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Choosing antioxidants for food and medical applications Dr Karen

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					               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.




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