Oxygen radical absorbance capacity
Encyclopedia
Oxygen Radical Absorbance Capacity (ORAC) is a method of measuring antioxidant
capacities in biological samples in vitro
.
A wide variety of foods has been tested using this method, with certain spice
s, berries and legumes rated highly. There exists no physiological proof in vivo
that free-radical theory
is valid. Consequently, the ORAC method, derived only in test tube
experiments, cannot currently be interpreted as relevant to human diets or biology
.
measures the oxidative degradation of the fluorescent molecule (either beta-phycoerythrin or fluorescein
) after being mixed with free radical generators such as azo-initiator compounds
. Azo-initiators are considered to produce the peroxyl radical by heating, which damages the fluorescent molecule, resulting in the loss of fluorescence. Antioxidant
s are considered to protect the fluorescent molecule from the oxidative degeneration. The degree of protection is quantified using a fluorometer
. Fluorescein is currently used most as a fluorescent probe. Equipment that can automatically measure and calculate the capacity is commercially available (Biotek, Roche Diagnostics).
The fluorescent intensity decreases as the oxidative degeneration proceeds, and this intensity is typically recorded for 35 minutes after the addition of the azo-initiator (free radical generator). So far, AAPH (2,2’-azobis(2-amidino-propane) dihydrochloride) is the sole free-radical generator used. The degeneration (or decomposition) of fluorescein is measured as the presence of the antioxidant slows the fluorescence decay. Decay curves (fluorescence intensity vs. time) are recorded and the area between the two decay curves (with or without antioxidant) is calculated. Subsequently, the degree of antioxidant-mediated protection is quantified using the antioxidant trolox
(a vitamin E analogue) as a standard. Different concentrations of trolox are used to make a standard curve, and test samples are compared to this. Results for test samples (foods) have been published as "trolox equivalents" or TE.
One benefit of using the ORAC method to evaluate substances' antioxidant capacity is that it takes into account samples with and without lag phases of their antioxidant capacities. This is especially beneficial when measuring foods and supplements that contain complex ingredients with various slow and fast acting antioxidants, as well as ingredients with combined effects that cannot be pre-calculated.
Drawbacks of this method are: 1) only antioxidant activity against particular (probably mainly peroxyl) radicals is measured; however, peroxyl radical formation has never been proven; 2) the nature of the damaging reaction is not characterized; 3) there is no evidence that free radicals are involved in this reaction; and 4) there is no evidence that ORAC values have any biological significance following consumption of any food. Moreover, the relationship between ORAC values and a health benefit has not been established.
Several modified ORAC methods have been proposed. Most of them employ the same principle (i.e. measurement of AAPH-radical mediated damage of fuorescein); however, ORAC-EPR, electron paramagnetic resonance (EPR) based ORAC method directly measures the decrease of AAPH-radical level by the scavenging action of the antioxidant substance.
s, which have antioxidant capacity in vitro
and so provide an artificial index of antioxidant strength—the ORAC measurement.
Other than for dietary antioxidant vitamins -- vitamin A
, vitamin C
and vitamin E
-- no food compounds have been proved with antioxidant efficacy in vivo
. Accordingly, regulatory agencies like the Food and Drug Administration
of the United States and the European Food Safety Authority
(EFSA) have published guidance disallowing food product labels to claim or imply an antioxidant benefit when no such physiological evidence exists. This guidance for the United States and European Union establishes that it is illegal to imply potential health benefits on package labels of products with high ORAC.
s are good antioxidant
s and probably influence the ORAC value, antioxidant effects in vivo
are probably negligible or absent. By non-antioxidant mechanisms still undefined, flavonoid
s and other polyphenols may reduce the risk of cardiovascular disease and cancer.
As interpreted by the Linus Pauling Institute
and EFSA, dietary polyphenols have little or no direct antioxidant food value following digestion. Not like controlled test tube conditions, the fate of polyphenols in vivo
shows they are poorly conserved (less than 5%), with most of what is absorbed existing as chemically-modified metabolites destined for rapid excretion.
The increase in antioxidant capacity of blood seen after the consumption of polyphenol-rich (ORAC-rich) foods is not caused directly by the polyphenols, but most likely results from increased uric acid
levels derived from metabolism of flavonoids. According to Frei, "we can now follow the activity of flavonoids in the body, and one thing that is clear is that the body sees them as foreign compounds and is trying to get rid of them."
have voluntarily published (i.e. USDA does not authorize these values) lists of ORAC values for plant foods commonly consumed by the U.S. population (fruits, vegetables, nuts, seeds, spices, grains, etc.). Values are expressed as the sum of the lipid soluble (e.g. carotenoid
) and water-soluble (e.g. phenolic
) antioxidant fractions (i.e., “total ORAC”) reported as in micromoles Trolox
equivalents (TE) per 100 gram sample, and are compared to assessments of total polyphenol content in the samples.
With nearly all vegetables, conventional boiling can reduce the ORAC value by up to 90%, while steaming retains more of the antioxidants.
Numerous health food and beverage companies and marketers have capitalized on the ORAC rating by promoting products claimed to be "high in ORAC". As most of these ORAC values have not been independently validated or subjected to peer review
for publication in scientific literature, they remain unconfirmed, are not scientifically credible, and may mislead consumers.
Antioxidant
An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When...
capacities in biological samples in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
.
A wide variety of foods has been tested using this method, with certain spice
Spice
A spice is a dried seed, fruit, root, bark, or vegetative substance used in nutritionally insignificant quantities as a food additive for flavor, color, or as a preservative that kills harmful bacteria or prevents their growth. It may be used to flavour a dish or to hide other flavours...
s, berries and legumes rated highly. There exists no physiological proof in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
that free-radical theory
Free-radical theory
The free-radical theory of aging states that organisms age because cells accumulate free radical damage over time. A free radical is any atom or molecule that has a single unpaired electron in an outer shell. While a few free radicals such as melanin are not chemically reactive, most...
is valid. Consequently, the ORAC method, derived only in test tube
Test tube
A test tube, also known as a culture tube or sample tube, is a common piece of laboratory glassware consisting of a finger-like length of glass or clear plastic tubing, open at the top, usually with a rounded U-shaped bottom....
experiments, cannot currently be interpreted as relevant to human diets or biology
Biology
Biology is a natural science concerned with the study of life and living organisms, including their structure, function, growth, origin, evolution, distribution, and taxonomy. Biology is a vast subject containing many subdivisions, topics, and disciplines...
.
Method
The assayAssay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
measures the oxidative degradation of the fluorescent molecule (either beta-phycoerythrin or fluorescein
Fluorescein
Fluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications....
) after being mixed with free radical generators such as azo-initiator compounds
Azo compound
Azo compounds are compounds bearing the functional group R-N=N-R', in which R and R' can be either aryl or alkyl. IUPAC defines azo compounds as: "Derivatives of diazene , HN=NH, wherein both hydrogens are substituted by hydrocarbyl groups, e.g. PhN=NPh azobenzene or diphenyldiazene." The more...
. Azo-initiators are considered to produce the peroxyl radical by heating, which damages the fluorescent molecule, resulting in the loss of fluorescence. Antioxidant
Antioxidant
An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When...
s are considered to protect the fluorescent molecule from the oxidative degeneration. The degree of protection is quantified using a fluorometer
Fluorometer
A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. These parameters are used to identify the presence and the amount of specific molecules in a medium...
. Fluorescein is currently used most as a fluorescent probe. Equipment that can automatically measure and calculate the capacity is commercially available (Biotek, Roche Diagnostics).
The fluorescent intensity decreases as the oxidative degeneration proceeds, and this intensity is typically recorded for 35 minutes after the addition of the azo-initiator (free radical generator). So far, AAPH (2,2’-azobis(2-amidino-propane) dihydrochloride) is the sole free-radical generator used. The degeneration (or decomposition) of fluorescein is measured as the presence of the antioxidant slows the fluorescence decay. Decay curves (fluorescence intensity vs. time) are recorded and the area between the two decay curves (with or without antioxidant) is calculated. Subsequently, the degree of antioxidant-mediated protection is quantified using the antioxidant trolox
Trolox
Trolox is Hoffman-LaRoche's trade name for 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-soluble derivative of vitamin E. It is an antioxidant, like vitamin E, and is used in biological or biochemical applications to reduce oxidative stress or damage.Trolox equivalent antioxidant...
(a vitamin E analogue) as a standard. Different concentrations of trolox are used to make a standard curve, and test samples are compared to this. Results for test samples (foods) have been published as "trolox equivalents" or TE.
One benefit of using the ORAC method to evaluate substances' antioxidant capacity is that it takes into account samples with and without lag phases of their antioxidant capacities. This is especially beneficial when measuring foods and supplements that contain complex ingredients with various slow and fast acting antioxidants, as well as ingredients with combined effects that cannot be pre-calculated.
Drawbacks of this method are: 1) only antioxidant activity against particular (probably mainly peroxyl) radicals is measured; however, peroxyl radical formation has never been proven; 2) the nature of the damaging reaction is not characterized; 3) there is no evidence that free radicals are involved in this reaction; and 4) there is no evidence that ORAC values have any biological significance following consumption of any food. Moreover, the relationship between ORAC values and a health benefit has not been established.
Several modified ORAC methods have been proposed. Most of them employ the same principle (i.e. measurement of AAPH-radical mediated damage of fuorescein); however, ORAC-EPR, electron paramagnetic resonance (EPR) based ORAC method directly measures the decrease of AAPH-radical level by the scavenging action of the antioxidant substance.
Regulatory guidance
In the following discussion, the term "antioxidant" refers mainly to non-nutrient compounds in foods, such as polyphenolPolyphenol
Polyphenols are a structural class of natural, synthetic, and semisynthetic organic chemicals characterized by the presence of large multiples of phenol structural units...
s, which have antioxidant capacity in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
and so provide an artificial index of antioxidant strength—the ORAC measurement.
Other than for dietary antioxidant vitamins -- vitamin A
Vitamin A
Vitamin A is a vitamin that is needed by the retina of the eye in the form of a specific metabolite, the light-absorbing molecule retinal, that is necessary for both low-light and color vision...
, vitamin C
Vitamin C
Vitamin C or L-ascorbic acid or L-ascorbate is an essential nutrient for humans and certain other animal species. In living organisms ascorbate acts as an antioxidant by protecting the body against oxidative stress...
and vitamin E
Vitamin E
Vitamin E is used to refer to a group of fat-soluble compounds that include both tocopherols and tocotrienols. There are many different forms of vitamin E, of which γ-tocopherol is the most common in the North American diet. γ-Tocopherol can be found in corn oil, soybean oil, margarine and dressings...
-- no food compounds have been proved with antioxidant efficacy in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
. Accordingly, regulatory agencies like the Food and Drug Administration
Food and Drug Administration
The Food and Drug Administration is an agency of the United States Department of Health and Human Services, one of the United States federal executive departments...
of the United States and the European Food Safety Authority
European Food Safety Authority
The European Food Safety Authority is an agency of the European Union that provides independent scientific advice and communication on existing and emerging risks associated with the food chain, created by European Regulation 178/2002....
(EFSA) have published guidance disallowing food product labels to claim or imply an antioxidant benefit when no such physiological evidence exists. This guidance for the United States and European Union establishes that it is illegal to imply potential health benefits on package labels of products with high ORAC.
Physiological context
Although research in vitro indicates that polyphenolPolyphenol
Polyphenols are a structural class of natural, synthetic, and semisynthetic organic chemicals characterized by the presence of large multiples of phenol structural units...
s are good antioxidant
Antioxidant
An antioxidant is a molecule capable of inhibiting the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When...
s and probably influence the ORAC value, antioxidant effects in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
are probably negligible or absent. By non-antioxidant mechanisms still undefined, flavonoid
Flavonoid
Flavonoids , are a class of plant secondary metabolites....
s and other polyphenols may reduce the risk of cardiovascular disease and cancer.
As interpreted by the Linus Pauling Institute
Linus Pauling Institute
The Linus Pauling Institute is a research institute located at Oregon State University with a focus on health maintenance. The mission statement of the institute is three-fold. First, to determine the functional roles of micronutrients and phytochemicals in promoting optimal health and to treat or...
and EFSA, dietary polyphenols have little or no direct antioxidant food value following digestion. Not like controlled test tube conditions, the fate of polyphenols in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
shows they are poorly conserved (less than 5%), with most of what is absorbed existing as chemically-modified metabolites destined for rapid excretion.
The increase in antioxidant capacity of blood seen after the consumption of polyphenol-rich (ORAC-rich) foods is not caused directly by the polyphenols, but most likely results from increased uric acid
Uric acid
Uric acid is a heterocyclic compound of carbon, nitrogen, oxygen, and hydrogen with the formula C5H4N4O3. It forms ions and salts known as urates and acid urates such as ammonium acid urate. Uric acid is created when the body breaks down purine nucleotides. High blood concentrations of uric acid...
levels derived from metabolism of flavonoids. According to Frei, "we can now follow the activity of flavonoids in the body, and one thing that is clear is that the body sees them as foreign compounds and is trying to get rid of them."
Food sources
Scientists with the United States Department of AgricultureUnited States Department of Agriculture
The United States Department of Agriculture is the United States federal executive department responsible for developing and executing U.S. federal government policy on farming, agriculture, and food...
have voluntarily published (i.e. USDA does not authorize these values) lists of ORAC values for plant foods commonly consumed by the U.S. population (fruits, vegetables, nuts, seeds, spices, grains, etc.). Values are expressed as the sum of the lipid soluble (e.g. carotenoid
Carotenoid
Carotenoids are tetraterpenoid organic pigments that are naturally occurring in the chloroplasts and chromoplasts of plants and some other photosynthetic organisms like algae, some bacteria, and some types of fungus. Carotenoids can be synthesized fats and other basic organic metabolic building...
) and water-soluble (e.g. phenolic
Polyphenol
Polyphenols are a structural class of natural, synthetic, and semisynthetic organic chemicals characterized by the presence of large multiples of phenol structural units...
) antioxidant fractions (i.e., “total ORAC”) reported as in micromoles Trolox
Trolox
Trolox is Hoffman-LaRoche's trade name for 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-soluble derivative of vitamin E. It is an antioxidant, like vitamin E, and is used in biological or biochemical applications to reduce oxidative stress or damage.Trolox equivalent antioxidant...
equivalents (TE) per 100 gram sample, and are compared to assessments of total polyphenol content in the samples.
| Food | Serving size | Antioxidant capacity per serving size. Units are Total Antioxidant Capacity per serving in units of micromoles of Trolox equivalents. |
|---|---|---|
| Raw unprocessed Cocoa bean | 100 grams | 28,000 |
| Small Red Bean | ½ cup dried beans | 13,727 |
| Wild blueberry Blueberry Blueberries are flowering plants of the genus Vaccinium with dark-blue berries and are perennial... |
1 cup | 13,427 |
| Red kidney bean | ½ cup dried beans | 13,259 |
| Pinto bean | ½ cup | 11,864 |
| Blueberry Blueberry Blueberries are flowering plants of the genus Vaccinium with dark-blue berries and are perennial... |
1 cup (cultivated berries) | 9,019 |
| Cranberry Cranberry Cranberries are a group of evergreen dwarf shrubs or trailing vines in the subgenus Oxycoccus of the genus Vaccinium. In some methods of classification, Oxycoccus is regarded as a genus in its own right... |
1 cup (whole berries) | 8,983 |
| Artichoke hearts | 1 cup, cooked | 7,904 |
| Blackberry Blackberry The blackberry is an edible fruit produced by any of several species in the Rubus genus of the Rosaceae family. The fruit is not a true berry; botanically it is termed an aggregate fruit, composed of small drupelets. The plants typically have biennial canes and perennial roots. Blackberries and... |
1 cup (cultivated berries) | 7,701 |
| Prune Prune A prune is any of various plum cultivars, mostly Prunus domestica or European Plum, sold as fresh or dried fruit. The dried fruit is also referred to as a dried plum... |
½ cup | 7,291 |
| Raspberry Raspberry The raspberry or hindberry is the edible fruit of a multitude of plant species in the genus Rubus, most of which are in the subgenus Idaeobatus; the name also applies to these plants themselves... |
1 cup | 6,058 |
| Strawberry Strawberry Fragaria is a genus of flowering plants in the rose family, Rosaceae, commonly known as strawberries for their edible fruits. Although it is commonly thought that strawberries get their name from straw being used as a mulch in cultivating the plants, the etymology of the word is uncertain. There... |
1 cup | 5,938 |
| Red Delicious Red Delicious The Red Delicious is a clone of apple cultigen, now comprising more than 50 cultivars, that was recognized in Madison County, Iowa, United States, in 1880... apple Apple The apple is the pomaceous fruit of the apple tree, species Malus domestica in the rose family . It is one of the most widely cultivated tree fruits, and the most widely known of the many members of genus Malus that are used by humans. Apple grow on small, deciduous trees that blossom in the spring... |
1 apple | 5,900 |
| Granny Smith Granny Smith The Granny Ramsey Smith green apple is a tip-bearing apple cultivar, which originated in Australia in 1868. It is named after Maria Ann Smith, who propagated the cultivar from a chance seedling. The tree is thought to be a hybrid of Malus sylvestris, the European Wild Apple, with the domestic... apple |
1 apple | 5,381 |
| Pecan Pecan The pecan , Carya illinoinensis, is a species of hickory, native to south-central North America, in Mexico from Coahuila south to Jalisco and Veracruz, in the United States from southern Iowa, Illinois, Missouri, and Indiana east to western Kentucky, southwestern Ohio, North Carolina, South... |
1 oz Ounce The ounce is a unit of mass with several definitions, the most commonly used of which are equal to approximately 28 grams. The ounce is used in a number of different systems, including various systems of mass that form part of the imperial and United States customary systems... |
5,095 |
| Sweet cherry | 1 cup | 4,873 |
| Black plum Plum A plum or gage is a stone fruit tree in the genus Prunus, subgenus Prunus. The subgenus is distinguished from other subgenera in the shoots having a terminal bud and solitary side buds , the flowers in groups of one to five together on short stems, and the fruit having a groove running down one... |
1 plum | 4,844 |
| Russet potato Russet Burbank potato The Russet Burbank potato is a large brown-skinned, white-fleshed cultivar of potato. Luther Burbank developed the Burbank potato in Lunenburg, Massachusetts, U.S., in the early 1870s. In 1875, Burbank sold his farm and the rights to his potato, and moved to Santa Rosa, California... |
1, cooked | 4,649 |
| Black bean Black bean Black bean can mean:* Black turtle bean, a small black variety of the common bean* Douchi, a kind of fermented soybean* Urad bean, also known as black gram* Castanospermum australe, also known as a blackbean... |
½ cup dried beans | 4,181 |
| Plum | 1 plum | 4,118 |
| Gala apple Gala (apple) Gala is a clonally propagated apple with a mild and sweet flavor. Gala apples ranked at number 2 in 2006 on the US Apple Association's list of most popular apples, after Red Delicious and before Golden Delicious, Granny Smith, and Fuji .... |
1 apple | 3,903 |
With nearly all vegetables, conventional boiling can reduce the ORAC value by up to 90%, while steaming retains more of the antioxidants.
Comparisons of ORAC values
When comparing ORAC data, care must be taken to ensure that the units and food being compared are similar. Some evaluations will compare ORAC units per gram of dry weight, others will evaluate ORAC units in wet weight and still others will look at ORAC units per serving. Under each evaluation, different foods can appear to have higher ORAC values. For example, although a raisin has no more antioxidant potential than the grape from which it was dried, raisins will appear to have a higher ORAC value per gram of wet weight than grapes due to their reduced water content. Likewise, large water content in watermelon can make it appear as though this fruit is low in ORAC. Similarly, the typical quantity of food used should be considered; herbs and spices may be high in ORAC, but are applied in much smaller quantities than for other foods.Numerous health food and beverage companies and marketers have capitalized on the ORAC rating by promoting products claimed to be "high in ORAC". As most of these ORAC values have not been independently validated or subjected to peer review
Peer review
Peer review is a process of self-regulation by a profession or a process of evaluation involving qualified individuals within the relevant field. Peer review methods are employed to maintain standards, improve performance and provide credibility...
for publication in scientific literature, they remain unconfirmed, are not scientifically credible, and may mislead consumers.
See also
- Polyphenol antioxidantPolyphenol antioxidantA polyphenol antioxidant is a type of antioxidant containing a polyphenolic substructure. Numbering over 4,000 distinct species, many of these compounds have antioxidant activity in vitro but are unlikely to have antioxidant roles in vivo...
s - Reduction potentialReduction potentialReduction potential is a measure of the tendency of a chemical species to acquire electrons and thereby be reduced. Reduction potential is measured in volts , or millivolts...
- List of antioxidants in food
- TroloxTroloxTrolox is Hoffman-LaRoche's trade name for 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, a water-soluble derivative of vitamin E. It is an antioxidant, like vitamin E, and is used in biological or biochemical applications to reduce oxidative stress or damage.Trolox equivalent antioxidant...
(TEACTrolox equivalent antioxidant capacityTrolox equivalent antioxidant capacity measures the antioxidant capacity of a given substance, as compared to the standard, Trolox. Most commonly, antioxidant capacity is measured using the ABTS Decolorization Assay...
)
External links
- ORAC levels of various foods These are "old" data from the ORAC assay used before 2004 and should be viewed only as qualitative rankings. A revised ORAC assay used since 2004 provides quantitative values several times higher than these measurements. See an abstract of the reference report at this link from PubMed.
- Database of ORAC values based on 2007 USDA study, sortable alphabetically or by ORAC level
- Effect of soaking, boiling, and steaming on total phenolic content and antioxidant activities of cool season food legumes, Feb 2, 2008.