Antioxidant capacity of BO-653, 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran, and uric acid as evaluated by ORAC method and inhibition of lipid peroxidation

The role of radical-scavenging antioxidant against oxidative stress has received much attention. The antioxidant capacity has been assessed by various methods. Above all, oxygen radical absorbance capacity (ORAC) has been frequently employed [Prior et.al., J. Agric. Food Chem.2005, 53, 4290]. In the present study, the antioxidant capacity of 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653) and uric acid was assessed by ORAC method using pyranine as a reference probe and compared with that against lipid peroxidation of human plasma. It was found that BO-653 was assessed to be much less potent than uric acid by ORAC method, whereas BO-653 exerted much higher antioxidant activity than uric acid against plasma lipid peroxidation. The reason for such discrepancy is discussed. The results suggest that ORAC method is suitable for the assessment of free radical scavenging capacity, but not for the assessment of antioxidant capacity against lipid peroxidation in plasma.     

Evaluation of plasma antioxidant activity in rats given excess EGCg with reference to endogenous antioxidants concentrations and assay methods

The contribution of (?)-epigallocatechin gallate (EGCg) intake to in vivo antioxidant activity is unclear, even with respect to plasma. In this study, we examined how administration of EGCg contributes to plasma antioxidant activity, relative to its concentration, endogenous antioxidants, and assay methods, namely oxygen radical absorbance capacity (ORAC) and ferric reducing/antioxidant power (FRAP). Administration of EGCg (500?mg/kg) to rats increased plasma EGCg (4μmol/L as free form) and ascorbic acid (1.7-fold), as well as ORAC (1.2-fold) and FRAP (3-fold) values. The increase in plasma ascorbic acid following EGCg administration was accompanied by its relocation from the adrenal glands and lymphocytes into plasma, and was related to the increase in FRAP. Plasma deproteinization and assays in plasma model solutions revealed that protein levels significantly contributed to ORAC values, where <3?μmol/L EGCg in the presence of protein exhibited minimal antioxidant activity, as measured by both FRAP and ORAC. As the concentration of plasma ascorbic acid was not influenced by deproteinization, differences in FRAP values with and without deproteinization were estimated to determine the contribution of enhanced ascorbic acid attributable to EGCg administration. These results will help to understand the points that should be considered when evaluating EGCg antioxidant activity in plasma.     

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.     

A device for the semiautomatic determination of oxygen-radical absorbance capacity

The oxygen-radical absorbance capacity (ORAC) assay has become a standard method to quantify the antioxidative properties of phytonutrients in fruit and vegetable extracts. However, it is usually not possible to determine directly the contribution of specific phytonutrients to the total ORAC value. Separation of the components in the plant extracts by HPLC followed by ORAC analyses of the column fractions might permit the determination of free radical-scavenging profiles. The accurate determination of ORAC values may require 1 to 2 h/sample. Considering the number of samples that would be generated by a single HPLC separation, a device was constructed which permits up to 45 simultaneous ORAC analyses. Varying degrees of automation were included in the design. Furthermore, since the assay has a Q(10) for peroxyl radical-scavenging of about 3, elevation of the assay temperature from the standard 37 to 47 degrees C significantly reduced the assay times. Relatively simple modifications would allow the apparatus to be used in a variety of time-dependent fluorescence and absorbance assays.     

基于ORAC评价多酚类化合物抗氧化活性

采用氧自由基清除能力(ORAC)方法考察20种多酚类化合物的抗氧化活性。结果表明,该方法具有较优的线性关系(R2=0.997）;检测限(LOD）和定量限(LOQ）为0.5～3.1 μmol·L-1,精密度＜18%,准确度91%～105%。比较20种多酚类化合物抗氧化活性,对羟基苯甲酸类化合物中,鞣花酸和没食子酸具有较强的抗氧化活性;对羟基肉桂酸类以咖啡酸及其衍生物抗氧化活性最高;在类黄酮组分中,黄烷-3-醇表现出优良的抗氧化特性,黄酮醇次之。ORAC可作为评价多酚类化合物抗氧化的简便、高效的标准化检测方法。     

Oxygen radical absorbance capacity of the phenolic compounds in plant extracts fractionated by high-performance liquid chromatography

The oxygen radical absorbance capacity (ORAC) assay has been used to quantify the antioxidative properties of phytonutrients in fruit and vegetable extracts. Using aqueous methanol extracts of tea and spinach as a model systems, separation of the components in the extracts by HPLC followed by semiautomatic ORAC analysis of the column fractions permitted the determination of peroxyl-radical-scavenging profiles, demonstrating the relative abilities of the individual extract components to scavenge peroxyl radicals. ORAC values for up to 80 HPLC fractions were measured, confirming the major contribution of epigallocatechin gallate in the peroxyl radical scavenging of green tea extracts. Although the flavonoids in spinach extracts provided resistance to peroxyl radicals, components that did not bind to the HPLC column and simple phenolic compounds may also be important contributors to the total ORAC activity of spinach leaf extracts. Application of these procedures to plants believed to provide certain human health benefits by reducing free radicals may allow the identification and characterization of the specific components responsible for the free-radical-scavenging activities.     

Identification and quantification of glycoside flavonoids in the energy crop Albizia julibrissin

Oxygen radical absorbance capacity (ORAC) values showed that methanolic extracts of Albizia julibrissin foliage displayed antioxidant activity. High performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques were utilized in the identification of the compounds. The analysis confirmed the presence of three compounds in A. julibrissin foliage methanolic extract: an unknown quercetin derivative with mass of 610 Da, hyperoside (quercetin-3-O-galactoside), and quercitrin (quercetin-3-O-rhamnoside). Fast performance liquid chromatography (FPLC) was employed to fractionate the crude A. julibrissin foliage methanolic extract into its individual flavonoid components. The flavonoids were quantified in terms of mass and their respective contribution to the overall ORAC value. Quercetin glycosides accounted for 2.0% of total foliage.     

Antioxidant capacity of human blood plasma and human urine: simultaneous evaluation of the ORAC index and ascorbic acid concentration employing pyrogallol red as probe

The oxygen radical absorbance capacity (ORAC) methodology has been employed to estimate the antioxidant capacity of human blood plasma and human urine using pyrogallol red (ORAC-PGR) as target molecule. Uric acid, reduced glutathione, human serum albumin, and ascorbic acid (ASC) inhibited the consumption of pyrogallol red, but only ASC generated an induction time. Human blood plasma and human urine protected efficiently pyrogallol red. In these assays, both biological fluids generated neat induction times that were removed by ascorbate oxidase. From these results, ORAC-PGR method could be proposed as a simple alternative to evaluate an ORAC index and, simultaneously, to estimate the concentration of ascorbic acid in human blood plasma or human urine.     

Niche overlap and diet composition of three sympatric coastal dolphin species in the southwest Atlantic Ocean

Sympatric species are expected to differ in ecological requirements to minimize niche overlap and avoid competition. Here we assess the trophic interactions among three coexisting dolphin species from southern Brazil: the franciscana dolphin (Pontoporia blainvillei), the Guiana dolphin (Sotalia guianensis), and the Lahille's bottlenose dolphin (Tursiops truncatus gephyreus). We evaluated temporal variation in carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values of bone collagen to examine potential dietary shifts resulting from increased fishing activity over the past three decades. We estimated the degree of niche overlap among these species and the contribution of potential prey sources to their diet. δ¹⁵N values were consistent among species and across years, while δ¹³C values increased for Guiana dolphins and decreased for bottlenose dolphins, suggesting changes in diet and/or foraging habitats through time. The similar δ¹³C and δ¹⁵N values and the high niche overlap between Guiana and bottlenose dolphins indicate that these species are primarily feeding on demersal prey. The franciscana diet is primarily composed of pelagic prey, resulting in a lower niche overlap in comparison with the other dolphin species. Our study provides further information about the foraging ecology of this unique dolphin community in southern Brazil with implications for its management and conservation.     

A novel and simple ORAC methodology based on the interaction of Pyrogallol Red with peroxyl radicals

Oxygen radicals absorbance capacities (ORAC) indexes are frequently employed to characterize the radical trapping capacity of pure compounds and their complex mixtures. A drawback of ORAC values obtained using phycoerythrin, fluorescein (FL) or c-phycocyanin as targets, makes it possible to conclude that for very reactive compounds they are much more related to stoichiometric factors than to the reactivity of the tested compound. In the present paper, we propose a simple methodology, based on the bleaching of Pyrogallol Red (PGR) absorbance that provides ORAC indexes that are almost exclusively determined by the reactivity of the tested compounds. This difference is due to the high reactivity of PGR and the high concentrations of this compound employed in the experiments.     

Application of the LUminometric Methylation Assay to ecological species: tissue quality requirements and a survey of DNA methylation levels in animals

The LUminometric Methylation Assay (LUMA) measures global DNA methylation. LUMA depends on digestion of DNA with methyl‐sensitive and methyl‐insensitive restriction enzymes, followed by pyrosequencing. Until recently, LUMA has been principally used for biomedical research. Here, we use chickens as a model to investigate sample quality issues relating to LUMA and then apply the method to ecological species. First, we assessed the effect of tissue storage conditions on DNA methylation values. This is an important consideration for ecological species because samples are not always ideally preserved and LUMA is sensitive to poor DNA quality. We found that good quality LUMA data could be obtained from chicken liver and brain tissues stored at 21 °C for at least 2 and 12 h, respectively. Longer storage times introduced nonspecific peaks to pyrograms which were associated with reduced DNA methylation. Repeatedly, freezing and thawing the tissues did not affect LUMA data. Second, we measured DNA methylation in 12 species representing five animal classes: amphibians (African and Western clawed frog), reptiles (green anole lizard), fish (yellow perch, goldfish, lake trout), mammals (American mink, polar bear, short‐beaked common dolphin, Atlantic white‐sided dolphin) and birds (chicken, Japanese quail). We saw a pattern of high DNA methylation in fish (84–87%), and intermediate levels in mammals (68–72%) and birds (52–71%). This pattern corresponds well with previous measures of DNA methylation generated by HPLC. Our data represent the first CpG methylation values to be reported in several species and provide a basis for studying patterns of epigenetic inheritance in an ecological context.     

In vivo total antioxidant capacity: comparison of different analytical methods

Several methods have been developed to measure the total antioxidant capacity of a biological sample. The use of peroxyl or hydroxyl radicals as pro-oxidants in the oxygen radical absorbance capacity (ORAC) assay makes it different and unique from the assays that involve oxidants that are not necessarily pro-oxidants. An improvement in quantitation is achieved in the ORAC assay by taking the reaction between substrate and free radicals to completion and using an area-under-curve technique for quantitation compared to the assays that measure a lag phase. The interpretation of the changes in plasma or serum antioxidant capacity becomes complicated by the different methods used in detecting these changes. The interpretation also depends upon the conditions under which the antioxidant capacity is determined because the measurement reflects outcomes in a dynamic system. An increased antioxidant capacity in plasma or serum may not necessarily be a desirable condition if it reflects a response to increased oxidative stress. Similarly, a decrease in plasma or serum antioxidant capacity may not necessarily be an undesirable condition if the measurement reflects decreased production of reactive species. Because of these complications, no single measurement of antioxidant status is going to be sufficient, but a "battery" of measurements, many of which will be described in Forum articles, will be necessary to adequately assess oxidative stress in biological systems.     

Synthesis,oxygen radical absorbance capacity,and tyrosinase inhibitory activity of glycosides of resveratrol,pterostilbene, and pinostilbene

The stilbene compound resveratrol was glycosylated to give its 4′-O-β-D-glucoside as the major product in addition to its 3-O-β-D-glucoside by a plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. This enzyme transformed pterostilbene to its 4′-O-β-D-glucoside, and converted pinostilbene to its 4′-O-β-D-glucoside as a major product and its 3-O-β-D-glucoside as a minor product. An analysis of antioxidant capacity showed that the above stilbene glycosides had lower oxygen radical absorbance capacity (ORAC) values than those of the corresponding stilbene aglycones. The 3-O-β-D-glucoside of resveratrol showed the highest ORAC value among the stilbene glycosides tested, and pinostilbene had the highest value among the stilbene compounds. The tyrosinase inhibitory activities of the stilbene aglycones were improved by glycosylation; the stilbene glycosides had higher activities than the stilbene aglycones. Resveratrol 3-O-β-D-glucoside had the highest tyrosinase inhibitory activity among the stilbene compounds tested.     

Response of plasma and red cell glutathione reductase of several mammalian species and domestic chickens to flavin adenine dinucleotide.

Glutathione reductase (GR) activity was determined in the plasma and red cells from a number of species (human, horse, cattle, sheep, chicken, rabbit, rat). The species with the highest plasma activity (horse and cattle) had the lowest red cell activity. Unlike red cell GR, plasma GR was not significantly stimulated by the addition of flavin adenine dinucleotide (FAD). In species with high plasma GR activity the determination of whole blood FAD activity coefficients may not be a good index of riboflavin status.     

Acute Effects of Dietary Glycemic Index on Antioxidant Capacity in a Nutrient‐controlled Feeding Study

Oxidative stress, caused by an imbalance between antioxidant capacity and reactive oxygen species, may be an early event in a metabolic cascade elicited by a high glycemic index (GI) diet, ultimately increasing the risk for cardiovascular disease and diabetes. We conducted a feeding study to evaluate the acute effects of low‐GI compared with high‐GI diets on oxidative stress and cardiovascular disease risk factors. The crossover study comprised two 10‐day in‐patient admissions to a clinical research center. For the admissions, 12 overweight or obese (BMI: 27–45 kg/m²) male subjects aged 18–35 years consumed low‐GI or high‐GI diets controlled for potentially confounding nutrients. On day 7, after an overnight fast and then during a 5‐h postprandial period, we assessed total antioxidant capacity (total and perchloric acid (PCA) protein‐precipitated plasma oxygen radical absorbance capacity (ORAC) assay) and oxidative stress status (urinary F_2α‐isoprostanes (F₂IP)). On day 10, we measured cardiovascular disease risk factors. Under fasting conditions, total antioxidant capacity was significantly higher during the low‐GI vs. high‐GI diet based on total ORAC (11,736 ± 668 vs. 10,381 ± 612 µmol Trolox equivalents/l, P = 0.002) and PCA‐ORAC (1,276 ± 96 vs. 1,210 ± 96 µmol Trolox equivalents/l, P = 0.02). Area under the postprandial response curve also differed significantly between the two diets for total ORAC and PCA‐ORAC. No diet effects were observed for the other variables. Enhancement in plasma total antioxidant capacity occurs within 1 week on a low‐GI diet, before changes in other risk factors, raising the possibility that this phenomenon may mediate, at least in part, the previously reported effects of GI on health.     

Glutamate dehydrogenase and glutamine synthetase activity in some organs of ruminants and monogastric animals.

A comparative study of glutamate dehydrogenase (GLDH 1.4.1.2) and glutamine synthetase (GS 6.3.1.2.) activity in liver, kidney and spleen homogenates from cattle, sheep, pigs and chickens showed that chicken liver contained on an average 3.5%, pig liver 8.3% and bovine liver 45.6% of the glutamate dehydrogenase activity present in sheep liver. Relatively low trace activity was found in the spleen and kidneys, except for the renal cortex of cattle (32% of activity in the liver). GS activity was the highest in chicken liver; in pigs it amounted to 33.40%, in cattle to 24.2% and in sheep to 19.7% of this activity. No marked interspecies differences were found in the values in the kidneys and spleen. It can be concluded from the results that the relatively high GLDH activity in the liver of ruminants compared with pigs and chicken is associated with the greater ability of ruminants to utilize ammonia. The higher GS activity and lower GLDH activity in chicken liver can be attributed to higher uric acid synthesis from ammonia via glutamine and purine bases and the lower ability of birds to utilize ammonia for protein synthesis. The presence of alanine dehydrogenase was not demonstrated in chicken liver, where the maximum oxidation of NADH after the addition to pyruvate and ammonia substrate was found.     

Natriuretic peptides in cetaceans: identification, molecular characterization and changes in plasma concentration after landing

Dolphins are aquatic animals free from gravity, and this may have imposed significant changes in their cardiovascular status and its hormonal regulation compared with terrestrial animals. This study molecularly characterized two major cardiovascular hormones, atrial and B-type natriuretic peptides (ANP and BNP) and measured their changes in dolphin plasma concentrations in relation to the cardiovascular status of the animal. We initially identified ANP and BNP in three species of dolphins (Lagenorhynchus obliquidens, Phocoenoides dalli and Tursiops truncatus). ANP precursors are highly conserved in most mammals, but dolphin BNP precursors were more variable. In molecular phylogenetic analyses, dolphin ANP and BNP precursors grouped with those of artiodactyls, particularly to the camel peptides. The chromatographic characterization of tissue and plasma molecular forms using specific radioimmunoassays showed that the predominant ANP and BNP in the atrium are prohormone and mature peptide, respectively, whereas mature ANP and BNP are circulating in the dolphin blood. A mass spectrometric analysis showed that atrial BNP consists of 26 amino acids, rather than the 32-amino-acid form detected in other mammals. Finally, changes in plasma ANP and BNP concentrations were examined in captive bottlenose dolphins (Tursiops truncatus) after their pool was drained. Plasma ANP and BNP concentrations did not change after landing, unlike terrestrial mammals. Plasma angiotensin II and cortisol concentrations did not change either, showing minor stress after landing. Since landed dolphins show a different cardiovascular status on land than terrestrial mammals, plasma ANP and BNP concentrations seem to reflect the cardiovascular status characteristic of dolphins.     

Antioxidant properties of prunes (Prunus domestica L.) and their constituents

Prunes contain large amounts of phenolics and show high antioxidant activity. The aim of this study is to clarify the contents of caffeoylquinic acid (CQA) isomers, and to estimate the contribution of these isomers to the antioxidant activity of prunes. Furthermore, structural elucidation and evaluation of antioxidant activity of prune components were also performed. CQA isomers in prunes were quantified by HPLC analysis, and it has become apparent that prunes contain relatively high amount of 4-O-caffeoylquinic acid. The contribution of CQA isomers to the antioxidant activity of prunes was revealed to be 28.4% on the basis of oxygen radical absorbance capacity (ORAC); hence, it was indicated that residual ORAC is dependent on unknown antioxidant components. Total 28 compounds were isolated and their structures were elucidated by NMR and MS analyses. Four abscisic acid related compounds, a chromanon, and a bipyrrole were novel. Each CQA isomer in prunes showed high antioxidant activities when measured by the oil stability index (OSI) method, O2- scavenging activity, and ORAC. Other isolated compounds such as hydroxycinnamic acids, benzoic acids, coumarins, lignans, and flavonoid showed high ORAC values. Furthermore, a novel chromanon indicated a remarkable synergistic effect on ORAC of CQA isomers.     

Aldehyde dehydrogenase activity in blood from various vertebrates

1. Aldehyde dehydrogenase activity was determined in whole blood samples from 17 selected vertebrates of 5 classes, using 3,4-dihydroxyphenylacetaldehyde (the aldehyde derived from dopamine) as substrate. 2. Aldehyde dehydrogenase activity in blood was widely but unevenly distributed among the species studied. 3. Mean aldehyde dehydrogenase activities in the range of 40-140 nmol/min.ml blood (measured at 37 degrees C, pH 8.8) were found in blood from man, monkey, rabbit, guinea pig and mouse (C57BL and NMRI strains), with the highest activity in rabbit blood. 4. Much lower aldehyde dehydrogenase activities (0.5-7.5 nmol/min.ml blood) were found in blood from Sprague-Dawley and Wistar rat, dog, cat, horse, pig, chicken, caiman, frog and rainbow trout, whereas the activities in blood from DBA mouse, cow, sheep and crucian carp were close to the detection limit.     

The Orexin System in the Enteric Nervous System of the Bottlenose Dolphin (Tursiops truncatus)

This study provides a general approach to the presence and possible role of orexins and their receptors in the gut (three gastric chambers and intestine) of confined environment bottlenose dolphin. The expression of prepro-orexin, orexin A and B and orexin 1 and 2 receptors were investigated by single immunostaining and western blot analysis. The co-localization of vasoactive intestinal peptide and orexin 1 receptor in the enteric nervous system was examined by double immunostaining. Also, orexin A concentration were measured in plasma samples to assess the possible diurnal variation of the plasma level of peptide in this species. Our results showed that the orexin system is widely distributed in bottlenose dolphin enteric nervous system of the all gastrointestinal tract examined. They are very peculiar and partially differs from that of terrestrial mammals. Orexin peptides and prepro-orexin were expressed in the main stomach, pyloric stomach and proximal intestine; while orexin receptors were expressed in the all examined tracts, with the exception of main stomach where found no evidence of orexin 2 receptor. Co-localization of vasoactive intestinal peptide and orexin 1 receptor were more evident in the pyloric stomach and proximal intestine. These data could suggest a possible role of orexin system on the contractility of bottlenose dolphin gastrointestinal districts. Finally, in agreement with several reports, bottlenose dolphin orexin A plasma level was higher in the morning during fasting. Our results emphasize some common features between bottlenose dolphin and terrestrial mammals. Certainly, further functional investigations may help to better explain the role of the orexin system in the energy balance of bottlenose dolphin and the complex interaction between feeding and digestive physiology.