Antioxidant Measurement and Applications: An Overview

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1 Chapter 1 Antioxidant Measurement and Applications: An Overview Downloaded via on December 9, 2018 at 23:57:09 (UTC). See for options on how to legitimately share published articles. Fereidoon Shahidi 1 and Chi-Tang Ho 2 1 Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1Β 3X9, Canada 2 Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ Antioxidants are added to fats, oils and fatty foods to prevent their oxidative deterioration. Antioxidants also protect the integrity of cellular structures and macromolecules from damage due to free radicals. Carotenoids and phenolic compounds are major dietary antioxidants. Because of the importance of antioxidant potential in foods and dietary supplements, it is necessary to have good and reliable methods for measuring antioxidant activity. Various in vitro and in vivo methods for assessing antioxidant activities are discussed. Introduction Autoxidation occurs widely in fats, oils and lipid-containing foods, and causes food quality deterioration with concomitant generation of loss of nutrients, unpleasant flavors, and even potentially toxic substances. Among the methods for preventing oxidation, addition of antioxidants is the most effective, convenient and economical one (7). Antioxidants are also important to human health. Antioxidant protection from damage due to free radicals is vital for the integrity of cellular structures American Chemical Society

2 and macromolecules (2,3). As we age, the system which utilizes antioxidants for our defense and protection also declines, and can be aggravated by the presence of various oxidative stresses caused by pollution, exercise, smoke exposure and radiation. This defense system operates through a series complex networks between vitamins C and E, carotenoids, zinc, copper, selenium, and magnesiumdependent enzyme antioxidants as well as other phytonutrients, which together perform highly involved recycling and regeneration reactions to optimize free radical protection. Deficiencies in any of the mentioned necessary components could potentially lead to a severely compromised defense system (4,5). Owing to the incomplete efficiency of our endogeneous defense systems, dietary antioxidants are needed to overcome the oxidative damage (5). 3 Dietary Antioxidants Carotenoids and phenolic compounds are major dietary antioxidants. Both of these groups of compounds contain hundred of members (6). Carotenoids are natural, fat-soluble pigments that provide bright coloration to plants and animals and act as antioxidants, which include the possibility of providing vitamin A activity. One defining characteristic of carotenoids is the chemical structure of what is often considered their backbone molecule, a 40- carbon polyene chain. The polyene backbone consists of a pattern of conjugated double bonds, which allows the carotenoids to take up excess energy from other molecules (7). This characteristic may be responsible for the antioxidant activity seen in biological carotenoids. In addition to scavenging free radicals, other health benefits related to this observed antioxidative activity include protection from sunburn and inhibition of the development of certain types of cancers (<S- 10). β-carotene is the most common carotenoid in food and the most potent of the provitamin A carotenoids. β-carotene is believed to have antioxidant activity. It has been shown to exhibit radical- trapping behavior only at partial pressure of oxygen significantly less than that in normal air (77). Such low oxygen partial pressures are found in most tissues under physiological conditions. At higher oxygen pressure it loses the antioxidant activity and shows a pro-oxidant effect (77). Lutein and its isomer, zeaxanthin, are yellow pigments that belong to the classes of non-provitamin A carotenoids. Unlike other carotenoids, hydroxyl groups are substituted on the ring structures at the end of the conjugated double bond chains of lutein and zeaxanthin. Lutein is naturally occurring and found predominantly in dark green, leafy vegetables such as spinach and kale. Zeaxanthin gives corn its yellow color. There is a growing body of evidence (including in vivo, in vitro and epidemiological studies) supporting the claim that

4 lutein and zeaxanthin contribute to health and delay age related macular degeneration of the eyes and, to a lesser extent, cancers and heart diseases (72-14).

3 4 lutein and zeaxanthin contribute to health and delay age related macular degeneration of the eyes and, to a lesser extent, cancers and heart diseases (72-14). The evidence for the role of lutein and zeaxanthin in eye health is very strong because of their exclusive presence in the ocular tissues and the high numbers of epidemiological studies that have been conducted. With a high accumulation in the macula of the eye, the area of highest visual acuity, lutein and zeaxanthin are proposed to have the ability to filter out harmful blue light, while at the same time acting as antioxidants to quench potentially damaging reactive oxygen species (ROS; 75). Lycopene, a carotenoid found in tomatoes, watermelon, papaya, apricot, orange and pink grapefruit, exhibits antioxidant and anticancer activities (76). About 80% of lycopene is consumed through tomatoes and tomato-related products. Numerous studies have suggested reduced risk of prostate cancer from the consumption of processed tomato products (77). Although, these beneficial health effects of lycopene are thought to be due to its antioxidant properties, evidence is accumulating to suggest other mechanisms of action like hormone and immune system modulation (18). Lycopene is the most abundant carotenoid in human plasma, which may imply its elevated level of importance in the human body compared with other carotenoids, such as β-carotene and lutein (19). Phenolic compounds occurring commonly in foods may be classified into simple phenols, hydroxybenzoic and hydrocinnamic acid derivatives, flavonoids, stibenes, lignans and hydrolysable as well as condensed tannins (20). Phenolics in foods may occur in the free, esterified, etherified and insoluble-bound forms. The most abundant phenolic compounds in food are flavonoids. Flavonoids are present in edible fruits, leafy vegetables, roots, tuber bulbs, herbs, spices, legumes, tea, coffee, cocoa, chocolate and red wine. They can be classified into seven groups: flavones, flavanones, flavonols, flavanonols, isoflavones, flavanols (catechins) and anthocyanidins. In general, the leaves, flowers and fruits or the plant itself contain flavonoid glycosides, woody tissues contain aglycones, and seeds may contain both (20). As a result of their ubiquity in plants, flavonoids are an integral part of the human diet. It is estimated that the average American's daily intake of the consumption of flavonols is close to mg/day (21). Almost all flavonoids possess several common biological and chemical properties: (a) antioxidant activity, (b) the ability to scavenge ROS, (c) the ability to scavenge electrophiles, (d) the ability to inhibit nitrosation, (e) the ability to chelate metals, (f) the potential to produce hydrogen peroxide in the presence of certain metals and (g) the capability to modulate certain cellular enzyme activities (22). It appears that diets rich in flavonoids may protect against cardiovascular diseases, neurodegenerative disorders and some forms of cancer.

5 Antioxidant Measurement The need to measure antioxidant activity is well documented; these are carried out for meaningful comparison of foods or commercial products and for provision of quality

4 5 Antioxidant Measurement The need to measure antioxidant activity is well documented; these are carried out for meaningful comparison of foods or commercial products and for provision of quality standards for regulatory issues and health claims (23). There are numerous methods for measureing antioxidant activity; these may be classified into two categories. The first category measures the ability of antioxidants in inhibiting oxidation in a model system by monitoring the associated changes using physical, chemical or instrumental means. Radical scavenging assays include methods based on hydrogen atom transfer (HAT) or electron transfer (ET) mechanisms. Oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP) and crocin bleaching assays are the major methods that measure HAT while Trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant antioxidant power (FRAP) and 2,2- diphenyl-l-picrglhydrozyl (DPPH) assays represent ET-based methods. Extensive relevant reviews are provided in the literature (23-25) as well as in this volume (26). It is interesting to note that DPPH radical is used to test antioxidant activity by its ability to abstract hydrogen atoms from polyphenols (27). Another stable radical, tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl radical, was developed as a good sensor to test the activity of polyphenols measuring their capacity to participate in electron transfer reactions (27). Antioxidant activity of a compound can also be evaluated in different cell culture assay for the prevention of carcinogenesis. Because oxidative DNA damage is considered to be relevant in carcinogenic process, one can evaluate the possible anticarcinogenic effect of dietary antioxidants by determining their effect on 12-Otetradecanoylphorbol 13-acetate (TPA)-inducing ROS generation, H scavenging, H induced apoptosis, xanthine oxidase activity, and lipopolysaccharide (LPS)-inducing NO generation. Details are discussed in this volume (28). Bioavailability of Dietary Antioxidants Bioavailability is the degree to which a drug, nutrient, dietary supplement or nutraceutical is available to the body. Bioavailability is influenced by how much of a substance is absorbed and circulated in the human body. Problems with bioavailability arise when trying to elucidate exactly what dose brings about the desired physiological response. Manufactures producing 500 mg vitamin C pills cannot claim that 500 mg of the vitamin are taken in and used by the body.

5 6 There are variations between different human subjects and their uptake of certain food-based chemicals. This means that two people taking the same dose could actually absorb different amounts of the same compound. One might only see the effect of 200 mg of a 500 mg pill while the other might see the effect of 100 mg. This disparity is due to variability in absorption, distribution, metabolism and excretion of the bioactives abbreviated as ADME. In addition, it is sometimes the case that the ingested chemical is not the final bioactive agent. Many molecules enter the digestive system in one form only to be broken down into smaller metabolites that interact through absorption. Science has yet to identify many of these breakdown reactions sufficiently to understand how these reactions affect the bioavailability of compounds. Studies have shown that 11% of caffeic acid and trace amounts of chlorogenic acid, present in coffee, are found in urine indicating that they failed to be fully absorbed through the gut barrier (29). The exact fate of the remaining caffeic acid is unknown. This is the problem of bioavailability and it translates to nearly every aspect of nutraceuticals. While it is difficult to say for sure how much of a compound will be taken in and used by an organism, there is some strong evidence showing how low some of the uptakes can be. In a study using chlorogenic acid, only 1.7% of it was recovered unchanged in the urine. In these cases, the colon could play a larger role in the metabolism of polyphenols (30). Polyphenols reaching the colon can be broken into smaller metabolites by colonic microbiota, the bacteria found in the colon (30). These bacteria are able to break down phenols, allowing absorption of these smaller metabolites by the kidneys, liver and other organs. Later, these smaller metabolites may find their way into the urine. Without a clear understanding of their chemical nature, we cannot screen for them in the urine. The same study also concluded that a large part of the ingested polyphenols will probably never enter the peripheral circulation as smaller metabolites (30). The problem of reduced antioxidant activity found in smaller metabolites of larger parent compounds increases the uncertainty of bioavailability studies. An organism is not likely to absorb an entire dose, and it is likely that the compound will be broken down into smaller, unidentified compounds. Further studies are warranted to identify these compounds. Considering these variables, it is very difficult to predict the total effect of an antioxidant on host cells. References 1. Wanasundara, P.K.J.P.D.; Shahidi, F. In Bailey's Industrial Oil & Fat Products, Sixth Edition, Vol. 1. Shahidi, F. (Ed.), Wiley Interscience, Hoboken, NJ, 2005, pp De la Fuente, M. Eur. J. Clin. Nutr. 2002, 56 Suppl. 3, S5-S8.

6 3. Grimble, R.F. New Horiz. 1994, 2, Calder, P.C.; Kew, S. Br. J. Nutr. 2002, 88 (Suppl. 2), S165-S Pietta, P.G. J. Nat. Prod. 2000, 63, Packer, L.; Hiramatsu, M.; Yoshikawa, T. Antioxidant Food Supplements in Human Health, Academic Press, San Diego, CA, Palozza, P.; Krinsly, Ν. I. Meth. Enzym. 1992, 213, Ito, Y.; Wakai, Κ.; Suzuki, Κ.; Tamakoshi, Α.; Seki, Ν.; Ando,M.; Nishino, Y.; Kondo, T.; Watanabe, Y.; Ozasa, K.; Ohno, Y. Cancer Sci. Jan. 2003, 94, Burrie, B.J. Nutr. Res. 1997, 17, Ziegler, R.G. J. Nutr. 1989, 19, Burton, G.W.; Ingold,K.U.Science 1984, 224, Tsao, R.; Wang, M.; Deng, Ζ. Chapter in this volume. 13. Chew, E.Y. Curr. Opin. Ophthalmol. 1995, 6, Snodderly, D.M. Am. J. Clin. Nutr. 1995, 62, 1448s-1461s. 15. Stahl, W. Dev. Ophthalmol. 2005, 38, Stahl, W.; Sies, H. Arch. Biochem. Biophys. 1996, 336, Clinton, S.K.; Emenhiser, C.; Schwartz, S.J.; Bostwick, D.G.; Williams, A.W.; Moore, B.J.; Erdman, J.W. Jr. Cancer Epidem. Biomarkers Prev. 1996, 5, Roa, A.V.; Agarwal, S. Nutr Res. 1999, 19, Sies, H.; Stahl, W. Proc. Soc. Exp. Biol. Med. 1998, 218, Shahidi, F.; Ho, C.-T. Phenolic Compounds in Foods and Natural Health Products, American Chemical Society, Washington, D.C., Manach, C.; Scalbert, Α.; Morand, C.; Ramesy, C.; Jimenez, L. Am. J. Clin. Nutr. 2004, 79, Huang, M.T.; Ferraro, T. In Phenolic Compounds in Food and Their Effects on Health II: Antioxidants & Cancer Prevention. Huang, M.T.; Ho, C.-T.; Lee, C.Y. (Eds.), American Chemical Society, Washington, D.C., 1992, pp Prior, R.L.; Wu, X.; Schaich, K. J. Agric. Food Chem. 2005, 53, Schlesier, M.; Harwat, M.; Böhm, V.; Bitsch, R. Free Rad. Res. 2002, 36, Shahidi, F.; Zhong. Y. In Bailey's Industrial Oil and Fat Products, Vol. 1, Shahidi, F., (Eds.), John Wiley & Sons, Ltd., Hoboken, NJ, 2005, pp Shahidi, F.; Zhong, Y. Chapter in this volume. 27. Jiménez, Α.; Selga, Α.; Torres, J.L.; Juliá, L. Org. Lett. 2004, 6, Pan, M.H.; Lai, C.S.; Ho, C.-T. Chapter in this volume. 29. Olthof, M.R.; Hollman, P.C.; Buijsman, M.N.; van Amelsvoort, J.M.; Katan, M.B. J. Nutr. 2003, 133, Olthof, M.R.; Hollman, P.C.; Katan, M.B. J. Nutr. 2001, 131,

Eligibility The NCSF online quizzes are open to any currently certified fitness professional, 18 years or older.

Eligibility The NCSF online quizzes are open to any currently certified fitness professional, 18 years or older. Deadlines Course completion deadlines correspond with the NCSF Certified Professionals certification