Free radical-induced oxidative stress has been hypothesized to be a major factor in the development of several degenerative chronic diseases. Oxidative stress can cause oxidative damage to large biomolecules such as lipids, proteins, and DNA, resulting in an increased risk for inflammatory diseases, cardiovascular disease (CVD), cancer, diabetes, Alzheimer’s disease, cataracts, and age-related functional decline. To prevent or slow the oxidative stress induced by free radicals, sufficient amounts of antioxidants need to be consumed (Liu, 2004).

Fruits, vegetables, whole grains, and other natural products contain a wide variety of antioxidant compounds (phytochemicals), such as phenolics, flavonoids, and carotenoids, and may help protect cellular systems from oxidative damage and also lower the risk of chronic diseases (Liu, 2004). The benefits of fruits, vegetables, and whole grains have been consistently supported by epidemiological studies reporting that regular consumption of these foods is associated with a reduced risk of developing chronic diseases such as cancer and CVD. Bioactive non-nutrient phytochemicals in fruits, vegetables, whole grains and other plant foods have been linked to the reduced risk for major chronic diseases, including cancer and CVD.

Due to the potential of antioxidants to decrease the risk of developing cancer and other chronic diseases, it is important to be able to measure antioxidant activity using biologically relevant assays. Antioxidant research has been expanded dramatically since the mid-1990s with the development of many chemical assays measuring phytochemical content and total antioxidant activity of pure compounds, foods, and dietary supplements. These assays include: total phenolics Folin-Ciocalteu (F-C) assay, oxygen radical absorbance capacity (ORAC), total radical absorption potentials (TRAP), total oxyradical scavenging capacity (TOSC), peroxyl radical scavenging capacity (PSC), trolox equivalent antioxidant capacity (TEAC), ferric reducing/antioxidant power (FRAP) and 2,2-diphenyl-picrylhydrazyl (DPPH) radical methods. However, none of these takes into account the bioavailability/uptake and metabolism of the antioxidants.

Biological systems are much more complex than the simple chemical mixtures employed and antioxidant compounds may operate via multiple mechanisms. The different efficacies of compounds in the various assays attest to the functional variation. In addition, the mechanisms of action of antioxidants go beyond the antioxidant activity scavenging free radicals in disease prevention and health promotion (Liu, 2004). The best measures are from animal models and human studies; however, these are expensive and timeconsuming and not suitable for initial antioxidant screening of foods and dietary supplements. Cell culture models provide an approach that is cost-effective, relatively fast, and addresses some issues of uptake, distribution, and metabolism.

Therefore, there is an urgent need for cell culture models to support antioxidant research prior to animal studies and human clinical trials, as indicated at the First International Congress on Antioxidant Methods (Liu and Finley, 2005). To address this need, we developed a cellular antioxidant activity (CAA) assay to measure the antioxidant activity of pure phytochemicals, dietary supplements, and foods (Wolfe and Liu, 2007 and 2008).

Principle of the Cellular Antioxidant Activity (CAA) Assay

The CAA assay utilizes 2′,7′-dichlorofluorescin diacetate (DCFH-DA) as a probe in cultured human HepG2 liver cancer cells (Wolfe and Liu, 2007). Non-polar DCFH-DA is taken up by HepG2 cells by passive diffusion and deacetylated by cellular esterases to form polar 2′,7′- dichlorofluorescin (DCFH), which is trapped within the cells. Peroxyl radicals generated from 2, 2′-azobis (2-amidinopropane) (ABAP) lead to the oxidation of DCFH to form a fluorescent compound dichlorofluorescein (DCF). The level of fluorescence formed within the cells is proportional to the level of oxidation (Wolfe and Liu, 2007). Pure phytochemical compounds, antioxidants and fruit extracts quench peroxyl radicals and inhibit the generation of fluorescent DCF. The decrease in cellular fluorescence compared to the control cells indicates the antioxidant capacity of the compounds.

Cellular antioxidant activity of CAA

Twenty-five common fruits consumed in the United States were evaluated for their antioxidant activity in the CAA assay (Wolfe et al, 2008). In general, the CAA values of the berries (wild blueberry, blackberry, strawberry, blueberry, raspberry, and cranberry) and pomegranate tended to be the highest. Wild blueberry had the highest CAA value, followed by pomegranate and blackberry, which had similar CAA values. Strawberry, blueberry, and raspberry were next, and were not significantly different from each other. These were followed by cranberry, plum, cherry, mango, apple, red grape, kiwifruit, pineapple, orange, lemon, grapefruit, peach, pear, nectarine, and honeydew. Honeydew, cantaloupe, and banana had the lowest activities of all the fruits tested in the CAA assay. Apples were found to be the largest contributors of fruit phenolics to the American diet, and apple and strawberries were the top providers of cellular antioxidant activity.

Fruits consumption to increase antioxidants intake

Antioxidant activity provided by fruits may be important in the prevention of cancer and other chronic diseases. Therefore, increasing fruit consumption is a logical strategy to increase antioxidants intake and decrease oxidative stress, and may lead to reduced risk of cancer.

Measuring the antioxidant activity of fruits in cell culture is an important step in screening for potential bioactivity and is more biologically representative than data obtained from Chemistry antioxidant activity assays. Further testing is needed to confirm the relationship between CAA values for fruits and their modulation of oxidative stress markers in vivo.