Free Radicals In Biological Systems Research Articles

Synthesis, DFT Study, and In Vitro Evaluation of Antioxidant Properties and Cytotoxic and Cytoprotective Effects of New Hydrazones on SH-SY5Y Neuroblastoma Cell Lines.

A series of ten new hydrazide-hydrazone derivatives bearing a pyrrole ring were synthesized and structurally elucidated through appropriate spectral characteristics. The target hydrazones were assessed for radical scavenging activity through 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) tests, with ethyl 5-(4-bromophenyl)-1-(2-(2-(4-hydroxy-3,5-dimethoxybenzylidene)hydrazine-yl)-2-oxoethyl)-2-methyl-1H-pyrrole-3-carboxylate (7d) and ethyl 5-(4-bromophenyl)-1-(3-(2-(4-hydroxy-3,5-dimethoxybenzylidene) hydra zine-yl)-3-oxopropyl)-2-methyl-1H-pyrrole-3-carboxylate (8d) highlighted as the best radical scavengers from the series. Additional density functional theory (DFT) studies have indicated that the best radical scavenging ligands in the newly synthesized molecules are stable, do not decompose into elements, are less polarizable, and with a hard nature. The energy of the highest occupied molecular orbital (HOMO) revealed that both compounds possess good electron donation capacities. Overall, 7d and 8d can readily scavenge free radicals in biological systems via the donation of hydrogen atoms and single electron transfer. The performed in vitro assessment of the compound's protective activity on the H2O2-induced oxidative stress model on human neuroblastoma cell line SH-SY5Y determined 7d as the most perspective representative with the lowest cellular toxicity and the highest protection.

An overview of recent advances in the detection of ascorbic acid by electrochemical techniques

Ascorbic acid is a water-soluble vitamin essential in human nutrition, an antioxidant, a scavenger of free radicals in biological systems, and a cofactor of several enzymes. The reference range for ascorbic acid in healthy people is 6 - 20 mg L-1. The variable concentration of ascorbic acid within biology fluids was found in clinical investigations to be a metric for assessing the exact amount of oxidative stress in the body's metabolism. Electroanalytical techniques are a group of methods in analytical chemistry, especially with extensive application in pharmaceutical industries. These techniques attracted further attention due to their unique characteristics, such as reduced sample or solvent con­sumption, high analysis speed, low operating cost, and high sensitivity, which made them suitable candidates for replacement or supplementation for spectrophotometry and separation approaches. The purpose of this article is to scrutinize the mechanisms and applications of current electroanalytical methods, including amperometric techniques, square wave voltammetry, differential pulse voltammetry and cyclic voltammetry, in their applications in pharmaceutical analysis for the detection of ascorbic acid. Related examples have been cited in the form of selected studies.

Benchtop X-band electron paramagnetic resonance detection of melanin and Nitroxyl spin probe in zebrafish

EPR spectroscopy is a technique that provides direct information about free radicals in biological systems. So far, X-band EPR was seldomly used for in vivo studies as the small resonator size and high power used to detect EPR signals were unsuitable for living organisms. Here, we report new solutions which lift some limitations and make X-band EPR suitable for an in vivo detection of free radicals in zebrafish - a small laboratory animal that is often used as a model for various studies related to free radicals. We designed specially-shaped glass and quartz capillaries to ensure the zebrafish's safety during the experiments. The optimal EPR spectrometer parameters, safe for zebrafish embryos and sufficient to obtain EPR spectrum, were 4 scans by 20s, 100G sweep, and 0.8 mW power. Combining the specially-shaped capillary with a multi-harmonic analyzer for the EPR spectrometer allowed increasing the time up to 16 scans by 11s and lowering the power to 0.25 mW. As a proof of principle, we demonstrate the detection of melanin radicals and the 5-DSA spin probe in zebrafish larvae. As fish survive the EPR scans, the possibility of performing multiple measurements of free radicals in living zebrafish offers new tools for studies aiming to understand redox biology and membrane-dependent functions in both health and disease.

A facile approach for synthesis of nano-CeO2 particles loaded co-polymer matrix and their colossal role for blood-brain barrier permeability in Cerebral Ischemia

A prospective resource of pharmacological treatment of ischemic brains stroke is rapid interference using potential neuroprotective materials. Cerium oxide nanoparticles have been shown to defend against blood brain barrier damage in cerebral ischemic brain stroke. While cerium oxide nanoparticles is highly permeable across the blood–brain barrier and also these nanoparticles are effective antioxidants, due to its ability to either donate or obtain electrons with alternative +3 and +4 valence states. This oxidation state of cerium oxide has shown efficiency in neutralizing generated free radicals in biological systems has been explored action for cerebral ischemic brain stroke. The nanoparticles are encapsulated on the poly-(lactide-co-glycolide)-polyethyleneglycol copolymer matrixes as nanoparticulate delivery vehicles and it can be enhanced brain targeted drug delivery. Furthermore, the results of spectroscopic and microscopic analysis confirmed that peripheral PEG-PLGA co-polymer chains provide excellent reactivity with nanoparticles which might improve the interface bonds of the nanocomposite formation. Mainly, neuroprotective properties of prepared CeO2-PEG/PLGA matrixes with and without nanoparticles are comparatively studied by using transient middle cerebral artery occlusion (MCAO) model of brain stroke. The prepared CeO2 nanoparticles combined with effective PEG/PLGA matrixes exhibited greater efficacy resulted in a lessening of focal ischemia by 60% and 78% decrease in brain edema in comparable to the control animals. The results are demonstrated that the neuroprotective efficiency of CeO2 nanoparticles with PEG/PLGA has enhanced and primarily protected the brain cortex areas from ischemic damage.

Stress-Generated Free Radicals Detected by Electron Paramagnetic Resonance Spectroscopy with Nitrone Spin Trap in Vicia faba Root

Electron paramagnetic resonance (EPR) spectroscopy is a promising technique for detection and quantification of short-lived free radicals in biological systems. In the present investigation, root of Viciafaba seedlings were subjected to 0.5 mM (T1) and 1 mM (T2) sodium azide (NaN3) in hydroponic medium. The stress-generated free radicals (superoxide and hydroxyl free radicals) in the root tips of the seedlings were analyzed by EPR spectroscopy using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) nitrone spin. Single cell gel electrophoresis (comet assay) and mitotic index along with the spectrophotometric measurement of the two major antioxidant enzymes, namely, superoxide dismutase (SOD) and catalase (CAT), in combination with analysis of lipid peroxidation product, malondialdehyde (MDA), were done to validate the EPR results. Data obtained from comet assay of root tip cells revealed significant increase in the olive moment in T1 and T2 as compared to control. Decreased mitotic index in T2 (60.63 ± 7.74%) as compared to control (88.23 ± 1.01%) in these cells as a consequence of increased reactive oxygen species (ROS) was depicted. Furthermore, the decrease in the activity of major antioxidant enzymes, SOD and CAT justified the intensified DMPO-OH specific peaks in T2 as compared to control obtained in the EPR spectra. The results establish that EPR spectroscopy can be used successfully at room temperature to monitor changes in the production of free radicals (combined superoxide and hydroxyl free radicals) in plant tissues using DMPO as a spin trap.

Electron Paramagnetic Resonance Spectrometry-based Assessment of Free Radicals Scavenging Potential of N-acetyl Tryptophan Glucoside

<p>Gamma radiation generates free radicals in biological system by inducing cellular water radiolysis. If not neutralised, free radicals oxidise vital bio-macromolecules causing structural and functional impairment and contribute to cell death. In present study, free radical scavenging activities of a novel bacterial secondary metabolite, N-acetyl tryptophan glucoside (NATG) was assessed against gamma-radiation induced damage in vitro and in vivo models. Effect of irradiated NATG (UV and gamma radiation 8 Gy and 20 Gy) on its free radical (DPPH radicals) and SOD-like activity was evaluated using EPR spectrometry. To assess the effect of NATG irradiation on its antioxidant potential, EPR based ascorbate, PBN and NO radicals scavenging activities were evaluated in blood and spleen tissue of strain A male mice. Results of the study indicated significant (p < 0.05) increase in DPPH radicals scavenging ability of irradiated NATG as compared to un-irradiated NATG. Similarly, irradiated NATG exhibited significant (p < 0.05) elevation in SOD-like activity as compared to control. Subsequently, NATG treatment displayed enhanced antioxidant activity as evident by significant (p < 0.05) decline in ascorbate, PBN and NO radicals at 1 h and 2h in blood and spleen tissues homogenate of treated mice as compared to control group. In conclusion, NATG possesses significant free radicals scavenging and radio protective capabilities against gamma radiation induced oxidative stress.</p>

Use of a cocktail of spin traps for fingerprinting large range of free radicals in biological systems.

It is well established that the formation of radical species centered on various atoms is involved in the mechanism leading to the development of several diseases or to the appearance of deleterious effects of toxic molecules. The detection of free radical is possible using Electron Paramagnetic Resonance (EPR) spectroscopy and the spin trapping technique. The classical EPR spin-trapping technique can be considered as a “hypothesis-driven” approach because it requires an a priori assumption regarding the nature of the free radical in order to select the most appropriate spin-trap. We here describe a “data-driven” approach using EPR and a cocktail of spin-traps. The rationale for using this cocktail was that it would cover a wide range of biologically relevant free radicals and have a large range of hydrophilicity and lipophilicity in order to trap free radicals produced in different cellular compartments. As a proof-of-concept, we validated the ability of the system to measure a large variety of free radicals (O-, N-, C-, or S- centered) in well characterized conditions, and we illustrated the ability of the technique to unambiguously detect free radical production in cells exposed to chemicals known to be radical-mediated toxic agents.

Cerium oxide based nanozymes: Redox phenomenon at biointerfaces.

Cerium oxide nanoparticles (nanoceria) are known to exhibit enzymelike activity, such as biological catalase, oxidase, superoxide dismutase, and peroxidase enzymelike activities. Catalytically active nanoceria offer several advantages over natural enzymes, such as controlled synthesis at low cost, tunable catalytic activities, as well as high stability against stringent physiological conditions. Exploiting these properties, several biomedical applications, such as biosensing, immunoassay, drug delivery, radiation protection, and tissue engineering, have been maneuvered. This review article provides a comprehensive summary of reported biological enzymelike activities of nanoceria and the possible mechanism of catalysis. It is also discussed that what physicochemical properties (surface charge, size, surface chemistry, and enzymelike activity) of nanoceria are altered when exposed to biologically relevant buffers and even in the mammalian cell cytoplasm. Based on various reports provided in the literature, these important issues need to be addressed. It has also been deliberated that in mammalian cells variations in properties of nanoceria observed are due to the intrinsic catalytic activity or as a result of downstream effects that could have emerged after the particles have interacted with biologic/environmental components. Such studies could be helpful in providing a better understanding the nanoceria's multienzyme-like activities and potential applications in the biomedical fields with special reference to damaging effects of free radicals in biological systems.

Evaluation of Cytotoxic Effect and Antioxidant Activity of Grape Seed Extract, Crocin, and Phenytoin.

Antioxidant compounds inhibit formation of free radicals, chelate catalytic metals, and scavenge free radicals in biological systems. In addition, antioxidants play a decisive role in prevention of numerous physiological dysfunctions, cancers, and metabolic disorders. This study sought to evaluate the antioxidant capacity and cytotoxic effect of grape seed extract (GSE), crocin (CRO), and phenytoin (PHEN) on a human breast cancer cell line (MCF-7). Methanol extracts of the three mentioned agents were prepared and their antioxidant activity was evaluated by diphenyl-1-picrylhydrazyl method, using Quercetine (QUER) as positive control. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the cytotoxic effect of the extracts on Michigan Cancer Foundation-7MCF-7 cell line, using doxorubicin hydrochloride (DOX) as the positive control. Given the results, greater scavenging activity was achieved by using GSE in comparison to CRO and PHEN. Further, a significant correlation was found between the antioxidant activity and cytotoxic effects of these agents, and GSE had the highest antioxidant capacity and cytotoxic effect in comparison to CRO and PHEN.

The Most Influential Scientist in the Development of Medical informatics (10): Marsden S. Blois.

The Most Influential Scientist in the Development of Medical informatics (10): Marsden S. Blois.

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures.

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.

Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps

Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-β-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated β-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.

Reactivity of catecholamine-driven Fenton reaction and its relationships with iron(III) speciation

Introduction: Fenton reaction is the main source of free radicals in biological systems. The reactivity of this reaction can be modified by several factors, among these iron ligands are important. Catecholamine (dopamine, epinephrine, and norepinephrine) are able to form Fe(III) complexes whose extension in the coordination number depends upon the pH. Fe(III)-catecholamine complexes have been related with the development of several pathologies.Methods: In this work, the ability of catecholamines to enhance the oxidative degradation of an organic substrate (veratryl alcohol, VA) through Fenton and Fenton-like reactions was studied. The initial VA degradation rate at different pH values and its relationship to the different iron species present in solution were determined. Furthermore, the oxidative degradation of VA after 24 hours of reaction and its main oxidation products were also determined.Results: The catecholamine-driven Fenton and Fenton-like systems showed higher VA degradation compared to unmodified Fenton or Fenton-like systems, which also showed an increase in the oxidation state of the VA degradation product. All of this oxidative degradation takes place at pH values lower than 5.50, where the primarily responsible species would be the Fe(III) mono-complex.Conclusion: The presence of Fe(III) mono-complex is essential in the ability of catecholamines to increase the oxidative capacity of Fenton systems.

Reaction enthalpies of O[sbnd]H bonds splitting-off in flavonoids: The role of non-polar and polar solvent

Flavonoids play important role in the scavenging of free radicals in biological systems. As the phenolic chain-breaking antioxidants they can act via three distinct mechanisms, namely hydrogen atom transfer (HAT), Single Electron Transfer–Proton Transfer (SET–PT) and Sequential Proton-Loss Electron-Transfer (SPLET). Therefore, it is inevitable to study the corresponding reaction enthalpies in solution-phase. For 10 naturally occurring flavonoids: apigenin, luteolin, fisetin, kaempferol, quercetin, epicatechin, taxifolin, tricetin, tricin and cyanidin, OH bond dissociation enthalpies, ionization potentials, proton dissociation enthalpies, proton affinities and electron transfer enthalpies were investigated using IEF-PCM B3LYP/6-311++G∗∗ method in benzene and water in order to: (i) identify the thermodynamically preferred mechanism and OH group in the two solvents and (ii) describe the solvent effect on the homolytic and heterolytic cleavage of OH groups in studied flavonoids.

Synthesis of a Fluorescence Labeled Spin Trap and Its ESR Study

Free radicals play a key role in physiological and pathological processes including aging and cancer development. To understand the biological role of free radicals,it is of critical importance to develop highly sensitive techniques for the detection and quantitation of free radicals being produced. Among the numbers of techniques used for the detection of free radicals,electron spin resonance( ESR)-spin trapping technique,owing to its relative higher specificity,is believed to be an indispensable method for the detection of free radicals in biological systems. However,the ESR-spin trapping technique was limited becauseit has the relative low sensitivity and it's hard to achieve real-time detection of free radicals in living cells. Considering that the fluorescence probes for labeling and tracking subcellular structures in living cells are easily available,a new spin trap conjugated with fluorescence moiety( DanPPN) was synthesized and characterized. The ESR study showed that the DanPPN could be used for the detection of superoxide anion,oxygen and carbon-centered radicals. Moreover,it was observed that the fluorescence intensity of DanPPN was decreased with the increase of the concentration of AIBN. The results suggested that the new spin trap may have a potential use for the detection of free radicals in biological systems.

Low-Oxygen Atmospheric Treatment Improves the Performance of Irradiation-Sterilized Male Cactus Moths Used in SIT

As part of sterile insect technique (SIT) programs, irradiation can effectively induce sterility in insects by damaging genomic DNA. However, irradiation also induces other off-target side effects that reduce the quality and performance of sterilized males. Thus, treatments that reduce off-target effects of irradiation on male performance while maintaining sterility can improve the feasibility and economy of SIT programs. Exposure to ionizing radiation induces the formation of damaging free radicals in biological systems that may reduce sterile male performance. Here, we test whether exposure to an anoxic environment for 1 h before and during irradiation improves male performance, while maintaining sterility in males of the cactus moth, Cactoblastis cactorum (Berg). We show that exposure to 1 h of anoxia increases the moth's antioxidant capacity and that irradiation in anoxia after 1 h of anoxic conditioning decreases irradiation-induced oxidative damage to the moth's lipids and proteins. Anoxia treatment that reduced oxidative damage after irradiation also produced moths with greater flight performance, mating success, and longevity, while maintaining F1 male sterility at acceptable levels for SIT. We conclude that anoxia pretreatment followed by irradiation in anoxia is an efficient way to improve the quality of irradiated moths and perhaps lower the number of moths needed for release SIT moth operations.

Physicochemical Origin for Free Radical Generation of Iron Oxide Nanoparticles in Biomicroenvironment: Catalytic Activities Mediated by Surface Chemical States

A wide range of nanoparticles (NPs) have been found to generate free radicals in biological systems. Hence, it is hypothesized that free radical generation may play a key role in the mechanism of nanomaterial toxicity in vivo. However, the main physicochemical basis for the free radical generation particularly in the biomicroenvironment has not yet been fully established. In this study, we performed comprehensive spectroscopic techniques to probe the physicochemical origin of free radical generation induced by iron oxide nanoparticles in biomicroenvironment. We demonstrated that α-Fe2O3 and γ-Fe2O3 NPs induced hydroxyl radical (•OH) via homogeneous and heterogeneous Fenton process, depending on the biological microenvironment of pH and reducing agent presence. The physicochemical structures such as chemical states of oxygen and iron on nanosurface are the key factors in the homogeneous and heterogeneous catalytic reactions. A noteworthy finding was that in situ surface reduction of Fe2O3 NPs occurred in t...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation, and ultimately to oxidative stress leading to cell injury or death. Superoxide radical anion (O2•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2•- in a process called uncoupling, which is believed to be caused by oxidation of heme or the co-factor, tetrahydrobiopterin (BH4). There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production. The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO, the phosphoryl-substituted DEPMPO, and the ester-substituted, EMPO and BMPO, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct.

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. Superoxide radical anion (O2•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2•- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8. There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9. The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

PROOXIDANT AND ANTIOXIDANT ACTIVITIES OF ROSMARINIC ACID

Rosmarinic acid is a fundamental compound of Rosmarinus officinalis. In this work, the intrinsic prooxidant (generation of H2O2 and free radicals by the action of peroxidase) and antioxidant (consumption of H2O2 and free radicals) properties of rosmarinic acid are characterized. In addition, its melanogenic prooxidant activity is characterized as it acts as a substrate of polyphenol oxidase to generate o-quinones, and its antimelanogenic and, so, antioxidant activity as it carries out the suicide inactivation of polyphenol oxidase. The balance between its pro- and antioxidant activities demonstrates that rosmarinic acid can be considered as a net antioxidant. PRACTICAL APPLICATIONS Antioxidants prevent oxidative damage by modulating the oxidative status of cells. The present results indicated that rosmarinic acid is an antioxidant and can act as scavenger of free radicals in biological systems, and could therefore provide health benefits to consumers. Moreover, rosmarinic acid can act as a suicide substrate of polyphenol oxidase and can inhibit the enzymatic browning of vegetables and fruits and melanogenesis in mammals.