Choosing antioxidants for food and medical applications Dr Karen Schaich, from Rutgers University in New Jersey, looks at choosing antioxidants for food and medical applications Interest in natural antioxidants for both health and for improved food stabilisation has intensified dramatically over the past ten years. Food as medicine is a current hot trend that is capturing everyone’s imagination with images of a new “magic bullet” or “fountain of youth”. Antioxidants that that have traditionally been used to inhibit oxidation in foods also quench dreaded free radicals and stop oxidation chains in vivo, so they have become viewed by many as nature’s answer to environmental and physiological stress, aging, atherosclerosis, and cancer. For the food industry, moving to natural antioxidants is a potential goldmine that offers a “green” label for food stabilisers plus intriguing new opportunities for formulating for health and specific medical benefits. In this context, our mothers’ admonitions to “Eat your fruits and vegetable!” and the old adage the “You are what you eat!” take on dramatic new meaning. The nutraceutical trend towards doubling the impact of natural antioxidants that stabilise food AND maximise health impact presents distinct challenges in evaluating antioxidant activity of purified individual compounds, mixed extracts, and endogenous food matrices and optimising applications. Determining antioxidant capacity has thus become a very active research topic, and an alphabet soup of assays has evolved to screen natural materials and identify likely candidates that will extend the shelf life and quality of both foods and human beings. The question is, what do these assays really tell us and which assay(s) will most accurately reflect antioxidant effectiveness in both foods and animals? The answer is still anything but clear. The most popular screening assays have been developed to be fast, easy, and use commonly available instrumentation, but they don’t all measure the same chemistry. ORAC (Oxygen Radical Absorbance Capacity)1-3, TRAP (Total Radical-Trapping Antioxidant Parameter)4-6, CL (chemiluminescence)7-9, TOSC (Total Oxidant Scavenging Capacity)10,11 and TAC (Total Antioxidant Capacity)12-14 assays measure abilities of compounds to quench radicals by transferring hydrogen atoms to reform the original compounds. In FRAP (Ferric Reducing Antioxidant Power)15-17 and CUPRAC (copper reduction)18,19 assays, compounds transfer electrons to reduce radicals to ions. These assays paradoxically also reveal pro-oxidant potential since reduced metals are active propagators of radical chains. To complicate matters further, TEAC (Trolox Equivalent Antioxidant Capacity)20- 23 and DPPH (diphenylpicrylhydrazyl)24-27 assays, based on reactions of stable free radicals, act by both mechanisms depending on the compound and the reaction conditions. Not surprisingly, tests of all kinds of plants with these assays have documented strong activity in brightly coloured red, purple, yellow, orange, and deep green materials that have high polyphenol concentrations3,28-37 (as mothers instinctively know). However, interpreting results superficially without careful consideration of reaction details in individual systems, and extrapolating results to more complex systems indiscriminately without considering critical differences, present several dilemmas that can limit the usefulness and accuracy of these assays beyond screening. Dilemma 1: Inconsistent antioxidant activity in different assays Antioxidant activity and mechanisms are system-dependent and vary with radical targets, individual and total antioxidant concentrations, solvent, antioxidant phase localisation26, presence of competing compounds including metals, sometimes pH, and presence of oxygen38. An antioxidant may act by one mechanism in system A and another mechanism at a different rate in System B; it may be catalytic at high concentration but protective at low levels. Mismatch between antioxidant mechanisms and assay reactions is one reason why total phenolic content often does not correlate with measured antioxidant capacity of natural extracts. However, such differences can be exploited to advantage. Integrating results from multiple assays with different endpoints can elucidate subtle but important differences in reactivity between compounds, as well as changes in reaction rates and mechanisms with solvent, environment, and antioxidant concentration. It can also reveal conditions under which antioxidants should not be used!39 Dilemma 2: Different activities in intact materials vs. mixed extracts vs. purified individual compounds There is a tendency to expect that if a compound is found to be the “active” component of a natural material by a given assay, it must be more effective if isolated and concentrated in pure form. However, individual antioxidants often behave differently in intact materials (e.g. ingested food), extracts containing multiple antioxidants with different solubilities and reactivities, and isolated form, so which is correct? When multiple components are synergistic, enhancing solubility and providing complementary reaction mechanisms, an antioxidant may be more effective in whole foods and extracts alone. In contrast, when multiple components are competitive, fighting for the same assay substrates and binding sites, activity of individual compounds increases with isolation. These differences need to be recognised and considered when interpreting results and developing applications. Dilemma 3: Results from different labs not comparable in format or values Even when established methods are supposedly followed, variations in details of operating procedures, methods of calculation, and reporting format from lab to lab contribute to inconsistent and contradictory reports of actual and relative antioxidant “capacities” of natural materials and make it often impossible to compare results between labs. The problem has become especially critical since manufacturers are now using ORAC values in advertising and product claims. ORAC units as area under the curve vary with each recorder and integrator, so unstandardised values are meaningless! International efforts to standardise assay methods in two International Congresses on Antioxidant Methods (2004 and 2005) are a step in the right direction40,41, but consistent and reproducible results will also require much more deliberate consideration of the chemistries involved in each reaction and system than are usually given.39,42 Dilemma 4: Assays often poorly predict antioxidant effectiveness in real systems in vitro It is tempting to extrapolate results of antioxidant assays to guide effective stabilisation of foods and cosmetics in vitro or to design nutraceuticals or pharmaceuticals for in vivo therapies. However, screening assays that monitor quenching of a single target radical under limited reaction conditions are poor models for antioxidants or antioxidant mixtures that must control multiple oxidative reactions simultaneously active in the complex systems of foods and biological tissues.39 Phase partitioning of radicals and antioxidants between lipid and water in real systems introduces further complications.42 In foods, the dominant radicals arise from oxidising lipids, but aqueous radicals may also arise from metals, photoinitiators, and perhaps also proteins. A very hydrophobic antioxidant will localise in the lipid phase and inhibit radical chains that are already active, but will not stop initiations. Hydrophilic antioxidants are more efficient in blocking hydroxyl radicals, superoxide anion, and other radicals in the aqueous phase but have little influence on reactions in the lipid phase once they are initiated. Some antioxidants partition between water and lipid and change their reaction depending on the solvent. Curcumins, for example, scavenge radicals rapidly in lipids but when water is present metal complexation dominates.43 Currently, only one version of the ORAC assay differentiates hydrophilic and lipophilic radical scavenging44, and no assay investigates solvent effects. Thus, predicting effectiveness in complex systems or designing applications strictly from assay results are often not successful. Chemistry is only a small part of antioxidant bioactivity. Moving up another level of complexity, chemical antioxidant assays conducted in the test tube are poor models of how antioxidants act in cells and tissues where radical generation is compartmentalised, antioxidants must be able to reach the radical source to be effective, and absorption processes thus become the controlling issue. Perhaps more importantly, phenolic antioxidants have many effects beyond free radical scavenging, so when the bioactivity being screened involves other mechanisms than, or in addition to, free radical scavenging, correlation with chemical assays is poor. Because of these disconnects, cell cultures should be viewed as their own separate level of antioxidant assay with their own quirks and advantages. Cells are particularly useful for monitoring how much of the antioxidant is taken up and by what pathway, determining reaction mechanisms and dose-response relationships – how much antioxidant is needed to induce an action and changes in response with dose level, and for observing the range of cellular responses to various challenges. Nonetheless, cell behaviour is closely linked to cell growth cycle, number of passes in cell culture, and source of cells especially for the popular Caco-2 intestinal cells where flavonoids alter proliferation and differentiation45, so problems with within lab and between-lab reproducibility can be significant. A final precaution -- neither chemical nor cell assays extrapolate to in vivo applications where what happens in the stomach and intestine determines antioxidant access to other tissues. Dilemma 5: Assays have questionable relevance and extrapolatability to bioactivity in vivo In vitro chemical assays of free radical scavenging are poor surrogates for biological activity in vivo because they provide no information about absorption, metabolism, tissue distribution, and excretion; they do not account for indirect action at a distance; and they assume that radical scavenging is the only antioxidant action while in fact it may be among the least important. Furthermore, when adapted to test antioxidant capacity of body fluids or tissues, they are plagued by interferences from cellular reducing agents and proteins and their interpretation is hampered by not knowing the sample composition. Cell cultures are only one step better: they do provide absorption and metabolism information45-49 but the doses applied directly are usually several orders of magnitude higher than could be expected to reach cells after absorption in vivo. Whether activity under such conditions accurately reflects what happens in vivo is thus open to question. Despite this shortcoming, antioxidant action in cell culture is cited in the literature almost universally as if were in vivo. An explosion of new research on antioxidant bioavailability and metabolism shows that while antioxidant vitamins are fully available, uptake of small phenols is lower and variable, and absorption of larger polyphenol molecules is very low to negligible in most cases, with most flavonoids remaining in the intestine.30,50,51 Phenolic compounds that are absorbed appear to be rapidly metabolised, appearing in the urine as methylated, glucuronidated, or sulfated conjugates within hours52-55. The greatest problem is caused by sloppy sensationalism in reporting and interpreting results. Contemporary technology allows very sensitive detection of ever tinier amounts – now picograms or less of material can be accounted for -- and mere “detection” is often presented without absorption calculations as if the full dose were absorbed. Surprisingly, molar concentrations (M) and mole amounts (mol) appear in to be used interchangeably all too frequently, so actual doses and concentrations are unclear. Absorption reported as concentrations in tissues, e.g. ng/ml plasma or g tissue are difficult to convert to total amounts absorbed, which would give a more precise and honest accounting. Methods for detecting trace levels of compounds may also need re-evaluation for accuracy since after-the-fact estimates of total absorption using average rat and human plasma volumes and tissue weights give yields substantially higher than doses in some studies. Clearly, standardisation is needed for in vivo methodology as well as chemical assays. This skepticism aside, critically needed scientifically rigorous studies of antioxidant absorption, distribution, metabolism, and excretion that are beginning to appear show a pattern of very low or selective absorption followed by rapid conjugation and elimination of what little gets through, particularly for flavonoids and other polyphenols.54,55 Improved analytical instrumentation and recent observations that sugars attached to flavonoids increase their absorption may change this picture somewhat. Nevertheless, these observations raise serious questions about the rationale currently underlying antioxidant testing: physiological responses55,56 ranging from inhibition of inflammation and edema, urinary tract infections, cancer, and aging are either exquisitely sensitive to a few molecules or they cannot be explained by direct action of the antioxidants and simple radical quenching alone. We thus need to look beyond traditional thinking to evaluate other mechanisms. To be sure, it is not easy to track the effects of antioxidants in living animals. Test tube and cell studies have revealed that, in addition to reduction of oxidative stress, (poly)phenols complex metals,57, 58 bind to proteins59, 60 and digestive secretions61, and both activate and inactivate enzymes that mediate a wide range of cell responses.62,63 These activities need to be verified in animals. In vivo, polyphenols block estrogen56,64,65 and other66 receptors, and binding to proteins in the intestinal epithelium may alter other receptors and unleash a signal transduction cascade67,68 that leads to systemic response, e.g. massive induction of endogenous antioxidants such as uric acid69 and tocopherol.70,71 Microbial flora digestion of polyphenols is also important,48,72,73 and absorption and bioactivity of these products need to be evaluated. Rational standardised protocols must be developed to determine the full role these various actions play in overall “antioxidant action” of individual and mixed antioxidant compounds and to establish the concentration limits controlling each. Eradicating the dilemmas Antioxidant research had its childhood in finding antioxidants in nearly all natural materials and its puberty in discovering that antioxidants have important bioactivities. Now it is time for antioxidant research to grow up as a field, to move beyond easy screening and shift focus to the more difficult work of systematically elucidating details of how antioxidants work so that when they are used in food formulations, their effectiveness in both foods and in vivo can be maximised. We can eliminate the five dilemmas of antioxidant research listed above first by thinking more about what information is really needed rather continuously running extracts through screening assays just to generate numbers for publications. Next, abandon the “quick and dirty” approach. The complexities of food and physiological applications of antioxidants, separately and combined, require rigorous consideration and analysis of all aspects of the (bio)chemistry, operative reaction mechanisms, and reaction/radical/target specificity in various test systems, as well as careful and accurate quantitation of all reactants and products involved. To do this, some old tests must be abandoned and some will remain useful if more depth and control is incorporated; but, in addition, new approaches must be adapted or developed to provide greater detail of action at the molecular level and account for the multiple complex actions of antioxidants in both foods and living systems. Accomplishing this will reveal the full power of antioxidants, in all its forms, and point the way to more effective utilisation of natural antioxidants in foods, nutrition, and medicine. References 1. 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