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by Neil H. Mermelstein

Determining Antioxidant Activity

ood and beverage companies are touting the presence of antioxidants in their products in response to consumer interest in the potential health benefits of antioxidants in the diet. For example, Lipton teas carry a logo, AOX, Naturally Protective Antioxidants, POM Wonderful pomegranate juice says its the real Antioxidant Superpower, and Hersheys Nuggets Special Dark Mildly Sweet Chocolate bears a logo stating Natural Source of Flavonol Antioxidants. A variety of in-vitro chemical methods are being used to determine the antioxidant activity of products and ingredients, but questions regarding whether the results have any bearing on effectiveness in the human body are leading to development of additional methods that may be more appropriate for screening potential antioxidant ingredients. vs time are recorded, and the area under the curves with and without addition of an antioxidant is calculated and compared to a standard curve generated using the antioxidant ()-6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid, a water-soluble vitamin E analog trademarked by HoffmanLaRoche as Trolox. TRAP, Total Radical-Trapping Antioxidant Parameter method. This method uses a luminescence spectrometer to measure the fluorescence decay of R-phycoerythrin during a controlled peroxidation reaction. TRAP values are calculated from the length of the lag-phase caused by the antioxidant compared to that of Trolox. TEAC, Trolox Equivalent Antioxidant Capacity method. This method, similar in principle to ORAC, uses a diode-array spectrophotometer to measure the loss of color when an antioxidant is added to the blue-green chromophore ABTS +, 2,2'-azino-bis(3-ethylbenzthiazoline6-sulfonic acid). The antioxidant reduces ABTS + to ABTS, decolorizing it. ABTS + is a stable radical not found in the human body.

Berries are high in antioxidant activity. Photo Matteo De Steffano/iStockphoto

InVitro Methods
The following are some of the most widely used in-vitro methods: ORAC, Oxygen Radical Absorbance Capacity method. When a free-radical generator such as

method is based on the reaction between peroxyl radicals and -keto--methiolbutyric acid (KMBA), which is oxidized to ethylene. Added antioxidant competes with KMBA for the peroxyl radicals, reducing the production of ethylene, which is generally measured by gas chromatography. Syft Technologies Ltd. (www.syft.com) has developed a Selected Ion Flow Tube Mass Spectrometric (SIFT-MS) test that is based on TOSC. PSC, Peroxyl Radical Scavenging Capacity method. This method,

Its clear that theres a group of foods that rank high in antioxidant capacity, but growing conditions, varieties, genetics, etc., can affect the antioxidant capacity measured in vitro.
an azo-initiator compound is added to a fluorescent molecule such as beta-phicoerythrin or fluorescein and heated, the azo-initiator produces peroxyl free radicals, which damage the fluorescent molecule, resulting in the loss of fluorescence. Curves of fluorescence intensity DPPH. This assay measures by spectrophotometer the ability of antioxidants to reduce 2,2- diphenylpicrylhydrazyl (DPPH), another radical not commonly found in biological systems. TOSC, Total Oxyradical Scavenging Capacity method. This also similar to ORAC, is based on the degree of inhibition of dichlorofluorescin oxidation by antioxidants that scavenge peroxyl radicals generated from thermal degradation of 2,2'-azobis(amidinopropane). FRAP, Ferric Reducing/Antioxidant Power method. This method
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Determining Antioxidant Activity continued...

are reduced by polyphenolic antioxidants such as gallic acid and catechin, resulting in a blue solution with maximal absorption at 765 nm. The standard curve is prepared using gallic acid, and results are reported as gallic acid equivalents. the digestive tract, he said. Some are absorbed, while others arent or are otherwise modified. There are enzymes in cells and tissues in the body that protect the body from excess oxidation. In addition, other components from those same foods and their metabolites may influence oxidation/antioxidant status once absorbed. There clearly must be interactions and synergies between antioxidants from foods and antioxidant enzymes in the body that affect the overall antioxidant status of the body, he said. Moreover, we dont know if the antioxidant will act as an antioxidant, act as a prooxidant at higher consumption levels, or have other roles in the body. Many epidemiological studies and clinical trials utilize in-vivo biomarkers of antioxidant stress as surrogate end-point markers, he added, but these markers dont predict or signify health status very well. More effort to directly link changes in antioxidant biomarkers to health outcomes is sorely needed. Ronald L. Prior (priorronaldl@ uams.edu), Research Chemist/ Nutritionist, USDA/ARS Arkansas Childrens Nutrition Center, Little Rock, Ark., said that antioxidant phytochemicals in fruits and vegetables are effective in protecting against free-radical damage in vitro but that additional research is needed on factors affecting their absorption and metabolism. Prior said that different radicals can give different test results, depending on their structure. ORAC uses the peroxyl radical, the most common free radical in the human body. A lot of methods give the same relative ranking of antioxidants, he said, but theres some variation depending on how the radicals react with components in food. Prior and his coworkers developed the automated ORAC method and the ORAC values for foods and beverages that appear in the USDA National Nutrient Database for Standard Reference, the 2007 ORAC Report, and the 2007 USDA Database

Relevance Questioned
Despite wide usage of these chemical antioxidant activity assays, their ability to predict in-vivo activity has not been demonstrated, according to Rui Hai Liu (rl23@cornell.edu), Associate Professor, Dept. of Food Science, Cornell University, Ithaca. Liu chaired a symposium entitled Nutrition Controversies: Moving Beyond Lab Chemical Methods for Antioxidant Assessment as Related to Health Benefits at the 2008 IFT Annual Meeting & Food Expo in New Orleans last July. He said that many of the chemical antioxidant assays being used do not reflect cellular physiological conditions and do not consider the bioavailability, uptake, and metabolism of the antioxidants. Liu said that the protocols of the chemical antioxidant activity assays often do not include the appropriate biological substrates to be protected, relevant types of oxidants encountered, the partitioning of compounds between water and lipid phases, and the influence of interfacial behavior. Biological systems are much more complex than the simple chemical mixtures employed, he said, and antioxidant compounds may operate via multiple mechanisms. As a result, the various in-vitro assays provide differing results. John W. Erdman Jr. (jwerdman@ illinois.edu), Professor of Food Science and Human Nutrition, University of Illinois at Urbana, said that in-vitro tests might be useful in comparing different foods for their antioxidant capacity but that the chemical antioxidant capacity of a food may not be relevant to what happens in the human body. We dont know if the antioxidant compounds from those foods survive

Cornell University professor Rui Hai Liu (seated) discusses with his assistant the cellculture method he and his coworkers have developed to measure antioxidant activity.

measures the ability of antioxidants to reduce ferric iron. It is based on the reduction of the complex of ferric iron and 2,3,5-triphenyl-1,3,4triaza-2-azoniacyclopenta-1,4-diene chloride (TPTZ) to the ferrous form at low pH. This reduction is monitored by measuring the change in absorption at 593 nm, using a diode-array spectrophotometer. Folin-Ciocalteau Total Phenolic Assay. This assay measures the change in color when metal oxides

Rules for Antioxidant Health Claims

ccording to the Food and Drug Administration (www.cfsan.fda.gov/~dms/hpotguid.html), a claim that describes the level of antioxidant nutrients present in a food is a nutrient content claim, and an antioxidant nutrient content claim can only be made for nutrients for which a Reference Daily Intake (RDI) has been established. The nutrient must have recognized antioxidant activity, i.e., there must be scientific evidence that after it is eaten and absorbed from the gastrointestinal tract the substance participates in physiological, biochemical, or cellular processes that inactivate free radicals or prevent free radical-initiated chemical reactions. The nutrient must also meet the requirements for nutrient content claims, such as high, good source, and more. For example, to say that a product is a good source of antioxidant beta-carotene, at least 10% of the RDI for vitamin A must be present as beta-carotene per serving. 64
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for the Flavonoid Content of Selected Foods. Its clear that theres a group of foods that rank high in antioxidant capacity, Prior said, but growing conditions, varieties, genetics, etc., can affect the antioxidant capacity measured in vitro. Also, we cant assume that something high on the list in in-vitro tests will be high in the body. It depends on whether the compounds are getting absorbed and are effective in the body. For example, berries with anthocyanins as their primary component, such as blueberries and blackberries, are generally high in antioxidants, but the anthocyanins dont appear to be stable in the body. As a result, the amount that needs to be consumed is higher than for other foods that have lower antioxidant levels. Prior said that were not really sure whether oxidative stress is a primary factor in development of disease or secondary to another factor, so we cant yet make dietary recommendations. Understanding more about whats going on in the body with regard to oxidative stress will require clinical studies.

are generally high in antioxidant activity, but apples contribute more antioxidants to the diet because of their higher rate of consumption. The challenge ahead, he said, is to correlate the CAA results to results of in-vivo methods to see if it can predict in-vivo results. He feels that CAA would be a better method for screening

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Cell-Culture Approach
Animal models and human studies are obviously the best methods for determining the actual efficacy of antioxidants in the body, Liu said, but they are expensive and time-consuming and not suitable for initial antioxidant screening of foods and dietary supplements. Accordingly, Liu and his coworkers have developed a cell-culture method, called the Cellular Antioxidant Activity (CAA) assay, that they feel is more biologically relevant than the chemical assays because it takes into account some aspects of cell uptake, distribution, and metabolism of antioxidant compounds. Dichlorofluorescin is a compound that once in human cells is easily oxidized to the fluorescent compound dichlorofluorescein (DCF). The CAA method measures the ability of antioxidants to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence compared to the control cells indicates the antioxidant capacity of the compounds. Using the method, Liu and coworkers have determined the antioxidant activity of various phytochemicals and fruit extracts (see sidebar). Among the pure compounds tested, quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin. Among 25 fruits tested, blueberry had the highest CAA value, followed in decreasing order by pomegranate, blackberry, strawberry, raspberry, and cranberry. Berries, Liu said,

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Determining Antioxidant Activity continued... Recent Papers on the Cellular Antioxidant Activity Assay
Wolfe, K.L. and Liu, R.H. 2007. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J. Agric. Food Chem. 55: 8896-8907. Wolfe, K. and Liu, R.H. 2008. Structureactivity relationships of flavonoids in the cellular antioxidant activity assay. J. Agric. Food Chem. 56: 8404-8411. Wolfe, K., Kang, X., He, X., Dong, M., Zhang, Q., and Liu, R.H. 2008. Cellular antioxidant activity of common fruits. J. Agric. Food Chem. 56: 8418-6426. antioxidants than current chemical methods. if the molecule behaves as a reducer (i.e., antioxidant), it is measured. But there are other antioxidant mechanisms that are not captured by the method, he added. Baugh said that he has always liked the ORAC method and admires the ongoing effort to keep it current. However, the Food and Drug Administrations recent position on antioxidants (see sidebar) seems to disregard the progress the method has made over the years. FDA is comfortable regulating single compounds, as it has been with pharmaceuticals, he said, so people wanting to pursue approval need to provide bioavailability and intervention data based on specific compounds. For those reasons, he prefers to use high-performance liquid chromatography for testing specific compounds, given the increasingly specific regulatory environment. He feels that this is the future as people move into clinical trials by showing bioavailability of specific compounds in the blood or urine and in-vivo activity of specific compounds. He feels that the cellculture method, combined with bioavailability data, is a good middle ground. FT
Neil H. Mermelstein, a Fellow of IFT, is Editor Emeritus of Food Technology nhmermelstein@comcast.net

An Industrial View
Steve Baugh (steveb@ chromadex.com), Manager of Analytical Services, ChromaDex Corp., prefers the Folin-Ciocalteau method over the ORAC method to measure the activity of the antioxidants and high-purity reference materialsvitamin C, anthocyanidins, proanthocyanidins, catechins, and othersthat his company offers for standardization of laboratory instruments. He said that he uses the Folin-Ciocalteau test to compare antioxidants because the source of the antioxidant is not an issue

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