Testing times

Detecting levels of the good and the bad in nutraceuticals, food and cosmetics

The luminescent material Pholasin is extrtacted from a marine mollusc, Pholas dactylus, the common piddock

Free radicals and other reactive oxygen containing species (ROS) are continually produced in the body and are continually destroyed by antioxidants . However, when ROS production gets out of control oxidative stress occurs. This is especially noticeable at sites of inflammation where billions of ROS-producing white blood cells accumulate. When this happens these reactive chemical species, together with enzymes released from granules within the white blood cells injure or even kill cells, damage DNA and attack enzymes and other compounds. There are however other occasions, during the course of normal cell respiration for example, when free radicals and ROS are produced as by-products of cellular metabolism. And here again, in the absence of sufficient quantities of appropriate antioxidants, oxidative stress and concomitant tissue damage can occur, sometimes over a very long period of time.
There are over 150 diseases, including heart disease, diabetes, neurodegenerative diseases and many cancers, in which these highly reactive oxidising species are implicated in one way or another. And it is generally agreed that there is a correlation between ageing and the accumulation of oxidatively damaged proteins, lipids and nucleic acids .

Oxidative stress is bad for the skin . Free radical damage can cause deterioration of the supportive connective tissues resulting in decreased elasticity and resilience. Exposure of skin to solar ultraviolet radiation starts photochemical reactions in the skin leading to ROS formation. Sun damage produces both skin cancers as well as photo-ageing which shows itself on the skin as wrinkling, scaling, dryness and mottled pigmentation.
Antioxidants play an important part in protective and repair mechanisms within the skin. The antioxidants may be consumed from food and supplements, manufactured in the skin or applied to the skin as topical preparations. The antioxidants of most significance in slowing down free-radical damage to the skin are: the vitamins A, C and E, the enzyme superoxide dismutase, the group of chemical compounds known as flavonoids, and the individual chemical substances beta carotene, glutathione, selenium and zinc.
The relevance here to the cosmetics industry is that there have been many studies demonstrating beneficial health effects resulting from topical application of preparations containing antioxidants. While the underlying mechanisms for these effects are not fully understood, enough is known about the distribution, activity and regulation of antioxidants in the skin to enable the development of effective pharmaceutical and cosmetic strategies involving antioxidant formulations. These developments are aimed at reducing the risk of UV induced cancers, photo ageing and desquamatory skin disorders.

Included among products commonly seen to have antioxidant properties are nutraceuticals, functional foods and cosmetics
Nutraceuticals are food-derived products containing ingredients with claimed health benefits. The term ‘functional food’ is used for any food or food ingredient that may provide a health benefit beyond the traditional nutrients it contains. The potential of functional foods to mitigate disease, promote health and reduce health care costs is fueling the fastest growing sector in the food industry - nutraceuticals. Major food processing companies (Unilever, Nestlé and Kraft) are creating high price-margin food and nutraceutical products with claimed health benefits. These products are addressing world markets worth $196 billion in 2005.
Cosmeceuticals are personal care products intended to have therapeutic effects when applied to the body. This fastest growing sector of the cosmetics industry has been forecast to be $5.8 billion by 2008 . It is therefore not surprising that so many companies are actively involved in research and development of new products to satisfy this growing demand.

Our normal source of antioxidants is our food, especially fruit and vegetables. However, to make sure that we have enough antioxidants in our diet even though we may not eat the recommended five portions of fruit and vegetables a day we may have recourse to sources other than normal food.
Consumer preferences in personal care products are being influenced by consumer trends in the food and beverage industry and vice versa. Natural organic products, vitamins and multipurpose products are gaining in popularity among consumers. And while the claims made for dietary supplements cannot be applied to cosmetics, the consumer perceives the same kind of benefit irrespective of whether she or he “eats it or wears it”.
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 being 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 much greater understanding of how best to use these phytochemicals prophylactically and therapeutically. What is fundamental to this whole story is that the body cannot manufacture these micronutrients so they must be provided in the diet.

Manufacturers beware! While measurement of the concentration of specific antioxidants in a product is one approach to quality assurance, this approach can be misleading because the sum of the contributions of the specific antioxidants in the product may not match its total antioxidant activity. This is because synergistic interactions between a complex mixture of molecules may lead to enhanced, or even diminished, antioxidant activity. It is therefore more revealing to use the total relative antioxidant capacity (RAC) of a unit weight of a product than the sum of the component activities. This is analogous to the specific activity of enzymes in which enzyme activity is expressed per weight of protein.
Factors affecting differences in antioxidant capacity of materials from different

Bottled Pholasin and light organs of the piddock

sources may be related to conditions of cultivation, storage and transportation prior to and during processing. In addition, the treatment of ingredients and finished products before, during and after manufacture affects capacity. For example, irradiation of ingredients as well as of the finished product will lead to loss of 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, which are molecules when attacked by free radicals produce even more free radicals, may be generated, sometimes from antioxidants that have lost their own activity .

There are a number of methods available for the measurement of antioxidants including: TEAC (Trolox-Equivalent Antioxidant Capacity); ORAC (Oxygen Radical Absorbance Capacity); TRAP (Total Peroxyl Radical Trapping), FRAP (Ferric Reducing Antioxidant Power). Each of these tests has its particular deficiencies: lack of reproducibility between replicates and between laboratories, time taken for completion, sensitivity, restriction on use of solvents, only clear solutions can be assayed and some only work at non-physiological conditions of pH and temperature. Moreover none of the tests 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. And not all antioxidants react with all known oxidants, so for a total analysis more than one system of analysis may be needed. All of these shortcomings are remedied in the ABEL-RAC methods introduced by Knight Scientific (KSL).
ABEL is an acronym for Analysis By Emitted Light: RAC for Relative Antioxidant Capacity. The five ABEL antioxidant tests  that quantify antioxidant and pro-oxidant capacity are all based on a substance that emits light in the presence of free radicals and oxidants. This luminescent material, Pholasin, is unique to these tests (and to Knight Scientific) and is extracted from a marine mollusc, Pholas dactylus, the common piddock.
In the ABEL assays samples containing unknown antioxidants are challenged with defined oxidants: superoxide (high concentration), superoxide (enzymatically produced continually at a precise concentration), hydroxyl radical, peroxynitrite or hypochlorous acid in the presence of Pholasin. Other ABEL assays can determine the activity of superoxide dismutase and peroxidase enzymes. The diminution in light emitted by Pholasin is related to antioxidant activity and can be quantified as ABEL-RAC scores for each type of challenge.
In the ABEL assays samples containing unknown antioxidants are challenged with defined ROS: superoxide (high concentration), superoxide (enzymatically produced), hydroxyl radical, peroxynitrite or hypochlorous acid, all in the presence of the light-emitting protein Pholasin. If the material has antioxidant capacity, it will compete with Pholasin for the particular ROS, reducing the amount of light emitted by Pholasin and sometimes delaying the time at which maximum light is detected.
By running a range of concentrations of material to be tested, the concentration of material able to reduce the light by half, the effective concentration (EC50) of the sample, is determined. The EC50 is converted to an ABEL-RAC mg score: (1/EC50) x 100 for each particular challenge.
It is also possible to determine from the kinetics, for example, if a material is acting as a chemical quencher of superoxide or as a mimetic of the enzyme superoxide dismutase. The results can in addition be expressed as equivalent values derived from sets of standards of pure antioxidant substances.
The most usual way of expressing the score is in terms of weight. Thus: ABEL RAC-mg. However, simple calculations will lead to the very useful parameters, ABEL RAC-cost and ABEL RAC-dose.
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
ABEL-RAC is easy to understand and enables comparisons to be readily made between different materials and batches as well as challenged with different ROS (Figure 5). 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

Manufacturers and suppliers of products based on natural materials need to be able to satisfy themselves and their customers that the products they are selling can be matched precisely, batch to batch, for their efficacy. The quantification of antioxidant activity can be used as a QA tool for parameters such as stability, uniformity of ingredients and finished products, possible changes during manufacture and shelf life of products. Quality assurance of nutraceutical products on which to base statements regarding their potential benefits to the consumers is the way forward for the fastest growing sector in the food industry.

By Dr Jan Knight.
Jan is MD and co-founder of Knight Scientific. She isd involved in applications of Pholasin to inflammation, free radical and oxidative stress research.

References
  1.Halliwell, B., Gutteridge, J (2000) Free Radicals in Biology and Medicine, Oxford University Press,  ISBN 0198500459
 2. Stadtman, E R (2001) Protein Oxidation in Aging and Age-Related Diseases Annals NY Acad Sci 928:22-38
 3. Thiele, J J (2001) Oxidative Targets in the Stratum Corneum: A New Basis for Antioxidative Strategies Skin Pharmacology and Applied Skin Plysiology 14: 87-91
 4. Pelle E, Muizzuddin N, Mannone T et al (1999) Protection against endogenous and UVB-induced oxidative damage in stratum corneum lipids by an antioxidant-containing cosmetic formulation. Photodermatol Photoimmunol Photomed 15(3-4) 115-9.
 5. Thiele, JJ (2001) Oxidative Targets in the Stratum Corneum Skin Pharmacology & Applied Skin Physiology 14:87-91.
 6. Thiele JJ, Schroeter C, Hsieh SN, Podda M, Packer L (2001) The Antioxidant Network of the Stratum Corneum. Curr Probl Dermatol 29:26-42.
7. Russo, T (2003) STAT-USA Market Research Reports (www.stat-usa.gov)
8.  Nutrition Business Journal, Burrill & Company
9. (2003) The US Market for Natural Personal Care Products: Beauty and Grooming for a New Age published by Packaged Facts  www.mindbranch.com.
10. Knight, J., Ganderton, M., Hothersall, J, Zitouni, K. & Nourooz-Zadeh, J. (2002). The ABEL® peroxynitrite antioxidant test with Pholasin® measures the antioxidant capacity of plasma to protect against peroxyl radical attack. In  Phil Stanley, Larry Kricka  Editors Bioluminescence & Chemiluminescence: Progress & Current Applications World Scientific Publishing Co. Ptc. Ltd. 257-260.
11.  Knight J, Armstrong K & Ganderton M (2006) ABEL Antioxidant Assays (download) www.knightscientific.com