A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds 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 when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.

Consumption of fruits and vegetables has been shown to be effective in the prevention of chronic diseases. These benefits are often attributed to the high antioxidant content of some plant foods. Apples are commonly eaten and are large contributors of phenolic compounds in European and North American diets. The peels of apples, in particular, are high in phenolics. During applesauce and canned apple manufacture, the antioxidant-rich peels of apples are discarded. To determine if a useful source of antioxidants is being wasted, the phytochemical content, antioxidant activity, and antiproliferative activity of the peels of four varieties of apples (Rome Beauty, Idared, Cortland, and Golden Delicious) commonly used in applesauce production in New York state were investigated. The values of the peels were compared to those of the flesh and flesh + peel components of the apples. Within each variety, the total phenolic and flavonoid contents were highest in the peels, followed by the flesh + peel and the flesh. Idared and Rome Beauty apple peels had the highest total phenolic contents (588.9 +/- 83.2 and 500.2 +/- 13.7 mg of gallic acid equivalents/100 g of peels, respectively). Rome Beauty and Idared peels were also highest in flavonoids (306.1 +/- 6.7 and 303.2 +/- 41.5 mg of catechin equivalents/100 g of peels, respectively). Of the four varieties, Idared apple peels had the most anthocyanins, with 26.8 +/- 6.5 mg of cyanidin 3-glucoside equivalents/100 g of peels. The peels all had significantly higher total antioxidant activities than the flesh + peel and flesh of the apple varieties examined. Idared peels had the greatest antioxidant activity (312.2 +/- 9.8 micromol of vitamin C equivalents/g of peels). Apple peels were also shown to more effectively inhibit the growth of HepG(2) human liver cancer cells than the other apple components. Rome Beauty apple peels showed the most bioactivity, inhibiting cell proliferation by 50% at the low concentration of 12.4 +/- 0.4 mg of peels/mL. The high content of phenolic compounds, antioxidant activity, and antiproliferative activity of apple peels indicate that they may impart health benefits when consumed and should be regarded as a valuable source of antioxidants.

Measurement of antioxidant activity using biologically relevant assays is important in the screening of fruits for potential health benefits. The cellular antioxidant activity (CAA) assay quantifies antioxidant activity in cell culture and was developed to meet the need for a more biologically representative method than the popular chemistry antioxidant capacity measures. The objective of the study was to determine the cellular antioxidant activity, total phenolic contents, and oxygen radical absorbance capacity (ORAC) values of 25 fruits commonly consumed in the United States. Pomegranate and berries (wild blueberry, blackberry, raspberry, and blueberry) had the highest CAA values, whereas banana and melons had the lowest. Apples were found to be the largest contributors of fruit phenolics to the American diet, and apple and strawberries were the biggest suppliers of cellular antioxidant activity. Increasing fruit consumption is a logical strategy to increase antioxidant intake and decrease oxidative stress and may lead to reduced risk of cancer.

Chemical antioxidant activity assays are used extensively to evaluate the potential bioactivity of plant foods and their phytochemical constituents, but they do not mimic the complexity of biological systems. The cellular antioxidant activity (CAA) activity assay was developed to be a more biologically relevant model to measure antioxidant activity. Structure-activity relationships of flavonoids have been determined in many chemistry antioxidant activity assays, and they vary with the protocols. The objective of this study was to determine structure-activity relationships of selected flavonoids in the CAA assay. The structures that conferred flavonoids with the most antioxidant activity in the CAA assay were a 3',4'- o-dihydroxyl group in the B-ring, a 2,3-double bond combined with a 4-keto group in the C-ring, and a 3-hydroxyl group. Isoflavones had no cellular antioxidant activity. Flavanols with a galloyl moiety had higher antioxidant activity than those without, and a B-ring 3',4',5'-trihydroxyl group further improved their efficacy. ORAC values for flavonoids were not related to their CAA values. Knowledge of structure-activity relationships in the CAA assay may be helpful in assessing potential in vivo antioxidant activity of flavonoids.

There is some evidence that chronic diseases, such as cancer and cardiovascular disease, may occur as a result of oxidative stress. Apple peels have high concentrations of phenolic compounds and may assist in the prevention of chronic diseases. Millions of pounds of waste apple peels are generated in the production of applesauce and canned apples in New York State each year. We proposed that a valuable food ingredient could be made using the peels of these apples if they could be dried and ground to a powder without large losses of phytochemicals. Rome Beauty apple peels were treated with citric acid dips, ascorbic acid dips, and blanches before being oven-dried at 60 degrees C. Only blanching treatments greatly preserved the phenolic compounds, and peels blanched for 10 s had the highest total phenolic content. Rome Beauty apple peels were then blanched for 10 s and dried under various conditions (oven-dried at 40, 60, or 80 degrees C, air-dried, or freeze-dried). The air-dried and freeze-dried apple peels had the highest total phenolic, flavonoid, and anthocyanin contents. On a fresh weight basis, the total phenolic and flavonoid contents of these samples were similar to those of the fresh apple peels. Freeze-dried peels had a lower water activity than air-dried peels on a fresh weight basis. The optimal processing conditions for the ingredient were blanching for 10s and freeze-drying. The process was scaled up, and the apple peel powder ingredient was characterized. The total phenolic content was 3342 +/- 12 mg gallic acid equivalents/100 g dried peels, the flavonoid content was 2299 +/- 52 mg catechin equivalents/100 g dried peels, and the anthocyanin content was 169.7 +/- 1.6 mg cyanidin 3-glucoside equivalents/100 g dried peels. These phytochemical contents were a significantly higher than those of the fresh apple peels if calculated on a fresh weight basis (p < 0.05). The apple peel powder had a total antioxidant activity of 1251 +/- 56 micromol vitamin C equivalents/g, similar to fresh Rome Beauty peels on a fresh weight basis (p > 0.05). One gram of powder had an antioxidant activity equivalent to 220 mg of vitamin C. The freeze-dried apple peels also had a strong antiproliferative effect on HepG(2) liver cancer cells with a median effective dose (EC(50)) of 1.88 +/- 0.01 mg/mL. This was lower than the EC(50) exhibited by the fresh apple peels (p < 0.05). Apple peel powder may be used in a various food products to add phytochemicals and promote good health.