactive hydroxyl radical (·OH) upon incubation with hydrogen peroxide (H₂O₂). The H₂O₂ appears to cause breakdown of the haem, releasing iron ions that convert H₂O₂ into ·OH outside the protein. Oxyleghaemoglobin (oxygenated ferrous protein) is more sensitive to attack by H₂O₂ than is metleghaemoglobin (ferric protein). The possibility of oxyleghaemoglobin breakdown by H₂O₂ and formation of damaging ·OH may explain why the root nodule is equipped with iron-storage proteins and enzymes that can remove H₂O₂.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Hydroxyl radical scavenging activity of flavonoids

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.