The antiradical activities of various antioxidants were determined using the free radical, 2,2-Diphenyl-1-picrylhydrazyl (DPPH*). In its radical form. DPPH* has an absorption band at 515 nm which dissappears upon reduction by an antiradical compound. Twenty compounds were reacted with the DPPH* and shown to follow one of three possible reaction kinetic types. Ascorbic acid, isoascorbic acid and isoeugenol reacted quickly with the DPPH* reaching a steady state immediately. Rosmarinic acid and δ-tocopherol reacted a little slower and reached a steady state within 30 min. The remaining compounds reacted more progressively with the DPPH* reaching a steady state from 1 to 6 h. Caffeic acid, gentisic acid and gallic acid showed the highest antiradical activities with a stoichiometry of 4 to 6 reduced DPPH* molecules per molecule of antioxidant. Vanillin, phenol, γ-resorcylic acid and vanillic acid were found to be poor antiradical compounds. The stoichiometry for the other 13 phenolic compounds varied from one to three reduced DPPH* molecules per molecule of antioxidant. Possible mechanisms are proposed to explain the experimental results.

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Use of a Free Radical Method to Evaluate Antioxidant Activity

Free radicals and antioxidants are widely discussed in the clinical and nutritional literature. Antioxidants are needed to prevent the formation and oppose the actions of reactive oxygen and nitrogen species, which are generated in vivo and cause damage to DNA, lipids, proteins, and other biomolecules. Endogenous antioxidant defenses (superoxide dismutases, H2O2-removing enzymes, metal binding proteins) are inadequate to prevent damage completely, so diet-derived antioxidants are important in maintaining health. Many dietary compounds have been suggested to be important antioxidants: The evidence for a key role of vitamins E and C is strong, but that for carotenoids and related plant pigments is weaker. Interest is also growing in the role of plant phenolics, especially flavonoids. Some antioxidants can exert prooxidant effects in vitro, but their physiological relevance is uncertain. Experimental approaches to the optimization of antioxidant nutrient intake are proposed.

Antioxidants in human health and disease

This article is a review of the fundamental chemistry of the tocopherols and tocotrienols relevant to their antioxidant action. Despite the general agreement that α-tocopherol is the most efficient antioxidant and vitamin E homologuein vivo, there was always a considerable discrepancy in its “absolute” and “relative” antioxidant effectivenessin vitro, especially when compared to γ-tocopherol. Many chemical, physical, biochemical, physicochemical, and other factors seem responsible for the observed discrepancy between the relative antioxidant potencies of the tocopherolsin vivo andin vitro. This paper aims at highlighting some possible reasons for the observed differences between the tocopherols (α-, β-, γ-, and δ-) in relation to their interactions with the important chemical species involved in lipid peroxidation, specifically trace metal ions, singlet oxygen, nitrogen oxides, and antioxidant synergists. Although literature reports related to the chemistry of the tocotrienols are quite meager, they also were included in the discussion in virtue of their structural and functional resemblance to the tocopherols.

The chemistry and antioxidant properties of tocopherols and tocotrienols

Free radicals and other reactive oxygen species (ROS) are constantly formed in the human body. Free-radical mechanisms have been implicated in the pathology of several human diseases, including cancer, atherosclerosis, malaria, and rheumatoid arthritis and neurodegenerative diseases. For example, the superoxide radical (O2·−) and hydrogen peroxide (H2O2) are known to be generated in the brain and nervous system in vivo, and several areas of the human brain are rich in iron, which appears to be easily mobilizable in a form that can stimulate free-radical reactions. Antioxidant defenses to remove O2·− and H2O2 exist. Superoxide dismutases (SOD) remove O2·− by greatly accelerating its conversion to H2O2. Catalases in peroxisomes convert H2O2 into water and O2 and help to dispose of H2O2 generated by the action of the oxidase enzymes that are located in these organelles. Other important H2O2-removing enzymes in human cells are the glutathione peroxidases. When produced in excess, ROS can cause tissue injury. However, tissue injury can itself cause ROS generation (e.g., by causing activation of phagocytes or releasing transition metal ions from damaged cells), which may (or may not, depending on the situation) contribute to a worsening of the injury. Assessment of oxidative damage to biomolecules by means of emerging technologies based on products of oxidative damage to DNA (e.g., 8-hydroxydeoxyguanosine), lipids (e.g., isoprostanes), and proteins (altered amino acids) would not only advance our understanding of the underlying mechanisms but also facilitate supplementation and intervention studies designed and conducted to test antioxidant efficacy in human health and disease.

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Free radicals, oxidative stress, and antioxidants in human health and disease

Phenolics in Food and Nutraceuticals is the first single-source compendium of essential information concerning food phenolics. This unique book reports the classification and nomenclature of phenolics, their occurrence in food and nutraceuticals, chemistry and applications, and nutritional and health effects. In addition, it describes antioxidant activity of phenolics in food and nutraceuticals as well as methods for analysis and quantification. Each chapter concludes with an extensive bibliography for further reading. Food scientists, nutritionists, chemists, biochemists, and health professionals will find this book valuable.

Phenolics in food and nutraceuticals

Scope of the Paper This paper cites the paradoxical actions of antioxidants in processed foods and in living biological systems. Essentially, it is a recognition of patterns of contradictory behavior. Limitations of space prevent detailed treatment of the paradoxes, their mechanistic basis, or citation of any more than one or two illustrative references. It is hoped that these will lead the interested reader to further substantiation of the paradoxes cited.

Paradoxical behavior of antioxidants in food and biological systems.

In this paper, I shall discuss the important trends of the past seven years in applications of antioxidants in foods. The nature and course of lipid autoxidation and antioxidant action have been treated in a previous paper of this Symposium (Symposium, 1979), as has the effect of water activity. Other papers will deal with the biological effects of antioxidant use, including antimicrobial action. Natural antioxidants will also be treated elsewhere. Therefore, I shall discuss primarily the use of the commonly added synthetic antioxidants (Fig. 1) and four naturally occurring ones — α-tocopherol and ascorbic (Fig. 2), citric and phosphoric acids, which common usage seems to segregate from the “natural” antioxidants. In addition, I shall handle many secondary antioxygenic compounds and treatments, such as oxygen exclusion or scavenging, browning antioxidants, and products produced by fermentation, smoking, nitrite curing, and hydrolysis of vegetable protein.

Recent Trends in Food Applications of Antioxidants

Ease of use and oxidative stability of fish and flaxseed oils were improved by encapsulation and antioxidants. The oils, containing various levels of vitamin E, rosmarinic acid, ethylenediaminetetraacetic acid and citric acid, plus carnosic acid and ascorbyl palmitate for fish oil, were encapsulated and tested weekly for 8 weeks by a front-face ratiometric fluorometric method applied to both powders and volatile-reacting polyamide plates. Regression analysis of fluorescence ratio versus time was used to calculate induction times and oxidation rate; significance of inhibition was additionally determined at each pull. Encapsulation increased the stability of both oils, most significantly for flaxseed oil. Encapsulated fish oil stability was increased by all antioxidants, most significantly by carnosic acid. Encapsulated unsupplemented flaxseed oil was more stable than fish oil, but its stability was diminished by most antioxidants. Sensory properties of high-carbohydrate performance bars supplemented with the encapsulated oils were comparable with those of an unsupplemented control.

PRACTICAL APPLICATIONS
Fortification of foods with omega-3 fatty acid-containing fish and flaxseed oils would improve nutritional quality through provision of docosahexaenoic acid, eicosapentaenoic acid, and α-linolenic acid. The oils in their native state not only lack functionality (i.e., are not suitable for addition to dry foods), but also are not shelf-stable because of their susceptibility to oxidation. Incorporation of the oils into a high-load powder through encapsulation produces an easily added ingredient for fortification; incorporation of effective antioxidants at optimal levels ensures oxidative shelf stability. Analysis of the encapsulated oils over several weeks and at high storage temperatures can indicate best antioxidants and concentrations to incorporate into long shelf-life food.

Effect of various antioxidants, antioxidant levels, and encapsulation on the stability of fish and flaxseed oils: assessment by fluorometric analysis

Inhibition of pyrazine formation by natural antioxidants and the foods containing them was measured in a microwaved glucose/glycine model system. Inhibition of lipid oxidation by the same materials was assayed in both bulk and emulsion systems. Pyrazines were determined by solid-phase micro extraction followed by GC. Lipid oxidation volatiles were assayed by polyamide fluorescence produced by either a bulk oil display or a hematin- or 2,2′-azobis-(2-amidino=propane) dihydrochloride-accelerated lecithin or fish oil emulsion. It was shown that (i) the inhibition of pyrazine formation depends on high concentrations of water-soluble antioxidants; (ii) such antioxidants occur naturally in some foods and are usually polyphenols; (iii) during pyrazine inhibition, oxidized polyphenols show enhanced nonfluorescing browning similar to enzymic browning products; (iv) monophenols, which structurally cannot form quinone polymers on oxidation, inhibit pyrazines with less browning; (v) during the final pyrazine-forming phase of the Maillard reaction, polyphenolics and reducing agents such as glutathione and ascorbic acid are partially consumed with some nutritional loss; (vi) fruit powders of grape seed, grape skin, and red wine are highly pyrazine-inhibitory, steeped blueberry strongly so, but plum purees are moderately pro-pyrazine, and freeze-dried vegetables strongly pro-pyrazine; and (vii) black and green tea infusions are highly inhibitory, whereas spices have mixed effects.

Modification of maillard browning in a microwaved glucose/glycine model system by water-soluble natural antioxidants and foods containing them

A non-destructive technique for examining the oxidative status of packaged, dry or intermediate moisture foods. In a preferred embodiment, the technique involves packaging the foods in an assembly comprising a container or pouch formed from a pair of sheets heat-sealed to one another along their peripheries. The sheets may each be a trilaminate comprising a polyethylene terephthalate outer layer, an aluminum foil middle layer and a polyolefin inner layer. One of the sheets is provided with a cut-out portion which serves as a light transmissive window. A device comprising a strip of polyethylene terephthalate which is coated on one side with a polyamide coating is mounted across the light transmissive window, with the polyamide coating facing in towards the interior of the pouch. The food within the pouch is stored within about 2 cm of the polyamide coating during oxidation. To test the oxidative status of the food, a beam of about 420 nm light is used to illuminate the polyamide coating through strip of polyethylene terephthalate, and the resultant fluorescence is observed through the strip of polyethylene terephthalate at a wavelength of approximately 464 nm.

William L. Porter

Papers

Paradoxical behavior of antioxidants in food and biological systems.

Recent Trends in Food Applications of Antioxidants

Effect of various antioxidants, antioxidant levels, and encapsulation on the stability of fish and flaxseed oils: assessment by fluorometric analysis

Modification of maillard browning in a microwaved glucose/glycine model system by water-soluble natural antioxidants and foods containing them

Technique for determining the oxidative status of packaged dry or intermediate moisture foods