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.     

Evaluation of antioxidant activity of vetiver (Vetiveria zizanioides L.) oil and identification of its antioxidant constituents

Antioxidant capacities of vetiver (Vetiveria zizanioides) oil were evaluated by two different in vitro assays: the DPPH* free radical scavenging assay and the Fe2+-metal chelating assay. Results showed that the vetiver oil (VO) possessed a strong free radical scavenging activity when compared to standard antioxidants such as butylated hydroxytoluene (BHT) and alpha-tocopherol. However, its metal chelating capacity was relatively weak. VO (10 microL/mL) dissolved in methanol exhibited approximately 93% free radical scavenging activity in the DPPH* assay and approximately 34% Fe2+ chelating activity in the metal chelating assay. By contrast, 10 mM BHT and 0.1 mM alpha-tocopherol exhibited 93 and 89% free radical scavenging activities in the DPPH* assay, respectively, and 1 mM EDTA exhibited approximately 97% activity in the metal chelating assay. Among the complex constituents in the crude VO, beta-vetivenene, beta-vetivone, and alpha-vetivone, which had shown strong antioxidant activities, were isolated and identified using various chromatographic techniques including silica gel open column chromatography, silica HPLC, and GC-MS. These results show that VO and some of its inherent components can be potential alternative natural antioxidants.     

Electron spin resonance estimation of hydroxyl radical scavenging capacity for lipophilic antioxidants

Hydroxyl radical scavenging capacity estimation for lipophilic antioxidants is a challenge due to their poor solubility in aqueous radical generating and measuring systems. In this study, an electron spin resonance (ESR) method was developed and validated for its application in estimating the relative hydroxyl radical (HO*) scavenging capacity for lipophilic antioxidants under physiological pH using a Fenton Fe2+/H2O2 system for radical generation and acetonitrile as a solvent. The Fenton Fe2+/H2O2 system generates a constant flux of pure HO* under the assay conditions. The method was validated by linearity, precision, and reproducibility using selected known lipophilic antioxidants including alpha-tocopherol, lutein, beta-carotene, and BHT. The potential effects of commonly used water-miscible and water-immiscible organic solvents on the Fenton Fe2+/H2O2 HO* generating system as well as their possible interactions with the fluorescent and spectroscopic probes were also reported. In addition, the limitation of the ESR assay was described.     

High-throughput methods to assess lipophilic and hydrophilic antioxidant capacity of food extracts in vitro

Assays comprising three probes for different mechanisms of antioxidant activity in food products have been modified to allow better comparison of the contributions of the different mechanisms to antioxidant capacity (AOC). Incorporation of a common format for oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), and iron(II) chelating activity (ICA) assays using 96-well microplates provides a comprehensive and high-throughput assessment of the antioxidant capacity of food extracts. The methods have been optimized for aqueous extracts and validated in terms of limit of quantification (LoQ), linearity, and precision (repeatability and intermediate reproducibility). In addition, FRAP and ORAC assays have been validated to assess AOC for lipophilic extracts. The relative standard deviation of repeatability of the methods ranges from 1.2 to 6.9%, which is generally considered to be acceptable for analytical measurement of AOC by in vitro methods. Radical scavenging capacity, reducing capacity, and iron chelating properties of olive mill wastewaters (OMWW), oregano, and parsley were assessed using the validated methods. OMWW showed the highest radical scavenging and reducing capacities, determined by ORAC and FRAP assays, respectively, followed by oregano and parsley. The ability to chelate Fe (2+) was, in decreasing order of activity ( p > 0.05) parsley congruent with oregano > OMWW. Total phenol content, determined by the Folin-Ciocalteu method, correlated to the radical scavenging and reducing capacities of the samples but not to their chelating properties. Results showed that the optimized high-throughput methods provided a comprehensive and precise determination of the AOC of lipophilic and hydrophilic food extracts in vitro.     

Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and other biological and food samples

Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORAC(FL)) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37 degrees C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 +/- 1.5 and 38.2 +/- 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples.     

Isoflavone composition and antioxidant capacity of modified-lipoxygenase soybeans grown in Maryland

Maryland-grown soybean lines modified for low lipoxygenase-1 (LOX-1) content and a traditional nonmodified cultivar were analyzed for fatty acid composition, total phenolic content (TPC), isoflavone composition, relative DPPH? scavenging capacity (RDSC), and hydroxyl radical scavenging capacity (HOSC). Soybean lines included black, brown, and yellow soybeans. TPC of all soybean lines ranged from 2.84 to 4.74 mg gallic acid equiv (GAE)/g flour. Total isoflavones were between 2.78 and 8.66 μmol/g flour. RDSC of all lines was between 0.48 and 14.62 μmol Trolox equiv (TE)/g flour, and HOSC ranged from 53.57 to 135.52 μmol TE/g flour. Some modified-LOX genotypes demonstrated antioxidant capacity and/or isoflavone content similar to or higher than those of the nonmodified cultivar (P < 0.05). Black soybeans demonstrated higher TPC and RDSC than most yellow soybean lines, although they did not have higher isoflavone content. The results demonstrate that modification of the LOX trait did not necessarily alter the antioxidant capacity or chemical composition of the experimental soybean lines when compared with a nonmodified cultivar. These soybean lines may be studied further for nutraceutical properties and use in functional foods.     

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.     

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.     

Enzymatic synthesis of lipophilic rutin and vanillyl esters from fish byproducts

Lipase-catalyzed synthesis of lipophilic phenolic antioxidants was carried out with a concentrate of n-3 polyunsaturated fatty acids (PUFAs), recovered from oil extracted from salmon ( Salmon salar ) byproduct. Vanillyl alcohol and rutin were selected for the esterification reaction, and obtained esters yields were 60 and 30%, respectively. The antioxidant activities of the esters were compared with those of commercial butylated hydroxytoluene (BHT) and α-tocopherol using DPPH radical scavenging and thiobarbituric acid assays. In the DPPH assay, rutin esters showed better activity than vanillyl esters, and on the contrary in lipophilic medium, vanillyl esters were found to be superior to rutin esters. In bulk oil system, the antioxidant activities of rutin and vanillyl derivatives were lower than that of BHT and α-tocopherol, but in emulsion, they showed better activity than α-tocopherol. By attaching to natural phenolics, the PUFAs are protected against oxidation, and PUFA improves the hydrophobicity of the phenolic, which could enhance its function in lipid systems.     

Carotenoids in antioxidant networks. Colorants or radical scavengers

Optical and electronic properties of carotenoids as also reflected in their colors have been fine-tuned through evolution, resulting in a structural diversity important for carotenoid properties as radical scavengers and as quenchers of electronically excited states. Carotenoids form antioxidant networks based on one-electron transfer with other carotenoids depending on the balance between ionization energy and electron affinity of the individual carotenoids as has been demonstrated by real-time kinetic studies and later confirmed by quantum mechanical calculations. The more hydrophilic xanthophylls serve as molecular wiring across membranes in these networks through anchoring in water/lipid interfaces resulting in synergism with more lipophilic carotenoids. Radical scavenging of such networks seems to be thermodynamically controlled according to a two-dimensional classification of potential antioxidants. Carotenoids in birds' plumage, as reflected by their color and color intensity, seem to be indicators of good antioxidant status and health of the bird, and such antioxidant networks appear to be in "equilibrium". Carotenoids are under other conditions involved in networks with other types of antioxidants as in egg yolk and in some fish. For the more hydrophilic (iso)flavonoids and their glycosides, antioxidant synergism through regeneration of the lipophilic carotenoids active as radical scavengers becomes kinetically controlled at interfaces. Carotenoids appear accordingly, and also in food, as antioxidants under two types of conditions: (i) in "equilibrium" with other antioxidants in thermodynamically controlled networks serving as color indicators of good antioxidant status and (ii) as antioxidants active through radical scavenging in networks with kinetically controlled regeneration.     

Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam.) leaves

Water, aqueous methanol, and aqueous ethanol extracts of freeze-dried leaves of Moringa oleifera Lam. from different agroclimatic regions were examined for radical scavenging capacities and antioxidant activities. All leaf extracts were capable of scavenging peroxyl and superoxyl radicals. Similar scavenging activities for different solvent extracts of each collection were found for the stable 1,1-diphenyl 2-picrylhydrazyl (DPPH(*)) radical. Among the three different moringa samples, both methanol and ethanol extracts of Indian origins showed the highest antioxidant activities, 65.1 and 66.8%, respectively, in the beta-carotene-linoleic acid system. Nonetheless, increasing concentration of all the extracts had significantly (P < 0.05) increased reducing power, which may in part be responsible for their antioxidant activity. The major bioactive compounds of phenolics were found to be flavonoid groups such as quercetin and kaempferol. On the basis of the results obtained, moringa leaves are found to be a potential source of natural antioxidants due to their marked antioxidant activity. This is the first report on the antioxidant properties of the extracts from freeze-dried moringa leaves. Overall, both methanol (80%) and ethanol (70%) were found to be the best solvents for the extraction of antioxidant compounds from moringa leaves.     

Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated beta-cyclodextrin as the solubility enhancer

We recently reported the improved oxygen radical absorbance capacity (ORAC) assay using fluorescein (FL) as the fluorescent probe. The current ORAC(FL) assay is limited in hydrophilic antioxidant due to the aqueous environment of the assay. Lipophilic antioxidants mainly include the vitamin E family and carotenoids, which play a critical role in biological defense systems. In this paper, we expanded the current ORAC(FL) assay to lipophilic antioxidants. Randomly methylated beta-cyclodextrin (RMCD) was introduced as the water solubility enhancer for lipophilic antioxidants. Seven percent RMCD (w/v) in a 50% acetone-H(2)O mixture was found to sufficiently solubilize vitamin E compounds and other lipophilic phenolic antioxidants in 75 mM phosphate buffer (pH 7.4). This newly developed ORAC assay (abbbreviated ORAC(FL-LIPO)) was validated through linearity, precision, accuracy, and ruggedness. The validation results demonstrate that the ORAC(FL-LIPO) assay is reliable and robust. For the first time, by using 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid as a standard (1.0), the ORAC values of alpha-tocopherol, (+)-gamma-tocopherol, (+)-delta-tocopherol, alpha-tocopherol acetate, tocotrienols, 2,6-di-tert-butyl-4-methylphenol, and gamma-oryzanol were determined to be 0.5 +/- 0.02, 0.74 +/- 0.03, 1.36 +/- 0.14, 0.00, 0.91 +/- 0.04, 0.16 +/- 0.01, and 3.00 +/- 0.26, respectively. The structural information of oxidized alpha-tocopherol obtained by liquid chromatography/mass spectrometry reveals that the mechanism for the reaction between the vitamin E and the peroxyl radical follows the hydrogen atom transfer mechanism, which is in agreement with the notion that vitamin E is the chain-breaking antioxidant.     

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.     

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.     

Antioxidant capacity of different broccoli (Brassica oleracea) genotypes using the oxygen radical absorbance capacity (ORAC) assay

Antioxidant capacity of hydrophilic and lipophilic extracts from eight broccoli genotypes was compared using the oxygen radical absorbance capacity (ORAC) assay. Each genotype was analyzed for carotenoid, tocopherol, ascorbic acid, and flavonoid content. Results indicate that the antioxidant capacity of hydrophilic extracts ranged from 65.8 to 121.6 micromol trolox equivalents (TE)/g of tissue, and the capacity of lipophilic extracts ranged from 3.9 to 17.5 micromol TE/g. Ascorbic acid and flavonoid content of the hydrophilic extracts did not explain the total variation in antioxidant capacity of those extracts, suggesting either the presence of other antioxidant components that have yet to be identified or that the known antioxidants are producing synergistic effects. The carotenoids did correlate with antioxidant capacity of the lipophilic extracts and accounted for the majority of the variability in that fraction. The variability in hydrophilic and lipophilic antioxidant capacity found among these genotypes suggests that potential efficacy from antioxidants will vary considerably from genotype to genotype.     

Free radical scavenging properties of wheat extracts

Three hard winter wheat varieties (Akron, Trego, and Platte) were examined and compared for their free radical scavenging properties and total phenolic contents (TPC). Free radical scavenging properties of wheat grain extracts were evaluated by spectrophotometric and electron spin resonance (ESR) spectrometry methods against stable 2,2-diphenyl-1-picryhydrazyl radical (DPPH*) and radical cation ABTS*+ (2,2'-azino-di[3-ethylbenzthiazoline sulfonate]). The results showed that the three wheat extracts differed in their capacities to quench or inhibit DPPH* and ABTS*+. Akron showed the greatest activity to quench DPPH radicals, while Platte had the highest capacity against ABTS*+. The ED50 values of wheat extracts against DPPH radicals were 0.60 mg/mL for Akron, 7.1 mg/mL for Trego, and 0.95 mg/mL for Platte under the experimental conditions. The trolox equivalents against ABTS*+ were 1.31 +/- 0.44, 1.08 +/- 0.05, and 1.91 +/- 0.06 micromol/g of grain for Akron, Trego, and Platte wheat, respectively. ESR results confirmed that wheat extracts directly reacted with and quenched free radicals. The TPC were 487.9 +/- 927.8 microg gallic acid equivalents/g of grain. No correlation was observed between TPC and radical scavenging capacities for DPPH* and ABTS*+ (p = 0.15 and p > 0.5, respectively).     

Antioxidant and antimelanogenic activities of polyamine conjugates from corn bran and related hydroxycinnamic acids

The antioxidant activity of three major polyamine conjugates, N,N'-dicoumaroyl-putrescine (DCP), N-p-coumaroyl-N'-feruloylputrescine (CFP), and N,N'-diferuloyl-putrescine (DFP) isolated from corn bran, and their related hydroxycinnamic acids, p-coumaric acid and ferulic acid, were evaluated by three antioxidant in vitro assay systems, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide and hydroxyl radicals generated by enzymatic and nonenzymatic reactions. Additionally, five phenolic compounds were evaluated for melanogenesis inhibitory activity using mushroom tyrosinase and B16 melanoma cells. Most of the phenolic compounds significantly scavenged DPPH, superoxide, and hydroxyl radicals in a dose-dependent manner. Particularly, DFP showed potent DPPH (IC50 = 38.46 microM) and superoxide (IC50 = 291.62 microM) radical scavenging activities, while DCP exhibited the strongest hydroxyl radical scavenging activity (IC50 = 120.55 microM). CFP also exerted moderate DPPH, superoxide, and hydroxyl radical scavenging activities. Meanwhile, DCP (IC50 = 181.73 microM) showed potent tyrosinase inhibitory activity toward l-tyrosine as the substrate, whereas DFP (IC50 = 733.64 microM) significantly inhibited melanin synthesis in B16 melanoma cells. These current results indicate that these three polyamine conjugates from corn bran may be useful potential sources of natural antioxidants and skin-whitening agents.     

Comparison of the concentrations of phenolic constituents in cane sugar manufacturing products with their antioxidant activities

Polyphenol content and free radical scavenging capacity of seven kinds of sugar manufacturing products (A sugars, clear juices, syrups, massecuite, and A, B, and C molasses) were studied. Seventy-two samples were collected at different stages of the process during two sugar harvests from a local sugar factory (Bois-Rouge, La Réunion). The total phenolic content of sugar products was determined according to the Folin-Ciocalteu assay. Polyphenols of sugar products were extracted with ethyl acetate and quantified by LC-UV-ESI-MS during all of the process. ABTS and DPPH assays were applied to aqueous solution of sugar products, which exhibited interesting free radical scavenging activity. Comparatively, ethyl acetate extracts exhibited higher antioxidant activity. Multivariate analyses (principal component analysis and canonical discriminant analysis) demonstrated a significant correlation between polyphenols and antioxidant activity. Moreover, it was observed that the sugar process results in an increase of the phenolic content and the free radical scavenging capacity of the different products. These products and especially molasses proved to be a rich source of natural antioxidants and may represent an interesting alternative to synthetic food antioxidants.     

Isolation and identification of compounds responsible for antioxidant capacity of Euryale ferox seeds

Euryale ferox seed is consumed medicinally or for food in China. The present study revealed it to contain significant antioxidant activity, which may be associated with its medical applications as a proteinuria inhibitor of diabetic nephropathy. This study resulted in the identification of 3 new sesquineolignans, named euryalins A-C (1-3), and 16 known compounds, which were all first isolated from this plant apart from 5,7,4-trihydroxy-flavanone. The antioxidant potential of the partial isolates was evaluated using the DPPH radical scavenging assay and mesangial cellular assay. Compounds 2, rel-(2α,3β)-7-O-methylcedrusin (4), syringylglycerol-8-O-4-(sinapyl alcohol) ether (5), and (+)-syringaresinol (7) were found to be most active on DPPH assay, whereas compounds 2, 4, 7, (1R,2R,5R,6S)-2-(3,4-dimethoxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, and buddlenol E could significantly inhibit high glucose-stimulated reactive oxygen species production in mesangial cells. The results suggested that E. ferox seed could be considered as an excellent source of natural antioxidants and is useful in the prevention of diabetic nephropathy.     

Vitamin C equivalent antioxidant capacity (VCEAC) of phenolic phytochemicals

To express the antioxidant capacity of plant foods in a more familiar and easily understood manner (equivalent to vitamin C mg/100 g), two stable radical species, ABTS(*)(-) and DPPH(*), commonly used for antioxidant activity measurements, were employed independently to evaluate their efficacies using apple polyphenolic extracts and seven polyphenolic standards including synthetic Trolox. Their antioxidant activities were expressed as vitamin C equivalent antioxidant capacity (VCEAC) in mg/100 g apple or mg/100 mL of the reference chemical compounds in 10 and 30 min using the ABTS(*)(-) and DPPH(*) scavenging assays, respectively. The antioxidant capacity of Gala apples and seven phenolic standards, determined by both ABTS(*)(-) and DPPH(*) scavenging assays, showed a dose-response of the first-order. Fresh Gala apples had a VCEAC of 205.4 +/- 5.6 mg/100 g using the ABTS assay, and the relative VCEACs of phenolic standards were as follows: gallic acid > quercetin > epicatechin > catechin > vitamin C > rutin > chlorogenic acid > Trolox. With the DPPH radical assay, the VCEAC of fresh Gala apples was 136.0 +/- 6.6 mg/100 g, and the relative VCEACs of seven phenolic standards were, in decreasing order, as follows: gallic acid > quercetin > epicatechin > catechin > or = vitamin C > Trolox > rutin > chlorogenic acid. Because the ABTS assay can be used in both organic and aqueous solvent systems, employs a specific absorbance at a wavelength remote from the visible region, and requires a short reaction time, it is a more desirable method than the DPPH assay. Therefore, it is recommended that antioxidant capacity be expressed as vitamin C mg/100 g equivalent (VCEAC) using the ABTS assay.