Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORAC(FL)) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37 degrees C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 +/- 1.5 and 38.2 +/- 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples.

Vegetables are the most important sources of phenolics in the Mediterranean diet. Phenolics, especially flavonoids, are suggested as being essential bioactive compounds providing health benefits. In this study, twenty-seven vegetables, fifteen aromatic herbs and some spices consumed in Central Italy (the Marches region) were studied to reveal total phenolic, flavonoid and flavanol content as well as their antioxidant capacity measured by the oxygen radical absorbance capacity (ORAC) method. A comparison in terms of antioxidant capacity was made between different salads, as well as between salads to which aromatic herbs had been added. Lemon balm and marjoram at a concentration of 1·5 % w/w increased by 150 % and 200 % respectively the antioxidant capacity of a salad portion. A 200 g portion of a salad enriched with marjoram corresponded to an intake of 200 (SD 10) mg phenolics and 4000 (SD 300) ORAC units (mmol Trolox equivalents). Olive oils and wine or apple vinegars were the salad dressings that provided the highest increase in antioxidant capacity. Among the spices tested, cumin and fresh ginger made the most significant contribution to the antioxidant capacity. The results are useful in surveying the antioxidant parameters of vegetables, herbs and spices produced and consumed in our geographical area as well as in quantifying the daily intake of phenolics and ORAC units. The results can be used in public health campaigns to stimulate the consumption of vegetables able to provide significant health protection in order to prevent chronic diseases.

The phenolic and oxygen radical absorbance capacity (ORAC) values have been measured in six fresh and frozen vegetables (beet green, spinach, broccoli, carrot, onion, and celery) from the same cultivar by analyzing the whole juice (WJ) and the acetonic extract of the squeezed pulp. To exploit the effect of the acid environment on the stability and recovery of the phenolics, perchloric acid (PCA) was added directly to WJ and to the pulp before the extraction with acetone. In both fresh and frozen vegetables, PCA markedly increased the recovery of phenolics extracted from the pulp, but PCA had no effect on the WJ. Four of six frozen vegetables showed lower phenolic and ORAC values than the fresh vegetables, whereas in the other two cases, values were significantly higher compared to fresh samples. Among the fresh vegetables, beet green showed the highest ORAC and phenolic values; however, when measured in two different cultivars of beet green, the ORAC value showed as much as 4.5-fold variation, whereas total phenolics and flavonoids showed 1.2- and 3.5-fold variations, respectively. The results show that total phenolics and ORAC, compared in fresh and frozen vegetables, represent an index of the mildness of blanching in the industry of frozen vegetables and provide a measure of the gap in antioxidants in the diet of people who consume frozen instead of fresh vegetables. The plant genotype is an important source of variability in the ORAC value, which can be conveniently used to increase the intake of antioxidants from vegetables.

In this study, the oxygen radical absorbance capacity (ORAC) of vegetable oils was investigated using a spectrofluorometric method, which measures the protection of the phenolic substances of the oil on the β-phycoerythrin fluorescence decay in comparison with Trolox. More than 97% of the phenolic substances was extracted from the oil using methanol, and the methanolic extract was then used for the ORAC and the total phenolics assay. We found a significant correlation between ORAC values of different olive oils and the total amount of phenolics. For extra-virgin olive oils, maximal ORAC values reached 6.20 ± 0.31 µmol Trolox equivalent/g, while refined and seed oils showed values in the 1-1.5 µmol Trolox equivalent/g range. Our method is useful to assess the quality of olive oils and to predict, in combination with the rancidity tests, their stability against oxidation.

The induction period (IP), determined using accelerated methods such as the Rancimat test, is a parameter that has been used to predict the shelf life of virgin olive oil. The oxygen radical absorbance capacity (ORAC) has recently been proposed as a quality index of virgin olive oil because it measures the efficiency of phenolic compounds in the protection against peroxyl radicals. This study aims to investigate relationships between the ORAC and IP values and proposes ORAC as a new parameter of virgin olive oil stability. The concentrations of phenolics, o-diphenols, tocopherol, β-carotene, lutein, and ORAC and IP values were determined in 33 virgin olive oils. Regression analyses showed that both ORAC and IP values correlate with total phenols and o-diphenols with highly significant indices, whereas the correlations of both ORAC and IP with tocopherols, β-carotene, and lutein were not significant. The ORAC values correlate with the IP values with low but significant indices (R = 0.42; P < 0.02). The results confirm the key role of phenolic compounds in accounting for the shelf life of virgin olive oil and suggest that the ORAC parameter may be used as a new index of quality and stability. Paper no. J10173 in JAOCS 79, 977-982 (October 2002). FIG. 2. Regression analysis between oxygen radical absorbance capacity (ORAC) values and total phenolics for 33 virgin olive oils. Values are the average of four determinations. TE, Trolox equivalents. The SD are less than 5%. FIG. 3. Regression analysis between ORAC values and o-diphenolic compounds for 33 virgin olive oils. Values are the average of four determinations. The SD are less than 5%. For abbreviations see Figure 2. FIG. 4. Regression analysis between the Rancimat induction periods and the total phenols (A) and o-diphenols (B). Values are the average of four determinations. The SD are less than 5%.