Antioxidants in foods have gained much attention in recent years and cocoa powders tend to have relatively high amounts. As cocoa makes its way from fresh beans to finished products like cocoa powder and chocolate, the concentration of antioxidant compounds can be affected by a variety of biological and processing conditions. Genetics can cause as much as a 4-fold difference in antioxidant content of fresh cocoa beans (3, 4). Fermentation of fresh cocoa beans, while critical for full cocoa flavor, also tends to decrease antioxidant content. Roasting of cocoa beans and treatment of cocoa powder with alkali can also decrease the final content of antioxidants. A study of the antioxidant content of various alkalized cocoa powders reveals that the antioxidant content decreases proportionally with the amount of alkalization(2). Due to the effect of all of these factors, it has become important to identify the specific types of antioxidants in cocoa products and develop analytical measurements for their contents.
There are several types of antioxidant compounds found in cocoa powder. There has recently been a great deal of interest in polyphenolic compounds, particularly flavonoids, as antioxidants. Flavonoids are synthesized by all vascular plants. As a result, fruits, vegetables, nuts, seeds, herbs, spices, and whole grains are sources of flavonoids in our diet(5). Cocoa beans are a concentrated source of antioxidants(6) and flavonoids with the flavan-3-ols and their derivatives being present in high concentrations(7).
The discovery of flavan-3-ols and their procyanidin polymeric forms in cocoa can be traced back as early as 1909(8). These flavan-3-ol compounds were later identified as catechins(9, 10). In 1939, leucoanthocyanin phenolic compounds were identified(11) and, in 1955, fractionation and characterization of these compounds was reported(12). The procyanidins in cocoa have more recently been fractionated into monomers through decamers with even higher forms existing (13). The most abundant polyphenols present in cocoa are the flavan-3-ol monomers, epicatechin and catechin(14), which also serve as building blocks for the polymeric procyanidin forms. The makeup of the polymeric forms is determined by the structure of the flavan-3-ol starter unit and its companion compound. Two primary forms of procyanidins occur: A-type and B-type which differ by the linkage between the individual compounds. The A-type procyanidins form 2-7 cross links and can be found in cranberries. The B-type procyanidins form 4-8 cross links. The B-1 through B-4 types differ only in the arrangement of their catechin and epicatechin units with procyandin B-1 found in grape, sorghum and cranberry, type B-2 in apple, cocoa and cherry, B-3 in strawberry and hops, and B-4 found in raspberry and blackberry(15).
The number of antioxidant compounds within a particular food is believed to be an important factor in determining nutritive value. Antioxidants can be measured indirectly by activity-based tests or directly by compound-based tests.
Activity-based tests do not measure specific phytonutrients; rather these tests measure the total capacity of compounds within a food/beverage to contribute to antioxidant activity. Most activity-based tests are conducted in test tubes. As a result, there is some question if antioxidant activity in a test tube is a reliable indicator of added antioxidant benefits in the body.
Activity-based tests are particularly useful when comparisons of foods/beverages are made because antioxidant activity represents a “common currency” measure. Activity-based testing cuts through the difference in phytochemical content of differing foods/beverages permitting scientists and marketers to make rough comparisons of foods/beverages based on the results of these tests.
Activity-based tests have various four to five letter names such as ORAC, FRAP, TEAC, etc. By far the most common measure in the US is an assay developed and perfected by USDA scientists called Oxygen Radical Absorbance Capacity (ORAC)(16). The most recent form of the test measures water-soluble as well as oil-soluble antioxidants.
ORAC is a measure of general antioxidant activity in the food/beverage and is commonly used in the US for comparison of different foods/beverages to one another. USDA has published databases of foods/beverages with ORAC measurements expressed on an equal weight basis and on a per serving basis. Cocoa powder and dark chocolate are in the top 3% and milk chocolate is in the top 10% of foods/beverages with antioxidant activity. ORAC is typically expressed as micrograms of Trolox equivalents per gram or serving of food/beverage.
Compound-based tests measure the actual amount of chemical compounds in foods/beverages. General compound tests measure broad classes of compounds, whereas specific compound tests measure individual or small groups of very specific compounds within the food/beverages. Specific measures focus on compounds that are either known to be important to human nutrition such as vitamins, lycopene, fiber or compounds implicated in health benefits. Examples of compound-based tests that are used to measure specific classes of antioxidant compounds in cocoa include total polyphenols, flavanol monomers, proanthocyandins (PACs), and 4-dimethylaminocinnamaldehye (DMAC).
Total Polyphenols is a general chemical measure of compounds that have a phenolic group associated with them(17). There are literally thousands of compounds identified in raw materials that will be measured by the total polyphenol test. Some of the compounds measured will have bioactivity while others will not be bioactive. Cocoa powder is a food that is high in polyphenols. Other foods that have high polyphenols include spices, tea, and coffee. Total polyphenols are typically expressed as mg of gallic acid or mg of epicatechin per gram or serving of food/beverage.
Flavanols are thought to be the chemicals that are, at least in part, responsible for some of the healthful benefits of cocoa and chocolate. Direct measurement of the flavanols in cocoa and other foods can be done by several means, each method focusing on various size classes of the flavanols. The basic sub-units of the flavanols are epicatechin and catechin, which can be linked into polymers. Direct measurement of the subunits of the flavanols can be done in which epicatechin and/or catechin monomers are isolated from the food and separated (19). In this case, results are expressed as mg of the specific monomeric unit using pure epicatechin and catechin as standards.
PACs – Flavanol polymers, sometimes called procyanidins, proanthocyanidins or PACs, can be measured in cocoa powder and chocolate as well as in other foods. These PACS can be isolated and separated into their component parts including the monomeric subunits, small polymers and long chain polymers. The various polymeric flavanols can be isolated by HPLC based on their size (20). These peaks are then quantified using chemical standards isolated from cocoa or other flavanol-rich plants. Data can be reported for individual polymeric units or as a sum of all monomers and polymers as mg of PACs. The availability of specific standards for each polymer has been lacking in the past and, as a result, much of the PAC data in the literature is based on differently prepared standards. This makes it difficult to compare data from different labs. Recently, pure polymeric standards have become available from dimers through decamers. The Hershey Company has recently chosen to report all new PAC data as the sum of flavanols including monomers through decamers in chain length (PAC-10).
DMAC is a more specific measure of the total proanthocyanidins (PACs) content and is based on the colorimetric reaction of a specific reagent, 4-dimethylaminocinnamaldehyde, or DMAC, with the proanthocyanidins in a food or beverage.(18) The Hershey Company and other collaborating labs have chosen to use the commercially-available flavanol dimer B2 as the standard for this test. Results are reported as mg of total proanthocyanidins on a B2 dimer basis, per gram of sample. This relatively new test allows the more accurate reporting of total proanthocyanidin content in a sample.
The antioxidant activity and compounds in various cocoa powders have been measured using techniques described in this section. Cocoa powders listed in the table below include HERSHEY’S Cocoa (a natural cocoa powder) and HERSHEY’S SPECIAL DARK Cocoa (a blend of natural and alkalized cocoa). Also included are a variety of commercial cocoa powders that have been alkalized to different degrees ranging from natural/non-alkalized (pH 5.56), to lightly alkalized (pH 6.97), to moderately alkalized (pH 7.35), and to heavily alkalized (pH 7.88).
| Cocoa Powder Description
||ORAC (micromoles TE/g)
||Total Polyphenols (mg/g)
||Flavanol Monomers (mg/g)
|HERSHEY’S SPECIAL DARK Cocoa
|Commercial Natural Cocoa
|Lightly Alkalized Cocoa
|Moderately Alkalized Cocoa
|Heavily Alkalized Cocoa
Data from Miller et al., 2008 (2)
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