Novel fluorometric assay for hydroxyl radical scavenging capacity (HOSC) estimation

A novel fluorometric method was developed and validated for hydroxyl radical scavenging capacity (HOSC) estimation using fluorescein as the probe. A constant flux of pure hydroxyl radical is generated under physiological pH using a Fenton-like Fe3+/H2O2 reaction. The generation of pure hydroxyl radicals under the experimental conditions was evaluated and confirmed using electron spin resonance with DMPO spin-trapping measurements. The hydroxyl radical scavenging capacity of a selected antioxidant sample is quantified by measuring the area under the fluorescence decay curve with or without the presence of the antioxidant and expressed as Trolox equivalents per unit of the antioxidant. The assay may be performed using a plate reader with a fluorescence detector for high-throughput measurements. The assay was validated for linearity, precision, accuracy, reproducibility, and its correlation with a popular peroxyl radical scavenging capacity assay using selected pure antioxidant compounds and botanical extracts. This method may provide researchers in the food, nutrition, and medical fields an easy to use protocol to evaluate free radical scavenging capacity of pure antioxidants and natural extracts in vitro against the very reactive hydroxyl radical, which may be linked to numerous degenerative diseases and conditions.     

The chemistry behind antioxidant capacity assays

This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays.     

Antioxidant activity of oregano, parsley, and olive mill wastewaters in bulk oils and oil-in-water emulsions enriched in fish oil

The antioxidant activity of oregano, parsley, olive mill wastewaters (OMWW), Trolox, and ethylenediaminetetraacetic acid (EDTA) was evaluated in bulk oils and oil-in-water (o/w) emulsions enriched with 5% tuna oil by monitoring the formation of hydroperoxides, hexanal, and t-t-2,4-heptadienal in samples stored at 37 degrees C for 14 days. In bulk oil, the order of antioxidant activity was, in decreasing order (p < 0.05), OMWW > oregano > parsley > EDTA > Trolox. The antioxidant activity in o/w emulsion followed the same order except that EDTA was as efficient an antioxidant as OMWW. In addition, the total phenolic content, the radical scavenging properties, the reducing capacity, and the iron chelating activity of OMWW, parsley, and oregano extracts were determined by the Folin-Ciocalteau, oxygen radical absorbance capacity, ferric reducing antioxidant power, and iron(II) chelating activity assays, respectively. The antioxidant activity of OMWW, parsley, and oregano in food systems was related to their total phenolic content and radical scavenging capacity but not to their ability to chelate iron in vitro. OMWW was identified as a promising source of antioxidants to retard lipid oxidation in fish oil-enriched food products.     

Antioxidant activity of knotwood extractives and phenolic compounds of selected tree species

The antioxidant potency and the radical scavenging capacity of superoxide and peroxyl radicals were assessed for 13 hydrophilic knotwood extracts of commercially important wood species, or fractions thereof, as well as for five pure wood-derived lignans and the flavonoid taxifolin. The chemical composition of the knotwood extracts was determined by gas chromatography combined with mass spectrometry. Most of the investigated wood species were rich in hydrophilic extractives (10-20% of the dry wood) with one or a few compounds dominating in each extract. All extracts had a high antioxidative potency and/or radical scavenging capacity as compared to the well-known antioxidants Trolox and butylated hydroxyanisole. The pure wood-derived lignans and taxifolin also had a high antioxidative potency and/or radical scavenging capacity. However, the antioxidant potency and/or radical scavenging capacity of several of the hydrophilic knotwood extracts were higher than that of the dominating compounds in pure form.     

Rapid peroxyl radical scavenging capacity (PSC) assay for assessing both hydrophilic and lipophilic antioxidants

This paper reports a simple, rapid, and sensitive assay for assessing peroxyl radical scavenging capacity (PSC) of both hydrophilic and lipophilic antioxidant compounds and food extracts. The assay is based on the degree of inhibition of dichlorofluorescin oxidation by antioxidants that scavenge peroxyl radicals, generated from thermal degradation of 2,2'-azobis(amidinopropane). For hydrophilic antioxidant activity, the dose required to cause a 50% inhibition of the reaction (EC(50)) ranged from 2.41 +/- 0.02 (EGCG) to 21.26 +/- 0.38 microM (ferulic acid). EC(50) values for the hydrophilic antioxidant activity of food extracts ranged from 309.2 +/- 3.63 (apple) to 3345.1 +/- 151.5 micromol of vitamin C equiv/100 g for wheat bran. The EC(50) values for lipophilic antioxidant activity were 1.58 +/- 0.11 (Trolox), 4.35 +/- 0.43 (alpha-tocopherol), 18.94 +/- 0.38 (BHA), and 182.69 +/- 13.7 microM (BHT). Whole grain lipophilic antioxidant activity ranged from 3.49 +/- 0.57 (wheat) to 8.79 +/- 1.98 micromol of alpha-tocopherol equiv/100 g of rice. Hydrophilic antioxidant activity contributed >98% of the total antioxidant activity (hydrophilic plus lipophilic) of whole grains tested. The PSC assay was accurate (86-108% recovery), precise (0.12-11% CV), and reproducible (12% RSD) and produced results comparable to those of similar published assays. The PSC assay can be routinely used to analyze or screen both hydrophilic and lipophilic antioxidants or food extracts and will be a valuable alternative biomarker for future epidemiological studies of chronic diseases.     

Novel low-density lipoprotein (LDL) oxidation model: antioxidant capacity for the inhibition of LDL oxidation

A novel model of peroxyl radical initiated low-density lipoprotein (LDL) oxidation (LDL oxidation model for antioxidant capacity, or LOMAC) was developed to assess the free radical scavenging capacity of antioxidants and the extracts of natural products. A water-soluble free radical initiator, 2,2'-azobis(amidinopropane) dihydrochloride, was used at physiological temperature (37 degrees C) to generate peroxyl radicals to catalyze lipid oxidation of LDL isolated from human plasma samples. Headspace hexanal, a major decomposition product of LDL oxidation, was measured by a headspace gas chromatograph as an indicator of antioxidant capacity of different concentrations of pure antioxidants (vitamins C and E) and the extracts of natural products (fresh apple phytochemical extracts). All vitamin C and E and apple extract concentrations tested resulted in increasing partial suppression and delay of LDL oxidation. On the basis of the median effective dose (EC(50)) calculated for each compound or extract tested, the LOMAC value of 100 g of apple against LDL oxidation was equivalent to 1470 mg of vitamin E or to 402 mg of vitamin C. This study shows that the LOMAC assay can be routinely used to analyze or screen antioxidants or phytochemical extracts against LDL oxidation to prevent cardiovascular disease. The food-specific LOMAC values will be very useful as a new alternative biomarker for future epidemiological studies of cardiovascular disease.     

Assessment of radical scavenging capacity and lipid peroxidation inhibiting capacity of antioxidant

The role of radical scavenging antioxidants against oxidative stress has received much attention, and the antioxidant capacity has been assessed by various methods. Among them, a method that measures the effect of antioxidant on decay of the probe is one of the most widely used methods. The present study was performed to compare the two methods to assess the antioxidant capacity, one to follow the decay of the probe and the other to measure lipid peroxidation products in human plasma. It was shown that the method following probe decay was suitable for assessment of radical scavenging capacity of antioxidant, but not for the capacity to inhibit lipid peroxidation in plasma. This is true whether a hydrophilic or lipophilic probe is used. Such different results arise from the fact that the efficacy of inhibition of lipid peroxidation by antioxidants depends on the fate of antioxidant-derived radical and interaction between antioxidants as well as the capacity of free radical scavenging. Thus, the capacity of antioxidants for inhibition of lipid peroxidation should be assessed from the effect on the extent of oxidation, not from the effect on probe decay.     

Phytic acid, phytase, minerals, and antioxidant activity in Canadian dry bean ( Phaseolus vulgaris L.) cultivars

Ten bean cultivars grown in southern Manitoba in 2006 were evaluated for variability in phytate, phenolic, and mineral contents, phytase activity, and antioxidant properties to elucidate the relationship of these components. Phytic acid content and phytase activity varied significantly among cultivars and market classes, ranging from 16.7 to 25.1 g/kg and from 224 to 361 phytase activity unit/kg of sample, respectively. The bean cultivars with total phenolic content ranging from 2.2 to 5.6 g of catechin equiv/kg of sample exhibited significant variation in antioxidant capacity [1.6-11.2 microM Trolox equiv (TE)/g of dry matter] and peroxyl radical scavenging activity (72-158 microM TE/g) using photochemiluminescence and fluorescence assays, respectively. Multivariate data analysis performed on 22 components analyzed in this study using principal component analysis and cluster methods demonstrate that differences in phytase, antioxidant activity, mineral contents, and bioavailability are much larger within market class than among bean cultivars.     

Simple assessment of radical scavenging capacity of beverages

The radical-scavenging antioxidants play an important role against oxidative stress in the defense system in vivo. The beneficial effects of antioxidants contained in foods and beverages have been well-accepted, and their antioxidant capacity has been assessed by various methods. In the present study, a simple method is proposed in which the total radical scavenging capacity is assessed from the bleaching of pyranine and pyrogallol red induced by free radicals generated from azo initiator. The total content of antioxidants contained in red wine, green tea, and cassis drink and their reactivities toward peroxyl radicals were measured from the lag phase and rate of bleaching using pyranine and pyrogallol red as a probe, respectively. It was found that this method to follow the bleaching of two probes by visible light spectrophotometer is convenient and applicable for assessment of total radical scavenging capacity of both content and activity of the antioxidants contained in beverages.     

Antioxidant capacity and other bioactivities of the freeze-dried Amazonian palm berry, Euterpe oleraceae mart. (acai)

The fruit of Euterpe oleraceae, commonly known as acai, has been demonstrated to exhibit significantly high antioxidant capacity in vitro, especially for superoxide and peroxyl scavenging, and, therefore, may have possible health benefits. In this study, the antioxidant capacities of freeze-dried acai fruit pulp/skin powder (OptiAcai) were evaluated by different assays with various free radical sources. It was found to have exceptional activity against superoxide in the superoxide scavenging (SOD) assay, the highest of any food reported to date against the peroxyl radical as measured by the oxygen radical absorbance capacity assay with fluorescein as the fluorescent probe (ORACFL), and mild activity against both the peroxynitrite and hydroxyl radical by the peroxynitrite averting capacity (NORAC) and hydroxyl radical averting capacity (HORAC) assays, respectively. The SOD of acai was 1614 units/g, an extremely high scavenging capacity for O2*-, by far the highest of any fruit or vegetable tested to date. Total phenolics were also tested as comparison. In the total antioxidant (TAO) assay, antioxidants in acai were differentiated into "slow-acting" and "fast-acting" components. An assay measuring inhibition of reactive oxygen species (ROS) formation in freshly purified human neutrophils showed that antioxidants in acai are able to enter human cells in a fully functional form and to perform an oxygen quenching function at very low doses. Furthermore, other bioactivities related to anti-inflammation and immune functions were also investigated. Acai was found to be a potential cyclooxygenase (COX)-1 and COX-2 inhibitor. It also showed a weak effect on lipopolysaccharide (LPS)-induced nitric oxide but no effect on either lymphocyte proliferation and phagocytic capacity.     

Determination of peroxyl radical scavenging activity of flavonoids and plant extracts using an automatic potentiometric titrator

A novel potentiometric method for evaluation of peroxyl radical scavenging activity of flavonoids and plant extracts was developed. The oxidation of potassium iodide (KI) was performed in acetonitrilephosphate buffer (1:1) containing antioxidant using 2,2'-azobis(2-amidinopropane) dihydrochloride as a peroxyl radical generator. The amount of iodine released from KI during a 20-min free radical oxidation was determined quantitatively using an automatic potentiometric titrator with sodium thiosulfate. The radical scavenging activity of the sample was expressed as the inhibition ratio for iodine release of the control group mediated by the radical. The results obtained from some authentic polyphenols correlated well with those of previous reports. This is a simple, time-saving method requiring less than 30 min and is useful in assessing the radical scavenging activity of antioxidants in plant extracts. We describe the radical scavenging activities of various flavonoids including 21 kinds of tea catechins and vegetable extracts by this method.     

Long-wavelength fluorimetric determination of food antioxidant capacity using Nile blue as reagent

A method for the determination of the antioxidant capacity using long-wavelength fluorescence measurements is described for the first time. This method is a modification of the conventional oxygen radical absorbance capacity (ORAC) method that uses fluorescein or phycoerythrin and the generator of peroxyl radicals, 2,2'-azo-bis-(2-methylpropionamidine) dihydrochloride (AAPH). The long-wavelength fluorophor nile blue is proposed as an analytical reagent alternative to these conventional fluorophores. Kinetic curves have been obtained by monitoring the fluorescence variation (λex, 620; λem, 680 nm) with time, using the 96-well microplate format. The vitamin E analogue 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) has been chosen as the model analyte, and the normalized area under the decay curve has been used as the analytical parameter. The dynamic range of the calibration curve is 0.8-8.0 μM, and the detection limit is 0.45 μM. The precision of the method, expressed as relative standard deviation and assayed using 1 and 5 μM Trolox concentrations, was 5.6 and 2.9%, respectively. The method has been applied to the analysis of fruit juices and wines, obtaining results that did not differ significantly from those provided using the ORAC method with fluorescein as reagent.     

Synthesis of lipophilic clovamide derivatives and their antioxidative potential against lipid peroxidation

Some N-(hydroxycinnamoyl)-L-tyrosine and L-DOPA alkyl esters were synthesized and evaluated as a variation of the clovamide (N-caffeoyl-L-3,4-dihydroxyphenylalanine) structure, a known antioxidant found in red clover. The amides were prepared in good yields starting from methyl and dodecylesters of L-tyrosine and L-DOPA by reacting with the N-hydroxysuccinimidyl esters of ferulic, sinapic, and acetyl-protected caffeic acid, respectively. In the DPPH* (2,2-diphenyl-1-picrylhydrazyl) and superoxide radical quencher assays they showed radical scavenging activity equal to or higher than those of the standard antioxidants ascorbic acid and tocopherol. The antioxidative potentials of the clovamide derivatives against bulk lipid oxidation, as determined by the accelerated autoxidation of oils, were equal to or higher than those of the standard antioxidants; some of the compounds were able to protect an emulsion of linoleic acid/beta-carotene against oxidation. N-Caffeoyl L-tyrosine methyl ester and the N-cinnamoyl L-DOPA alkyl esters especially were potent antioxidants in bulk lipids and moderate protectants in emulsions.     

Evaluation of the antioxidant capacity of limonin, nomilin, and limonin glucoside

The antioxidant capacity (AOC) of three representative citrus limonoids, limonin, nomilin, and limonin glucoside, was examined by the oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC), beta-carotene-linoleic acid bleaching, and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assays. Pure compounds and proper negative (cinnamic acid) and positive (2,6-di-tert-butyl-4-methylphenol (BHT) and ascorbic acid) controls were used to remove any ambiguity in interpreting results. In all cases, limonin and nomilin gave results equivalent to those of cinnamic acid, indicating that they do not possess any inherent AOC and should not be considered antioxidants. Similar results were observed for limonin glucoside, with the exception of an anomalous result obtained from the beta-carotene-linoleic acid bleaching assay. Limonin glucoside was deemed not to be an antioxidant on the basis of the three unequivocal assays.     

Antioxidant capacity of oat (Avena sativa L.) extracts. 1. Inhibition of low-density lipoprotein oxidation and oxygen radical absorbance capacity

Milled oat groat pearlings, trichomes, flour, and bran were extracted with methanol and the fractions tested in vitro for antioxidant capacity against low-density lipoprotein (LDL) oxidation and R-phycoerythrin protein oxidation in the oxygen radical absorbance capacity (ORAC) assay. The oxidative reactions were generated by 2,2'-azobis(2-amidinopropane) HCl (AAPH) or Cu(2+) in the LDL assay and by AAPH or Cu(2+) + H(2)O(2) in the ORAC assay and calibrated against a Trolox standard to calculate Trolox equivalents (1 Trolox equivalent = 1 TE = activity of 1 micromol of Trolox). The antioxidant capacity of the oat fractions was generally consistent with a potency rank of pearlings (2.89-8.58 TE/g) > flour (1.00-3.54 TE/g) > trichome (1.74 TE/g) = bran (1.02-1.62 TE/g) in both LDL and ORAC assays regardless of the free radical generator employed. A portion of the oat antioxidant constituents may be heat labile as the greatest activity was found among non-steam-treated pearlings. The contribution of oat tocols from the fractions accounted for <5% of the measured antioxidant capacity. AAPH-initiated oxidation of LDL was inhibited by the oat fractions in a dose-dependent manner, although complete suppression was not achieved with the highest doses tested. In contrast, Cu(2+)-initiated oxidation of LDL stimulated peroxide formation with low oat concentrations but completely inhibited oxidation with higher doses. Thus, oats possess antioxidant capacity most of which is likely derived from polar phenolic compounds in the aleurone.     

Antioxidant properties of pearled barley fractions

Two barley varieties (Falcon and AC Metcalfe) were separated by pearling into seven fractions and subsequently extracted with 80% methanol. The extracts, after solvent removal, were evaluated for their radical scavenging efficacy using Trolox equivalent antioxidant capacity (TEAC). The radical scavenging capacity of the extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, oxygen radical absorbance capacity (ORAC(FL)), and superoxide radical assays and a photoinduced chemiluminescence technique. In both barley varieties the outermost fraction (F1) yielded the highest phenolic content. In general, Falcon had a significantly higher total phenolic content than AC Metcalfe. A similar trend was observed for TEAC, DPPH, and superoxide radical scavenging capacities of the extracts. The contents of water-soluble antioxidants of Falcon and AC Metcalfe were 1.15-12.98 and 2.20-12.25 micromol of Trolox equiv/(g of defatted material), while the corresponding lipid-soluble counterparts varied from 1.44 to 4.70 micromol of alpha-tocopherol equiv/(g of defatted material). Phenolic acids, namely, vanillic, caffeic, p-coumaric, ferulic, and sinapic acids, were identified by HPLC in barley fractions.     

Evaluation of antioxidant and prooxidant activities of bamboo Phyllostachys nigra var. Henonis leaf extract in vitro

Solvent-extracted bamboo leaf extract (BLE) containing chlorogenic acid, caffeic acid, and luteolin 7-glucoside was evaluated in vitro for free radical scavenging and antioxidant activities using a battery of test methods. BLE exhibited a concentration-dependent scavenging activity of DPPH radical. BLE prolonged the lag phase and suppressed the rate of propagation of liposome peroxidation initiated by peroxyl radical induced by 2,2'-azobis(2-amidinopropane dihydrochloride (AAPH) at 37 degrees C. BLE also prevented human low-density lipoprotein oxidation, mediated by Cu(2+), which was monitored by the lower formation of conjugated diene and fluorescence and a reduced negative charge of apo-B protein. Finally, BLE protected supercoiled DNA strand against scission induced by AAPH-mediated peroxyl radical. Prooxidant activity of BLE was seen in a Cu(2+)-induced peroxidation of structured phosphatidylcholine liposome, indicating catalytic peroxidation due to a relatively high reducing power of BLE. It was concluded that the BLE has both antioxidant activity and prooxidant activity; the antioxidant activity was attributed to free radical scavenging activity, and the prooxidant activity, albeit minor, resulted from the reducing power of plant phenolics in the presence of transitional metal ions.     

Radical-scavenging activities of citrus essential oils and their components: detection using 1,1-diphenyl-2-picrylhydrazyl

Thirty-four kinds of citrus essential oils and their components were investigated for radical-scavenging activities by the HPLC method using 1,1-diphenyl-2-picrylhydrazyl (DPPH). To examine the oils' relative radical-scavenging activities compared with that of a standard antioxidant, Trolox was employed. All of the essential oils were found to have scavenging effects on DPPH in the range of 17. 7-64.0%. The radical-scavenging activities of 31 kinds of citrus essential oils were comparable with or stronger than that of Trolox (p < 0.05). The oils of Ichang lemon (64.0%, 172.2 mg of Trolox equiv/mL), Tahiti lime (63.2%, 170.2 mg of Trolox equiv/mL), and Eureka lemon (61.8%, 166.2 mg of Trolox equiv/mL) were stronger radical scavengers than other citrus oils. Citrus volatile components such as geraniol (87.7%, 235.9 mg of Trolox equiv/mL), terpinolene (87.4%, 235.2 mg of Trolox equiv/mL), and gamma-terpinene (84.7%, 227.9 mg of Trolox equiv/mL) showed marked scavenging activities on DPPH (p < 0.05).     

Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative study

A total of 927 freeze-dried vegetable samples, including 111 white cabbages, 59 carrots, 51 snap beans, 57 cauliflower, 33 white onions, 48 purple onions, 130 broccoli, 169 tomatoes, 25 beets, 88 peas, 88 spinach, 18 red peppers, and 50 green peppers, were analyzed using the oxygen radical absorption capacity (ORAC) and ferric reducing antioxidant capacity (FRAP) methods. The data show that the ORAC and FRAP values of vegetable are not only dependent on species, but also highly dependent on geographical origin and harvest time. The two antioxidant assay methods, ORAC and FRAP, also give different antioxidant activity trends. The discrepancy is extensively discussed based on the chemistry principles upon which these methods are built, and it is concluded that the ORAC method is chemically more relevant to chain-breaking antioxidants activity, while the FRAP has some drawbacks such as interference, reaction kinetics, and quantitation methods. On the basis of the ORAC results, green pepper, spinach, purple onion, broccoli, beet, and cauliflower are the leading sources of antioxidant activities against the peroxyl radicals.     

Fast and simple method for the simultaneous evaluation of the capacity and efficiency of food antioxidants in trapping peroxyl radicals in an intestinal model system

A simple oxygraphic method, for which the theoretical and experimental bases have been recently revised, has been successfully applied to evaluate the peroxyl radical chain-breaking characteristics of some typical food antioxidants in micelle systems, among which is a system that reproduces conditions present in the upper part of the digestive tract, where the absorption and digestion of lipids occur. This method permits one to obtain from a single experimental run the peroxyl radical trapping capacity (PRTC, that is, the number of moles of peroxyl radicals trapped by a given amount of food), the peroxyl radical trapping efficiency (PRTE, that is, the reciprocal of the amount of food that reduces to half the steady-state concentration of peroxyl radicals), and the half-life of the antioxidant ( t(1/2)) when only a small fraction of peroxyl radicals reacts with the antioxidants present in foods. Examples of application of the method to various types of foodstuffs have been reported, assessing the general validity of the method in the simple and fast evaluation of the above-reported fundamental antioxidant characteristics of foods.