The Scientific World Journal

The Scientific World Journal / 2014 / Article

Research Article | Open Access

Volume 2014 |Article ID 931581 | 12 pages |

A Modified Method for Studying Behavioral Paradox of Antioxidants and Their Disproportionate Competitive Kinetic Effect to Scavenge the Peroxyl Radical Formation

Academic Editor: A. A. Iglesias
Received15 Aug 2013
Accepted24 Nov 2013
Published05 Feb 2014


We have described a modified method for evaluating inhibitor of peroxyl radicals, a well-recognized and -documented radical involved in cancer initiation and promotion as well as diseases related to oxidative stress and ageing. We are reporting hydrophilic and lipophilic as well as natural and synthetic forms of antioxidants revealing a diversified behaviour to peroxyl radical in a dose-dependent manner (1 nM–10 μM). A simple kinetic model for the competitive oxidation of an indicator molecule (ABTS) and a various antioxidant by a radical (ROO) is described. The influences of both the concentration of antioxidant and duration of reaction (70 min) on the inhibition of the radical cation absorption are taken into account while determining the activity. The induction time of the reaction was also proposed as a parameter enabling determination of antioxidant content by optimizing and introducing other kinetic parameters in 96-well plate assays. The test evidently improves the original PRTC (peroxyl radical trapping capacity) assay in terms of the amount of chemical used, simultaneous tracking, that is, the generation of the radical taking place continually and the kinetic reduction technique (area under curve, peak value, slope, and ).

1. Introduction

Peroxyl radicals (ROO), the chain carrying analogs of perhydroxyl radical (HOO) where the H atom is replaced by an organic group (R), are formed due to the oxidation of proteins and lipids [14]. Activation of neutrophil during oxidative stress related inflammation also produces peroxyl radicals [5, 6]. They are stable (reduction potential [+] 0.77–[+] 1.44 V) and do not dissociate into oxygen because of the stable C–O bond. Its formation depends on the concentration of oxygen and other reactants as well as the hydrophobic/hydrophilic environment in which the reaction occurs [79]. These radicals not only occur in a cell but also transpire in aquatic systems (lakes, rivers, streams, and oceans) and in atmosphere (water droplets). Such successive reactions involving free radicals in biological systems lead to many physiological and pathological progressions [1018].

In study allied to human disease, lipid peroxidation is correlated with peroxyl radical arbitrated reaction [7, 19, 20], induction of DNA damage by superoxide is additively boosted by peroxyl radicals which is implicated in modification of protein, lipid, and DNA cleavage, nevertheless arsenic-mediated ROS generation produces dimethylarsinic peroxyl radicals [7, 21]. The peroxyl radical, besides playing an outstanding character in radicals dependent shabbiness of membranes and proteins, is also fretful in the pathogenesis of a number of human diseases and disorders involving autoxidation of lipids, inactivation of certain enzymes, cleavage of phosphodiester bond resulting in single and double strand breaks, oxidizing thymidine resulting in mutagenic 5-methyl oxidation products, and causing transversion at deoxyguanosine [2226]. Involvement of peroxyl radicals in carcinogenesis relevant to tumor initiation and promotion is well reported. Investigation related to cancer and redox biology proved that diminutive consideration has been given to peroxyl radicals and family of oxygen centred free radicals [27]. Modest information has been demeanour for peroxyl reactions, in spite of its importance in anticancer irradiation therapy, neurodisorders, ischaemia, and other oxidative stress related diseases and disorders [28].

A number of ways have been utilized for averting the constant expression of peroxyl radicals generated by water soluble 2,2′ azobis (2-amidinopropane) dihydrochloride (AAPH) or lipid soluble 2,2′-azobis 2, 4-dimethylvaleronitrile (AMVN) [28, 29] and the interaction of peroxyl radicals with antioxidants/unknown compounds can be analyzed by an assortment of indicator molecule/model systems as given in Table 1. These reported methods evaluate how the substrate protects a compound from being degraded by peroxyl radicals, using an array of molecules as reactive targets/analysis of end products by different techniques. We employed ABTS as target molecule to assess the reactivity of antioxidants towards AAPH/ABAP-derived peroxyl radical formation in an easy controlled mode at 37°C in aqueous media.

Name Reference

BODIPY 581/591 C11[29]
cis-Parinaric acid[30]
Luminescence/chemiluminescence of luminal[3436]
Pyrogallol red[37]
Analysis of lipid hydroperoxides by HPLC[38]
Malondialdehyde-thiobarbituric assay [39, 40]
Electron spin resonance, a spin-trapping technique[41]

As depicted in Figure 1, there is a unimolecular decomposition of AAPH/ABAP resulting in the formation of two carbon-centred radicals and nitrogen; the former reacts with oxygen to produce peroxyl radicals. We have modified the method of Bartosz et al. (1998) [43] and developed a 96-well plate assay procedure in which decomposition of AAPH (the source of peroxyl and alkoxyl radicals generated at a defined rate in aqueous solution) was measured at 414 nm with ABTS forming a green colored complex. In addition to this juncture, the overall reaction depends on temperature, solvent form, time, and pH, propped up by kinetic studies carried out with the help of spectrophotometric analysis, thus clarifying the paradox behaviour of a series of antioxidants (Table 2). We therefore have a simple high throughput access system and/or technique to identify and explore novel scavenger/inhibitor of peroxyl radicals.

Pubchem IDMolecular formulaMolecular weight (g/mol)XLogP3−H-bond donorH-bond acceptor

Trolox4063C14H18O4250.29032 AA: 2.824
α-Tocopherol14985C29H50O2430.7061 AA: 10.712
Nicotinic acid982C6H5NO2123.110.413
β Carotene5280489C40H56536.87264 13.500
Ascorbic acid54670067C6H8O6176.12412 −1.646
n-Propyl gallate4947C10H12O5212.19928 1.835
BHA8456C11H16O2180.24354 3.212
BHT31404C15H24O220.35046 AA: 5.311
TBHQ16043C10H14O2166.21696 2.822
tBHP6410C4H10O290.121 AA: 0.612
Hydrogen peroxide784H2O234.01468 −0.922

2. Materials and Methods

2.1. Chemicals

2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) also known as 2,2′-azobis (2-amido propane) (ABAP), 2,2′azoino-bis(3-ethyl benzothiazoline-6-sulfonic acid) diammoniumsalt (ABTS) 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), hydrogen peroxide (H2O2), L-ascorbic acid, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich Chemical Company, USA, while butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), tert-butyl hydroperoxide (tBHP), butylated hydroxyanisole (BHA), tocopherol, n-propyl gallate, quercetin, and β-carotene were purchased from Himedia, India. Di-sodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), ethanol, methanol, and acetone were purchased from Merck India Ltd. All the chemicals, solvents and reagents used were either of analytical grade or higher.

2.2. Experimental

Stock solution of 0.1 M sodium phosphate buffer (pH 7.0) was made by mixing solutions of Na2HPO4 (0.1 M) and NaH2PO4 (0.1 M) while stock solution of ABTS (5 mM) and AAPH (200 mM) was made in deionized water. 10 mM stock solution of tBHP, BHT, L-ascorbic acid, trolox, nicotinic acid, and hydrogen peroxide was made in deionized water, tocopherol, BHA and n-propyl gallate was made in ethanol, TBHQ in methanol, quercetin in DMSO, and β-carotene in acetone. Dilution was done in 0.1 M sodium phosphate buffer. The concentrations used for the analysis were 100 μM, 10 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM.

Colorimetric peroxyl radical averting assay (PRAA) was done following method described by Bartosz et al. (1998) [43] with modification including increase in the time period from 10 min to 70 min and reduction in the volume of the reaction cocktail by 20 times. The additional feature of the modified method comprises kinetic reduction technique (area under curve, peak value, slope, time of half maximum, mean, and ). Total reaction volume (150 μL) of the modified method in 96-well plate contains 15 μL of samples (10x), 15 μL AAPH (200 mM), 4.5 μL ABTS (5 mM), and rest phosphate buffer. The phosphate buffer was preheated to 37°C for 30 min followed by the addition of other components including AAPH (source of peroxyl radical) added at the last and the absorbance was taken at 414 nm in a kinetic mode by presetting the spectrophotometer at 37°C for 0–70 min. From the kinetic curve, time-dependent increase in the absorbance was read for each compound along with control. Appropriate solvent blank was run in each assay.

2.3. Assay Validation, Data Scrutiny, and Statistical Analysis

Data represented are mean/average ± standard deviation of three independent experiments in duplicate. IC50 values were calculated from dose-responsive curve by using Table Curve 2D Windows version 4.07 (SPSS Inc., Chicago, IL, USA). Using the advanced kinetic reduction technique available in SoftMax Pro Microplate Data Acquisition and Analysis Software Version 5.3 (Molecular Devices Corporation, Sunnyvale CA, USA) various kinetic parameters were calculated: percent values for (milli OD/min), peak, slope, and time of half maximum, mean, and area under curve using the formula: (()/Control*100). Pearson and RSQ values were also calculated for different parameters at higher tested concentration.

3. Results and Discussion

In the present work, a competitive kinetic method using ABTS as an indicator molecule was employed to estimate the reactivity of antioxidants towards AAPH/ABAP-derived peroxyl radical formation. The results obtained indicate that the relative protection afforded by a given compound strongly depends upon the experimental conditions employed and emphasize the role of secondary reactions of the phenol-derived radicals initially formed. Herein we are reporting hydrophilic and lipophilic as well as natural and synthetic forms of antioxidants revealing a diversified behaviour to peroxyl radical in a dose-dependent manner (1 nM–10 μM). Hydrogen peroxide and tert-butyl hydroperoxide were used as checks. The influence of concentration of the antioxidants, duration of reaction system, and inhibition of the radical (cation or structure of antioxidant) absorption were taken into account while determining the scavenging potential.

Figures 2(a)2(l) were plotted with control which showed a linear gradient in absorbance (optical density) from 0.06 to 0.6 within 70 min at 414 nm which gradually reaches maximum (0.8–1.0) in 24 h. The time period of 70 min (10 time increase in OD) was chosen in order to get a clear picture of the compound which was not apparent in 10 min incubation time. For IC50 calculation, the percent scavenging value of (milli absorbance/min) was chosen over the lag time period earlier reported to determine the scavenging capacity. It is evident from our plotted curves that not only lag time period but other features should also be considered while evaluating the ability of a particular compound as an effective scavenger of peroxyl radicals. The behaviour of the compounds varies which can be manifested by the graph not following the pattern of the control and the echelon of the off-track hints the individual ability of both concentration and time-dependent.

A comprehensible concentration-dependent (1 nM–10 μM) diminishing interaction with peroxyl radical was observed in trolox (Figure 2(a)). Maximum kinetic inhibition was detected at 10 μM and percent inhibition (in terms of , milli absorbance/min) was found to be in the range of −8.2 to 67.2 proving it as an effective scavenger of peroxyl radical formation in a dose-dependent manner. Trolox, a cell-permeable, water soluble imitative of vitamin E with compelling antioxidant assets [44, 45], is frequently used as a standard or positive control in antioxidant assays. It is also used to gauge the job of oxidative injury in cell death and ageing [46, 47] and as an effective therapy in the treatment of certain cancers [48]. Noteworthy effect was monitored in α-tocopherol (Figure 2(b)) at 10 μM and 1 μM concentration. Percent inhibition was found to be in the range of −8.0 to 81.5 compared to control. α-Tocopherol is the most imperative (90%) among eight natural tocopherol, as peroxyl radicals scavenger/repressor of lipid peroxidation. Mechanistic study reveals that hydrogen atom is abstracted from the OH group in α-tocopherol by a lipid peroxyl radical [AOO] producing fairly inert tocopheroxyl radical [TocO], which may then react with a second radical [AOO] to yield a nonradical product, AOO-Toc, thus destroying two radicals and terminating the radical chain reactions thereby contributing two electrons as a chain breaking antioxidant [49]. Inspite of scavenging peroxyl radicals, they are unable to act as a potent scavenger of hydroxyl, alkoxyl, nitrogen dioxide, and thiyl radicals in vivo [50]. Behaviour of nicotinic acid (NA) was uncanny till 40 min and afterwards trifling scavenging started with time (Figure 2(c)). Percent inhibition was found to be in the range of −6.1 to 37.4 as the concentration increases from 1 nM to 10 μM. NA, a colorless, water soluble derivative of pyridine with a carboxyl group at the 3-position, is a cofactor for NAD and NADP acting as coenzymes for more than thousand hydrogenases involved in almost every aspect of cell metabolism [51, 52]. Additionally, it also reduces LDL, VLDL-C, and triglycerides but effectively increases HDL [53] and affects vascular endothelial oxidative and inflammatory events [54]. Much litigious β-carotene showed good scavenging effect at 10 μM while at other concentrations the effect was equivalent to basal values (Figure 2(d)). Percent inhibition was found to be in the range of −4.4 to 40.5. Belonging to carotenoid family, β-carotene also known as provitamin A has an unsaturated and long aliphatic hydrocarbon chain ultimately splitting into two molecules of vitamin A. Unlike phenolic antioxidants, they do not have reactive hydrogen to contribute to radicals, which make it difficult to use conventional probes for the assessment of their radical scavenging capacity [55]. Earlier investigators [56, 57] observed that it inhibits peroxyl radical-initiated autoxidation of both tetralin and methyl linoleate and it is more effective antioxidant at 15 torr oxygen concentration than at 150 torr. Others have noticed a reticent increase in its action against liposomes at 15 torr [56, 58] and at about 4 torr in microsomes [56, 59]. β-Carotene eventually form a resonance-stabilized, carbon-centered radical adduct [AOO-β-C]. Interaction of second peroxyl radical to the adduct produces a nonradical product and results in an overall trapping of two peroxyl radicals per β-carotene consumed [56]. L-Ascorbic acid was found as an excellent scavenger at 10 μM and 1 μM, an effect which decreases at lower concentrations (Figure 2(e)). Percent inhibition was found to be in the range of −2.5 to 97.7. Also known as vitamin C, ascorbic acid is an important water soluble antioxidant in extracellular fluid present in its deprotonated state under most physiologic conditions [6062]. It is effective scavenger of superoxide anion radical, hydrogen peroxide, hypochlorite, the hydroxyl radical, and peroxyl radicals [60, 6369]. Comparatively, it is more effective in inhibiting lipid peroxidation initiated by a peroxyl radical than other human plasma components, such as protein thiols, urate, bilirubin, and α-tocopherol [60, 70]. Ascorbic acid can also protect membranes against peroxidation by enhancing the action of tocopherol and thereby reestablishing the radical scavenging activity [60, 7174]. Quercetin showed a preeminent scavenging of peroxyl radical formation both at 10 μM and 1 μM concentration (Figure 2(f)) and the percent scavenging effect was found to be in the range of 11.48 to 98.3 in a concentration-dependent manner. It is a flavonoid phytochemical naturally occurring in the rind and bark of numerous plants. Chemically it is 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one where B-ring is the active center for scavenging and stabilizing the free radicals [7577]. Best effect of averting action of peroxyl radical by n-propyl gallate was detected at 10 μM with marginal to no scavenging was found at 0.1 μM, 1 μM, 10 nM, and 1 nM concentration (Figure 2(g)). Percent averting action was found to be in the range of 17.17 to 79.57. Propyl gallate, obtained from natural gallic acid (3,4,5-trihydroxybenzoic acid, C6H2(OH)3COOH), is one of the most effective antioxidant-based antimicrobials for the food industry. It has two functional groups hydroxyl and carboxyl and its two analogues were more effective than trolox in preventing cell lysis of human erythrocytes induced by peroxyl radical initiator [78]. Butylated hydroxyanisole (BHA) and butyl hydroxytoluene (BHT) are synthetic antioxidants exhibiting diverse effect. Significant inhibition was observed at 10 μM and 1 μM which decreases at lower dose (Figures 2(h) and 2(i)). Percent inhibition was found to be in the range of 23.75 to 96.6 and −10.9 to 96.2 for BHA and BHT, respectively. BHA consists of a mixture of two isomeric organic compounds, 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. It is a waxy solid used as a food additive (E320), as an antioxidant and preservative in food, food packaging, animal feed, cosmetics, rubber, and petroleum products to prevent from rancidity and developing objectionable odors. It is also used in medicines, such as isotretinoin, lovastatin, and simvastatin [79]. BHT is a lipophilic organic compound which behaves as a synthetic analogue of vitamin E, primarily acting as a terminating agent that suppresses autoxidation by converting peroxyl radicals to hydroperoxides through donating a hydrogen atom [80]. EC50 value for BHA and BHT was found to be and μM, respectively, in an ABAP generated peroxyl radicals, measured by inhibition of dichlorofluorescein oxidation [33]. Like BHT, the conjugated aromatic ring of BHA is able to stabilize free radicals through sequestration. Tertiary butylhydroquinone (TBHQ) flaunted good forestalling action at 10 μM while fairly negligible effects were seen at other concentration (Figure 2(j)). Percent inhibition was found to be in the range of 19.97 to 61.37 in a concentration-dependent manner. TBHQ, a derivative of hydroquinone, substituted with tert-butyl group reacts with peroxyl radicals to form a semiquinone resonance hybrid which undergo different reactions to form more stable products reacting with one another to form dimers, dismutate, and regenerate as semiquinones before finally counteracting with another peroxy radical [81]. Interaction with peroxyl radicals exhibited no effect with tert-butyl hydroperoxide (Figure 2(k)) and hydrogen peroxide (Figure 2(l)). Both tert-butyl hydroperoxide (tBuOOH/tBHP) and hydrogen peroxide (H2O2) were used as check compound for seeing the effect in peroxyl radical formation. tBuOOH/tBHP used in a variety of oxidation processes depletes GSH, induces lipid peroxidation, and tempts ROS formation, involved in PLA(2) activation in hepatocyte injury [82], responsible for K+ leakage [83]. Hydrogen peroxide (H2O2) is a potent oxidant and is even more toxic to cells than superoxide radicals removed by enzymes, such as catalase, glutathione peroxidases, and cysteinyl peroxidase [49].

Antioxidants deactivate free radicals either by reduction via electron or by hydrogen atom. The end point result is the same regardless of the mechanism but the kinetics differ. For kinetic analysis we have used software in which kinetic data reduction options were present. We tried different options present to get a vivid picture of our experimental observations. All the kinetic parameters were calculated with respect to control as percent inhibition. The advantage of calculating using percent inhibition brought all kinetic parameters on the same platform in order to judge the kinetic behaviour characteristics independently. Percent inhibition of (milli OD/min) of the kinetic curve was plotted for all twelve compounds (Figure 3) and IC50 values were calculated from table curve as given in Table 3. is the maximum slope of the kinetic display of mOD/min, calculated by measuring the slopes of a number of straight lines, where points determine the number of contiguous points over which each straight line is defined. This is an alternative method for analyzing nonlinear kinetic reactions that reports the elapsed time until the maximum reaction rate is reached, rather than reporting the maximum rate itself. rate is reported as signal/min (milli-OD/min) for a kinetic read. It is calculated using a linear curve fit, . A creeping iteration is performed using points and the slope of the steepest line segment is reported as rate. The first slope is calculated for a line drawn beginning at the first reading as defined by lag time and ending at a total number of readings equal to the points setting. The second and any subsequent slopes are calculated beginning at the second time point and ending at a total number of readings. The steepest positive or negative slope is reported as . Decreasing order for the highest concentration used (10 μM) is quercetin > L-ascorbic acid > BHA > BHT > α-tocopherol > trolox > TBHQ > β-carotene > n-propyl gallate > nicotinic acid > tBHP > H2O2.

NameIC50 (µM)Lag time (min) Concentration

Trolox0.1193010  M
α-Tocopherol0.289351  M
Nicotinic acidNDNDND
β-Carotene91.951510  M
Ascorbic acid1.38151  M
Quercetin0.239200.1  M
n-Propyl gallate5.84510  M
BHA0.21350.1  M
BHT0.885401  M
TBHQ8.931510  M
Hydrogen peroxideNDNDND

ND: not detected. Each data is mean average values of three independent experiments.

Percent inhibition of area under curve is represented in Figure 4. Defined by the data within the reduction limits, plots are treated as a series of trapezoids with vertices at successive data points and at the -axis coordinates of the data points. The areas defined by each of the trapezoids are then computed and summed. Order was found to be trolox > quercetin > n-propyl gallate > α-tocopherol > L-ascorbic acid > β carotene > BHA > TBHQ > nicotinic acid > H2O2 > tBHP > BHT. Figure 5 signifies the percent slope (rate) of the time-dependent kinetics with respect to control. The slope reduction option determines the slope of the combined plot using all visible time points in the reduction window. Slope is the same as rate when rate is set to the same number of points as the run but is different if we have modified points. Order noticed was trolox > L-ascorbic acid > BHA > quercetin > n-propyl gallate > α-tocopherol > nicotinic acid > β-carotene > BHT > tBHP > TBHQ > H2O2.

Implication of the percent values with respect to control was also calculated on the basis of time to half maximum of the kinetic curve (Figure 6). It denotes half of maximum OD to the time that falls within the reduction limits. The software determines the kinetic point that has maximum OD, divided by 2 thus getting maximum values; further it finds the time at this maximum value. Order of the percent values was recorded as BHT > quercetin > trolox > TBHQ > L-ascorbic acid > nicotinic acid > α-tocopherol > tBHP > BHA > H2O2 > β carotene > n-propyl gallate. Figure 7 designates percent mean values with respect to control, representing the average values (OD) generated during the specified time. Order was found to be BHT > trolox > L-ascorbic acid > quercetin > BHA > TBHQ > n-propyl gallate > α-tocopherol > β-carotene > nicotinic acid > H2O2 > tBHP. Figure 8 represents percent inhibition calculated from peak values of the kinetic data with respect to control, representing maximum absorbance of the compound at 414 nm. Order was found to be BHT > trolox > quercetin > L-ascorbic acid > BHA > TBHQ > n-propyl gallate > α-tocopherol > nicotinic acid > β-carotene > H2O2 > tBHP. Pearson “r” denotes the Pearson product moment correlation coefficient (Table 4). RSQ “” returns the square of the Pearson product moment correlation coefficient through the given data points. “” value is interpreted as the proportion of the variance in attributable to the variance in , where and represent different parameters in sequence. “” is the square of this correlation coefficient presented in Table 5.

PeakSlopeMean : (1/2) maxArea

: (1/2) max0.450.36−0.010.391.00−0.01

PeakSlopeMean : (1/2) maxArea

: (1/2) max0.

4. Conclusion

Redox biology, an inescapable field known for its beneficial/detrimental property is being studied extensively. Radicals can wreak devastation on macromolecules/metabolites and may cause short/long term effects on cell signalling. Lipid peroxidation has been the issue of far-reaching scrutiny of mechanistic cell signalling and its involvement in human diseases/disorders. The development of a high-throughput absorbance assay for monitoring kinetics of peroxyl radical reactions in vitro is described in this paper where the evolution of the increase in absorbance values over time provides a rapid, facile method to conduct competitive kinetic studies in the presence of different antioxidants. A quantitative treatment formulated for the temporal evolution of the kinetic interpretation in terms of different parameters is presented. Combined, competitive kinetic assay and the data analysis provides a new method to obtain, in a rapid, parallel format, relative antioxidant capacity to retard the formation of peroxyl radicals. These data underpin the key role which the lipid environment plays in modulating the rate of reaction of antioxidants characterized by different inherent chemical reactivity/membrane mobility. The accuracy of these measurements depends mainly on the pH of buffer, solvent form, temperature, AAPH/ABAP and ABTS solution preparation. Amalgamation of AAPH/ABAP and ABTS is a highly accurate combination as ABTS solution does not react with the compounds in absence of AAPH/ABAP, is not light sensitive, and does not require sophisticated techniques. On the whole, this method with kinetic analysis part is a simple way of analyzing and interpreting character and behaviour of the molecule. Altogether, a novel, facile method of study, new insights, and a quantitative understanding of the critical role in modulating peroxyl radical formation by antioxidants are reported.

Conflict of Interests

The authors declare no conflict of interests.


The authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India, for a Senior Research Fellowship to the first author (Nusrat Masood) and financial assistance (BSC0121). Research work on plant based drug discovery in our laboratory is supported by Science and Engineering Research Board, Department of Science and Technology, Ministry of Science and Technology, Government of India, Council of Science and Technology, Government of Uttar Pradesh and Indian Council of Medical Research, New Delhi.


  1. L. J. Marnett, “Peroxyl free radicals: potential mediators of tumor initiation and promotion,” Carcinogenesis, vol. 8, no. 10, pp. 1365–1373, 1987. View at: Google Scholar
  2. M. Kappler, A. B. Gerry, E. Brown, L. Reid, D. S. Leake, and S. P. Gieseg, “Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity,” Biochimica et Biophysica Acta, vol. 1773, no. 6, pp. 945–953, 2007. View at: Publisher Site | Google Scholar
  3. M. J. Davies, S. Fu, and R. T. Dean, “Protein hydroperoxides can give rise to reactive free radicals,” Biochemical Journal, vol. 305, no. 2, pp. 643–649, 1995. View at: Google Scholar
  4. U. P. Steinbrecher, S. Parthasarathy, D. S. Leake, J. L. Witztum, and D. Steinberg, “Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids,” Proceedings of the National Academy of Sciences of the United States of America, vol. 81, no. 12, pp. 3883–3887, 1984. View at: Google Scholar
  5. C. N. Oliver, “Inactivation of enzymes and oxidative modification of proteins by stimulated neutrophils,” Archives of Biochemistry and Biophysics, vol. 253, no. 1, pp. 62–72, 1987. View at: Google Scholar
  6. A. J. Kettle and C. C. Winterbourn, “Myeloperoxidase: a key regulator of neutrophil oxidant product,” Redox Report, vol. 3, no. 1, pp. 3–15, 1997. View at: Google Scholar
  7. M. Valko, C. J. Rhodes, J. Moncol, M. Izakovic, and M. Mazur, “Free radicals, metals and antioxidants in oxidative stress-induced cancer,” Chemico-Biological Interactions, vol. 160, no. 1, pp. 1–40, 2006. View at: Publisher Site | Google Scholar
  8. P. C. Burcham, “Genotoxic lipid peroxidation products: their DNA damaging properties and role in formation of endogenous DNA adducts,” Mutagenesis, vol. 13, no. 3, pp. 287–305, 1998. View at: Publisher Site | Google Scholar
  9. N. A. Porter, S. E. Caldwell, and K. A. Mills, “Mechanisms of free radical oxidation of unsaturated lipids,” Lipids, vol. 30, no. 4, pp. 277–290, 1995. View at: Google Scholar
  10. J. M. Gebicki, J. Collins, C. Gay, S. Duggan, and S. Gieseg, “The dissection of oxidative changes in human blood serum and U937 cells exposed to free radicals,” Redox Report, vol. 5, no. 1, pp. 55–56, 2000. View at: Google Scholar
  11. S. Gieseg, S. Duggan, and J. M. Gebicki, “Peroxidation of proteins before lipids in U937 cells exposed to peroxyl radicals,” Biochemical Journal, vol. 350, no. 1, pp. 215–218, 2000. View at: Publisher Site | Google Scholar
  12. J. Du and J. M. Gebicki, “Proteins are major initial cell targets of hydroxyl free radicals,” International Journal of Biochemistry and Cell Biology, vol. 36, no. 11, pp. 2334–2343, 2004. View at: Publisher Site | Google Scholar
  13. R. T. Dean, S. Fu, R. Stocker, and M. J. Davies, “Biochemistry and pathology of radical-mediated protein oxidation,” Biochemical Journal, vol. 324, no. 1, pp. 1–18, 1997. View at: Google Scholar
  14. J. A. Simpson, S. Narita, S. Gieseg, S. Gebicki, J. M. Gebicki, and R. T. Dean, “Long-lived reactive species on free-radical-damaged proteins,” Biochemical Journal, vol. 282, no. 3, pp. 621–624, 1992. View at: Google Scholar
  15. S. Gebicki and J. M. Gebicki, “Crosslinking of DNA and proteins induced by protein hydroperoxides,” Biochemical Journal, vol. 338, no. 3, pp. 629–636, 1999. View at: Publisher Site | Google Scholar
  16. M. B. Hampton, P. E. Morgan, and M. J. Davies, “Inactivation of cellular caspases by peptide-derived tryptophan and tyrosine peroxides,” FEBS Letters, vol. 527, no. 1–3, pp. 289–292, 2002. View at: Publisher Site | Google Scholar
  17. N. A. Porter, S. E. Caldwell, and K. A. Mills, “Mechanisms of free radical oxidation of unsaturated lipids,” Lipids, vol. 30, no. 4, pp. 277–290, 1995. View at: Google Scholar
  18. E. Niki, Y. Yoshida, Y. Saito, and N. Noguchi, “Lipid peroxidation: mechanisms, inhibition, and biological effects,” Biochemical and Biophysical Research Communications, vol. 338, no. 1, pp. 668–676, 2005. View at: Publisher Site | Google Scholar
  19. J. M. C. Gutteridge, “Lipid peroxidation and antioxidants as biomarkers of tissue damage,” Clinical Chemistry, vol. 41, no. 12, pp. 1819–1828, 1995. View at: Google Scholar
  20. E. Cadenas and H. Sies, “The lag phase,” Free Radical Research, vol. 28, no. 6, pp. 601–609, 1998. View at: Google Scholar
  21. K. Yamanaka, F. Takabayashi, M. Mizoi, Y. An, A. Hasegawa, and S. Okada, “Oral exposure of dimethylarsinic acid, a main metabolite of inorganic arsenics, in mice leads to an increase in 8-oxo-2′-deoxyguanosine level, specifically in the target organs for arsenic carcinogenesis,” Biochemical and Biophysical Research Communications, vol. 287, no. 1, pp. 66–70, 2001. View at: Publisher Site | Google Scholar
  22. K. U. Ingold, “Peroxy radicals,” Accounts of Chemical Research, vol. 2, no. 1, pp. 1–9, 1969. View at: Google Scholar
  23. L. J. Marnett, “Polycyclic aromatic hydrocarbon oxidation during prostaglandin biosynthesis,” Life Sciences, vol. 29, no. 6, pp. 531–546, 1981. View at: Google Scholar
  24. C. A. Gee, K. J. Kittridge, and R. L. Willson, “Peroxy free radicals, enzymes and radiation damage: sensitisation by oxygen and protection by superoxide dismutase and antioxidants,” British Journal of Radiology, vol. 58, no. 687, pp. 251–256, 1985. View at: Google Scholar
  25. M. Martini and J. Termini, “Peroxy radical oxidation of thymidine,” Chemical Research in Toxicology, vol. 10, no. 2, pp. 234–241, 1997. View at: Publisher Site | Google Scholar
  26. M. R. Valentine, H. Rodriguez, and J. Termini, “Mutagenesis by peroxy radical is dominated by transversions at deoxyguanosine: evidence for the lack of involvement of 8-oxo-dG1 and/or abasic site formation,” Biochemistry, vol. 37, no. 19, pp. 7030–7038, 1998. View at: Publisher Site | Google Scholar
  27. L. Zennaro, M. Rossetto, P. Vanzani et al., “A method to evaluate capacity and efficiency of water soluble antioxidants as peroxyl radical scavengers,” Archives of Biochemistry and Biophysics, vol. 462, no. 1, pp. 38–46, 2007. View at: Publisher Site | Google Scholar
  28. S. Lussignoli, M. Fraccaroli, G. Andrioli, G. Brocco, and P. Bellavite, “A microplate-based colorimetric assay of the total peroxyl radical trapping capability of human plasma,” Analytical Biochemistry, vol. 269, no. 1, pp. 38–44, 1999. View at: Publisher Site | Google Scholar
  29. Y. M. A. Naguib, “A fluorometric method for measurement of peroxyl radical scavenging activities of lipophilic antioxidants,” Analytical Biochemistry, vol. 265, no. 2, pp. 290–298, 1998. View at: Publisher Site | Google Scholar
  30. F. A. Kuypers, J. J. M. van den Berg, C. Schalkwijk, B. Roelofsen, and J. A. F. O. D. Kamp, “Parinaric acid as a sensitive fluorescent probe for the determination of lipid peroxidation,” Biochimica et Biophysica Acta, vol. 921, no. 2, pp. 266–274, 1987. View at: Google Scholar
  31. R. J. DeLange and A. N. Glazer, “Phycoerythrin fluorescence-based assay for peroxy radicals: a screen for biologically relevant protective agents,” Analytical Biochemistry, vol. 177, no. 2, pp. 300–306, 1989. View at: Google Scholar
  32. E. A. Lissi, M. Pizarro, A. Aspee, and C. Romay, “Kinetics of phycocyanine bilin groups destruction by peroxyl radicals,” Free Radical Biology and Medicine, vol. 28, no. 7, pp. 1051–1055, 2000. View at: Publisher Site | Google Scholar
  33. K. K. Adom and H. L. Rui, “Rapid peroxyl radical scavenging capacity (PSC) assay for assessing both hydrophilic and lipophilic antioxidants,” The Journal of Agricultural and Food Chemistry, vol. 53, no. 17, pp. 6572–6580, 2005. View at: Publisher Site | Google Scholar
  34. T. Metsä-Ketelä, “Luminescent assay for total peroxyl radical trapping capability of plasma,” in Bioluminescence and Chemiluminescence Current Status, P. E. Stanley and L. J. Kricka, Eds., pp. 389–392, Wiley & Sons, Chichester, UK, 1991. View at: Google Scholar
  35. E. Lissi, C. Pascual, and M. D. Del Castillo, “Luminol luminescence induced by 2,2'-Azo-bis(2-amidinopropane) thermolysis,” Free Radical Research Communications, vol. 17, no. 5, pp. 299–311, 1992. View at: Google Scholar
  36. H.-J. Freisleben and L. Packer, “Free-radical scavenging activities, interactions and recycling of antioxidants,” Biochemical Society Transactions, vol. 21, no. 2, pp. 325–330, 1993. View at: Google Scholar
  37. C. López-Alarcón and E. Lissi, “Interaction of pyrogallol red with peroxyl radicals. A basis for a simple methodology for the evaluation of antioxidant capabilities,” Free Radical Research, vol. 39, no. 7, pp. 729–736, 2005. View at: Publisher Site | Google Scholar
  38. N. Noguchi, N. Gotoh, and E. Niki, “Dynamics of the oxidation of low density lipoprotein induced by free radicals,” Biochimica et Biophysica Acta, vol. 1168, no. 3, pp. 348–357, 1993. View at: Publisher Site | Google Scholar
  39. T. F. Slater, “Overview of methods used for detecting lipid peroxidation,” Methods in Enzymology, vol. 105, pp. 283–293, 1984. View at: Google Scholar
  40. D. R. Janero, “Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury,” Free Radical Biology and Medicine, vol. 9, no. 6, pp. 515–540, 1990. View at: Publisher Site | Google Scholar
  41. B. Kalyanaraman, C. Mottley, and R. P. Mason, “A direct electron spin resonance and spin-trapping investigation of peroxyl free radical formation by hematin/hydroperoxide systems,” The Journal of Biological Chemistry, vol. 258, no. 6, pp. 3855–3858, 1983. View at: Google Scholar
  42. R. Lamrini, P. Lacan, A. Francina et al., “Oxidative decarboxylation of benzoic acid by peroxyl radicals,” Free Radical Biology and Medicine, vol. 24, no. 2, pp. 280–289, 1998. View at: Publisher Site | Google Scholar
  43. G. Bartosz, A. Janaszewska, D. Ertel, and M. Bartosz, “Simple determination of peroxyl radical-trapping capacity,” Biochemistry and Molecular Biology International, vol. 46, no. 3, pp. 519–528, 1998. View at: Google Scholar
  44. P. Cos, N. Hermans, M. Calomme et al., “Comparative study of eight well-known polyphenolic antioxidants,” Journal of Pharmacy and Pharmacology, vol. 55, no. 9, pp. 1291–1297, 2003. View at: Publisher Site | Google Scholar
  45. B. Tadolini, C. Juliano, L. Piu, F. Franconi, and L. Cabrini, “Resveratrol inhibition of lipid peroxidation,” Free Radical Research, vol. 33, no. 1, pp. 105–114, 2000. View at: Google Scholar
  46. H. J. Jang, S. Hwang, K. Y. Cho, D. K. Kim, K.-O. Chay, and J.-K. Kim, “Taxol induces oxidative neuronal cell death by enhancing the activity of NADPH oxidase in mouse cortical cultures,” Neuroscience Letters, vol. 443, no. 1, pp. 17–22, 2008. View at: Publisher Site | Google Scholar
  47. M. G. Benedetti, A. L. Foster, M. C. Vantipalli et al., “Compounds that confer thermal stress resistance and extended lifespan,” Experimental Gerontology, vol. 43, no. 10, pp. 882–891, 2008. View at: Publisher Site | Google Scholar
  48. Z. Diaz, A. Laurenzana, K. K. Mann, T. A. Bismar, H. M. Schipper, and W. H. Miller Jr., “Trolox enhances the anti-lymphoma effects of arsenic trioxide, while protecting against liver toxicity,” Leukemia, vol. 21, no. 10, pp. 2117–2127, 2007. View at: Publisher Site | Google Scholar
  49. R. Wolf, D. Wolf, and V. Ruocco, “Vitamin E: the radical protector,” Journal of the European Academy of Dermatology and Venereology, vol. 10, no. 2, pp. 103–117, 1998. View at: Publisher Site | Google Scholar
  50. E. Niki, “Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence,” Free Radical Biology Medicine, vol. 8, no. 66, pp. 3–12, 2014. View at: Publisher Site | Google Scholar
  51. P. Wan, S. Moat, and A. Anstey, “Pellagra: a review with emphasis on photosensitivity,” British Journal of Dermatology, vol. 164, no. 6, pp. 1188–1200, 2011. View at: Publisher Site | Google Scholar
  52. N. Ishii and Y. Nishihara, “Pellagra among chronic alcoholics: clinical and pathological study of 20 necropsy cases,” Journal of Neurology Neurosurgery and Psychiatry, vol. 44, no. 3, pp. 209–215, 1981. View at: Google Scholar
  53. T. C. Villines, A. S. Kim, R. S. Gore, and A. J. Taylor, “Niacin: the evidence, clinical use, and future directions,” Current Atherosclerosis Reports, vol. 14, no. 1, pp. 49–59, 2012. View at: Publisher Site | Google Scholar
  54. V. S. Kamanna, S. H. Ganji, and M. L. Kashyap, “Recent advances in niacin and lipid metabolism,” Current Opinion in Lipidology, vol. 24, no. 3, pp. 239–245, 2013. View at: Google Scholar
  55. M. Takashima, M. Shichiri, Y. Hagihara, Y. Yoshida, and E. Niki, “Capacity of peroxyl radical scavenging and inhibition of lipid peroxidation by β-carotene, lycopene, and commercial tomato juice,” Food Function, vol. 3, pp. 1153–1160, 2012. View at: Google Scholar
  56. T. A. Kennedy and D. C. Liebler, “Peroxyl radical scavenging by β-carotene in lipid bilayers. Effect of oxygen partial pressure,” The Journal of Biological Chemistry, vol. 267, no. 7, pp. 4658–4663, 1992. View at: Google Scholar
  57. G. W. Burton and K. U. Ingold, “β-Carotene: an unusual type of lipid antioxidant,” Science, vol. 224, no. 4649, pp. 569–573, 1984. View at: Google Scholar
  58. R. Stocker, Y. Yamamoto, and A. F. McDonagh, “Bilirubin is an antioxidant of possible physiological importance,” Science, vol. 235, no. 4792, pp. 1043–1046, 1987. View at: Google Scholar
  59. G. F. Vile and C. C. Winterbourn, “Inhibition of adriamycin-promoted microsomal lipid peroxidation by β-carotene, α-tocopherol and retinol at high and low oxygen partial pressures,” FEBS Letters, vol. 238, no. 2, pp. 353–356, 1988. View at: Google Scholar
  60. H. Sies and W. Stahl, “Vitamins E and C, β-carotene, and other carotenoids as antioxidants,” The American Journal of Clinical Nutrition, vol. 62, no. 6, pp. 1315S–1321S, 1995. View at: Google Scholar
  61. U. Moser and A. Bendich, “Vitamin C,” in Handbook of Vitamins, U. Machlin, Ed., pp. 195–232, Marcel Dekker, New York, NY, USA, 1991. View at: Google Scholar
  62. R. Stocke and B. Frei, “Endogenous antioxidant defense in human blood plasma,” in Oxidative Stress: Oxidants and Antioxidants, H. Sies, Ed., pp. 213–243, Academic Press, London, UK, 1991. View at: Google Scholar
  63. M. Nishikimi, “Oxidation of ascorbic acid with superoxide anion generated by the xanthine xanthine oxidase system,” Biochemical and Biophysical Research Communications, vol. 63, no. 2, pp. 463–468, 1975. View at: Google Scholar
  64. R. S. Bodannes and P. C. Chan, “Ascorbic acid as a scavenger of singlet oxygen,” FEBS Letters, vol. 105, no. 2, pp. 195–196, 1979. View at: Publisher Site | Google Scholar
  65. D. E. Cabelli and B. H. J. Bielski, “Kinetics and mechanism for the oxidation of ascorbic acid/ascorbate by HO2/O2- radicals. A pulse radiolysis and stopped-flow photolysis study,” The Journal of Physical Chemistry, vol. 87, no. 10, pp. 1809–1812, 1983. View at: Google Scholar
  66. A. Bendich, U. MachIm, O. Scandurra, G. W. Burton, and D. D. M. Wayner, “The antioxidant role of vitamin C,” Advances in Free Radical Biology and Medicine, vol. 2, no. 2, pp. 419–444, 1986. View at: Google Scholar
  67. B. Halliwell, M. Wasil, and M. Grootveld, “Biologically significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by ascorbic acid. Implications for antioxidant protection in the inflamed rheumatoid joint,” FEBS Letters, vol. 213, no. 1, pp. 15–17, 1987. View at: Google Scholar
  68. A. Dwenger, M. Funck, B. Lueken, G. Schweitzer, and U. Lehmann, “Effect of ascorbic acid on neutrophil functions and Hypoxanthine/Xanthine Oxidase-Generated, oxygen-derived radicals,” European Journal of Clinical Chemistry and Clinical Biochemistry, vol. 30, no. 4, pp. 187–191, 1992. View at: Google Scholar
  69. B.-M. Kwon and C. S. Foote, “Chemistry of singlet oxygen. 50. Hydroperoxide intermediates in the photooxygenation of ascorbic acid,” Journal of the American Chemical Society, vol. 110, no. 19, pp. 6582–6583, 1988. View at: Google Scholar
  70. B. Frei, L. England, and B. N. Ames, “Ascorbate is an outstanding antioxidant in human blood plasma,” Proceedings of the National Academy of Sciences of the United States of America, vol. 86, no. 16, pp. 6377–6381, 1989. View at: Google Scholar
  71. E. Niki, A. Kawakami, Y. Yamamoto, and Y. Kamiya, “Oxidation of lipids. VIII. Synergistic inhibition of oxidation of phosphatidylcholine liposome in aqueous dispersion by vitamin E and vitamin C,” Bulletin of the Chemical Society of Japan, vol. 58, no. 7, pp. 1971–1975, 1985. View at: Google Scholar
  72. D. D. M. Wayner, G. W. Burton, K. U. Ingold, L. R. C. Barclay, and S. J. Locke, “The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma,” Biochimica et Biophysica Acta, vol. 924, no. 3, pp. 408–419, 1987. View at: Google Scholar
  73. T. Doba, G. W. Burton, and K. U. Ingold, “Antioxidant and co-antioxidant activity of vitamin C. The effect of vitamin C, either alone or in the presence of vitamin E or a water-soluble vitamin E analogue, upon the peroxidation of aqueous multilamellar phospholipid liposomes,” Biochimica et Biophysica Acta, vol. 835, no. 2, pp. 298–303, 1985. View at: Publisher Site | Google Scholar
  74. P. Lambelet, F. Saucy, and J. Loliger, “Chemical evidence for interactions between vitamins E and C,” Experientia, vol. 41, no. 11, pp. 1384–1388, 1985. View at: Google Scholar
  75. H.-Y. Zhang, L.-F. Wang, and Y.-M. Sun, “Why B-ring is the active center for genistein to scavenge peroxyl radical: a DFT study,” Bioorganic and Medicinal Chemistry Letters, vol. 13, no. 5, pp. 909–911, 2003. View at: Publisher Site | Google Scholar
  76. W. Bors, W. Heller, C. Michel, and M. Saran, “Flavonoids as antioxidants: determination of radical-scavenging efficiencies,” Methods in Enzymology, vol. 186, pp. 343–355, 1990. View at: Publisher Site | Google Scholar
  77. P. J. O'Malley, “The reaction profile for hydrogen atom transfer from phenol to peroxyl free radicals,” Chemical Physics Letters, vol. 364, no. 3-4, pp. 318–322, 2002. View at: Publisher Site | Google Scholar
  78. J. Wu, H. Sugiyama, L.-H. Zeng, D. Mickle, and T.-W. Wu, “Evidence of Trolox and some gallates as synergistic protectors of erythrocytes against peroxyl radicals,” Biochemistry and Cell Biology, vol. 76, no. 4, pp. 661–664, 1998. View at: Google Scholar
  79. L. K. T. Lam, R. P. Pai, and L. W. Wattenberg, “Synthesis and chemical carcinogen inhibitory activity of 2-tert-butyl-4-hydroxyanisole,” Journal of Medicinal Chemistry, vol. 22, no. 5, pp. 569–571, 1979. View at: Google Scholar
  80. G. W. Burton and K. U. Ingold, “Autoxidation of biological molecules. 1. The antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in vitro,” Journal of the American Chemical Society, vol. 103, no. 21, pp. 6472–6477, 1981. View at: Google Scholar
  81. P. K. J. P.D. Wanasundara and F. Shahidi, “Antioxidants: science, technology, and applications,” in Bailey’s Industrial Oil and Fat Products, F. Shahidi, Ed., pp. 431–489, John Wiley & Sons, Hoboken, NJ, USA, 2005. View at: Google Scholar
  82. C. Martín, R. Martínez, R. Navarro, J. I. Ruiz-Sanz, M. Lacort, and M. B. Ruiz-Larrea, “tert-Butyl hydroperoxide-induced lipid signaling in hepatocytes: involvement of glutathione and free radicals,” Biochemical Pharmacology, vol. 62, no. 6, pp. 705–712, 2001. View at: Publisher Site | Google Scholar
  83. J. van der Zee, J. van Steveninck, J. F. Koster, and T. M. A. R. Dubbelman, “Inhibition of enzymes and oxidative damage of red blood cells induced by t-butylhydroperoxide-derived radicals,” Biochimica et Biophysica Acta, vol. 980, no. 2, pp. 175–180, 1989. View at: Google Scholar

Copyright © 2014 Nusrat Masood et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

More related articles

1069 Views | 490 Downloads | 1 Citation
 PDF  Download Citation  Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly and safely as possible. Any author submitting a COVID-19 paper should notify us at to ensure their research is fast-tracked and made available on a preprint server as soon as possible. We will be providing unlimited waivers of publication charges for accepted articles related to COVID-19. Sign up here as a reviewer to help fast-track new submissions.