Assessing antioxidant and prooxidant activities of phenolic compounds

Methods for determining primary antioxidant activity were evaluated. A beta-carotene bleaching method and a free radical method using 2, 2-diphenyl-1-picrylhydrazyl (DPPH(*)) were modified to rapidly test samples for potential antioxidant activity. Malonaldehyde production in a linoleic acid emulsion system assayed by an HPLC method was also used to determine antioxidant and prooxidant activities initiated by a metal catalyst (Cu(2+)). All methods were used to assess activity of selected phenolic compounds including several anthocyanidins/anthocyanins and selected berry extracts. Most phenolic compounds had prooxidant activity at low concentrations, unlike synthetic antioxidants (BHA and BHT). Compounds with similar structures exhibited comparable trends in antioxidant activity. Antioxidant activity usually increased with an increase in the number of hydroxyl groups and a decrease in glycosylation. The antioxidant activity of many phenolic compounds and extracts was comparable to those of synthetic antioxidants using the beta-carotene bleaching and HPLC methods.     

Reactivity of epicatechin in aqueous glycine and glucose maillard reaction models: quenching of C2, C3, and C4 sugar fragments

Mechanisms of how epicatechin alters the pathways of the Maillard reaction were investigated. Carbon-13 and nitrogen-15 labeling studies were utilized to define the reactivity of epicatechin with glucose, glycine, and/or reaction products in an aqueous model (pH 7, 125 degrees C for 30 min) via GC, GC/MS and HPLC/MS analysis. Quantification of the volatile reaction product isotopomers by GC/MS from a 1:1 labeled to unlabeled glucose (carbohydrate module labeling technique) plus glycine model system indicated the formation of 2,3-butanedione and acetol were primarily formed via intact C4 and C3 sugar fragments, whereas pyrazine, methylpyrazine, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, and cyclotene were primarily formed via intact C2/C2, C2/C3, C3/C3, C3/C3, and C3/C3 sugar fragment pairs, respectively. The formation of these seven compounds was also reported by GC analysis to be dramatically inhibited when epicatechin was added to the glucose/glycine model system (observed 9-113-fold reduction). HPLC/MS analysis of both the glucose-labeled and glycine-labeled model systems with and without epicatechin indicated that epicatechin reacted directly with C2, C3, and C4 sugar fragments, while epicatechin did not report any direct reactivity with glycine. In conclusion, the quenching of sugar fragmentation products via epicatechin was correlated with the observed inhibition on volatile compound formation when epicatechin was added to a glucose/glycine aqueous reaction model system.     

Individual and combined antioxidant effects of seven phenolic agents in human erythrocyte membrane ghosts and phosphatidylcholine liposome systems: importance of the partition coefficient

Antioxidant activities of seven phenolic agents against Fe(2+)-induced lipid oxidation were compared with alpha-tocopherol, beta-carotene, and vitamin C in human erythrocyte membrane ghosts and liposome systems. The antioxidant activity of five test flavonoids followed the order catechin > epicatechin > rutin > quercetin > myricetin in both systems (p < 0.05), which was negatively correlated with their partition coefficients. The antioxidant interaction of these phenolic agents with alpha-tocopherol, beta-carotene, or vitamin C in inhibiting Fe(2+)-induced lipid oxidation was examined. Synergistic effects were present in the combinations of alpha-tocopherol plus caffeic acid, catechin, or epicatechin as well as in all combinations of vitamin C plus phenolic antioxidants. On the basis of the stronger individual and combined effects present in caffeic acid, catechin, and epicatechin, the application of these three phenolic agents with or without alpha-tocopherol, beta-carotene, and vitamin C may provide stronger protective benefits against lipid oxidation, which may be helpful for oxidation-related diseases prevention.     

Chemical composition of the volatile extract and antioxidant activities of the volatile and nonvolatile extracts of Egyptian corn silk (Zea mays L.)

A total of 36 compounds, which comprised 99.4% of the extract, were identified by gas chromatography and mass spectrometry (GC-MS) in the volatile dichloromethane extract obtained from Egyptian corn silk. The main constituents of the volatile extract were cis-alpha-terpineol (24.22%), 6,11-oxidoacor-4-ene (18.06%), citronellol (16.18%), trans-pinocamphone (5.86%), eugenol (4.37%), neo-iso-3-thujanol (2.59%), and cis-sabinene hydrate (2.28%). Dried Egyptian corn silk was also directly extracted with petroleum ether, ethanol, and water. All extracts from solvent extraction and the volatile extract described above exhibited clear antioxidant activities at levels of 50-400 microg/mL in the 2,2-diphenyl-1-picrylhydrazyl (DPPH)/linoleic acid assay. The ethanol extract inhibited DPPH activity by 84% at a level of 400 microg/mL. All samples tested via the beta-carotene bleaching assay also exhibited satisfactory antioxidant activity with clear dose responses. This study indicates that corn silk could be used to produce novel natural antioxidants as well as a flavoring agent in various food products.     

Effect of high-pressure-moderate-temperature processing on the volatile profile of milk

The effects of high hydrostatic pressure on volatile generation in milk were investigated in this study. Raw milk samples were treated under different pressures (482, 586, and 620 MPa), temperatures (25 and 60 degrees C), and holding times (1, 3, and 5 min). Samples submitted to heat treatments alone (25, 60, and 80 degrees C for 1, 3, and 5 min) were used for comparison. Trace volatile sulfur compounds were analyzed using solid-phase microextraction (SPME) and gas chromatography (GC) with pulsed-flame photometric detection (PFPD), whereas the rest of the volatile compounds were analyzed using SPME-GC with flame ionization detection (FID). Multivariate analysis of variance (MANOVA) and principal component analysis (PCA) were used to study the effect of pressure, temperature, and time on volatile generation. Relative concentration increases of 27 selected volatile compounds were compared to an untreated sample. It was found that pressure, temperature, and time, as well as their interactions, all had significant effects (P < 0.001) on volatile generation in milk. Pressure and time effects were significant at 60 degrees C, whereas their effects were almost negligible at 25 degrees C. The PCA plot indicated that the volatile generation of pressure-heated samples at 60 degrees C was different from that of heated-alone samples. Heat treatment tended to promote the formation of methanethiol, hydrogen sulfide, methyl ketones, and aldehydes, whereas high-pressure treatment favored the formation of hydrogen sulfide and aldehydes.     

Stabilization of Lipids in a Biodegradable Zein‐Oleate Film by Incorporation of Antioxidants

Zein has been studied extensively for use as an edible, biodegradable food film. To function as such, as much as 40–50% oleic acid is added as a plasticizing agent. Over the course of time, these films became discolored and developed off‐odors. It was speculated that this was due to oxidation of incorporated oleic acid. This study sought to reduce oxidative changes in film lipids by incorporation of antioxidants. Incorporation of 4,000 ppm of BHA into the films eliminated peroxide formation during accelerated UV storage conditions. Gas chromatography and headspace (GC‐HS) chromatography revealed that total volatile components of UV‐treated control films were 9× higher than fresh control volatiles. Comparison of the profiles led to the demonstration of major differences in peaks of a specific region, suggesting the presence of oxidation products in that region. Differences in UV‐treated BHA films and UV‐treated control films appeared in the same specific region of the chromatogram. Solid‐phase microextraction analysis of volatile components of the various film components and films also demonstrated a major increase in peaks associated with oxidation due to UV treatment and reduction of oxidative products in UV‐treated BHA films. Antioxidant treatment was thus found to protect the films against oxidative deterioration.     

Lipoperoxidation and cyclooxygenases 1 and 2 inhibitory compounds from Iryanthera juruensis

Plants from Iryanthera genus have been traditionally used as food supplements by South American Indians. The MeOH extract of leaves of Iryanthera juruensis, one of the plants endemic to the Amazon region and consumed in Brazil, and the hexane extract from its seeds inhibited lipid peroxidation (LPO) and cyclooxygenase (COX-1 and -2)) enzymes in in vitro assays. Further analyses of these extracts yielded 5-deoxyflavones (1-5) from the leaf extract and sargachromenol (6), sargaquinoic acid (7), a novel juruenolic acid (8), omega-arylalkanoic acids (9a-c), and the lignan guaiacin (10) from the seed extract. Compounds 3-5 inhibited LPO by 86%, 77%, and 88% at 10 ppm, respectively, and compounds 6 and 9a-c showed inhibition at 76% and 78% at 100 ppm, respectively. However, compounds 7 and 8 were inactive and lignan 10 exhibited LPO inhibitory activity by 99% at 100 ppm compared to commercial antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and vitamin E. The flavones 1-5 also inhibited COX-1 and -2 enzymes by 50-65% at 100 ppm. Compound 6 showed high but nonselective inhibition of COX-1 and COX-2 enzymes, when compared to aspirin and Celebrex, a nonsteroidal anti-inflammatory drug (NSAID). Compounds 7 and 10 inhibited COX-1 by 60% and 65% and COX-2 by 37% and 18%, respectively, whereas compounds 8 and 9a-c showed little or no activity against these enzymes.     

Real-time monitoring of thermal flavor generation in skim milk powder using atmospheric pressure chemical ionization mass spectrometry

The feasibility of monitoring volatile flavor compounds formed by thermal treatment of skimmed milk powder in real time by atmospheric pressure chemical ionization mass spectrometry (APCIMS) was established. Skim milk powder samples were heated isothermally (70 to 120 degrees C) at different moisture contents (2.2 and 12.7 g water/100 g dry solids). Headspace was sampled and analyzed continuously in full scan mode (30-180 amu) by APCIMS. The identity of the volatile compounds monitored by APCIMS was confirmed by coupled GC-EI-APCIMS. The concentration measured by the APCIMS was the net effect of three processes, namely formation of the compound, partition from the skim milk powder into the gas phase, and dilution due to the headspace sampling method used. Preliminary experiments established that the technique could follow the effects of heating temperature and moisture content on the formation of selected compounds from skim milk powder.     

Effect of antioxidants on oxidative stability of edible fats and oils: thermogravimetric analysis

Thermogravimetric analysis was used to determine the oxidative stability of various edible oils (olive oil, milkfat) and triacylglycerides (triolein, trilinolein), while the effect of natural (alpha-tocopherol, ascorbic acid) and synthetic antioxidants (butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tertiary butyl hydroquinone were evaluated by addition to trilinolein. Oil resistance to oxidation was obtained by measuring the increase in sample weight due to the uptake of molecular oxygen, the temperature at maximum sample weight, and the temperature at the onset of oxidation. When comparing sample weight increase, trilinolein proved to be oxidatively less stable than triolein, olive oil, and milk fat, while triolein was less stable than olive oil and milk fat. Olive oil showed significantly higher stability than milkfat when comparing the temperature at the onset of oxidation. When comparing effectiveness of antioxidants, a combination of 0.01% BHA and 0.01% BHT increased trilinolein stability the most.     

Ionizing radiation and antioxidants affect volatile sulfur compounds, lipid oxidation, and color of ready-to-eat Turkey bologna

Bologna was processed from ground turkey breast meats containing one of four antioxidant treatments (none, rosemary extract, sodium erythorbate, and sodium nitrite). After it was cooked, the bologna was sliced, sealed in gas impermeable bags, exposed to 0, 1.5, and 3.0 kGy gamma-radiation, and then stored at 5 degrees C for up to 8 weeks. Thiobarbuturic acid reactive substances (TBARS), color, and volatile sulfur compounds were measured every 2 weeks during storage. Irradiation had no consistent effect on TBARS values. The rosemary extract and sodium nitrite inhibited, while erythorbate increased, TBARS values, independent of radiation dose or storage time. Irradiation promoted redness and reduced yellowness of the control (no antioxidant) bologna at weeks 0 and 2. The use of nitrite and rosemary extract inhibited the changes in color due to irradiation. Several volatile sulfur compounds (hydrogen sulfide, methanethiol, methyl sulfide, and dimethyl disulfide), measured using a pulsed flame photometric detector, increased with radiation dose. However, none of the antioxidants had any substantial effect on volatile sulfur compounds induced by irradiation. Our results suggest that antioxidants did not consistently affect irradiation-induced volatile sulfur compounds of turkey bologna although they did significantly impact color and lipid oxidation.     

Chemical composition and antioxidant activity of phenolic compounds from wild and cultivated Sclerocarya birrea(Anacardiaceae) leaves

A quantitative study of the phenolic constituents of wild and cultivated leaves of Sclerocarya birrea(Anacardiaceae) was carried out by HPLC-UV/PDA and LC-MS. Phytochemical analysis of the methanol extract of wild plants led to the isolation of one new flavonol glycoside, quercetin 3-O-alpha-l-(5' '-galloyl)-arabinofuranoside (1), and eight known phenolic compounds; two epicatechin derivatives were also isolated from the same extract of the cultivated species. The antioxidant activity of all isolated compounds was determined by measuring free radical scavenging effects using the Trolox equivalent antioxidant capacity assay and the coupled oxidation of beta-carotene and linoleic acid (autoxidation assay).     

Evaluation of extracts from Gevuina avellana hulls as antioxidants

The antioxidant activity of the extracts from Gevuina avellana hulls was evaluated and compared with that of BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole), using the beta-carotene bleaching assay, the accelerated oxidation of crude soybean oil, and the 2,2-diphenyl-beta-picrylhydrazyl (DPPH) radical scavenging method. Solvents of different polarity were used to obtain the extracts. Both the extraction yield and the antioxidant activity were strongly dependent on the solvent. The ethanol and diethyl ether soluble fractions were the most active with the beta-carotene assay. Ethanol and methanol extracts were the most active in hydrogen radical scavenging activity. Water and methanol inhibited more efficiently the oxidation of soybean oil at 70 and 80 degrees C, respectively. As a general trend, increased antioxidant activity was observed for increased extract concentration. Except the acetone extracts, all were stable after 6 months storage at 4 degrees C. The ethanol solubles from G. avellana hulls present antioxidant activity similar to that of synthetic antioxidants and to other reported residual agroindustrial materials.     

Antioxidizing potency of phenol compounds in olive oil mill wastewater

The antioxidizing potency of phenol compounds contained in olive oil mill wastewater (OOMWW) has been elucidated. Commercially available phenol standards at varying concentrations and the Rancimat oxidation test have been used. Refined purified olive oil was utilized as an oxidation lipid substrate. Synthetic antioxidants, such as 2,3-tert-butyl-4-hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxytoluene (BHT), l-ascorbic acid, and gallates (commonly used as food preservatives), and other known chemicals endowed with antioxidizing properties have been employed as reference compounds. The OOMWW phenol compounds have been classified into different groups depending on their antioxidizing potency. This was significantly affected by the tested concentrations of the standards. Mixtures of phenol standards and other antioxidants (l-proline, chlorophyll a, chlorophyll b, and alpha-, gamma-, and delta-tocopherol) have also been tested. Many phenol compounds present in OOMWW showed antioxidizing potency higher compared to that of the less safe synthetic antioxidants and could therefore replace these in the industrial preservation of food items. They could also be used in combination with other natural antioxidants (e.g., tocopherols). In fact, some mixtures of antioxidants, owing also to the synergistic phenomena, showed strong antioxidizing potency.     

Formation of methanethiol and dimethyl disulfide in crushed tissues of broccoli florets and their inhibition by freeze-thawing

The formation of methanethiol and dimethyl disulfide in crushed, homogenized, and frozen-thawed tissues of broccoli florets was investigated. These volatile sulfur compounds were produced in crushed florets, but their formation was inhibited in frozen-thawed tissues. Only dimethyl disulfide was formed in homogenized tissues. High pH treatment triggered the release of dimethyl disulfide in frozen-thawed tissues and also enhanced the action of cysteine sulfoxide lyase in all disrupted tissues. Methyl methanethiosulfinate and methyl methanethiosulfonate were not detected in crushed florets; thus, the favored mechanism for the formation of methanethiol and dimethyl disulfide is the chemical disproportionation of methanesulfenic acid. In contrast, the formation of dimethyl disulfide in frozen-thawed and homogenized tissues occurs from the chemical disproportionation of methyl methanethiosulfinate that was detected in these tissues. The inhibition of dimethyl disulfide production during freeze-thawing must be caused by a sudden drop in the pH of the tissue, adherence of dimethyl disulfide on the tissue surfaces, and weakening of the cysteine sulfoxide lyase activity under acidic conditions.     

Selective determination of catechin among phenolic antioxidants with the use of a novel optical fiber reflectance sensor based on indophenol dye formation on nano-sized TiO₂

The optical sensor for "tea catechins" was built by immobilizing 2,2'-(1,4-phenylenedivinylene)bis-8-hydroxyquinoline (PBHQ) on TiO? nanoparticles (NPs). The sensor worked by "indophenol blue" dye formation on PBHQ-immobilized TiO? NPs as a result of p-aminophenol (PAP) autoxidation with dissolved O? at pH 10. Among quercetin, rutin, naringenin, naringin, gallic acid, caffeic acid, ferulic acid, p-coumaric acid, catechin, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, and trolox, only catechin group antioxidants delayed the color formation on NPs, as measured by the reflectance signal at 710 nm. For quantitative analysis, reflectance signal versus time was recorded, and the difference between the areas under curve (ΔAUC) in the presence and absence of catechin was correlated (r = 0.98) to catechin concentration. The selectivity of the sensor for catechins was shown in tea infusions compared to other plant extracts and was ascribed to the nonplanar structure of catechin interfering with the formation of perfectly conjugated indophenol blue on TiO? surface.     

Activity and concentration of polyphenolic antioxidants in apple juice. 3. Stability during storage

Kinetic data are reported describing the stability of various classes of polyphenolic antioxidants in an apple juice enriched in these compounds as a function of storage temperature and oxygen concentration. The most thermally sensitive compounds were the various quercetin glycosides and epicatechin, whereas phloridzin and chlorogenic acid were more stable. The quercetin glycosides showed differences in their stability: quercetin galactoside approximately quercetin rhamnoside > quercetin glucoside/rutinoside > quercetin arabinoside. The effect of the presence of oxygen on the degradation rates was clear for only quercetin and to a lesser extent for epicatechin. Accelerated shelf-life testing of enriched apple juice during 4 days at 80 degrees C showed decreases in the antioxidant activity of 20-40%. The parameters obtained were used to predict the stability at different storage conditions. Calculations showed that polyphenolic antioxidants and antioxidant activity of enriched apple juice will be quite stable at ambient or refrigerated storage conditions up to half a year.     

Isolation and structure determination of new antioxidative ferulic acid glucoside esters from the rhizome of Alpinia speciosa, a Zingiberaceae plant used in Okinawan food culture

An assay-guided isolation gave three antioxidants including two newly identified compounds from the rhizomes of Alpinia speciosa, which is used as an important plant in the food culture of the Okinawa area of Japan. Spectroscopic analysis of the two new compounds revealed them to be new glucoside esters of ferulic acid. The antioxidant activity of the esters was measured using two different methods. Both compounds showed greater activity than that of Trolox in the TLC method; however, one of the compounds showed weaker inhibitory activity than that of Trolox and epicatechin against AMVN-induced methyl linoleate oxidation.     

Structure and function of the oxidation products of polyphenols and identification of potent lipoxygenase inhibitors from Fe-catalyzed oxidation of resveratrol

Polyphenols have recently attracted much attention as potent antioxidants and related bioactive substances. These potent antioxidative polyphenols are very oxidizable due to their chemical properties, and their oxidation products must accumulate in the oxidizing foods when they are contained as the active ingredients. In this investigation, 30 polyphenols and related phenolics were oxidized with oxygen in the presence of a catalytic amount of Fe ions. Piceatannol, catechin, epicatechin, hydroxytyrosol, carnosol, and carnosic acid were oxidized very quickly. Sinapic acid, caffeic acid, chlorogenic acid, rosmarinic acid, gallic acid, propyl gallate, α-tocopherol, quercetin, and nordihydroguaiaretic acid were moderately oxidized. Protocatechuic acid, syringic acid, taxifolin, resveratrol, gentisic acid, secoisolariciresinol, and ellagic acid were oxidized for 19-20 days; however, their oxidation was very slow and did not complete. The other phenolics were not oxidized. The obtained oxidation products were next subjected to a lipoxygenase inhibition assay and the results compared to those of the corresponding phenols. Very interestingly, the oxidation product from resveratrol showed a high inhibitory activity, whereas resveratrol itself had no activity and its oxidation efficiency was low. To clarify the inhibition principle of the oxidation product, an LC-MS analysis was carried out on the oxidation product. The analytical results showed that they are the oligomeric and degraded compounds of resveratrol. Among them, the structures of three dimeric compounds were successfully identified, and their activity data clarified that the closed ring dimers were potent lipoxygenase inhibitors, whereas the opened ring dimer was not. It should be noted that resveratrol had almost no lipoxygenase inhibitory activity, contrary to some researchers' findings.     

On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.)

An on-line HPLC-DPPH screening method for phenolic antioxidants in apple methanol/water (80:20, v/v) extracts was applied. The determination of antioxidants was based on a decrease in absorbance at 515 nm after postcolumn reaction of HPLC-separated antioxidants with the 2,2'-diphenyl-1-picrylhydrazyl radicals (DPPH*). Each of the antioxidants separated by the HPLC column was observed as a negative peak corresponding to its antioxidative activity. The on-line method was applied for quantitative analysis of the antioxidants. A linear dependence of negative peak area on concentration of the reference antioxidants was observed. For validation of the on-line method the limit of detection, LOD (microg/mL), and the limit of quantification, LOQ (microg/mL), of the phenolic compounds were determined. Comparison of the UV and DPPH radical quenching chromatograms with authentic compounds identified catechin, chlorogenic acid, caffeic acid, epicatechin, and phloridzin in the apple cultivars (Lobo, Golden Delicious, and Boskoop), and the distribution of total antioxidant activity was calculated.     

Characterization of phenolic compounds in virgin olive oil and their effect on the formation of carcinogenic/mutagenic heterocyclic amines in a model system

Mutagenic heterocyclic amines (HAs) are formed at low levels during cooking of meat and fish, and some of them are considered to be possible human carcinogens. The formation of HAs may be affected by the presence of synthetic or naturally occurring antioxidants. In the present study the effect of virgin olive oil (VOO) phenolic compounds, identified and quantified by LC-MS, on the formation of HAs in a model system was evaluated. An aqueous solution of creatinine, glucose, and glycine was heated in the presence of two samples of VOO differing only in the composition of phenolic compounds. The addition of VOO to the model system inhibited the formation of 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) by between 30 and 50% compared with the control. Fresh-made olive oil, which contained a high amount of dihydroxyphenylethanol derivatives, inhibited HA formation more than a 1-year-old oil did. The inhibition of HA formation was also verified using phenolic compounds extracted from VOO.