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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 it’s “the real Antioxidant Superpower™,” and Hershey’s 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
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,
It’s clear that there’s 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
11.08 • www.ift.org
while others aren’t or are otherwise modified.html). Many epidemiological studies and clinical trials utilize in-vivo biomarkers of antioxidant stress as surrogate end-point markers.cfsan. A lot of methods give the same relative ranking of antioxidants. Prior (priorronaldl@ uams. John W. and metabolism of the antioxidants..edu). a claim that describes the level of antioxidant nutrients present in a food is a nutrient content claim. their ability to predict in-vivo activity has not been demonstrated. There are enzymes in cells and tissues in the body that protect the body from excess oxidation. such as “high. USDA/ARS Arkansas Children’s Nutrition Center.edu). and an antioxidant nutrient content claim can only be made for nutrients for which a Reference Daily Intake (RDI) has been established. but these markers don’t predict or signify health status very well.” For example. 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. As a result. The nutrient must also meet the requirements for nutrient content claims. and results are reported as gallic acid equivalents..fda. uptake. More effort to directly link changes in antioxidant biomarkers to health outcomes is sorely needed.3. 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.” at least 10% of the RDI for vitamin A must be present as beta-carotene per serving. i. Moreover. This assay measures the change in color when metal oxides Rules for Antioxidant Health Claims A ccording to the Food and Drug Administration (www. the various in-vitro assays provide differing results. to say that a product is a “good source of antioxidant beta-carotene. Research Chemist/ Nutritionist. This reduction is monitored by measuring the change in absorption at 593 nm. and the 2007 USDA Database Relevance Questioned Despite wide usage of these chemical antioxidant activity assays.org pg . 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. we don’t know if the “antioxidant” will act as an antioxidant. The standard curve is prepared using gallic acid.08 • www. he said. The nutrient must have recognized antioxidant activity. measures the ability of antioxidants to reduce ferric iron. biochemical. Prior said that different radicals can give different test results.” “good source. Liu said that the protocols of the chemical antioxidant activity assays often do not include the appropriate biological substrates to be protected. Erdman Jr.[LABORATORY] Determining Antioxidant Activity continued. or cellular processes that inactivate free radicals or prevent free radical-initiated chemical reactions. act as a prooxidant at higher consumption levels. depending on their structure.” and “more. Ark. Associate Professor. 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. (jwerdman@ illinois. Professor of Food Science and Human Nutrition. In addition. of Food Science. University of Illinois at Urbana. the digestive tract. the partitioning of compounds between water and lipid phases.4-diene chloride (TPTZ) to the ferrous form at low pH. and antioxidant compounds may operate via multiple mechanisms. the 2007 ORAC Report. he said. other components from those same foods and their metabolites may influence oxidation/antioxidant status once absorbed. 64 11.ift. there must be scientific evidence that after it is eaten and absorbed from the gastrointestinal tract the substance participates in physiological. relevant types of oxidants encountered. he said. 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. Cornell University.edu). Dept.. ORAC uses the peroxyl radical.gov/~dms/hpotguid. according to Rui Hai Liu (rl23@cornell. the most common free radical in the human body. he added.5-triphenyl-1.4triaza-2-azoniacyclopenta-1. and the influence of interfacial behavior. but there’s some variation depending on how the radicals react with components in food.e. Some are absorbed. Little Rock. We don’t 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. are reduced by polyphenolic antioxidants such as gallic acid and catechin. Biological systems are much more complex than the simple chemical mixtures employed.. It is based on the reduction of the complex of ferric iron and 2. resulting in a blue solution with maximal absorption at 765 nm. Ithaca. He said that many of the chemical antioxidant assays being used do not reflect cellular physiological conditions and do not consider the bioavailability. • Folin-Ciocalteau Total Phenolic Assay. Ronald L.3. or have other roles in the body. using a diode-array spectrophotometer. he said.
It’s clear that there’s a group of foods that rank high in antioxidant capacity.08 • www. berries with anthocyanins as their primary component. The decrease in cellular fluorescence compared to the control cells indicates the antioxidant capacity of the compounds.ift.. blackberry. can affect the antioxidant capacity measured in vitro. Liu said. Prior said. and metabolism of antioxidant compounds. Dichlorofluorescin is a compound that once in human cells is easily oxidized to the fluorescent compound dichlorofluorescein (DCF). called the Cellular Antioxidant Activity (CAA) assay. but they are expensive and time-consuming and not suitable for initial antioxidant screening of foods and dietary supplements. Accordingly. such as blueberries and blackberries. and luteolin. Prior said that we’re not really sure whether oxidative stress is a primary factor in development of disease or secondary to another factor.2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. Liu and his coworkers have developed a cell-culture method. Berries. genetics. so we can’t yet make dietary recommendations. followed in decreasing order by pomegranate. Among the pure compounds tested. Among 25 fruits tested.org 65 pg . epigallocatechin gallate (EGCG). 11.for the Flavonoid Content of Selected Foods. Using the method. etc. As a result. distribution. blueberry had the highest CAA value. The CAA method measures the ability of antioxidants to prevent the formation of DCF by 2. we can’t assume that something high on the list in in-vitro tests will be high in the body. but growing conditions. Also. Understanding more about what’s going on in the body with regard to oxidative stress will require clinical studies. For example. but the anthocyanins don’t appear to be stable in the body. raspberry. and cranberry. followed by kaempferol. the amount that needs to be consumed is higher than for other foods that have lower antioxidant levels. varieties. strawberry. Liu said. quercetin had the highest CAA value. He feels that CAA would be a better method for screening AD 1/2 ISL 4C Cell-Culture Approach Animal models and human studies are obviously the best methods for determining the actual efficacy of antioxidants in the body. is to correlate the CAA results to results of in-vivo methods to see if it can predict in-vivo results. It depends on whether the compounds are getting absorbed and are effective in the body. he said. are generally high in antioxidants. but apples contribute more antioxidants to the diet because of their higher rate of consumption. that they feel is more biologically relevant than the chemical assays because it takes into account some aspects of cell uptake. The challenge ahead. Liu and coworkers have determined the antioxidant activity of various phytochemicals and fruit extracts (see sidebar). are generally high in antioxidant activity. myricetin.
H. Agric. combined with bioavailability data. However.L. catechins. M..com). He feels that the cellculture method. Kang. 56: 8404-8411. proanthocyanidins.. Agric. and dietary supplements..ift. He. Mermelstein. K.[LABORATORY] Determining Antioxidant Activity continued. Wolfe.H. J.H. foods. Zhang. he added. he said. 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. antioxidant). anthocyanidins. R. Food Chem... J. X. But there are other antioxidant mechanisms that are not captured by the method. a Fellow of IFT.. if the molecule behaves as a reducer (i. and Liu.08 • www. Recent Papers on the Cellular Antioxidant Activity Assay Wolfe. Agric. antioxidants than current chemical methods. He said that he uses the Folin-Ciocalteau test to compare “antioxidants” because the source of the antioxidant is not an issue— AD 1/2 Horiz Bleed pg 66 11. 55: 8896-8907... 2008. the Food and Drug Administration’s recent position on antioxidants (see sidebar) seems to disregard the progress the method has made over the years. X. FDA is comfortable regulating single compounds. Cellular antioxidant activity (CAA) assay for assessing antioxidants. prefers the Folin-Ciocalteau method over the ORAC method to measure the activity of the antioxidants and high-purity reference materials—vitamin C. and Liu. is Editor Emeritus of Food Technology • nhmermelstein@comcast. R.. it is measured. he prefers to use high-performance liquid chromatography for testing specific compounds. 56: 8418-6426. is a good middle ground.e. Food Chem. Dong. given the increasingly specific regulatory environment. so people wanting to pursue approval need to provide bioavailability and intervention data based on specific compounds. ChromaDex Corp. R. For those reasons. as it has been with pharmaceuticals.org . Cellular antioxidant activity of common fruits.net An Industrial View Steve Baugh (steveb@ chromadex. K. K. J. FT Neil H. Structureactivity relationships of flavonoids in the cellular antioxidant activity assay. Baugh said that he has always liked the ORAC method and admires the ongoing effort to keep it current. 2007. Q. and others—that his company offers for standardization of laboratory instruments. Wolfe. Food Chem. Manager of Analytical Services. 2008. and Liu.
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