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 method to measure antioxidant activity in organic media: application to lipophilic vitamins

The 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS(.+)) can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase in an organic medium. The ABTS radical is easily generated in acidified ethanol medium in about 100 s with a stability of 1.7 x 10(-3) (-deltaabs/min) monitored at 730 nm. Other organic solvents, such as methanol or acetone, have lower radical generation times but the radical is less stable. The addition of Trolox or a lipophilic antioxidant such as alpha-tocopherol or beta-carotene produces a decrease in absorbance that can be used to estimate antioxidant capacity. Using a spectrophotometric end-point method and microplate-reader equipment, we have developed a method that estimates the antioxidant activity of different lipophilic vitamins. The use of Trolox as an antioxidant standard led to a limit of detection of 0.08 nmoles and limit of quantitation of 0.28 nmoles, while similar values were obtained for alpha-tocopherol and beta-carotene. The relative antioxidant activity values obtained by different antioxidants showed that alpha-tocopherol has a similar antioxidant potential to Trolox and that beta-carotene has 2.6 times the antioxidant potential of Trolox. In our opinion, this method can be useful for estimating the antioxidant activity in lipophilic samples and as a complement to other methods that measure antioxidant activity in aqueous media.     

A method to measure the oxidizability of both the aqueous and lipid compartments of plasma.

The lipophilic radical initiator (MeO-AMVN) and the fluorescent probe C11BODIPY581/591 (BODIPY) were used to measure the lipid compartment oxidizability of human plasma. Aqueous plasma oxidizability was initiated by the aqueous peroxyl radical generator, AAPH, and 2',7'-dichlorodihydrofluorescein (DCFH) was employed as the marker of the oxidative reaction. The distribution in aqueous and lipid compartments of the two radical initiators was determined by measuring the rate of consumption of the plasma hydrophilic and lipophilic endogenous antioxidants. In the presence of AAPH (20 mM), the order of consumption was: ascorbic acid > alpha-tocopherol > uric acid > beta-carotene, indicating a gradient of peroxyl radicals from the aqueous to the lipid phase. When MeO-AMVN was used (2mM), beta-carotene was consumed earlier than uric acid and almost at the same time as alpha-tocopherol, reflecting the diffusion and activation of MeO-AMVN in the lipophilic phase. The rate of BODIPY oxidation (increase in green fluorescence) significantly increased after the depletion of endogenous alpha-tocopherol and beta-carotene, whereas it was delayed for 180 min when AAPH was used instead of MeO-AMVN. The measurement of lipid oxidation in plasma was validated by adding to plasma the two lipophilic antioxidants, alpha-tocopherol and beta-carotene, whose inhibitory effects on BODIPY oxidation were dependent on the duration of the preincubation period and hence to their lipid diffusion. DCFH oxidation induced by AAPH only began after uric acid, the main hydrophilic plasma antioxidant, was consumed. In contrast, when MeO-AMVN was used, DCFH oxidation was delayed for 120 min, indicating its localization in the aqueous domain. In summary, the selective fluorescence method reported here is capable of distinguishing the lipophilic and hydrophilic components of the total antioxidant capacity of plasma.     

GC-MS analysis and in vitro antioxidant and enzyme inhibitory activities of essential oil from aerial parts of endemic Thymus spathulifolius Hausskn. et Velen

We investigated the antioxidant and enzyme inhibitory activities and chemical composition of the hydro-distilled essential oil (0.35% yield) from aerial parts of Thymus spathulifolius. Antioxidant capacity of the oil was assessed by different methods including free radical scavenging (DPPH and ABTS), reducing power (FRAP and CUPRAC) and phosphomolybdenum assay. Inhibitory activities were analyzed against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-amylase, α-glucosidase, and tyrosinase. Twenty-one constituents were identified representing 97.2% of the total oil with thymol (50.5%), borneol (16.7%) and carvacrol (7.7%) as the major components. The essential oil exhibited good antioxidant activity with IC₅₀ values of 3.82 and 0.22?mg/mL determined by free radical scavenging DPPH and ABTS, respectively. EC₅₀ values of FRAP and CUPRAC were found to be 0.12 and 0.34?mg/mL, respectively. The results of the present study support the uses of T. spathulifolius essential oil as a source of natural antioxidants and bioactivities for functional foods and phytomedicines.     

Assessment of antioxidant activity by using different in vitro methods

In this study, six common tests for measuring antioxidant activity were evaluated by comparing four antioxidants and applying them to beverages (tea and juices): Trolox equivalent antioxidant capacity assay (TEAC I-III assay), Total radical-trapping antioxidant parameter assay (TRAP assay), 2,2-diphenyl- l -picrylhydrazyl assay (DPPH assay), N , N -dimethyl- p -phenylendiamine assay (DMPD assay), Photochemiluminescence assay (PCL assay) and Ferric reducing ability of plasma assay (FRAP assay). The antioxidants included gallic acid representing the group of polyphenols, uric acid as the main antioxidant in human plasma, ascorbic acid as a vitamin widely spread in fruits and Trolox ® as water soluble vitamin E analogue. The six methods presented can be divided into two groups depending on the oxidising reagent. Five methods use organic radical producers (TEAC I-III, TRAP, DPPH, DMPD, PCL) and one method works with metal ions for oxidation (FRAP). Another difference between these tests is the reaction procedure. Three assays use the delay in oxidation and determine the lag phase as parameter for the antioxidant activity (TEAC I, TRAP, PCL). They determine the delay of radical generation as well as the ability to scavenge the radical. In contrast, the assays TEAC II and III, DPPH, DMPD and FRAP analyse the ability to reduce the radical cation (TEAC II and III, DPPH, DMPD) or the ferric ion (FRAP). The three tests acting by radical reduction use preformed radicals and determine the decrease in absorbance while the FRAP assay measures the formed ferrous ions by increased absorbance. Gallic acid was the strongest antioxidant in all tests with exception of the DMPD assay. In contrast, uric acid and ascorbic acid showed low activity in some assays. Most of the assays determine the antioxidant activity in the micromolar range needing minutes to hours. Only one assay (PCL) is able to analyse the antioxidant activity in the nanomolar range. Black currant juice showed highest antioxidant activity in all tests compared to tea, apple juice and tomato juice. Despite these differences, results of these in vitro assays give an idea of the protective efficacy of secondary plant products. It is strongly recommended to use at least two methods due to the differences between the test systems investigated.     

Phenolics as potential antioxidant therapeutic agents: mechanism and actions

Accumulating chemical, biochemical, clinical and epidemiological evidence supports the chemoprotective effects of phenolic antioxidants against oxidative stress-mediated disorders. The pharmacological actions of phenolic antioxidants stem mainly from their free radical scavenging and metal chelating properties as well as their effects on cell signaling pathways and on gene expression. The antioxidant capacities of phenolic compounds that are widely distributed in plant-based diets were assessed by the Trolox equivalent antioxidant capacity (TEAC), the ferric reducing antioxidant power (FRAP), the hypochlorite scavenging capacity, the deoxyribose method and the copper-phenanthroline-dependent DNA oxidation assays. Based on the TEAC, FRAP and hypochlorite scavenging data, the observed activity order was: procyanidin dimer > flavanol > flavonol > hydroxycinnamic acids > simple phenolic acids. Among the flavonol aglycones, the antioxidant propensities decrease in the order quercetin, myricetin and kaempferol. Gallic acid and rosmarinic acid were the most potent antioxidants among the simple phenolic and hydroxycinnamic acids, respectively. Ferulic acid displayed the highest inhibitory activity against deoxyribose degradation but no structure–activity relationship could be established for the activities of the phenolic compounds in the deoxyribose assay. The efficacies of the phenolic compounds differ depending on the mechanism of antioxidant action in the respective assay used, with procyanidin dimers and flavan-3-ols showing very potent activities in most of the systems tested. Compared to the physiologically active (glutathione, -tocopherol, ergothioneine) and synthetic (Trolox, BHA, BHT) antioxidants, these compounds exhibited much higher efficacy. Plant-derived phenolics represents good sources of natural antioxidants, however, further investigation on the molecular mechanism of action of these phytochemicals is crucial to the evaluation of their potential as prophylactic agents.     

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.     

Postprandial increases in serum antioxidant capacity in older women.

Eight women were recruited for studying the effects of a meal on overall antioxidant status. Subjects resided in a metabolic research unit for two 36-h periods. During period A, subjects fasted overnight (12 h) and were then given a breakfast, a lunch, a snack, and a dinner. During period B, subjects fasted for 23 h and were then given a dinner. These meals were designed to contain negligible antioxidants. Blood samples were collected for analyzing total antioxidant capacity (TAC) and individual antioxidants. The results showed that serum TAC significantly increased by up to 23% after the consumption of the lunch and dinner during period A. Serum TAC did not increase until after the consumption of the dinner during period B. Among the antioxidants (vitamin C, alpha-tocopherol, bilirubin, and uric acid) examined, serum uric acid was the only one that showed a significant postprandial increase, which was also parallel to the postprandial response in serum TAC. These results indicate that food intake, even if low in antioxidants, can increase the serum total antioxidant activity.     

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.     

Phytochemical and antioxidative potential of orange,red, yellow,rainbow and black coloured tropical carrots (<Emphasis Type="Italic">Daucus carota</Emphasis> subsp. <Emphasis Type="Italic">sativus</Emphasis> Schubl. &amp; Martens)

This study was executed to determine phytochemical content i.e. total carotenoids, phenolics and flavonoids, and antioxidant ability expressed in the form of FRAP, CUPRAC and ABTS activity among different coloured tropical carrots (orange, red, yellow, rainbow and black carrot) developed at ICAR-IIVR, Varanasi, Uttar Pradesh, India. Overall, within different colour group, the extent of variation for various phytochemical content and antioxidant potentiality is narrow i.e. ranged from 1.04- to 3.21-fold; but at the same time, the genotypic variability across genotypes is too wide which varied 20.90- to 57.92-fold for phytochemical and antioxidants is an indication of broad genetic base of carrot germplasm. Among all the carrots, black carrot had an exceptionally high content of total phenolics and flavonoids, and thereby led to the highest antioxidant ability in the terms of FRAP, CUPRAC and ABTS activity expressing about 76–83% relative potentiality followed by rainbow carrot, and least in orange, red and yellow carrot (black carrot?>?rainbow carrot?>?red carrot?≈?orange carrot?≈?yellow carrot). The content of phenolics and flavonoids were highly correlated with antioxidant activity (0.955** to 0.992**). However, the most cultivated and consumed carrots, orange and red one, possessed higher amount of carotenoids. The content of carotenoids negatively correlated with total phenolics, flavonoids and antioxidants activity (??0.612** to ??0.627**). Broad genetic base and selection based on total phenolics content could be pivotal in the future breeding to harness the genetic wealth of carrot efficiently.     

Total antioxidant performance: a validated fluorescence assay for the measurement of plasma oxidizability

The antioxidant capacity of human plasma was determined by following the oxidation kinetics of the lipid-soluble fluorescent marker BODIPY using 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the lipophilic radical initiator. The results are expressed as a total antioxidant performance (TAP) value based on the inhibition of BODIPY oxidation, as determined by the appearance of green fluorescence, with respect to a control sample (phosphatidylcholine with or without delipidized human serum). The suitability of the assay was evaluated on the basis of its precision, reproducibility, and specificity. The intra- and interassay coefficients of variation both were less than 5%. The addition of a representative substrate of plasma peroxidation, phosphatidylcholine, up to 750mug/ml did not induce significant changes in the TAP value. Also, BODIPY photooxidation was not observed during the experimental time course (220min). The TAP values of 6 plasma samples from healthy donors were measured and correlated with the main plasma water- and lipid-soluble antioxidants (uric acid and ascorbic acid, alpha-tocopherol, and carotenoids) and lipid profiles. Significant correlations were found between TAP and uric acid (R=0.97, P<0.05) and cholesterol-adjusted alpha-tocopherol (R=0.93, P<0.01). The results confirm that the TAP assay is suitable to measure the antioxidant activity of plasma antioxidants localized in both the lipophilic and hydrophilic compartments.     

Comparative evaluation of antioxidant reactivity within obstructed and control rabbit urinary bladder tissue using FRAP and CUPRAC assays

Partial urinary bladder outlet obstruction mediates cyclic ischemia and reperfusion resulting in the generation of both reactive oxygen species and reactive nitrogen species. It is theorized that with an increase in the level of free radicals, the level of protective antioxidants should decrease. To test this hypothesis, two electron transfer assays, the FRAP method and the CUPRAC method, were used to determine the level of antioxidant reactivity of obstructed and control bladder tissue. The results showed that the CUPRAC assay detected a significant decrease in the reactivity of antioxidants found within the obstructed bladder tissue as compared to the control bladder tissue in both the muscle and mucosa. The FRAP assay did not detect any difference between the muscle and mucosa of the obstructed and control bladder tissue.     

The inhibition of radical-initiated peroxidation of microsomal lipids by both alpha-tocopherol and beta-carotene.

Rat liver microsomal lipids in hexane solution were exposed to the lipid-soluble radical initiator, azobis-isobutyronitrile (AIBN), and the antioxidant activities of alpha-tocopherol and beta-carotene have been compared. Lipid peroxidation was monitored both by conjugated diene formation at 233 nm, and by malondialdehyde (MDA) formation in the thiobarbituric acid assay at 535 nm. Diene formation was continuous for at least 120 min in the presence of 85 micrograms/ml lipid and 4 mM AIBN. Both alpha-tocopherol and beta-carotene acted as chain-breaking antioxidants, suppressing lipid peroxidation and producing an induction period at concentrations as low as 0.5 and 8 microM, respectively. When both of these lipid-soluble antioxidants were present together, the oxidation was strongly suppressed and the induction period was the sum of the individual antioxidants, alpha-Tocopherol and beta-carotene also inhibited MDA generation. In the presence of 170 micrograms/ml lipid and 8 mM AIBN, beta-carotene exhibited an IC50 of 1.1 microM and inhibited completely at 15 microM. Using beta-carotene, an induction period was observed, although much less pronounced than with alpha-tocopherol. Furthermore, beta-carotene inhibited MDA production in a concentration-dependent manner and exhibited an IC50 of 50 microM. In addition, added beta-carotene delayed the radical-initiated destruction of the endogenous alpha-tocopherol and gamma-tocopherol in this system.     

Measurement of relative antioxidant activityof compounds: a methodological note

Abstract

The relativeThe 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.     

红花桑寄生叶提取物的抗氧化活性及酚类物质分析

采用DPPH法、TEAC法、FRAP法对红花桑寄生叶不同溶剂提取物的抗氧化活性进行体外评价,并测定其总酚、总黄酮含量。结果表明,溶剂种类对红花桑寄生叶提取物的得率、总酚、总黄酮及抗氧化活性影响显著。在3种评价方法中,不同溶剂提取物的抗氧化活性均表现出不同程度的量效依赖关系。3种溶剂提取物的抗氧化活性强弱依次为丙酮提取物 >甲醇提取物 >水提取物,其中80%丙酮提取物(总酚含量最高,达276.83mg/g)抗氧化活性最强,清除DPPH自由基能力EC₅₀值为0.247,FRAP值(FeSO₄ mmol/100g)为115.81,浓度为1.0mg/ml时,TEAC值为2.04。     

Influence of treatment of diabetic rats with combinations of pycnogenol, beta-carotene, and alpha-lipoic acid on parameters of oxidative stress

Treatment with antioxidants may act more effectively to alter markers of free radical damage in combinations than singly. This study has determined whether treatment with combinations of pycnogenol, beta-carotene, and alpha-lipoic acid was more effective at reducing oxidative stress in diabetic rats than treatment with these antioxidants alone. It is not feasible, based on this study, to assume that there are interactive effects that make combinations of these antioxidants more effective than any one alone to combat oxidative stress. Female Sprague-Dawley rats, normal and streptozotocin-induced diabetic, were treated (10 mg/kg/day ip for 14 days) with pycnogenol, beta-carotene, pycnogenol + beta-carotene, or pycnogenol + beta-carotene + alpha-lipoic acid; controls were untreated. Concentrations of thiobarbituric acid reactive substances, glutathione and glutathione disulfide, and activities of glutathione reductase, glutathione peroxidase, superoxide dismutase, and catalase were measured in liver, kidney, and heart. Four types of effects were observed: (1) treatment with beta-carotene alone either reversed (cardiac glutathione disulfide) or elevated (cardiac glutathione, hepatic glutathione peroxidase activity) levels seen in diabetic animals; (2) beta-carotene alone produced no effect, but pycnogenol both alone and in combinations elevated (renal glutathione peroxidase and glutathione reductase activities, hepatic glutathione reductase activity and glutathione disulfide) or depressed (cardiac glutathione disulfide) levels seen in untreated diabetic animals; (3) all treatments with antioxidants, either alone or in combination, either normalized (lipid peroxidation in all tissues), elevated (hepatic GSH, cardiac glutathione peroxidase activity), or had no effect on (activities of hepatic catalase and superoxide dismutase in all tissues) levels seen in diabetic animals; (4) in only one case (cardiac glutathione reductase activity) levels in diabetic animals treated with combinations of antioxidants were normal, but elevated in animals treated with either antioxidant alone. Antioxidant effects seem to be dependent on the nature of the antioxidant used and not on combination effects.     

Plasma total antioxidant capacity is associated with dietary intake and plasma level of antioxidants in postmenopausal women

Increased plasma total antioxidant capacity (TAC) has been associated with a high consumption of fruits and vegetables. However, limited information is available on whether plasma TAC reflects the dietary intake of antioxidants and the levels of individual antioxidants in plasma. By using three different assays, the study aimed to determine if plasma TAC can effectively predict dietary intake of antioxidants and plasma antioxidant status. Forty overweight and apparently healthy postmenopausal women were recruited. Seven-day food records and 12-h fasting blood samples were collected for dietary and plasma antioxidant assessments. Plasma TAC was determined by vitamin C equivalent antioxidant capacity (VCEAC), ferric-reducing ability of plasma (FRAP) and oxygen radical absorbance capacity (ORAC) assays. TAC values determined by VCEAC were highly correlated with FRAP (r=0.79, P<.01) and moderately correlated with ORAC (r=0.34, P<.05). Pearson correlation analyses showed that plasma TAC values by VCEAC and ORAC had positive correlation with plasma uric acid (r=0.56 for VCEAC; r=0.49 for ORAC) and total phenolics (r=0.63 for VCEAC; r=0.36 for ORAC). However, TAC measured by FRAP was correlated only with uric acid (r=0.69). After multivariate adjustment, plasma TAC determined by VCEAC was positively associated with dietary intakes of γ-tocopherol (P<.001), β-carotene (P<.05), anthocyanidins (P<.05), flavones (P<.05), proanthocyanidins (P<.01) and TAC (P<.05), as well as with plasma total phenolics (P<.05), α-tocopherol (P<.001), β-cryptoxanthin (P<.05) and uric acid (P<.05). The findings indicate that plasma TAC measured by VCEAC reflects both dietary and plasma antioxidants and represents more closely the plasma antioxidant levels than ORAC and FRAP.     

Antioxidants and total oxyradical scavenging capacity during grass shrimp, Palaemonetes pugio, embryogenesis

During embryogenesis in grass shrimp the capacity to scavenge oxyradicals increased as measured by the Total Oxyradical Scavenging Capacity (TOSC) assay. The increase in TOSC during embryogenesis was associated with increasing concentrations of a number of antioxidants, including coenzyme Q (ubiquinone), alpha-tocopherol and reduced glutathione. Glutathione concentrations ranged from 0.004 to 0.005 nmol/embryo in early embryo stages and reached concentrations between 0.16 to 0.23 nmol/embryo in late embryo stages. Ascorbate remained essentially constant (0.16-0.20 nmol/embryo) throughout embryogenesis and may provide the preponderance of TOSC during early embryo development. Carotenoids were associated with yolk lipovitellin and these antioxidants decreased as yolk was absorbed during embryogenesis. Astaxanthin and beta-carotene were identified in embryos with astaxanthin always the principal carotenoid. In early embryo stages there are maternally derived antioxidants but as embryogenesis proceeds there is an assembly of a complex antioxidant system by newly formed cells and tissues.     

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.     

Evaluation of the copper(II) reduction assay using bathocuproinedisulfonic acid disodium salt for the total antioxidant capacity assessment: The CUPRAC-BCS assay

There is heightened interest in determining antioxidant status of individuals in experimental and clinical studies investigating progression of diseases or diverse aspects of oxidative stress, among others. The aim of this study was to evaluate the copper(II) reduction assay with bathocuproinedisulfonic acid disodium salt as chelating agent (the CUPRAC-BCS assay) for the total antioxidant capacity (TAC) assessment in human plasma and urine. Samples from 20 individuals were determined with four spectrophotometric assays—CUPRAC-BCS, ferric reducing ability of plasma (FRAP), trolox equivalent antioxidant capacity (TEAC), and 1,1-diphenyl-2-picrylhydrazyl assay (DPPH)—to compare these methods. CUPRAC-BCS was significantly correlated with FRAP and TEAC for plasma and urine samples (r > 0.5, P < 0.05 for all) and with DPPH for urine samples (r = 0.925, P < 0.001) but not with DPPH for plasma samples (r = 0.366, P = 0.112). However, the four methods do not agree given that lines of equality and regression were not matched up. The imprecision of the method is less than 6%, the detection limit is 41.8 μmol trolox equivalents/L, it is linear up to 2 mM trolox, and ethylenediaminetetraacetic acid dihydrate disodium salt (EDTA) binds to Cu(II), avoiding the formation of Cu(I)-BCS complex. This study shows that CUPRAC-BCS is a simple, fast, inexpensive, and suitable method for TAC assessment in human urine and heparinized plasma samples.