Hydroxyl Radical Trapping Research Articles

Reevaluation of Quantitative ESR Spin Trapping Analysis of Hydroxyl Radical by Applying Sonolysis of Water as a Model System

Abstract Electron spin resonance (ESR) spin trapping using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is commonly applied for quantitative analysis of hydroxyl radical. For better understanding of the analysis, we investigated kinetics related to formation and decay of hydroxyl radical spin adduct of DMPO compared with that of 3,3,5,5-tetramethyl-1-pyrroline N-oxide (M4PO) and 5-(diphenylphosphinoyl)-5-methyl-1-pyrroline N-oxide (DPPMPO). In our study where hydroxyl radical was generated by sonolysis of water, we found that (1) DMPO-OH formation was saturated even though hydroxyl radical was generated continuously and (2) the concentration of DMPO-OH decreased in inverse proportion to time after cessation of ultrasound irradiation, suggesting that the decay is a second-order reaction. Similar results were obtained in an experiment of DMPO-OH generated by photolysis of H2O2. Other than DMPO, M4PO, and DPPMPO also trapped hydroxyl radical but the spin trap efficiency was less than that of DMPO. Furthermore, M4PO-OH and DPPMPO-OH decayed more quickly than DMPO-OH. From this, we conclude that DMPO is more suitable for quantification of hydroxyl radical than M4PO and DPPMPO but results from the quantitative analysis must be interpreted with consideration of the kinetics related to formation and decay of DMPO-OH, especially in quantification of large amount of hydroxyl radical generation.

Antioxidant activity of mandarin (Citrus reticulata) peel

Mandarin peel, a waste product coming from juice production, was extracted by conventional extraction with 70% acetone. Content of flavonoids in mandarin peel extract (MPE) was determined by HPLC. Hesperidin was the most dominant flavonoid. Free radical scavenging activity of MPE on stable DPPH radicals and reactive hydroxyl radicals was also evaluated. EC50 value determined in spectrophotometrical DPPH radical assay was 0.179 mg/ml, while this value in ESR spin trapping hydroxyl radical assay was 0.415 mg/ml. Also, MPE showed protective effects in stabilising sunflower oil during accelerated storage. The results indicated that mandarin peel can be a valuable source of natural antioxidants.

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.

Antioxidant Activity of Caffeic Acid through a Novel Mechanism under UVA Irradiation

Effect of caffeic acid on the formation of hydroxyl radicals was examined during xanthone-mediated photosensitization. The reaction was performed on irradiation (λ = 365 nm) of the standard reaction mixture containing 15 µM xanthone, 0.1 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 20 mM phosphate buffer (pH 7.4) using electron paramagnetic resonance (EPR) with spin trapping. Caffeic acid inhibited the formation of hydroxyl radicals. Caffeic acid hardly scavenged both hydroxyl radicals and superoxide radicals under conditions employed in this paper in spite of its ability to act as a hydrogen donor or a reagent for the aromatic hydroxylation, because high concentration of DMPO trapped hydroxyl radicals overwhelmingly. Furthermore, caffeic acid inhibited the formation of hydroxyl radicals in the standard reaction mixture with EDTA under UVA irradiation. Accordingly, the inhibitory effect of caffeic acid on the formation of hydroxyl radicals in the standard reaction mixture under UVA irradiation is not due to its ability to chelate iron. Thus, the inhibitory effect of caffeic acid seems to occur in the standard reaction mixture under UVA irradiation through a novel antioxidation activity, i.e., ability to quench the exited xanthone.

Establishment of activity indicator of TiO 2 photocatalytic reaction—Hydroxyl radical trapping method

In this study, a new, low cost and easy method, hydroxyl radical trapping method, was employed to investigate the photo-activity of UV/TiO 2 photocatalytic reaction. The Taguchi method was utilized to optimize the preparation of titanium dioxide (TiO 2) thin-film reactor through the modified chemical vapor deposition (CVD) method. The optimal yield of hydroxyl radicals was then evaluated by calculating the conversion ratio of salicylic acid under the optimal conditions. In the experiments, salicylic acid was used as the free-radical scavenger and the formation of three different intermediates were examined to shed light on the trend and kinetics of reaction of hydroxyl radical with organic substance under different operation conditions. The results indicated that the yield of hydroxyl radicals increased with increasing irradiation intensity and dissolved oxygen level. The optimal experimental conditions obtained in this study were irradiation with intensity of 2.9 mW cm −2 on salicylic acid at concentration of 250 mg L −1 by both agitation and aeration processes (dissolved oxygen level = 8.2 mg O 2 L −1) at pH 5. Such conditions could achieve the optimal hydroxyl radical yield of 5.1 × 10 −17 M.

Solid-phase synthesis of aryl, heteroaryl, and sterically hindered alkyl amines using the Curtius rearrangement

An efficient method for the solid-phase synthesis of aryl amines, heteroaryl amines, and sterically hindered alkyl amines has been developed. The key step in this process was the formation of resin-bound carbamates ( B) by the Curtius rearrangement of aryl carboxylic acids with Wang resin providing the trapping hydroxyl group. N-Alkylation reactions of B gave secondary amines in good yield. Some biaryl amines, which are found widely in biologically active substances, were also prepared by the Suzuki reaction of resin-bound carbamates of 2-iodoaniline ( 16) or 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline ( 21). The developed methods can be applied to the preparation of libraries containing aryl, heteroaryl, and sterically hindered alkyl amine structures as the pharmacophores.

フェノール性二量体の抗酸化活性と抗菌活性

We studied the antioxidant and antibacterial activities of monomeric and dimeric phenol compounds. Dimeric compounds had higher antioxidant activities than monomeric compounds. Electron spin resonance spin-trapping experiments showed that phenol compounds with an allyl substituent on their aromatic rings directly scavenged superoxide, and that only eugenol trapped hydroxyl radicals. We developed a generation system of the hydroxyl radical without using any metals by adding L-DOPA and DMPO to PBS or MiliQ water in vitro. We found that eugenol trapped hydroxyl radicals directly and is metabolized to a dimer. On the other hand, dipropofol, a dimer of propofol, has strong antibacterial activity against Gram-positive bacteria. However, it lacks solubility in water and this property is assumed to limit its efficacy. We tried to improve the solubility and found a new solubilization method of dipropofol in water with the addition of a monosaccharide or ascorbic acid.

Detection and characterization of cyclic hydroxylamine adducts by mass spectrometry

Two cyclic hydroxylamines (cHA) bearing pyrrolidine (CPH) and piperidine moieties (TMTH) were evaluated to trap hydroxyl, peptide and phospholipid free radicals using mass spectrometry for their detection. The cHA ionized as [M+H]+ ions, showing higher relative abundances when compared to the DMPO, probably due to higher ionization efficiency. In the presence of hydroxyl radicals, both cHA generated new ions that could be attributed to loss of •H and •CH3, most likely deriving from decomposition reactions of the nitroxide spin adduct. Addition of cHA to Leucine-enkephalin and palmitoyl-lineloyl-glycerophosphatidylcholine free radicals promoted the formation of cHA biomolecule adducts, which were confirmed by MS/MS data. Results suggest that the cHA are not suitable for hydroxyl radical trapping but can be used for trapping biomolecule radicals, having the advantage, over the most used cyclic nitrones, of being water soluble. The biomolecule adducts identified by MS are ESR silent, evidencing the importance of MS detection.

ESR Measurement Using 2-Diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-Oxide (DPhPMPO) in Human Erythrocyte Ghosts

Abstract Competitive spin-trapping behavior of 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) and 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) indicated that the affinity of DPhPMPO for hydroxyl radicals was 1.7 times higher than that of DMPO. Human erythrocyte ghosts (HEGs) with encapsulated FeII catalysts (HEG–Fe) were prepared for ESR measurement. By trapping the hydroxyl radical (OH•) in the presence of polyethylene glycol (PEG-4000) outside the HEG–Fe, the generation of OH• inside the catalyst was confirmed. The trapping of hydroxyl radicals generated in HEG was accomplished using DPhPMPO, and the permeability of the erythrocyte membrane to DPhPMPO was 2.4 times greater than that of DMPO.

Effects of operational parameters on the decolorization of C.I. Reactive Red 198 in UV/TiO 2-based systems

The effects of pH, dye concentration, electron acceptor (hydrogen peroxide, persulfate and copper ions) and hydroxyl radical trap (ethanol) on the decolorization of C.I. Reactive Red 198 using a UV/TiO 2 system were determined. Decolorization rate increased as the concentration of persulfate ions added increased. The k values followed the order: pH 4 > pH 7 > pH 10 for the UV/TiO 2, UV/TiO 2/H 2O 2 and UV/TiO 2/Na 2S 2O 8 systems. Hydrogen peroxide, persulfate and copper ions trapped photogenerated electrons and inhibited the recombination of electron/hole pairs in UV/TiO 2, thereby accelerating decolorization rate; persulfate ions trapped photogenerated electrons and generated sulfate free radicals. The addition of ethanol inhibited decolorization, suggesting that the predominant decolorization pathway involves hydroxyl radicals and that direct oxidation by photogenerated holes is also significant.

Synthesis and ESR studies of a novel cyclic nitrone spin trap attached to a phosphonium group-a suitable trap for mitochondria-generated ROS?

In this study we report the synthesis and biological application of a novel cyclic nitrone spin trap containing a phosphonium cation. This new spin trap ([4-(2-methyl-1-oxy-3, 4-dihydro-2H-pyrrole-2-carbonyloxy)-butyl]-triphenyl-phosphonium bromide, MitoBMPOBr) is a derivative of the cyclic nitrone, 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO). MitoBMPOBr forms radical adducts upon trapping of superoxide and hydroxyl radicals that exhibit highly distinct and characteristic EPR spectra. The stability of these adducts is comparable to those of BMPO. Because of the presence of a positively-charged phosphonium moiety, MitoBMPOBr may be suitable for trapping reactive oxygen species (ROS) in the mitochondria.

Antioxidant properties of different fractions of tubers from Pueraria tuberosa Linn

Pueraria tuberosa Linn. (PT), Leguminosae, is a perennial climber, growing throughout tropical parts of India. In the Ayurvedic system of medicine, it is used as a drug of choice to manage pain, inflammation and other related diseases. The antioxidant potency of P. tuberosa was investigated for the first time. Total antioxidant capacity was determined using an ABTS ∗+ assay. Lipid peroxidation was assessed in terms of thiobarbituric acid-reactive substances by using egg-yolk homogenates as lipid-rich media. Superoxide radical-scavenging was measured using riboflavin-light-nitro blue tetrazolium (NBT) assay. Hydroxyl radical trapping potential was determined by evaluating hydroxyl radical induced deoxyribose degradation using thiobarbituric acid method. In order to assess the metal chelation property, hydroxyl radical induced deoxyribose degradation was evaluated in the absence of ethylenediaminetetraacetic acid. Both hexane and methanol fractions inhibited lipid peroxidation and also chelated the iron, showing potent antioxidant property.

Halogenated Derivatives of Aromatic Amino Acids Exhibit Balanced Antiglutamatergic Actions: Potential Applications for the Treatment of Neurological and Neuropsychiatric Disorders

Glutamate, the major excitatory neurotransmitter, is critical for normal brain development and function. Both extremes of glutamate receptor activity are detrimental for the brain. Glutamate's role in excitotoxicity has driven the search for receptor antagonists as neuroprotective agents, most of which have failed to achieve clinical, i.e. efficacious and safe, neuroprotection. High selectivity and potency provide potential explanations for this failure. For example, targeting individual glutamate receptor subtypes leaves other pathways of glutamatergic excitotoxicity intact. Furthermore, potent depression of glutamate receptor activity causes clinical side effects, such as the symptoms of schizophrenia produced by NMDA receptor antagonists. To produce efficacious neuroprotection devoid of significant side effects, it may be necessary to normalize the function of all components of the glutamatergic system, instead of blocking a single type of glutamate receptors. Halogenated derivatives of aromatic amino acids modulate glutamatergic activity via multiple pre- and postsynaptic actions with moderate efficacy. In addition, these compounds may trap hydroxyl radicals and facilitate hydroxyl radical-impaired glutamate uptake. Their balanced polyvalent action may overcome the limitations of previously tested glutamatergic agents and provide a basis for their use in the treatment of neurological and neuropsychiatric disorders. The properties of this class of compounds and relevant patents are reviewed in this article.

Antioxidant properties of different fractions of Vitex negundo Linn.

Vitex negundo Linn. (VN), belonging to family Verbenaceae, is an aromatic shrub distributed throughout India. In the ayurvedic system of medicine it is used as a drug of choice to manage pain, inflammation and other related diseases. It contains many polyphenolic compounds, terpenoids, glycosidic iridoids and alkaloids. Since polyphenolic compounds have high antioxidant potential, the antioxidant potency of V. negundo was investigated by employing various established in vitro systems, such as 2,2′-azino-bis 3-ethyl benzothiazoline-6-sulfuric acid (ABTS ∗+)/Lipid Peroxides (LPO)/Superoxide/Hydroxyl radical scavenging and iron ion chelation. Total antioxidant capacity was determined by the assay based on the preformed radical monocation ABTS ∗+. Lipid peroxidation was assessed in terms of thiobarbituric acid reactive substances by using egg yolk homogenates as lipid rich media. Superoxide radical scavenging assay was based on the riboflavin-light-Nitro blue tetrazolium (NBT) system. Hydroxyl radical trapping potential was determined by evaluating hydroxyl radical induced deoxyribose degradation using the thiobarbituric acid method. In order to assess the metal chelation properties, hydroxyl radical induced deoxyribose degradation was evaluated in the absence of Ethylenediamine tetra acetic acid (EDTA). All the polar fractions significantly showed trapping of free radicals, and thereby inhibition of lipid peroxidation, and also chelated the iron ion. Interestingly, the hexane fraction did not show any activity against superoxides radicals and it had minimum trapping potential for other free radical (FR) species also. Thus, it may be concluded that the polar fractions of VN possess potent antioxidant properties, which may be mediated through direct trapping of the free radicals and also through metal chelation. Therefore its reported anti-inflammatory properties, could be through the down regulation of the free radical mediated pathway of inflammation.

Edaravone (3-Methyl-1-Phenyl-2-Pyrazolin-5-one), A Novel Free Radical Scavenger, for Treatment of Cardiovascular Diseases

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a strong novel free radical scavenger, is used for treatment of patients with acute brain infarction. Edaravone has preventive effects on myocardial injury following ischemia and reperfusion in patients with acute myocardial infarction. Antioxidant actions of edaravone include enhancement of prostacyclin production, inhibition of lipoxygenase metabolism of arachidonic acid by trapping hydroxyl radicals, inhibition of alloxan-induced lipid peroxidation, and quenching of active oxygen, leading to protection of various cells, such as endothelial cells, against damage by reactive oxygen species (ROS). Recently, we have shown that edaravone improves endothelial function through a decrease in ROS in smokers. From a clinical perspective, it is important to select an appropriate drug that is effective in improving endothelial function in patients with cardiovascular diseases. The novel free radical scavenger edaravone may represent a new therapeutic intervention for endothelial dysfunction in the setting of atherosclerosis, chronic heart failure, diabetes mellitus, or hypertension. This review focuses on clinical findings and on putative mechanisms underlying the beneficial effects of the antioxidative agent edaravone on the artherosclerotic process in patients with cardiovascular diseases.

Genetically Modified Hairy Roots ofWithania somniferaDunal: A Potent Source of Rejuvenating Principles

Transgenic hairy roots were induced from Withania somnifera Dunal, by infecting leaf explants with Agrobacterium rhizogenes. Polymerase chain reaction for rol A gene and Southern blot confirmed the integration of T-DNA in the genome. Cultures were grown in Murashige and Skoog solid as well as in liquid medium. The antioxidant activity was assayed in roots grown in solid media and liquid media. Hairy roots grown in liquid media found to possess highly significant activity in 1,1-diphenyl-2-pecryl-hydrazyl radical, beta-carotene linoleic acid model system. The activity was 57.34%, 75.64%, and 93.41% in case DPPH model and 55.3%, 76.3%, and 90.5% in case of b-CLAMS in 25, 50, and 100 mg L(-1) concentration, respectively. In case of hydroxyl radical trapping and brain lipid peroxidation assay, the activity was more significant in hairy roots grown on solid medium in comparison with commercial formulation prepared using normal roots and standard withanaloids. Root extract grown in solid medium has shown 93.2% hydroxyl radical trapping activity at 100 mg L(-1) concentration, and 500 mg L(-1) has shown 83.6% in case of brain lipid peroxidation assay. High-performance liquid chromatography analysis demonstrated the presence of withanaloids in the hairy root extracts. The results of the study clearly indicate that there is enhancement of secondary metabolites in hairy roots, which is indicated through significant enhancement of the antioxidant activity, since these are the major constituents responsible for the activity. This is the first report on the presence of antioxidant principles in genetically modified roots of W. somnifera. These results of the present study may aid in utilization of the W. somnifera hairy roots for its rejuvenating principles.

Trapping Effect of Eugenol on Hydroxyl Radicals Induced by L-DOPA in Vitro

Many researchers have stated that eugenol might inhibit lipid peroxidation at the stage of initiation, propagation, or both, and many attempts have been made to elucidate the mechanism of its antioxidant activity. Nevertheless, details of its mechanism are still obscure. This study was carried out to investigate the trapping effect of eugenol on hydroxyl radical generated from L-3,4-dihydroxyphenylalanine (DOPA) in MiliQ water and the generation mechanism of the hydroxyl radical by this system which uses no metallic factor. This was studied by adding L-DOPA and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to phosphate buffered saline (PBS) or MiliQ water, and the generation of hydroxyl radical was detected on an ESR spectrum. By this method, the effect of antioxidants was detected as a modification of ESR spectra. We found that the eugenol trapped hydroxyl radicals directly, because it had no iron chelating action, did not trap L-DOPA semiquinone radical and inhibited hydroxyl radicals with or without iron ion.

Determination of the antioxidant capacity of different food natural products with a new developed flow injection spectrofluorimetry detecting hydroxyl radicals

This paper presents an automatic spectrofluorimetric method (flow injection analysis spectrofluorimetry) for detecting hydroxyl radicals. Based on H 2O 2 catalyzed by Co 2+ yielding HO , the method utilized sodium terephthalate to trap hydroxyl radicals and obtained sodium 2-hydroxyterephthalate by aromatic hydroxylation, which resulted in an increase in the fluorescence intensity. The relative fluorescence intensity was proportional to the concentration of hydroxyl radicals. Hydroxyl radicals could be determined indirectly, based on the increase of fluorescence. It was a simple, rapid, precise, sensitive and automatic technique for the determination of HO . The relative standard deviation of 11 determinations was 0.57%. The method can be used to sieve antioxidant medicines and will have theoretical and practical guidance on the mechanism of hydroxyl radicals-damaging biology.

UV or visible light induced photodegradation of AO7 on TiO 2 particles: the influence of inorganic anions

In this paper, the effects of various inorganic anions (NO 3 −, Cl −, SO 4 2−, HCO 3 −, H 2PO 4 −) on the photodegradation of an azo dye, acid orange 7 (AO7) were investigated under UV or visible light irradiation in the presence of TiO 2 particles. The effects of inorganic anions on the photodegradation of AO7 under visible light irradiation were significantly different from UV irradiation pathway. In TiO 2/UV system, inorganic anions inhibited the photodegradation of the dye by their trapping hydroxyl radicals. In TiO 2/Vis system, the observed inhibition effects of inorganic anions can be interpreted by competitive adsorption. In addition, the results indicated that the photodegradation of AO7 took place mainly in the bulk solution under UV light irradiation, while under visible light irradiation, the reaction occurred on the catalyst surface.

External pressure modulates spin-trapping rates in solution as studied with electron spin resonance

Using a competitive spin-trapping method, we determined the effect of high static pressure on the trapping rate of hydroxyl and phenyl radical in solution. Under various pressures, hydroxyl or phenyl radicals were produced in solution in the presence of two different spin-trapping compounds, and the yield of trapped products was used to calculate relative trapping rates. We found that external pressure increases the phenyl-radical trapping rates by approximately 40% at the pressure of 490 bar, while there was no pressure effect in hydroxyl-radical trapping rates. We interpreted the pressure effect based on the reactivity difference of phenyl and hydroxyl radicals to the spin trap which is mainly caused by the variation in the volume of the group.