Antioxidant activity applying an improved ABTS radical cation decolorization assay.

A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants. The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First, the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC assay. Third, it is applicable to both aqueous and lipophilic systems.     

A simple, post-additional antioxidant capacity assay using adenosine triphosphate-stabilized 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation in a G-quadruplex DNAzyme catalyzed ABTS-H2O2 system

The scavenging of 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation (ABTS(+)) by antioxidants has been widely used in antioxidant capacity assay. Because of ABTS(+) disproportionation, however, this radical cannot be prepared on a large scale and stored long-term, making it unsuitable for high-throughput detection and screening of antioxidants. We developed a modified "post-additional" antioxidant capacity assay. This method possessed two remarkable features: First, instead of natural peroxidases, an artificial enzyme, G-quadruplex DNAzyme, was used for the preparation of ABTS(+), thus greatly reducing the cost of the assay, and eliminating the strict demand for the storage of enzymes. Second, an ABTS(+) stabilizer, adenosine triphosphate (ATP), was used. In the presence of ATP, the disproportionation of ABTS(+) was effectively inhibited, and the lifetime of this radical cation was prolonged about 6-fold (12 days versus 2 days), making the large-scale preparation of ABTS(+) possible. Utilizing this method, the antioxidant capacities of individual antioxidants and real samples can be quantified and compared easily. In addition, this method can be developed as a high-throughput screening method for antioxidants. The screening results could even be judged by the naked eye, eliminating the need for expensive instruments.     

Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: the CUPRAC method

BACKGROUND: Tests measuring the combined antioxidant effect of the nonenzymatic defenses in biological fluids may be useful in providing an index of the organism's capability to counteract reactive species known as prooxidants, resist oxidative damage and combat oxidative stress-related diseases. The selected chromogenic redox reagent for the assay of human serum should be easily accessible, stable, selective, respond to all types of biologically important antioxidants such as ascorbic acid, alpha-tocopherol, beta-carotene, reduced glutathione (GSH), uric acid and bilirubin, regardless of chemical type or hydrophilicity. Currently, there is no rapid method for total antioxidant assay of human serum meeting the above criteria.METHODS: Our recently developed cupric reducing antioxidant capacity (CUPRAC) spectrophotometric method for a number of polyphenols and flavonoids using the copper(II)-neocuproine reagent in ammonium acetate buffer was now applied to a complete series of plasma antioxidants for the assay of total antioxidant capacity (TAC) of serum, and the resulting absorbance at 450 nm was recorded either directly (e.g. for ascorbic acid, alpha-tocopherol and glutathione) or after incubation at 50 degrees C for 20 min (e.g. for uric acid, bilirubin and albumin), quantitation being made by means of a calibration curve. The lipophilic antioxidants, alpha-tocopherol and beta-carotene, were assayed in dichloromethane (DCM). Lipophilic antioxidants of serum were extracted with n-hexane from an ethanolic solution of serum subjected to centrifugation. Hydrophilic antioxidants of serum were assayed after perchloric acid precipitation of proteins in the centrifugate.Results: The molar absorptivities, linear ranges and trolox equivalent antioxidant capacity (TEAC) coefficients of the serum antioxidants were established with respect to the CUPRAC spectrophotometric method, and the results (TEAC, or TEAC coefficients) were evaluated in comparison to the findings of the ABTS/TEAC reference method using persulfate as oxidant. As for hydrophilic phase, a linear correlation existed between the CUPRAC and ABTS findings (r=0.58), contrary to current literature reporting that either serum ORAC or serum ferric reducing antioxidant potency (FRAP) does not correlate at all with serum TEAC. The analytical responses of serum antioxidants were shown to be additive, enabling a TAC assay. The intra- and inter-assay CVs were 0.7 and 1.5%, respectively, for serum.Conclusions: The CUPRAC assay proved to be efficient for glutathione and thiol-type antioxidants, for which the FRAP test was nonresponsive. The findings of CUPRAC completely agreed with those of ABTS-persulfate for lipophilic phase. The additivity of absorbances of all the tested antioxidants confirmed that antioxidants in the CUPRAC test did not chemically interact among each other so as to cause an intensification or quenching of the theoretically expected absorbance. As a distinct advantage over other electron-transfer based assays (e.g. Folin, FRAP, ABTS, DPPH), CUPRAC is superior in regard to its realistic pH close to the physiological pH, favourable redox potential, accessibility and stability of reagents and applicability to lipophilic antioxidants as well as hydrophilic ones.     

Rapid screening and characterisation of antioxidants of Cosmos caudatus using liquid chromatography coupled with mass spectrometry

Ulam raja (Cosmos caudatus) is used traditionally for improving blood circulation. In this study, it was found that ulam raja had extremely high antioxidant capacity of about 2,400 mg l-ascorbic acid equivalent antioxidant capacity (AEAC) per 100 g of fresh sample. Antioxidant peaks in extract of ulam raja were firstly characterized using free radical spiking test through high performance liquid chromatography coupled with mass spectrometry (MS). Upon reaction with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, intensities of antioxidant peaks will be significantly reduced. HPLC/MS(n) was further applied to elucidate the chemical structures of antioxidant peaks characterized in the spiking test. More than twenty antioxidants were identified in ulam raja, and their chemical structures were proposed. The major antioxidants in ulam raja were attributed to a number of proanthocyanidins that existed as dimers through hexamers, quercetin glycosides, chlorogenic, neo-chlorogenic, crypto-chlorogenic acid and (+)-catching. High content of antioxidants antioxidants contained in ulam raja could be partly responsible for its ability to reduce oxidative stress.     

Doxorubicin inhibits oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by a lactoperoxidase/H(2)O(2) system by reacting with ABTS-derived radical

The effect of doxorubicin on oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by lactoperoxidase and hydrogen peroxide has been investigated. It was found that: (1) oxidation of ABTS to its radical cation (ABTS*(+)) is inhibited by doxorubicin as evidenced by its induction of a lag period, duration of which depends on doxorubicin concentration; (2) the inhibition is due to doxorubicin hydroquinone reducing the ABTS*(+) radical (stoichiometry 1: 1.8); (3) concomitant with the ABTS*(+) reduction is oxidation of doxorubicin; only when the doxorubicin concentration decreases to a near zero level, net oxidation of ABTS could be detected; (4) oxidation of doxorubicin leads to its degradation to 3-methoxysalicylic acid and 3-methoxyphthalic acid; (5) the efficacy of doxorubicin to quench ABTS*(+) is similar to the efficacy of p-hydroquinone, glutathione and Trolox C. These observations support the assertion that under certain conditions doxorubicin can function as an antioxidant. They also suggest that interaction of doxorubicin with oxidants may lead to its oxidative degradation.     

Antioxidant activity: what do we measure?

Inhibition of oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by free radicals generated by decomposition of 2,2'-azobis(2-amidopropane) (ABAP) by antioxidants and biological material was studied. A correlation was found between the ability of various substances to delay the onset of ABTS oxidation and their rapid reduction of the ABTS+* cation radical, and between the ability to reduce the maximal rate of ABTS oxidation and slow reduction of ABTS+*. The length of the lag period of ABTS oxidation was found to be independent of ABTS concentration. Similar decrease of peroxynitrite-induced ABTS+* formation by antioxidants was observed when the antioxidants were added before and after peroxynitrite. All these findings indicate that the main effect of antioxidants in this system is reduction of ABTS+* and not prevention of its formation. Reduction of oxidation products rather than inhibition of their formation may be the predominant mode of action of antioxidants in various assays of antioxidant activity.     

Endogenous and dietary indoles: a class of antioxidants and radical scavengers in the ABTS assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS*+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like L-tryptophan and derivatives (N-acetyltryptophan, L-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-beta-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9 mM, usually higher than that for Trolox and ascorbic acid (1 mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS*+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic beta-carbolines (pyridoindoles), that did not scavenge ABTS*+. Radical scavenger activity of indoles against ABTS*+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

Measurement of relative antioxidant activity of compounds: a methodological note

The relative activities of some hydrogen-donating antioxidants were assessed by comparing their activities with that of Trolox (Trolox equivalent antioxidant capacity, TEAC) for scavenging the ABTS radical cation (ABTS.+) generated in the aqueous phase. We have verified, however, that TEAC values may change with the concentration of compounds and with the measuring times used. Not withstanding, TEAC values do not differ significantly if the compounds have kinetic curves of ABTS.+ formation similar to that of Trolox. This is the case with ascorbic acid, whose TEAC values, determined by using five concentrations at three different measuring times, are very close. For the flavonoids studied (catechin, rutin, naringenin and silibinin) which have kinetic curves of ABTS.+ formation different from that of Trolox, the TEAC values decrease with increasing concentrations of the compounds for each measuring time, and increase with increasing measuring times for each concentration. In the present study, we conclude that, in order to evaluate relative antioxidant activities of compounds by the ABTS assay, it is essential to perform kinetic studies to assess scavenging of ABTS.+ by these compounds. Therefore, when the TEAC values of compounds are determined for more than one measuring time, we may be sure that all the antioxidant potential of compounds is being considered and whether or not it is possible to establish a hierarchy for their antioxidant activities.     

Evaluation of antioxidants: Scope, limitations and relevance of assays

Peroxidation of lipids, particularly polyunsaturated fatty acid residues (PUFA) of phospholipids and cholesterol esters, is a process of marked implications: it shortens the shelf-life of food and drugs, it causes fragmentation of DNA, it damages cellular membranes and it promotes the genesis of many human diseases. Much effort is therefore devoted to a search for "potent antioxidants", both synthetic and from natural sources, mostly plants. This, in turn, requires a reliable, simple, preferably high throughput assay of the activity of alleged antioxidants. The most commonly used assays are based on measurements of the total antioxidant capacity (TAC) of a solution, as evaluated either by determining the rate of oxidation of the antioxidant or by measuring the protection of an easily determined indicator against oxidation by the antioxidants. The commonly used assays utilized for ranking antioxidants share three common problems: (i) They usually evaluate the effects of those antioxidants that quench free radicals, which constitute only a part of the body's antioxidative network, in which enzymes play the central role. (ii) Both the capacity and potency of antioxidants, as obtained by various methods, do not necessarily correlate with each other. (iii) Most estimates are based on methods conducted in solution and are therefore not necessarily relevant to processes that occur at the lipid-water interfaces in both membranes and micro emulsions (e.g. lipoproteins). Given this "state of art", many researchers, including us, try to develop a method based on the formation of hydroperoxides (LOOH) upon peroxidation of PUFA in lipoproteins or in model membranes, such as liposomes. In these systems, as well as in lipoproteins, the most apparent effect of antioxidants is prolongation of the lag time preceding the propagation of a free radical chain reaction. In fact, under certain conditions both water soluble antioxidants (e.g. vitamin C and urate) and the lipid soluble antioxidant tocopherol (vitamin E), promote or even induce peroxidation. Based on the published data, including our results, we conclude that terms such as 'antioxidative capacity' or 'antioxidative potency' are context-dependent. Furthermore, criteria of the efficacy of antioxidants based on oxidation in solution are not necessarily relevant to the effects of antioxidants on peroxidation in biological systems or model lipid assemblies, because the latter processes occur at water/lipid interfaces. We think that evaluation of antioxidants requires kinetic studies of the biomarker used and that the most relevant characteristic of 'oxidative stress' in the biological context is the kinetics of ex vivo peroxidation of lipids. We therefore propose studying the kinetics of lipid-peroxidation in the absence of the studied antioxidant and in its presence at different antioxidant concentrations. These protocols mean that antioxidants are assayed by methods commonly used to evaluate oxidative stress. The advantage of such evaluation is that it enables quantization of the antioxidants' efficacy in a model of relevance to biological systems. In view of the sensitivity of the lag time preceding peroxidation, we propose studying how much antioxidant is required to double the lag observed prior to rapid peroxidation. The latter quantity (C(2lag)) can be used to express the strength of antioxidants in the relevant system (e.g. LDL, serum or liposomes).     

Antioxidant properties of 2-O-beta-D-glucopyranosyl-L-ascorbic acid

The antioxidant activity of a provitamin C agent, 2-O-beta-D-glucopyranosyl-L-ascorbic acid (AA-2betaG), was compared to that of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) and ascorbic acid (AA) using four in vitro methods, 1,1-diphenyl-picrylhydrazyl (DPPH) radical-scavenging assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(*+))-scavenging assay, oxygen radical absorbance capacity (ORAC) assay, and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis inhibition assay. AA-2betaG slowly and continuously scavenged DPPH radicals and ABTS(*+) in roughly the same reaction profiles as AA-2G, whereas AA quenched these radicals immediately. In the ORAC assay and the hemolysis inhibition assay, AA-2betaG showed similar overall activities to AA-2G and to AA, although the reactivity of AA-2betaG against the peroxyl radical generated in both assays was lower than that of AA-2G and AA. These data indicate that AA-2betaG had roughly the same radical-scavenging properties as AA-2G, and a comprehensive in vitro antioxidant activity of AA-2betaG appeared to be comparable not only to that of AA-2G but also to that of AA.     

In vitro antioxidant activity of Hedyotis corymbosa (L.) Lam. aerial parts

The methanolic extract of the aerial part of Hedyotis corymbosa (L.) Lam. (Rubiaceae) was screened for antioxidant activity using 1,1-diphenyl-2-picryl hydroxyl (DPPH) quenching assay, 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS) cation decolorization test, ferric reducing power (FRP), scavenging capacity towards hydroxyl ion (OH*) radicals and nitric oxide (NO) radical inhibition activity using established assay procedures. Total phenolics and total flavonoid contents were, also determined. The plant yielded 210 mg gallic acid equivalent/100 g phenolic content and 55 mg quercetin equivalent/100 g flavonoid content. The extract exhibited high antiradical activity against DPPH, ABTS, nitric oxide and hydroxyl radicals with EC50 value of 82, 150, 130, and 170 microg/ml, respectively. The FRP increased with increasing concentration of the sample. The antioxidant activity of the extract was comparable with that of the standard butylated hydroxyl toluene (BHT). High correlation between total phenolic/flavonoid contents and scavenging potential of different reactive oxygen species (R2 = 0.785-0.998) indicated the polyphenols as the main antioxidants.     

In vitro antioxidant activity of 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), a vitamin E metabolite

2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) has been identified as a major water-soluble metabolite of vitamin E, which circulates in the blood and is excreted with the urine. The aim of this study was to assess the antioxidant activity of alpha-CEHC using several methods with different prooxidant challenges. In the Oxygen Radical Absorbance Capacity assay, a fluorescent protein acts as a marker for oxidative damage induced by peroxyl radicals. In the Trolox Equivalent Antioxidant Capacity (TEAC) assay, a stable free radical, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS.+) is reduced directly by antioxidants. Scavenging properties vs. reactive nitrogen species were studied measuring the effects on tyrosine nitration after reaction with peroxynitrite. Trolox, alpha-tocopherol, ascorbic acid, and (-)-epicatechin were simultaneously tested in order to compare their antioxidant activities. In all mentioned systems, alpha-CEHC exhibited antioxidant properties similar to those of Trolox. We conclude that alpha-CEHC is a molecule with good antioxidant activity, having the advantage over Trolox of being a naturally occurring compound. These properties might be useful for research or industrial purposes.     

Antioxidative properties of ascorbigen in using multiple antioxidant assays

The antioxidative properties of ascorbigen, one of the major indole-derived compounds of Brassica vegetables, were systematically evaluated using multiple assay systems with comparison to the well-known antioxidants ascorbic acid and Trolox. We first performed assays using model radicals, DPPH radical, galvinoxyl radical, and ABTS radical cation (ABTS^?+). Ascorbigen showed stronger activity than that of ascorbic acid in the ABTS^?+-scavenging assay but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays. In the ABTS^?+-scavenging assay, the indole moiety of ascorbigen contributed to scavenging of the radicals to produce indole-3-aldehyde as one of the final reaction products. The activity of ascorbigen was then evaluated by an oxygen radical absorbance capacity assay and an oxidative hemolysis inhibition assay using physiologically relevant peroxyl radicals, AAPH-derived radicals. Ascorbigen showed much stronger antioxidant activity than did ascorbic acid and Trolox. Therefore, antioxidant activity of ascorbigen might be more beneficial than has been thought for daily health care.     

An antioxidant screening assay based on oxidant-induced growth arrest in Saccharomyces cerevisiae

This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants.     

Tannic acid interferes with the commonly used laccase-detection assay based on ABTS as the substrate

Laccase enzymatic activity in biological samples is usually detected spectrophotometrically through its capacity to oxidize several specific aromatic compounds. One of the most commonly used substrates is the compound 2-2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), which becomes green-blue coloured when it is oxidized by laccase. In this work we study the interference of tannic acid with the spectrophotometric assay to detect laccase by using ABTS as the substrate. Our data show that under the normal reaction conditions of this assay, but in the absence of any catalyst, tannic acid is able to carry out the chemical reduction of the oxidized specie of ABTS, thus decreasing the overall detectable laccase-activity values observed when this enzyme is present in the reaction mixture. Therefore, our results represent an important warning concerning a commonly used method for measuring, detecting or screening laccases in biological samples that may content tannic acid or structural-related molecules.     

The identification of antioxidants in dark soy sauce

Soy sauce is a traditional fermented seasoning in Asian countries, that has high antioxidant activity in vitro and some antioxidant activity in vivo. We attempted to identify the major antioxidants present, using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay as a guide. 3-Hydroxy-2-methyl-4H-pyran-4-one (maltol) was one of several active compounds found in an ethyl acetate extract of dark soy sauce (DSS) and was present at millimolar concentrations in DSS. However, most of the antioxidant activity was present in colored fractions, two of which (CP1 and CP2) were obtained by gel filtration chromatography. Their structural characteristics based on nuclear magnetic resonance (NMR) and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) analysis suggest that carbohydrate-containing pigments such as melanoidins are the major contributors to the high antioxidant capacity of DSS.     

A novel 19F-NMR method for the investigation of the antioxidant capacity of biomolecules and biofluids.

A new assay for the measurement of the antioxidant capacity of biomolecules by high resolution 19F-NMR spectroscopy is presented here. This method is based on the use of trifluoroacetanilidic detectors, namely trifluoroacetanilide, N-(4-hydroxyphenyl)-trifluoroacetamide and 2-hydroxy-4-trifluoroacetamidobenzoic acid. Upon hydroxyl radical attack, such fluorinated detectors yield trifluoroacetamide and trifluoroacetic acid that can be quantitatively determined by 19F-NMR spectroscopy. Trifluoroacetamide was found to be a reliable reporter of hydroxyl radical attack on the fluorinated detectors, whereas N-(4-hydroxyphenyl)-trifluoroacetamide was found to be the most sensitive detector amongst the ones considered. Therefore, N-(4-hydroxyphenyl)-trifluoroacetamide has been used in competition experiments to assess the antioxidant capacity of a number of low and high molecular weight antioxidants. The antioxidant capacity of a given compound has been scaled in terms of an adimensional parameter, kF, that represents the ratio between the scavenger abilities of the fluorinated detector and the competitor. kF values obtained for low-molecular-mass compounds fall in the range 0.17 < kF < 1.5 and are in good agreement with second order rate constants (k2OH) for the reaction of the antioxidant with hydroxyl radicals. The kF value for serum albumin is much larger (46.9) than that predicted from the reported k2OH value. This finding supports the view that the protein can very effectively scavenge hydroxyl radicals as well as secondary radicals. Human blood serum showed that its antioxidant capacity is even higher than that shown by aqueous solutions of albumin at physiologic concentration suggesting a further contribution from other macromolecular serum components.     

In vitro antioxidant activity of banana (Musa spp. ABB cv. Pisang Awak)

The methanolic extract of Musa ABB cv Pisang Awak was investigated for the polyphenolic contents and antioxidant activity. The total phenol and flavonoid contents of the fruit extract were found to be 120 mg gallic acid equivalents (GAE) and 440 mg quercetin equivalents (QE)/100 g of sample dry weight, respectively. The antioxidant activity of the Pisang Awak methanol extract (PAME) (20-500 microg/ml) was determined using 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, reducing capacity, 2-2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS) radical cation decolourization and hydroxyl radical scavenging capacity (OH*). The EC50 values of DPPH, ABTS and OH* activities of the PAME and butylated hydroxy toluene (BHT) were found to be 65 and 9 microg/ml, 29 and 6 microg/ml, 36 and 42 microg/ml respectively. The reducing capacity increased with increasing concentration (31.5-1000 mg/ml) of the fruit extract and the activity was comparable with the standard BHT. The high performance thin layer chromatography (HPTLC) analysis of the extract revealed the presence of polyphenols. The strong and positive correlations were obtained between total phenol/flavonoid contents (R2 = 0.693-1.0) and free radical scavenging ability was attributed to the polyphenols as the major antioxidants.     

Measuring circulating antioxidants in wild birds

Antioxidants protect against free radical damage, which is associated with various age-related pathologies. Antioxidants are also an important buffer against the respiratory burst of the immune system. This protection presumably has costs and therefore might underlie important life-history trade-offs. Studying such trade-offs in a comparative context requires field-applicable methods for assessing antioxidant capacity in wild animals. Here, we present modifications to a simple spectrophotometric assay (the TEAC or TAS assay) that can be applied to miniscule amounts of blood plasma to determine circulating antioxidant capacity. Additionally, uric acid, the most abundant circulating antioxidant, should be measured independently. Uric acid in birds is derived from amino acid catabolism, perhaps incidentally to its antioxidant function. The assay was validated in experimental studies on chickens showing effects of diet on antioxidant capacity, and in field measurements on 92 species of birds, which demonstrate substantial species differences in constitutive antioxidant capacity. Furthermore, most wild birds demonstrate a dramatic change in antioxidant capacity due to stress. These results show that this technique detects variation appropriate for both interspecific and intraspecific studies, and that antioxidants and uric acid change in response to conditions of interest to field ecologists, such as diet and stress.     

Pitfalls in a Method for Assessment of Total Antioxidant Capacity

A relatively simple and widely applied method for quantitating the total antioxidant capacity of body fluids and drug solutions based on the absorbance of the ABTS radical cation was evaluated. In this assay, the end-point is an antioxidant-induced decrease in absorbance at a fixed time. This decrease is used as an index of total antioxidant capacity. It is shown that Trolox, potassium cyanide and quercetin all decrease the absorbance of ABTS radical cations at a fixed time, but by different mechanisms. Trolox scavenges the ABTS radical, potassium cyanide inhibits radical formation, while quercetin acts by both mechanisms. Using this method antioxidant capacity may be overestimated, due to both a scavenger effect and an effect on the rate of ABTS oxidation. To distinguish between these effects, a post-addition assay was used in which the sample is added when the formation of radicals is stable. Using post- addition assay conditions enables discrimination between effects on radical scavenging and on the radical formation, two major mechanisms for antioxidant action. In extrapolating the results to an ill vivo situation it should be questioned: (i) whether the peroxidase process does indeed mimic the process of radical formation in vivo, and (ii) whether the ABTS radicals do resemble the radical species involved in an in vivo situation. Results obtained in the ABTS radical-based methods should therefore be reviewed critically before the antioxidant capacity can be assessed.