Nutraceuticals that boost antioxidant intake are a growing niche but producers must ensure on-pack claims measure up, as Jan and Robert Knight of Knight Scientific explain
Reactive oxygen species (ROS) and free radicals are continually produced in the body and are continually destroyed by substances collectively known as antioxidants. ROS have important functions in the body, both good and bad. But when the production of these highly reactive molecules exceeds the supply of antioxidants necessary to keep them under control oxidative stress occurs.1
Oxidative stress occurs at sites of inflammation when billions of ROS-producing white blood cells accumulate and, instead of confining their activity to ridding the body of pathogens, an excess of free radicals and ROS injure and kill healthy tissue, damage DNA and attack enzymes. ROS are implicated in more than 150 pathological conditions including cancer, heart disease and diabetes.
Much effort is presently focused on changing oxidative status through diet and supplements. Epidemiological studies over the past 20 years have shown that diets rich in fruits, vegetables and grain products can lead to significant reductions in chronic diseases, in particular heart disease and certain cancers.2 The protective effects have been largely attributed to antioxidants.
The principle antioxidants derived from food are vitamin E, beta-carotene and vitamin C. In addition, the trace element selenium is required for the proper functioning of the antioxidant enzyme glutathione peroxidase. There is a whole host of other antioxidant phytochemicals, such as lycopene from tomatoes and allylic sulphides from garlic, that are credited with reducing the risk of cancer and heart disease. And the understanding that vitamin E is a generic term encompassing the family of tocopherols and tocotrienols is leading to a greater understanding of how best to use these phytochemicals prophylactically and therapeutically.
Fundamental to this whole story is that the body cannot manufacture these micronutrients so they must be provided in the diet; as a result such nutraceutical products are addressing markets worth billions of dollars.3
However, to meet the aspirations of discerning consumers the industry must be able to produce products with quantifiable and reproducible activity. It is fundamental that industry demonstrates adequate quality control of materials before, during and after processing, and quality assurance of finished products.
Measurement of the concentration of specific antioxidants in a product is one approach to quality assurance. However, this approach can be misleading, because the sum of the contributions of the specific antioxidants in the product may not match the total antioxidant activity of the product. This is because certain members of the complex mixture of molecules in the product may act in synergy leading to enhanced, or perhaps diminished, antioxidant capacity. It is therefore more revealing to use the total relative antioxidant capacity (RAC) of a unit weight of the product as well as that of its components.
Factors affecting differences in antioxidant capacity of a product may be related to the source of the constituent materials and their conditions of cultivation, transportation and storage prior to processing as well as to the conditions of processing and the treatment of nutraceutical products after manufacture. For example, irradiation of ingredients (see figure 1) as well as of the finished product will reduce antioxidant activity because free radicals produced during irradiation may attack some or even all of the antioxidants present in the sample.
And if all the antioxidants are attacked but free radical production continues then pro-oxidants - molecules that when attacked by free radicals produce even more free radicals, - may be generated, sometimes, and perversely, from antioxidants that have lost their own activity.4
During the manufacture of nutraceuticals free radicals can be produced from: grinding, compacting, exposure to UV light, drying, heating and especially by sonication. It is therefore desirable to monitor the RAC of the ingredients and of the finished product throughout the various stages of manufacture and if necessary to change procedures to avoid losses.
Many analytical methods exist for the measurement of the concentration and purity of individual ingredients. Other methods aim to measure the functional capacity of a material, for example, its capacity to neutralise free radicals. Functional analysis gives the best measure of the final biochemical actions of the material when it has been absorbed into the body.
In most analysis methods a sample is exposed to free radicals or oxidants and the amount of quenching is measured and quantified against a standard of known concentration. Such tests include: TEAC (Trolox-Equivalent Antioxidant Capacity), involving production of a coloured intermediate that is decolourised on exposure to antioxidants; ORAC (Oxygen Radical Absorbance Capacity), used extensively to score foods for their antioxidant capacity; and TRAP (Total Peroxyl Radical Trapping) in which peroxyl radicals are generated and used in the presence of a fluorescent molecule that decays when attacked by radicals.
All the tests have their deficiencies: lack of reproducibility, especially between laboratories; time taken to complete; sensitivity. Moreover, none of these identifies pro-oxidants, or the formation of pro-oxidants as a function of concentration. Knowledge of the latter is fundamental to a consideration of formulations and dosages. Moreover, not all antioxidants react with all known oxidants, so for a total analysis more than one system of analysis may be needed.
Knight Scientific (KSL) has developed five tests for quantifying antioxidant and pro-oxidant capacity,5 all based on the use of a substance that emits light in the presence of free radicals and oxidants.6 In KSL's ABEL assays samples containing unknown antioxidants are challenged with defined oxidants: superoxide (high concentration), superoxide (enzymatically produced), hydroxyl radical, peroxynitrite and hypochlorous acid while in the presence of the luminescent material. The resulting light is related to the antioxidant activity of the sample.
The luminescent material, which is unique to both these tests and to KSL, is Pholasin, a protein extracted from a marine bioluminescent mollusc, Pholas dactylus.
The RAC of a material can be calculated in terms of weight, cost and dose. A sample is challenged with a particular free radical or oxidant in the presence of the light-emitting protein Pholasin. If the material has antioxidant capacity, it will compete with Pholasin for the free radical or oxidant, reducing the amount of light emitted by Pholasin and sometimes delaying the time at which maximum light from Pholasin is detected. By running a range of concentrations of the material to be tested, the effective concentration (EC50) of the sample (ie, the concentration of material able to reduce the light by 50%, normalised to g/L or mg/mL), is determined (see figure 2). The greater the amount of material required to reduce the light by half, the weaker the antioxidant capacity. Thus a high EC50 value indicates a low antioxidant capacity.
ABEL-RAC scores are the reciprocal of the EC50 multiplied by 100; the higher the ABEL-RAC score, the higher the antioxidant capacity of the sample. Moreover, for greater precision, the score may be expressed in terms of the oxidant used in the test, ABEL-RAC peroxynitrite, for example.
The most usual way of expressing the score is in terms of weight. Thus: ABEL-RACmg. However, simple calculations will lead to the very useful parameters, ABEL-RACcost and ABEL-RACdose.
ABEL-RAC scores of ingredients can be used to predict the score of the finished product. However, the score of the finished product must also be measured because of the possibility of synergy between ingredients leading to an enhanced or diminished score. Both positive and negative synergy have been recognised (see table 1).
ABEL-RAC enables comparisons to be readily made between different materials and batches. The assays can be used to assess antioxidant capacity at different concentrations and to identify those ingredients that do not follow typical dose responses but are pro-oxidant at some concentrations and antioxidant at others. Such unusual behaviour is known in toxicology as hormesis (see figure 3).
KSL's ABEL antioxidant assays may be run on either microplate or tube luminometers. The results are highly reproducible with a cv of less than 3% within and between assays. Testing of samples may be carried out in-house using the commercially available ABEL kits. Alternatively, samples may be sent for analysis to KSL or other contract testing laboratories.
ABEL assays have been used:
- to demonstrate batch-to-batch uniformity of natural ingredients,
- to exclude materials that may have lost antioxidant capacity during irradiation and other treatments,
- to assess synergistic effects when ingredients are mixed in products,
- to understand how activity of a product changes with increasing or decreasing concentration of its constituents, and
- to monitor changes occurring during manufacture.