Protection by estrogens of biological damage by 2,2'-azobis(2-amidinopropane) dihydrochloride

We examined by using 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) as a radical generator the ability of estrogens to scavenge carbon-centered and peroxyl radicals. Electron spin resonance signals of carbon-centered radicals from AAPH were diminished by catecholestrogens but not by phenolic estrogens, showing that catecholestrogens efficiently scavenged carbon-centered radicals. However, fluorescent decomposition of R-phycoerythrin by AAPH-derived peroxyl radicals was inhibited by catecholestrogens and phenolic estrogens. Evidently, peroxyl radicals were scavenged by catecholestrogens and by phenolic estrogens. However, the scavenging ability of 4-hydroxyestradiol was less than 2-hydroxyestradiol. Strand break of DNA induced by AAPH was inhibited by catecholestrogens, but not by phenolic estrogens under aerobic and anaerobic conditions. Inactivation of lysozyme induced by AAPH was completely blocked by 2-hydroxyestradiol under aerobic and anaerobic conditions, and by 4-hyroxyestradiol only under anaerobic conditions. Peroxidation of arachidonic acid by AAPH was strongly inhibited by catecholestrogens at low concentrations. Only large amounts of phenolic estrogens markedly inhibited lipid peroxidation. These results show that catecholestrogens were antioxidant against AAPH-induced damage to biological molecules through scavenging both carbon-centered and peroxyl radicals, but phenolic estrogens partially inhibited AAPH-induced damage because they scavenged only peroxyl radicals.     

The effects of nitroxide radicals on oxidative DNA damage

The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2, 6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1-0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H(2)O(2). The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.     

Peroxyl radical mediated oxidative DNA base damage: implications for lipid peroxidation induced mutagenesis

Endogenous DNA damage induced by lipid peroxidation is believed to play a critical role in carcinogenesis. Lipid peroxidation generates free radical intermediates (primarily peroxyl radicals, ROO(*)) and electrophilic aldehydes as the principal genotoxicants. Although detailed information is available on the role of aldehyde base adducts in mutagenesis and carcinogenesis, the contribution of peroxyl radical mediated DNA base damage is less well understood. In the present study we have mapped oxidative base damage induced by peroxyl radicals in the supF tRNA gene and correlated this information with peroxidation-induced mutations in several human fibroblast cell lines. Nearly identical patterns of oxidative base damage were obtained from reaction of DNA with either peroxidizing arachidonic acid (20:4omega6) or peroxyl radicals generated by thermolysis of ABIP in the presence of oxygen. Oxidative base damage primarily occurred at G and C. Transversions at GC base pairs in the supF gene were the major base substitution detected in all cell lines. Peroxyl radical induced tandem mutations were also observed. Many mutation hot spots coincided with sites of mapped oxidative lesions, although in some cases hot spots occurred adjacent to the damaged base. Evidence is presented for the involvement of 8-oxodG in the oxidation of DNA by ROO(*). These results are used to interpret some key features of previously published mutation spectra induced by lipid peroxidation in human cells.     

Aromatic and aliphatic mono- and bis-nitroxides: a study on their radical scavenging abilities

Nitroxide radicals are an emerging class of interesting compounds with versatile antioxidant and radioprotective properties. All literature studies have so far concentrated on compounds bearing only one nitroxide function. Here, we now investigate and compare the radical scavenging behaviour and antioxidant activity of aromatic indolinonic and aliphatic piperidine bis-nitroxides, i.e compounds bearing two nitroxide functions. Their corresponding mono-derivatives were also studied for comparison. Radical scavenging activity was investigated using EPR and UV-Vis spectroscopy by following spectral changes in acetonitrile of the nitroxides in the presence of alkyl and peroxyl radicals generated, respectively, under anoxic or aerobic conditions from thermal decomposition of AMVN [2,2'-azobis(2,4-di-methylvaleronitrile)]. Antioxidant activity of the nitroxides was evaluated by monitoring conjugated dienes (CD) formation during methyl linoleate micelles peroxidation and by measuring carbonyl content in oxidized bovine serum albumin (BSA). The results show that: (a) each nitroxide moiety in bis-nitroxides scavenges radicals independent of each other; (b) aliphatic nitroxides do not scavenge peroxyl radicals, at least under the experimental conditions used here, whereas indolinonic aromatic ones do: their stoichiometric number is 1.14 and 2.17, respectively, for mono- and bis-derivatives; (c) bis-nitroxides are roughly twice more efficient at inhibiting lipid peroxidation compared to their corresponding mono-derivatives. Although this study provides only comparative information on the relative radical-scavenging abilities of mono- and bis-nitroxides, it helps in understanding further the interesting reactivity of these compounds especially with regards to peroxyl radicals where many controversies in the literature exist.     

Do stable nitroxide radicals catalyze or inhibit the degradation of hyaluronic acid?

Reactive oxygen-derived species and particularly OH radicals can degrade hyaluronic acid (HA), resulting in a loss of viscosity and a subsequent decrease in its effectiveness as a joint-lubricating agent. The production of OH in the vicinity of HA can be catalyzed by bound redox-active metals, which participate in the Haber-Weiss reaction. Damage to HA can also occur as a result of hypochlorite formed by myeloperoxidase (MPO). The protective reagents commonly used to inhibit oxidative stress-induced degradation of HA include antioxidative enzymes, such as SOD and catalase, chelators that coordinate metal ions rendering them redox-inactive, and scavengers of radicals, such as OH, as well as nonradical reactive species. In recent years, stable cyclic nitroxides have also been widely used as effective antioxidants. In many cases, nitroxide antioxidants operate catalytically and mediate their protective effect through an exchange between their oxidized and reduced forms. It was anticipated, therefore, that nitroxides would protect HA from oxidative degradation as well. On the other hand, nitroxides serve as catalysts in many oxidation reactions of alcohols, sugars and polysaccharides, including hyalouronan. Such opposite effects of nitroxides on oxidative degradation are particularly intriguing and the aim of the present study was to examine their effect on HA when subjected to diverse forms of oxidative stress. The results indicate that nitroxides protect HA from OH radicals generated enzymatically or radiolytically. The protective effect is attributable neither to the scavenging of OH nor to the oxidation of reduced metal, but to the reaction of nitroxides with secondary carbohydrate radicals-most likely peroxyl radicals.     

Aromatic and aliphatic mono- and bis-nitroxides: A study on their radical scavenging abilities

Nitroxide radicals are an emerging class of interesting compounds with versatile antioxidant and radioprotective properties. All literature studies have so far concentrated on compounds bearing only one nitroxide function. Here, we now investigate and compare the radical scavenging behaviour and antioxidant activity of aromatic indolinonic and aliphatic piperidine bis-nitroxides, i.e compounds bearing two nitroxide functions. Their corresponding mono-derivatives were also studied for comparison. Radical scavenging activity was investigated using EPR and UV–Vis spectroscopy by following spectral changes in acetonitrile of the nitroxides in the presence of alkyl and peroxyl radicals generated, respectively, under anoxic or aerobic conditions from thermal decomposition of AMVN [2,2′-azobis(2,4-di-methylvaleronitrile)]. Antioxidant activity of the nitroxides was evaluated by monitoring conjugated dienes (CD) formation during methyl linoleate micelles peroxidation and by measuring carbonyl content in oxidized bovine serum albumin (BSA). The results show that: (a) each nitroxide moiety in bis-nitroxides scavenges radicals independent of each other; (b) aliphatic nitroxides do not scavenge peroxyl radicals, at least under the experimental conditions used here, whereas indolinonic aromatic ones do: their stoichiometric number is 1.14 and 2.17, respectively, for mono- and bis-derivatives; (c) bis-nitroxides are roughly twice more efficient at inhibiting lipid peroxidation compared to their corresponding mono-derivatives. Although this study provides only comparative information on the relative radical-scavenging abilities of mono- and bis-nitroxides, it helps in understanding further the interesting reactivity of these compounds especially with regards to peroxyl radicals where many controversies in the literature exist.     

An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant’s scavenging capacity against AAPH-derived free radicals

A new method is proposed for the evaluation of oxygen radical absorbance capacity (ORAC). The current fluorescence-based ORAC assay (ORAC-FL) is an indirect method that monitors the antioxidant’s ability to protect the fluorescent probe from free radical-mediated damage, and an azo-radical initiator, AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), has been used as a thermal free radical source. The new ORAC assay employs a short in situ photolysis of AAPH to generate free radicals. The electron paramagnetic resonance (EPR) spin trapping method was employed to identify and quantify AAPH radicals. In the presence of antioxidant, the level of AAPH radicals was decreased, and ORAC-EPR values were calculated following a simple kinetic formulation. Alkyl-oxy radical was identified as the sole decomposition product from AAPH; therefore, we concluded that ORAC-FL is the assay equivalent to alkyl-oxy radical scavenging capacity measurement. ORAC-EPR results for several antioxidants and human serum indicated that the overall tendency is in agreement with ORAC-FL, but absolute values showed significant discrepancies. ORAC-EPR is a rapid and simple method that is especially suitable for thermally labile biological specimens because the sample heating is not required for free radical production.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

Nitroxides block DNA scission and protect cells from oxidative damage.

The protective effect of cyclic stable nitroxide free radicals, having SOD-like activity, against oxidative damage was studied by using Escherichia coli xthA DNA repair-deficient mutant hypersensitive to H2O2. Oxidative damage induced by H2O2 was assayed by monitoring cell survival. The metal chelator 1,10-phenanthroline (OP), which readily intercalates into DNA, potentiated the H2O2-induced damage. The extent of in vivo DNA scission and degradation was studied and compared with the loss of cell viability. The extent of DNA breakage correlated with cell killing, supporting previous suggestions that DNA is the crucial cellular target of H2O2 cytotoxicity. The xthA cells were protected by catalase but not by superoxide dismutase (SOD). Both five- and six-membered ring nitroxides, having SOD-like activity, protected growing and resting cells from H2O2 toxicity, without lowering H2O2 concentration. To check whether nitroxides protect against O2.(-)-independent injury also, experiments were repeated under hypoxia. These nitroxides also protected hypoxic cells against H2O2, suggesting alternative modes of protection. Since nitroxides were found to reoxidize DNA-bound iron(II), the present results suggest that nitroxides protect by oxidizing reduced transition metals, thus interfering with the Fenton reaction.     

Interaction and reactivity of urocanic acid towards peroxyl radicals

The capacity of urocanic acid to interact with peroxyl radicals has been evaluated in several systems: oxidation in the presence of a free radical source (2,2'-azobis(2-amidinopropane; AAPH), protection of phycocyanin bleaching elicited by peroxyl radicals, and Cu(II)- and AAPH-promoted LDL oxidation. The results indicate that both isomers (cis and trans) are mild peroxyl radical scavengers. For example, trans-urocanic acid is nearly 400 times less efficient than Trolox in the protection of the peroxyl radical promoted bleaching of phycocyanin. Regarding the removal of urocanic acid by peroxyl radicals, nearly 100 muM trans-urocanic acid is required to trap half of the produced radicals under the employed conditions (10 mM AAPH, 37 degrees C). Competitive experiments show that the cis-isomer traps peroxyl radicals 30% less efficiently than the trans-isomer. Given the high concentrations that trans-urocanic acid reaches in skin, its capacity to trap peroxyl radicals could contribute to the protection of the tissue towards ROS-mediated processes. Furthermore, both isomers, and particularly the cis-isomer, protect LDL from Cu(II)-induced oxidation.     

Lipid oxidation predominates over protein hydroperoxide formation in human monocyte-derived macrophages exposed to aqueous peroxyl radicals

In U937 and mouse myeloma cells, protein hydroperoxides are the predominant hydroperoxide formed during exposure to AAPH or gamma irradiation. In lipid-rich human monocyte-derived macrophages (HMDMs), we have found the opposite situation. Hydroperoxide measurements by the FOX assay showed the majority of hydroperoxides formed during AAPH incubation were lipid hydroperoxides. Lipid hydroperoxide formation began after a four hour lag period and was closely correlated with loss of cell viability. The macrophage pterin 7,8-dihydroneopterin has previously been shown to be a potent scavenger of peroxyl radicals, preventing oxidative damage in U937 cells, protein and lipoprotein. However, when given to HMDM cells, 7,8-dihydroneopterin failed to inhibit the AAPH-mediated cellular damage. The lack of interaction between 7,8-dihydroneopterin and AAPH peroxyl radicals suggests that they localize to separate cellular sites in HMDM cells. Our data shows that lipid peroxidation is the predominant reaction occurring in HMDMs, possibly due to the high lipid content of the cells.     

Lipid oxidation predominates over protein hydroperoxide formation in human monocyte-derived macrophages exposed to aqueous peroxyl radicals

In U937 and mouse myeloma cells, protein hydroperoxides are the predominant hydroperoxide formed during exposure to AAPH or gamma irradiation. In lipid-rich human monocyte-derived macrophages (HMDMs), we have found the opposite situation. Hydroperoxide measurements by the FOX assay showed the majority of hydroperoxides formed during AAPH incubation were lipid hydroperoxides. Lipid hydroperoxide formation began after a four hour lag period and was closely correlated with loss of cell viability. The macrophage pterin 7,8-dihydroneopterin has previously been shown to be a potent scavenger of peroxyl radicals, preventing oxidative damage in U937 cells, protein and lipoprotein. However, when given to HMDM cells, 7,8-dihydroneopterin failed to inhibit the AAPH-mediated cellular damage. The lack of interaction between 7,8-dihydroneopterin and AAPH peroxyl radicals suggests that they localize to separate cellular sites in HMDM cells. Our data shows that lipid peroxidation is the predominant reaction occurring in HMDMs, possibly due to the high lipid content of the cells.     

The hydroxyl free radical reactions of ascorbyl palmitate as measured in various in vitro models.

The OH(*) free radical scavenging properties of ascorbyl palmitate (AP), water-solubilized in the presence of a surfactant (Brij 35), were tested in various systems: (1) The inhibition of polymerization of bovine serum albumin by OH(*) free radicals generated by the Fenton reaction indicated AP exerts a considerable protective effect against polymerization by scavenging the OH(*) free radicals. (2) ESR spin trapping comparisons of DMPO with AP were conducted. Using the Fenton reaction as a source of OH(*) free radicals, AP was 1 order of magnitude faster in scavenging these radicals than DMPO. (3) Oxidative modification of BSA by (60)Co-gamma irradiation of 80 krad, results in a strong increase in protein carbonyl content. AP inhibits carbonyl formation very efficiently, indicating that AP may be utilized as a biological OH(*) free radical scavenger in human therapy.     

Inhibition of protein hydroperoxide formation by protein thiols

Abstract

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2′ azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by β-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Inhibition of protein hydroperoxide formation by protein thiols

Studies on plasma and cells exposed to hydroxyl and peroxyl radicals have indicated that there are few inhibitors of protein hydroperoxide formation. We have, however, observed a small variable lag period during bovine serum albumin (BSA) oxidation by 2-2' azo-bis-(2-methyl-propionamidine) HCl (AAPH) generated peroxyl radicals, where no protein hydroperoxide was formed. The addition of free cysteine to BSA during AAPH oxidation also produced a lag phase suggesting protein thiols could inhibit protein hydroperoxide formation. The selective reduction of thiols on BSA by beta-mercaptoethanol treatment caused the appearance of a lag period where no protein hydroperoxide was formed during the AAPH mediated oxidation. Increasing free thiol concentration on the BSA increased the lag period. Protein hydroperoxide formation began when the protein thiol concentration dropped below one thiol per BSA molecule. It is unlikely that the lag period is due to gross structural alteration of the reduced protein since blocking the free thiols with N-ethyl maleimide eliminated the lag in protein hydroperoxide formation. Protein thiols were found to be ineffective in inhibiting hydroxyl radical-mediated protein hydroperoxide formation during X-ray radiolysis. Evidence is given for protein thiol oxidation occurring via a free radical mediated chain reaction with both free cysteine and protein bound thiol. The data suggest that reduced protein thiol groups can inhibit protein hydroperoxide formation by scavenging peroxyl radicals.     

Antioxidant protection of human serum albumin by chitosan

Inhibition of protein oxidation by reactive oxygen species (ROS) would confer benefit to living organisms exposed to oxidative stress, because oxidized proteins are associated with many diseases and can propagate ROS-induced damage. We measured the ability of 2800Da chitosan, D-glucosamine and N-acetyl glucosamine to protect human serum albumin from oxidation by peroxyl radicals derived from 2,2'-azobis(2-amidinopropane)dihydrochloride and N-centered radicals from 1,1'-diphenyl-2-picrylhydrazyl and from 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). Comparison with the antioxidant action of vitamin C showed that, on a molar basis, chitosan was equally effective in preventing formation of carbonyl and hydroperoxide groups in human serum albumin exposed to peroxyl radicals. It was also a potent inhibitor of conformational changes in the protein, assessed by absorption spectrum and intrinsic fluorescence. D-glucosamine was much less effective and N-acetyl glucosamine was not a useful antioxidant. Protection of the albumin from peroxyl radicals was achieved by scavenging of peroxyl radical. Chitosan was also a good scavenger of N-centered radicals, with glucosamine and N-acetyl glucosamine much less effective. The results suggest that administration of low molecular weight chitosans may inhibit neutrophil activation and oxidation of serum albumin commonly observed in patients undergoing hemodialysis, resulting in reduction of oxidative stress associated with uremia.     

Protective effects of carnosine, homocarnosine and anserine against peroxyl radical-mediated Cu,Zn-superoxide dismutase modification

Carnosine (beta-alanyl-L-histidine), homocarnosine (gamma-amino-butyryl-L-histidine) and anserine (beta-alanyl-1-methyl-L-histidine) have been proposed to act as anti-oxidants in vivo. The protective effects of carnosine and related compounds against the oxidative damage of human Cu,Zn-superoxide dismutase (SOD) by peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) were studied. The oxidative damage to Cu,Zn-SOD by AAPH-derived radicals led to protein fragmentation, which is associated with the inactivation of enzyme. Carnosine, homocarnosine and anserine significantly inhibited the fragmentation and inactivation of Cu,Zn-SOD by AAPH. All three compounds also inhibited the release of copper ions from the enzyme and the formation of carbonyl compounds in AAPH-treated Cu,Zn-SOD. These compounds inhibited the fragmentation of other protein without copper ion. The results suggest that carnosine and related compounds act as the copper chelator and peroxyl radical scavenger to protect the protein fragmentation. Oxidation of amino acid residues in Cu,Zn-SOD induced by AAPH were significantly inhibited by carnosine and related compounds. It is proposed that carnosine and related dipeptides might be explored as potential therapeutic agents for pathologies that involve Cu,Zn-SOD modification mediated by peroxyl radicals.     

Edarabone scavenges nitric oxide

Oxygen free radicals have been proposed to be major causative agents in secondary brain damage in traumatic and ischemic brain injury. Edarabone (3-methyl-1-phenyl-2-pyrazolin-5-one), a powerful antioxidative radical scavenger, is the only drug currently available in clinical practice for the treatment of cerebral infarction. There has been increasing interest in the role of nitric oxide (NO(*)) as a causative agent in brain injury. In the present study, we investigated the scavenging effect of Edarabone on nitric oxide (NO(*)), using an electron spin resonance (ESR) method. NO(*) was generated from 1-hydroxy-2-oxo-3-(N-3-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7), and analyzed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy (carboxy-PTI) produced from the reaction between 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (carboxy-PTIO) and NO(*). Edarabone directly scavenged NO(*) in a dose-dependent manner. These ESR studies indicate that Edarabone has a direct NO(*) scavenging activity and the additional possibility of novel neuroprotective activities against brain injury and focal cerebral ischemia.     

Antioxidant inhibition of oxygen radicals for measurement of total antioxidant capacity in biological samples

Few methods for assessing total antioxidant capacity (TAC) include both the percentage of inhibition and the length of inhibition in the measurement. Available methods require above ambient constant temperature incubation, reaction preheating, and/or separate assays for testing hydrophilic and hydrophobic samples. We describe a high-throughput method, antioxidant inhibition of oxygen radicals (AIOR), that overcomes these difficulties. AIOR uses peroxyl radicals to trigger a decrease in fluorescence of the indicator molecule, uroporphyrin I, which is delayed by the presence of antioxidants. The area under the curve is measured by a fluorescence spectrophotometer in a 96-well microplate format, and TAC results are expressed as millimole/liter Trolox equivalents. AIOR is performed at ambient temperature and is applicable to samples in either aqueous or common organic solvents. The reaction between uroporphyrin I and the peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was found to be of first-order kinetics with a mean rate constant (k) of 0.0254. Applications to measure antioxidant capacity are demonstrated on individual chemicals and biological samples. The method has good linearity, within- and between-assay precision, and recovery.     

Free radical induced damages to rat liver subcellular organelles: inhibition by Andrographis paniculata extract

Aqueous extract of Andrographis paniculata was examined for antioxidant activity using rat liver subcellular organelles as model systems. The study deals with two important biological oxidative agents, ascorbate-Fe(+2) and AAPH generating hydroxyl and peroxyl radical, respectively. Oxidative damage was examined against the inhibition of membrane peroxidation, protein oxidation and restoration in decreased SOD and catalase activity. The antimutagenic activity of Ap was examined following inhibition in AAPH induced strand breaks in plasmid pBR322 DNA. Extract was a potent scavenger of DPPH, ABTS radicals, exemplified by ESR signals, O2-*, *OH and H2O2, displayed excellent reducing power, FRAP potentials to reduce Fe (III) --> Fe (II) and had considerable amount of phenolics/ flavonoids contents, an effective antioxidant index. The observed antioxidant effect might be primarily due to its high scavenging ability for ROS. Effect was confirmed ex vivo following inhibition in peroxidation, restoration in SOD enzyme, SOD band intensity and protein degradation in Ap fed liver homogenate. Based on these results, it was concluded that the aqueous extract of Andrographis paniculata might emerge as a potent antiradical agent against various pathophysiological oxidants.