Edwin N. Frankel

The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants

The activity of antioxidants in foods and biological systems is dependent on a multitude of factors, including the colloidal properties of the substrates, the conditions and stages of oxidation and the localisation of antioxidants in different phases. When testing natural antioxidants in vitro, it is therefore important to consider the system composition, the type of oxidisable substrate, the mode of accelerating oxidation, the methods to assess oxidation and how to quantify antioxidant activity. Antioxidant effectiveness is also determined by the heterogeneity and heterophasic nature of the system, the type of lipid substrate, including its physicochemical state and degree of unsaturation, the types of initiators, notably transition metals, other components and their possible interaction. For this reason there cannot be a short-cut approach to determining antioxidant activity. Each evaluation should be carried out under various conditions of oxidation, using several methods to measure different products of oxidation. Because most natural antioxidants and phytochemicals are multifunctional, a reliable antioxidant protocol requires the measurement of more than one property relevant to either foods or biological systems. Several recent studies on natural phytochemical compounds produced conflicting results because non-specific one-dimensional methods were used to evaluate antioxidant activity. There is a great need to standardise antioxidant testing to minimise the present chaos in the methodologies used to evaluate antioxidants. Several methods that are more specific should be used to obtain chemical information that can be related directly to oxidative deterioration of food and biological systems.

Inhibition of in vitro human LDL oxidation by phenolic antioxidants from grapes and wines

Current research suggests that wine contains substances that may reduce the mortality rate from coronary diseases. The oxidation of low-density lipoprotein (LDL) is thought to be a key step in the development of atherosclerosis. Phenolic fractions of a Petite Syrah wine were evaluated for their antioxidant activity in inhibiting LDL oxidation in vitro. The more active fractions contained components of the catechin family. The catechin oligomers and the procyanidin dimers (B 2 , B 3 , B 4 , B 6 , B 8 ) and trimers (C 1 , C 2 ) were extracted, isolated and purified from grapes seeds. These compounds were tested for their inhibition of LDL oxidation, along with other monomeric wine phenolics. The procyanidin dimers B 2 and B 8 , and trimer C 1 , and the monomers catechin, epicatechin and myricetin had the highest antioxidant activity. The procyanidin dimers B 3 , B 4 and C 2 and the monomers gallic acid, quercetin, caffeic acid, and rutin, and a group of compounds that included the dimer B 6 , ellagic acid, sinapic acid, cyanidin had lower antioxidant activity and α-tocopherol had the least activity. Thus, the numerous phenolic compounds found in wine are potent antioxidants in inhibiting LDL oxidation in vitro.

Antioxidants in lipid foods and their impact on food quality

Abstract This paper reviews the antioxidant properties of tocopherols and ascorbic acid in edible oils, and the impact of interfacial phenomena on their activities in emulsions and the effects of edible oil processing. Tocopherols are the most important natural antioxidants found in vegetable oil-derived foods. These antioxidants can interrupt lipid autoxidation by interfering with either the chain propagation or the decomposition processes. α-Tocopherol at high concentrations inhibits hydroperoxide decomposition but promotes hydroperoxide formation. The effect of antioxidants in inhibiting hydroperoxide decomposition may thus be critical in preserving food quality by reducing rancidity due to aldehyde formation. Ascorbic acid can regenerate α-tocopherol, inactivate metal initiators and reduce hydroperoxides. The activity of natural antioxidants is greatly affected by complex interfacial phenomena in emulsions and multi-component foods. In an oil-in-water emulsion system, the lipophilic antioxidants α-tocopherol and ascorbyl palmitate are more effective than in bulk oil, while the opposite trend is found for the hydrophilic antioxidants Trolox and ascorbic acid. The methodology to evaluate natural antioxidants must be carefully interpreted depending on whether oxidation is carried out in bulk oils or in emulsions, and what method is used to measure lipid oxidation.

Antioxidant interactions of catechin, cyanidin, caffeic acid, quercetin, and ellagic acid on human LDL oxidation

Abstract Flavonoids and phenolic acids are currently believed to exert cardioprotective effects in humans via their ability to inhibit oxidation of low-density lipoprotein (LDL). The influence of chemical structure on antioxidant activity of catechin, quercetin, cyanidin, caffeic acid, and ellagic acid was evaluated by measuring inhibition of copper-catalysed human LDL oxidation in vitro . The five plant phenols investigated all possess a similar o -dihydroxy moiety. The order of antioxidant activity was catechin > cyanidin ≈ caffeic acid > quercetin > ellagic acid. The observed differences in activities are discussed in terms of structural dissimilarities of the compounds. Potential synergistic or antagonistic effects between catechin, cyanidin, caffeic acid, quercetin, and ellagic acid were investigated by measuring the antioxidant activities on LDL of 20 different combinations of two/three of these phenols. All the antioxidant effects were additive except for combinations including ellagic acid with catechin, where ellagic acid exerted a significant antagonistic effect. It is proposed that the mechanism behind this antagonistic interaction is due to hydrogen bonding between carbonyls in ellagic acid and o -dihydroxyl groups in catechin.

Evaluation of Antioxidant Activity of Rosemary Extracts, Carnosol and Carnosic Acid in Bulk Vegetable Oils and Fish Oil and Their Emulsions

The antioxidant effectiveness of two rosemary extracts, carnosol and carnosic acid, was significantly influenced by the type of system tested (bulk oils vs oil-in-water emulsions), by the oil substrates, the methods used to follow oxi-dation, and the concentrations of test compounds. The rosemary extracts and compounds effectively inhibited conjugated diene hydroperoxide formation in corn oil, soya bean oil, peanut oil and fish oil, when tested in bulk. Test compounds also inhibited hexanal formation in bulk vegetable oils, and propanal and pentenal formation in bulk fish oils. In contrast, these test compounds were either inactive or promoted oxidation in the corresponding vegetable oil-in-water emulsions. In fish oil emulsions, however, the rosemary compounds inhibited the formation of conjugated diene and pentenal but not that of propanal. Interfacial phenomena may explain why the hydrophilic rosemary antioxidants afford more protection in the bulk oil systems by being oriented in the air–oil interface, and less protection in the oil-in-water emulsion systems by partitioning into the water phase.

How to standardize the multiplicity of methods to evaluate natural antioxidants.

A great multiplicity of methods has been used to evaluate the activity of natural antioxidants by using different techniques of inducing and catalyzing oxidation and measuring the end point of oxidation for foods and biological systems. Antioxidant in vitro protocols for foods should be based on analyses at relatively low levels of oxidation under mild conditions and on the formation and decomposition of hydroperoxides. For antioxidant in vivo protocols, widely different methods have been used to test the biological protective activity of phenolic compounds. Unfortunately, many of these protocols have been based on questionable methodology to accurately measure oxidative damage and to assess relevant changes in biological targets. Many studies testing the ex vivo activity of phenolic compounds to inhibit human low-density lilpoprotein (LDL) oxidation have been difficult to evaluate because of the structural complexity of LDL particles and because a multitude of markers of oxidative damage have been used. Although studies with animal models of atherosclerosis have demonstrated the antioxidant effect of phenolic compounds in delaying the progress of this disease, human clinical trials of antioxidants have reported inconsistent and mixed results. Complex mixtures of plant polyphenols have been shown to be absorbed to varying degrees as metabolites in the intestine, but little is known about their interactions, bioavailability, and their in vivo antioxidant activity. Several metabolites identified in human plasma after consuming flavonoids need to be tested for possible nonantioxidant activities. More research and better-designed human studies are required to clarify the complex questions of bioavailability of polyphenols and the factors affecting their in vivo activities. Until we know what relevant in vivo activities to measure, any claims on the biological and health protective effects of natural polyphenolic compounds in our diet are premature.

Chemistry of Extra Virgin Olive Oil: Adulteration, Oxidative Stability, and Antioxidants

Much analytical work has been published on the chemistry of extra virgin olive oil (EVOO) as a basis for the detection and quantitative analyses of the type and amount of adulteration with cheaper vegetable oils and deodorized olive oils. The analysis and authentication of EVOO represent very challenging analytical chemical problems. A significant amount of literature on EVOO adulteration has depended on sophisticated statistical approaches that require analyses of large numbers of samples. More effort is needed to exploit reliable chemical and instrumental methods that may not require so much statistical interpretation. Large assortments of methods have been used to determine lipid oxidation and oxidative stability and to evaluate the activity of the complex mixtures of phenolic antioxidants found in EVOO. More reliable chemical methods are required in this field to obviate excessive dependence on rapid antiradical methods that provide no information on the protective properties of antioxidants. The extensive literature on olive oil sensory tests, using many descriptors varying in different countries, should be supplemented by more precise gas chromatographic analyses of volatile compounds influencing the odor and flavors of EVOO.

Effect of natural antioxidants in virgin olive oil on oxidative stability of refined, bleached, and deodorized olive oil

The factors influencing the oxidative stability of different commercial olive oils were evaluated. Comparisons were made of (i) the oxidative stability of commercial olive oils with that of a refined, bleached, and deodorized (RBD) olive oil, and (ii) the antioxidant activity of a mixture of phenolic compounds extracted from virgin olive oil with that of pure compounds andα-tocopherol added to RBD olive oil. The progress of oxidation at 60°C was followed by measuring both the formation (peroxide value, PV) and the decomposition (hexanal and volatiles) of hydroperoxides. The trends in antioxidant activity were different according to whether PV or hexanal were measured. Although the virgin olive oils contained higher levels of phenolic compounds than did the refined and RBD oils, their oxidative stability was significantly decreased by their high initial PV. Phenolic compounds extracted from virgin olive oils increased the oxidative stability of RBD olive oil. On the basis of PV, the phenol extract had the best antioxidant activity at 50 ppm, as gallic acid equivalents, but on the basis of hexanal formation, better antioxidant activity was observed at 100 and 200 ppm.α-Tocopherol behaved as a prooxidant at high concentrations (>250 ppm) on the basis of PV, but was more effective than the other antioxidants in inhibiting hexanal formation in RBD olive oil.o-Diphenols (caffeic acid) and, to a lesser extent, substitutedo-diphenols (ferulic and vanillic acids), showed better antioxidant activity than monophenols (p- ando-coumaric), based on both PV and hexanal formation. This study emphasizes the need to measure at least two oxidation parameters to better evaluate antioxidants and the oxidative stability of olive oils. The antioxidant effectiveness of phenolic compounds in virgin olive oils can be significantly diminished in oils if their initial PV are too high.

Formation of headspace volatiles by thermal decomposition of oxidized fish oilsvs. oxidized vegetable oils

To understand the reasons for differences in oxidative stability among edible oils, the temperature dependence was investigated for the development of volatile lipid oxidation products in fish oils and in vegetable oils. A rapid headspace capillary gas chromatographic method was developed to determine volatile oxidation products of omega-6 (n-6) polyunsaturated fats (pentane and hexanal) and omega-3 (n-3) polyunsaturated fats (propanal) at different decomposition temperatures. Headspace gas chromatographic analyses of partially oxidized menhaden, bonita and sardine oils could be performed at 40°C, whereas soybean, canola, safflower, high-oleic sunflower and high-oleic safflower oils required temperatures greater than 100°C. Volatile formation by thermal decomposition of oxidized oils had lower apparent activation energies in fish oils than in vegetable oils, and significantly higher apparent activation energies in high-oleic oils than in polyunsaturated oils. The activation energy data on headspace volatiles provided another dimension toward a better understanding of the thermal stability of flavor precursors in unsaturated fish and vegetable oils.

Rapid headspace gas chromatography of hexanal as a measure of lipid peroxidation in biological samples.

A rapid, sensitive and convenient capillary gas chromatographic-headspace method was developed to determine hexanal as an important volatile decomposition product of hydroperoxides formed from n−6 polyunsaturated fatty acids in rat liver samples. Total volatiles were also determined as a measure of overall lipid peroxidation. Samples of headspace taken from sealed serum bottles incubated at 37°C were injected into a gas chromatograph. It was possible to make 15 determinations per hour. This method is convenient because no special sample manipulations are necessary. The addition of 0.5 mM ascorbic acid prior to gas chromatographic, analysis significantly increased hexanal production. The applicability of the method was demonstrated in studies of the effect of iron in the presence or absence of hydroperoxides of methyl linoleate and methyl linolenate andtert-butyl hydroperoxide on rat liver homogenates, slices and microsomes. A rapid silica cartridge chromatographic procedure was used to purify hydroperoxides from autoxidized methyl linoleate and methyl linolenate, and hydroperoxy epidioxides (cyclic peroxides) from autoxidized methyl linolenate in 20–40 mg quantities. The hydroperoxides and hydroperoxy epidioxides of methyl linolenate were, effective inducers n−6 polyunsaturated fatty acid peroxidation in liver homogenates. Hexanal and thiobarbituric acid-reacting substances were signficantly correlated in liver homogenates and microsomes but not in slices. This specific method for hexanal, a known product of peroxidation of n−6 polyunsaturated fatty acids, can be used as a good measure of lipid peroxidation.