1 General introduction – food-based natural antioxidants
The dictionary definition of an antioxidant is “any substance that retards deterioration by oxidation, especially of fats, oils, foods, petroleum products or rubber.” This article will concentrate on natural antioxidants, rather than synthetic antioxidants like BHT that are added to food to extend its shelf life. The term “food-based” is used to distinguish between antioxidants present naturally in the food we eat, and antioxidant supplements, that are often extracts/concentrates prepared from foods, which at the time of writing have not been found to be beneficial. Food-based natural antioxidants (FBNA) are thought to be useful in inducing the balanced neutralisation of harmful excesses of reactive oxygen species (ROS) such as free radicals and peroxides found in all the organs, tissues and fluids of the body. ROS are the natural by-products of necessary energy generating metabolic processes that, in moderate concentrations, are employed beneficially by the immune system to kill pathogens, and in general, to act as cell signalling chemicals. Antioxidants entering the body from digested food pass into the blood stream (absorption) from where they travel to target sites (bodily organs, tissues, etc.) undergoing “suicidal” neutralisation reactions with any excess ROS. Those that are not absorbed may well be beneficial in reacting with excess ROS in the lower gut (colon). High concentrations of phenolic acid antioxidants have been found in the colon. The measure of the imbalance between the production of ROS and the body’s ability to detoxify the reactive intermediates or to easily repair the resulting damage caused to proteins, lipids, DNA, etc. is called oxidative stress. Degenerative diseases such as atherosclerosis, Parkinson’s disease, stroke, high blood pressure, coronary heart disease (CHD), Altzheimer’s disease, chronic fatigue syndrome, certain cancers, and ageing are thought to be the result of oxidative stress. Clearly, the control of oxidative stress is of major importance, and the mechanisms involved are being actively studied. Not only is there a delicate balance between the helpful and harmful roles of the ROS, but also, as minute changes to the physiological environment occur, the chemical status of the circulating antioxidants can change into pro-oxidants enabling them to fulfil quite the opposite chemical task. The important message is that the roles of the players in this piece may be constantly changing. The reduction of oxidative stress is a major objective, but only one of many biochemical processes to be kept in equilibrium for the whole system to perform efficiently. This detailed introduction shows the complexity of interactions at the molecular level, and hence the danger that exists in trying to over simplify the behaviour of food-based natural antioxidants. There is much that isn’t understood, even though the popular articles on human nutrition give the impression that it is all done and dusted! On now to the positive information that is available based on reliable epidemiological evidence gathered from large-scale studies of different populations. Some time ago, the World Health Organisation (1991) recommended to consumption of 400 g, five 80 g portions of fruit and vegetables per day, towards a healthy diet. The Committee on the Medical Aspects of Food and Nutrition (COMA) in 1994 announced that this diet also protected against heart disease. More recently, The US National Cancer Institute suggested an increase to nine portions (2-4 fruit and 3-5 vegetables) in order to provide adequate protection from oxidative stress. It is unlikely therefore that many of the population are in danger of consuming too much antioxidant, although in principle it might have the effect of neutralising too many ROS leaving the body susceptible to attack from foreign bodies. Consuming too little, on the other hand, is entirely feasible, allowing ROS to damage proteins, DNA, etc. And so the question is “what is the optimum amount”? Considering that scientists cannot agree about the mechanism of the antioxidant/free radical interaction, it may be wise at this point to withdraw from the arena: taking refuge in the knowledge that diets rich in fruits, vegetables, nuts and grains help ward off degenerative diseases, improve well being and prolong life. So, until more is known about the mechanisms involved, and, even if antioxidants are not the active compounds, it might be a good idea to take out some “life insurance” by ensuring that you eat five to nine portions a day of the recommended foods.

2 Antioxidants – Good, bad or indifferent?
Currently, the debate continues among nutritionists over the question, are organically grown crop foods better for you than chemically assisted crop foods? Perhaps it would be helpful to review the latest thinking about one aspect of the controversy – the antioxidant content. While there is good epidemiological evidence to link human health and well being to diets rich in fruits and vegetables, e.g. the Mediterranean diet, and fruits and vegetables are good sources of antioxidants, it is by no means certain that antioxidants are the biologically active agents. Furthermore, the Mediterranean diet alone cannot be separated from other known factors contributing to the well being of the people who practise it, viz. - cooking in olive oil (high in monounsaturated fats) - eating a diet rich in fish (high in omega-3 polyunsaturated fatty acids) rather than meat - drinking wine (rich in polyphenol antioxidants) in moderation in preference to other drinks e.g. beer, - living in a sunny clime (facile vitamin D production) - eating more local and organic produce (higher levels of vitamins and phytonutrients) - slower, less stressful pace of life, and particularly the attitude that eating should celebrated and not rushed. And after all these additional variables have been taken into account, the best scientific evidence favours the view that, if antioxidants are the “vitamin-like” components of the diet that protect us from degenerative diseases, create general well being and increase life span, then, it is most likely to be a combination that provides the efficacious result. That being so, the job of proving which combinations of the known chemical antioxidant compounds has only just begun. Some examples of synergistic combinations of antioxidants are: - rutin and resveratrol - quercetin and vitamin A and some synergistic food combinations are: - chicken and broccoli (thought to be down to the selenium and sulforaphane content respectively) - spinach and orange juice (vitamin C from the orange juice enhances the release of non-haem iron from the spinach) If, as is suspected, only certain combinations of potential bioactive components are effective, the problems for the analyst intensify. There are many thousands of natural food-based antioxidants occupying several different chemical classes: vitamins, minerals, flavanoids (polyphenols, anthocyanidins, catechins, proanthocyanidins) and carotenoids. Before these compounds were gathered together and labelled as antioxidants, they had other properties of interest to us, the consumer of plant crops. Many of the carotenoid and anthocyanin classes are brightly coloured, making fruits and vegetables attractive. Others, such as the proanthocyanins are employed by plants as astringent repellents to deter “browsing” fauna that may otherwise consume fatal quantities of leaves and stems. So, immediately a conflict arises among the known properties of the astringent proanthocyanidin tannins. They are included in the tally of helpful antioxidants; they are known to behave as antinutritional binding agents for proteins and minerals, preventing them from being absorbed into the blood stream, and they have recently been found to protect the large colon from attack from ROS. Multifunctional roles such as these may well be found for many of the candidate beneficial antioxidants. Evidence from large case-control studies has been reported implies that individual antioxidant supplements are ineffective and that beta carotene actually increases mortality. However, opponents (Linus Pauling Institute) to the statistical analytical methods employed argue that impartial choice of data sources among the hundreds of studies published in the scientific literature – comprising the Mega study - was not adhered to. And so while the debate goes on, the proven fact is that diets rich in fruits and vegetables (along with other factors) have been shown to have beneficial effects on human well being, health and longevity. Whether food-based antioxidants will be shown to provide protection directly, or trigger other processes such as the activation of endogenous antioxidants, to reduce oxidative stress, or maybe some entirely different mechanism has to be postulated, such as the binding by pectin of the protein galectin-3, implicated in the progression of cancer, the subject is currently of great interest to the scientific community.

3 Availability of food-based natural antioxidants - chemical analysis
Researchers studying the natural products of plants in the late 19th and early 20th centuries discovered many of the compounds classes now collectively called natural antioxidants. Many of them were also classed as secondary plant metabolites and considered (at the time) to be superfluous to the essential biochemical processes being studied – the primary plant metabolites. The use of classical chemical methodology received a fillip in the form of gas and liquid chromatography, subsequently enhanced by using mass spectrometry, for detection and quantification of complex gas and liquid extracts from plants. These techniques rapidly separated and identified these mixtures from samples of plant material, especially the food plants. Huge databases of spectroscopic data are now available and easily accessed for comparison with unknowns as they continue to appear from the higher sensitivity analytical instruments in modern laboratories. Thus, equally large databases have been built containing lists of compound classes and individual compounds known to be present in a wide variety of food plants. The next step was to determine the antioxidant potency of each individual compound. This was possible for those compounds that could be synthesised to provide samples for analysis, but huge numbers of trace compounds were recognised and the task became more difficult. Exacerbated by the unknown effect of potency of various quantitative and qualitative combinations present in individual plants proved too daunting and a compromise was accepted, to measure the total antioxidant potency (capacity) of each food. The complete data for around 170 foods are available at the USDA nutrition database. [A convenient abridged list of 100 common UK foods can be found at Total antioxidants ] Now that we are advised to take more of our five-a-day as vegetables than fruits, it is noteworthy that vegetables generally contain less total antioxidants than fruit. This advice is given as a compromise arising out of the higher acidity of fruit. The total antioxidant capacity is a good starting point, but it remains to be proven whether 100 g walnuts with a total antioxidant capacity (TAC) of 13,540 are 10 times as potent as 100 g cabbage with a TAC of 1360. In the test tube it is so, but all antioxidants do not have the same bioavailability, and even for those that are equally well absorbed from the gut, there is no knowledge of the extent to which conflicting physiological roles in the body (in vivo) change the in vitro figures. Comparing the positions of foods in the table of total antioxidant capacity raises further analytical questions. Tomatoes, with their high concentration of the carotenoids, lycopene and others, ought to deserve a higher ranking than 340. However, it is known that lycopene is difficult to extract, and therefore the technique used may not go to completion. Optimisation of extraction is notoriously difficult, especially if a wide variety of textures are encountered, as is the case with crop food structures (from lettuce leaves to Brazil nuts) and therefore a large margin for error should be included in any comparative study. Once again, because of the shear complexity of the analytical task, the picture is incomplete and refinements are expected.

4 Bioavailability of food-based natural antioxidants – the five-star "Michelin" guide
Bioavailability is a measure of the success of the digestion and absorption processes to transfer nutrients from food via the blood plasma to the target sites in the body for utilisation. There are growing numbers of experiments designed to measure the bioavailability of antioxidants in antioxidant-rich meals, either enhanced by supplements or as selected high concentration food items. And there are several steps on the way from digestion of a known amount of the sample to a recorded positive therapeutic response, some time later. A five star system has been devised to simplify the recording of the steps on the way to an antioxidant being proved to be therapeutic: 1 star – the substance is shown to be an in vitro antioxidant (chemical analysis) 2-star – the antioxidant is absorbed and detected in the blood plasma (in vivo) 3-star – the absorbed material is traced to the target site 4-star – oxidative stress is reduced 5-star – a positive therapeutic response is recorded Biochemical assays have shown hundreds of chemical compounds to possess in vitro antioxidant capacity (1 star). The methods employed to measure the oxygen radical absorption capacity (ORAC) are reviewed at Chemical analysis There are many reports in the scientific literature describing the analysis of blood plasma samples, using the same analytical methods, taken at intervals after the ingestion of a meal containing a known amount of the antioxidant compound of interest (COI). From time zero, the amount of COI reaching the plasma is plotted on a graph which records the rise to a maximum value and then the fall back to zero again (2 star). Figures for the rise and fall of the absorbed antioxidant might be 2-4 hours. This figure is of interest if it is found that a continuous supply of plasma antioxidant is the most effective treatment, since the optimum interval between “meals” can then be set. This is currently the area of greatest activity and some popular antioxidants have already been shown to be poorly absorbed. Some of the flavanoids in particular decompose during absorption and therefore raise more questions about whether or not the degradation products are absorbed and if so, whether they have antioxidant capacity. It is also worth remembering that food preparation methods can help improve the absorption rate. Some kinds of antioxidants deteriorate as the food ages, therefore, fresh is better than stored; while the carotenoids in tomatoes become more available from cooked and processed foods (e.g. sun dried, fried, sauces, ketchups) because the processing helps to release the strongly bound compounds from the matrix. Some experiments have recorded the COI at the target site (3 star), but the linkage to oxidative stress (4 star) does not seem to have been made yet. Therefore the therapeutic effect of an individual antioxidant or a known amount of antioxidant present in the food eaten at a meal (5 star) is not known. If as is suggested at this time (January 2009), a combined mixture of antioxidants will be required for the reduction of oxidative stress, then no individual compound will earn 4 or 5 stars, but the category should be retained for defined mixtures of antioxidants as they are shown to reduce oxidative stress.

5 Alternative theories
At the time of writing (January 2009), the scientific press is not consistent about the popular notion, that antioxidants are good for you because they reduce oxidative stress, the yardstick for the body’s susceptibility to degenerative diseases, certain cancers and ageing. It may be helpful to discuss some alternatives until more concrete evidence turns up. First, a list of the uncertainties extant with the current antioxidant theory. Most of them have been mentioned previously, but in summary are: - antioxidants administered singly may not be bioavailable - not all antioxidants are bioavailable, but perhaps some of their breakdown products might be? - some of those that are bioavailable are of unknown potency - potency increases synergistically when certain combinations of antioxidants are ingested. (see Article 2) - some, taken as supplements, in certain cases, actually appear to increase oxidative stress - do exogenous (food-based) antioxidants, once absorbed, trigger the action of endogenous cellular antioxidants such as uric acid? - do food-based antioxidants trigger the production of endogenous antioxidant enzymes, e.g., superoxide dismutase? The advantage of this route is that enzymes can regenerate after neutralising a free radical and can continue to do this for 1000s of interactions. Thus such a mechanism would be effective in smoothing out the response to excesses of free radicals over a sustained period, in contrast to the food-based equivalents that “commit suicide” while neutralising a single ROS, which would mean, at best, that protection would wax and wane governed by the intervals between meals. With these provisos in mind, it is rewarding to examine the hypothesis afresh. Apparently, from epidemiological evidence, 5-9 portions of fruit and vegetables (plus nuts and grains) per day provide the body with the basic materials to combat the negative effects caused by life’s traumas and stresses and the ingestion of toxic substances (voluntarily, or otherwise) while necessarily eating food to generate energy for living: technically reducing oxidative stress.
Edo ergo sum – Hippocrates
At times in the past, Man’s food was unrefined to the point that toxic components (poisons) were present in significant quantities. So, meals were a balance of the nutritional versus the antinutritional components. The lower the toxicity level the more nutritious the meal became, and the successful experimenters thrived. Thus diets improved by trial and error, using the senses of smell and taste to select acceptable, nourishing foods. Eventually, a limited collection of botanical species, containing lower levels of toxins, became staple human foods. To the present day when possibly some diets are over refined, losing completely the bitter flavanoids and glucosinolates for example, some of which are thought to be beneficial. This might remind some of us of the parental guidance “to eat up our greens”, and the adage “you will eat a peck of dirt before you die”. Alternative theories may be postulated around these selection criteria. Astringency and bitterness are the qualities associated with foods containing e.g., glucosinolates and vegetable tannins, (proanthocyanins), which are now thought to be therapeutic, providing the sources of anticancer and anti-inflammatory agents respectively. On the other hand, glucosinolates can cause goitre and vegetable tannins can bind essential mineral elements, vitamins, and major nutrients like proteins and carbohydrates, removing them as unabsorbed complexes. Currently, modified pectins have been implicated in the binding of the recombinant form of the protein galectin-3, inhibiting it in its role of cancer progression and metastasis. If bioactive fragments of pectin can bind to antagonists, providing relief from degenerative diseases, possibly other large anthocyanins (flavanoid/carbohydrate polymeric molecules) might be capable of binding ROS to lower the oxidative stress. Even if they can’t, they can act as slimming agents by occluding nutrients (for the developed world only). However, a serious concern about high tannin antioxidant diets is the binding of essential mineral nutrients.