ORAC 6.0™

Antioxidant Capacity Measurements of Botanicals and the Use of Multiple Radical Sources Using the Oxygen Radical Absorbance Capacity Assay (ORAC)

Several thousand polyphenolic compounds exist in plants and many of these have antioxidant capac­ity (AOC). Because of the difficulty of quantitating the individual antioxidant compounds, a method which provides a “sum” of the antioxidant components in plants and biological samples is useful. These types of assays are often referred to as “total” antioxidant capacity assays. However, there is in actuality no single assay which provides a “total” measurement of antioxidant capacity. In think­ing about antioxidant capacity methods, one has to consider the oxidant source and the mechanism of reaction with potential antioxidants. Experimental evidence has suggested that there are six major reactive oxygen species (ROS) causing oxidative damage in the human body. These species include: superoxide anion (O₂·^–); peroxyl radicals (ROO·); hydroxyl radicals (HO·); singlet oxygen (¹O₂); peroxynitrite (ONOO^–), and hypochlorite. The peroxyl radical is the most abundant free radical in the human body. Another one of the more relevant radicals in biological regulation is superoxide anion radical. The superoxide anion is formed by the reduction of molecular oxygen in the process of energy metabolism.

Accumulated evidence indicates that reactive oxygen species, such as per­oxyl radicals (ROO·), hydroxyl radicals (HO·), the superoxide anion (O^2-·), singlet oxygen (¹O₂), and hypochlorite, are involved in the pathophysiology of aging and a multitude of diseases. To counteract the damage of the reactive oxygen species on living cells, a defense system is designed biologically to neutralize the reactive oxygen species or to prevent the reactive oxygen species from being generated in the first place. Depending on the reaction mechanisms, antioxidants are often classified into two major categories: radical chainbreaking antioxidants and preventive antioxidants. Chainbreak­ing antioxidants convert reactive free radicals (e.g., HO·) to stable and thus nonaggressive molecules through hydrogen atom transfer reactions between HO· and the antioxidants. As a result, the autoxi­dation chain reactions between the free radicals and the cellular molecules are terminated. Preven­tive antioxidants inhibit the oxidation reaction from occurring by either converting the precursors of the reactive oxygen species to unreactive species or inhibiting the oxidation reaction. To counteract the assault of the superoxide anion reactive species, living cells have a biological defense system composed of enzymatic antioxidants to convert reactive oxygen species or reactive nitrogen species to harmless species. In contrast, no enzymatic action is known to scavenge ROO·, HO·, ¹O₂, and ONOO^–, so the burden of defense relies on a variety of non-enzymatic antioxidants such as vitamin C, and vitamin E, and many phytochemicals that have the property of scavenging oxidants and free radicals. To comprehensively evaluate the oxidant-scavenging capacity of a food sample, assays have to be designed to include these reactive oxygen species. However, so far the majority of assays are designed to measure a sample’s capacity to react with one oxidant (either organic radical or redox active metal complex). The peroxyl radical has been the most frequently used reactive oxygen species in antioxidant capacity assays because it is the most relevant radical in lipid autoxidation and can be generated conveniently from azo compounds. The peroxyl radical therefor was the first radical source used in the Oxygen Radical Absorbance Capacity (ORAC) assay development.

It is evident that the antioxidant defense “team” in living cells contains individual antioxidants that function in very differ­ent tasks in the battles against oxida­tive stress and reactive oxygen species. Therefore, it is imperative that to compre­hensively evaluate the antioxidant capacity of food nutrients in vitro, we need a broad range of assays that can cover all aspects of antioxidant capacity. It is impossible to have a one-fits-for-all assay. Although there is a validated assay for peroxyl radi­cal absorbance capacity (ORAC) (1-5), no such assay had been developed for any other of the reactive oxygen species, until Brunswick Laboratories first published methods for the analysis of antioxidant capacity using the hydroxyl, and superoxide anion radicals (6, 7). Brunswick Laboratories has subsequently addressed the other oxygen radical species [singlet oxygen (¹O₂); peroxynitrite (ONOO^–)], and hypochlorite (ClO^–) by developing assays that assess their contribution to total antioxidant capacity.

A sample of available data is presented in Figure 1 and 2 on fruits and vegetables. From this data, it is evident that there is little or no correlation among the different radical sources used to assess antioxidant capacity. One example that stands out is with tomatoes, which has a very low antioxidant capacity as measured with the peroxyl radical, but much higher antioxidant capacity using the singlet oxygen radical (Figure 2). Furthermore, the original peroxyl ORAC represents no more than 27% of the antioxidant potential of selected fruits and vegetables. The other radical assays added to the ORAC suite represent the preponderance of antioxidant potential.

By measuring all six primary reactive oxygen species, ORAC 6.0 provides new opportunities which can be used in the formulation of nutritional products that deliver quantifiable, maximal protection against multiple radical sources.

References 1. Cao, G.; Prior, R. L., Measurement of Total Antioxidant Capacity in Nutritional and Clinical Studies. In Handbook of Antioxidants, Cadenas, E.; Packer, L., Eds. Marcel Dekker, Inc.: New York, 2001; pp 47-55. 2. Ou, B.; Hampsch-Woodill, M.; Prior, R. L., Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J. Agric. Food Chem. 2001, 49, 4619-4926. 3. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J.; Prior, R. L., High-Throughput assay of oxy­gen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format. J. Agric. Food Chem. 2002, 50, 4437-4444. 4. Prior, R. L.; Hoang, H.; Gu, L.; Wu, X.; Bacchiocca, M.; Howard, L.; Hampsch-Woodill, M.; Huang, D.; Ou, B.; Jacob, R., Assays for hydrophilic and lipophilic antioxidant capacity (Oxygen Radical Absorbance Capacity (ORACFL)) of plasma and other biological and food samples. J. Agric. Food Chem. 2003, 51, 3273 -3279. 5. Wu, X.; Beecher, G.; Holden, J.; Haytowitz, D.; Gebhardt, S. E.; Prior, R. L., Lipophilic and hydrophilic antioxidant capacities of common foods in the U.S. J. Agric. Food Chem. 2004, 52, 4026-4037. 6. Ou, B.; Hampsch-Woodill, M.; Flanagan, J.; Deemer, E. K.; Prior, R. L.; Huang, D., Novel fluoro­metric assay for hydroxyl radical prevention capacity using fluorescein as the probe. J. Agric. Food Chem. 2002, 50, 2772-2777. 7. Ou, B.; Huang, D.; Prior, R. L.; Hampsch-Woodill, M.; Flanagan, J. A. In A high throughput assay for superoxide radical absorbance capacity (S-ORAC) of botanicals, First International Congress on Antioxidant Methods, Orlando, FL, 2004; Orlando, FL, 2004.