Although the term phytochemical could be applied to any chemical constituent of plants, the term is used in this chapter to describe biologically active organic substances found in plants used by humans as food, which may be beneficial for health, but for which no specific human deficiency disorder has been identified. Thus nutrients are excluded from the discussion by definition, as are, for practical reasons, the carbohydrate polymers comprising dietary fibre. In general, phytochemicals are secondary plant metabolites; that is, substances synthesised by plant cells, but which serve some function beyond the primary needs of the cell, and contribute to the survival of the whole plant as a functional organism. Some phytochemicals confer colour or scent, others act as signalling molecules, either within the plant itself, or in interactions with other organisms, and many are believed to function as natural pesticides. Some of these substances are pharmacologically active, whilst others are either profoundly unpalatable or highly toxic. Obviously these properties exclude many classes of secondary plant metabolite from the human food chain, but thousands of food-borne phytochemicals are consumed in significant quantities, even in Western economies that cultivate and consume only a relatively small number of plant varieties as food.
Fruits, vegetables and cereals have long been recognised as important sources of vitamins and mineral micronutrients, but interest in the potentially beneficial effects of phytochemicals on human health began with epidemiological studies showing protective effects of plant foods against several of the chronic diseases of old age, and particularly against cancer. One of the most influential studies was the review of Block and colleagues (Block et al., 1992), who collated and summarised the published evidence for a relationship between fruit and vegetable consumption and the risk of cancer at many sites. Overall they observed strong evidence for a protective effect against a range of different cancers in many populations, and concluded that on average, individuals in the lowest population quartile for fruit and vegetable intake experienced about twice the risk of cancer compared to those in the highest quartile. Steinmetz and Potter (Steinmetz et al., 1991) came to similar conclusions, as did the World Cancer Research Fund in its 1997 report on Food, Nutrition and the Prevention of Cancer, which described ‘convincing’ evidence for protective effects of fruits and vegetables against cancers of the upper aerophagic tract, stomach and lung, and of vegetables against cancers of the colon and rectum (World Cancer Research Fund, 1997). This rising tide of evidence, coupled with improved analytical procedures and growing interest in the interactions between plant secondary metabolites and mammalian cells stimulated interest in the possibility that plants might confer health benefits beyond those that could be attributed to their nutrient content alone, and triggered a surge in research on the biological properties of phytochemicals (Johnson et al., 1994).
Clearly, if phytochemicals are to be of benefit to human health, they must reach their target tissues in physiologically significant quantities. Some secondary plant metabolites may act entirely within the lumen of the alimentary tract, perhaps by functioning as quenching agents for free radicals, or by interacting directly with gut epithelial cells, without ever crossing the intestine and entering the blood stream. This type of localised activity could perhaps account for protective effects of some fruits, vegetables or functional foods against digestive diseases such as gastric or colorectal cancer, but if phytochemicals play a larger role in human health, they must first cross the gut and enter the circulation in active forms. The complex issue of bioavailability is discussed in the next section of this chapter. Assuming that the necessary active concentrations are achieved, the next question is how do these various nonessential but nevertheless beneficial substances act to preserve human health? During the early stages of research on phytochemicals it was assumed that since so many could act as antioxidants in vitro, this would prove to be their most important role in human metabolism – indeed the terms phytochemical and antioxidant remain almost synonymous in the minds of consumers and some commercial marketing departments. However over the last decade or so it has become clear from studies in vitro and with animal models that phytochemicals interact with mammalian physiology and metabolism in many unexpected ways that might benefit human health, whilst in parallel with these developments, the true significance of phytochemicals as antioxidants has had to be re-evaluated. Sections 3 and 4 of this chapter will provide a critical discussion of these issues.
One very important characteristic of phytochemicals that distinguishes them from organic micronutrients is the lack of any evidence for specialised adaptations of the human body that might serve to maximise their absorption and delivery to the tissues. Indeed many phytochemicals are transferred only sparingly across the intestinal mucosa, and those compounds that do cross the intestinal surface in significant quantities tend to be rapidly metabolised by the Phase II enzymes, which convert potentially toxic molecules to water soluble conjugates. Much of this metabolism occurs in the gut mucosa, and a large fraction of the products are actively secreted back into the gut lumen (Petri et al., 2003), there to be either metabolised further by the gut microflora, or lost in the faeces. In many cases, any unmetabolised compounds that do enter the circulation undergo metabolic conversions during their first pass through the liver, so that it is the modified forms that reach the target tissues, not the native compound found in the plant. Unfortunately, much of the evidence linking phytochemicals to the health benefits of fruit and vegetables has come from in vitro research, in which isolated cells and tissue preparations have been exposed to unrealistically high concentrations of pure, unmetabolised compounds. In this section, the current state of knowledge with regard to the bioavailability to humans of the main classes of phytochemicals will be briefly reviewed.
The terpenes form a large class of organic compounds based upon the isoprene unit, which has the molecular formula C5H8. All terpenes have the general formula (C5H8)n, but their isoprene constituents may be present as linear chains, or as a combination of both rings and chains. Chemically modified terpenes are very common in nature, and are termed terpenoids or isoprenoids. Terpenes and their derivatives occur widely in the plant kingdom, often as components of resins and essential oils. They are often coloured or pungently scented, and they enter the human food chain as constituents of citrus fruits, or as aromatic food ingredients, such as ginger, cinnamon and cloves. The carotenoids are a particular class of terpenoids, based on eight isoprene units, which will be considered separately below.
Because of their highly lipophylic behaviour, terpenes and their derivatives are likely to cross biological membranes readily by passive diffusion. However their solubility in the aqueous phase of the gut lumen will be low; and their bioavailability will probably depend upon emulsification and partioning into the micellar phase during gastrointestinal lipid digestion. Apart from the carotenoids, discussed in section 3.2.3, studies on the intestinal transport of terpenoids have been relatively few in number. One important exception however is the compound d-limonene, which is a monoterpene (C10H16) based on two isoprene units containing a single ring. Citrus peel oils contain about 90% d-limonene and significant quantities are present in conventional citrus juices. Average intakes have been estimated to be about 0.27 mg/kg body weight per day in the USA, but may range up to 1 mg/kg per day in heavy consumers of citrus juices (FAEM Association, 1991). D-Limonene has attracted considerable attention because of its anticarcinogenic effects in rodent models of skin, stomach and mammary cancer. As with many other phytochemicals, the native food-borne compound does not appear in high concentrations in plasma, but the major metabolite perillic acid has been shown to be biologically active. Chow et al. ( 2002) have argued that citrus juices containing a high level of peel are important constituents of Mediterranean diets and that heavy consumption of such juices may contribute to low levels of certain cancers in countries such as Spain and Southern Italy.
Crowell et al. (1994) determined the plasma concentrations of d-limonene, perillic acid, dihydroperillic acid and limonene-1,2-diol in human volunteers after administration of d-limonene (100 mg/kg body weight) in the form of orange oil incorporated into a food product. Only traces of unmetabolised d-limonene were detected in plasma, but average concentrations of perillic acid, dihydroperillic acid and limonene-1,2-diol reached 35, 33 and 16 micromolar respectively. The pharmacokinetics of perillic acid after consumption of what was described as a Mediterranean-style lemonade made from whole lemons and containing up to 596 mg of d-limonene per 40 oz dose were investigated by Chow et al. (2002). The concentration of perillic acid peaked at one hour after consumption, indicating rapid absorption of d-limonene in the proximal gastrointestinal tract, and ranged between 4.5 and 14.0 microM. Subsequent work has shown that d-limonene consumed in this way is deposited to a significant extent in human adipose tissue (Miller et al., 2010). Evidently d-limonene, and presumably many other terpenoids with similar physical properties, are absorbed and metabolised to a significant extent from commonly consumed foods, but the biological significance of this for human beings remains largely unexplored.
Much of the complexity of the problems associated with the bioavailability of phytochemicals in general can be judged from the growing literature on the absorption and metabolism of food-borne polyphenols. Amongst this huge group of food-borne substances, the anthocyanins and flavanols have received particular attention. Anthocyanins are a large group of phenolic compounds found abundantly in fruit juices, berries of various types, and wine. Manach et al. (2005) reviewed published data on the absorption and metabolism of anthocyanins in humans, and concluded that from most food sources, only a very small fraction is absorbed, that the small amount of absorption that does occur takes place very rapidly in the stomach and upper intestine, and that excretion of the absorbed fraction is rapid and efficient. In most human bioavailability studies, the administered doses were in the range of hundreds of milligrams, and resulted in peak plasma concentrations in the 10–50 nmol/L range. The average bioavailability of the anthocyanins has been reported in many studies to be less than 1%. Unlike other polyphenols, unmetabolised anthocyanin glycosides are often detected in blood and urine, but there is evidence that anthocyanin glucuronides and sulphates are unstable in urine (Felgines et al., 2003) and that as a consequence their abundance, and hence the total absorption and excretion of the anthocyanins, may have been underestimated in many studies that did not allow for this.
Like the anthocyanins, flavonols are also present in plants as a mixture of water soluble glycosides, and this is also the form in which they are released into the alimentary tract during digestion. They too are commonly found in fruits and vegetables used for human consumption, although they tend to be present in the diet at lower concentrations than the anthocyanins. One of the first mechanistic studies on the absorption of flavonols in humans was conducted by Hollman and colleagues (Hollman et al., 1995), using volunteers who had previously undergone surgery for the removal of a diseased large intestine, and whose small intestine emptied via a permanent orifice at the abdominal surface (ileostomists). Because the digestive residues from the small intestine can be collected and analysed, such patients are often used to study the digestion of food constituents before they are exposed to the intense metabolic activity of the colonic microflora. Hollman et al. measured the disappearance of quercetin glycosides from test-meals of fried onion during their passage through small intestinal lumen, and compared it with the disappearance of pure quercetin aglycone. Their study showed that the absorption of the quercetin glucosides found in food was more efficient than the absorption of quercetin aglycone, and the absorption of the rhamnoglycoside, rutin was even less efficient. It was argued that this was evidence for absorption of the intact glucosides via the specialised glucose transport channels of the small intestinal epithelial cells. This study generated considerable interest and stimulated further research using in vitro systems, animal models and human volunteers, both to test the hypothesis, and to elucidate the metabolic fate of quercetin and other polyphenols in humans. As a result it is now well established that although flavonol glucosides do interact with the glucose transporters of intestinal epithelial cells, their effect is mainly to act as weak inhibitors of glucose absorption (Gee et al., 2000). In practice, most quercetin glycosides are readily hydrolysed by the digestive enzyme lactase phlorizin hydrolase (LPH), which is localised at the epithelial surface. A small fraction of one quercetin glucoside commonly found in foods, quercetin-4’-glucoside, may remain intact long enough to cross the epithelium via the glucose transporter, but the similarly abundant compound quercetin-3-glucoside appears to be absorbed entirely by the passage of free quercetin following hydrolysis by LPH (Day et al., 2003). In any case, unmodified flavonoid glycosides do not reach the human circulation. Only the metabolised flavonoids (e.g. glucuronides, sulphates) are found in the blood, and it is these compounds that must be studied in order to fully define and understand the physiological effects of flavonoids in the human body. The absorption of quercetin is somewhat slower than that of the anthocyanins, as is its excretion in urine. Prolonged dietary supplementation with quercetin can lead to plasma concentrations in the 1–2 µmol/L range (Conquer et al., 1998).
Another aspect of polyphenol metabolism that is poorly understood, but should not be neglected, is the large proportion of the ingested dose that remains in the gut lumen, but which is broken down to simpler, often more readily absorbable compounds, by the gut microflora (Forester et al., 2009). Bacterial metabolism of polyphenols includes ring-fission, and leads to a complex range of metabolites including aldehydes and phenolic acids. Many of these compounds are taken up into the circulation by passive absorption across the colon, and may also exert local anti-inflammatory activity in the gut lumen, which could be important for the maintenance of mucosal homeostasis and health (Larrosa et al., 2009).
Carotenoids are terpenoids containing forty carbon atoms, and are found throughout the plant kingdom, mainly as components of chloroplasts, where they occur as pigments in close association with the photosynthetic apparatus. The two main classes of carotenoids are the carotenes, which contain no oxygen atoms, and the xanthophylls, which do. There are about 600 known carotenoids in nature, but relatively few are thought to be of nutritional significance for humans. The provitamin A carotenoids (beta-carotene, alpha-carotene, gamma-carotene and beta-cryptoxanthin) are important because they are converted in the human intestinal mucosa to vitamin A. Beta-carotene and other carotenoids are potent antioxidants, and certain compounds, including the xanthophyll lutein, accumulate in the macula lutea of the human eye and the corpus luteum of the ovaries, where they are thought to plan an important protective role against free-radical mediated damage.
Because of their well established nutritional role in vitamin A metabolism, and their putative function as phytochemicals in their own right, the bioavailability of carotenoids has received much attention. Being both hydrophobic and tightly bound within robust intracellular structures, the bioavailability of carotenoids depends upon their physical release from the plant tissue, and incorporation into a suitable lipid phase, either during food processing or in the intestinal lumen during digestion. The details of this initial stage, termed bioaccessability, vary markedly between different food sources. The release of carotenoids from the cells of fruit and vegetable tissues is greatly facilitated by thermal processing, but also exposes the molecules to the possibility of chemical degradation. For example, lycopene is released from tomatoes by thermal processing, but becomes susceptible to cis-isomerisation, which may modify its biological activity (Schierle et al., 1997).
Having been released into the gut lumen as an emulsion, the absorption of carotenoids occurs via the mixed micelle phase formed in the presence of bile salts during lipid absorption. The presence of adequate quantities of lipid in the digesta is thus an essential prerequisite for uptake of carotenoids, and their bioavailability depends on the contemporaneous intake of dietary fat. This is an excellent example of the extent to which the micronutrient or phytochemical content of the diet may be an inadequate predictor of its biological effects if the issue of bioavailability is ignored. In an interesting study, Unlu et al. (2005) explored the effects of the lipid-rich fruit avocado, or extracted avocado oil, on the bioavailability to humans of carotenoids from salsa or salads. The addition of 150 g of avocado to salsa enhanced the area under the plasma concentration vs time curve (AUC) for lycopene and beta-carotene by 4.4-fold and 2.6-fold respectively. Similarly 150 g of avocado or 24 g of avocado oil added to salad increased the uptake of alpha-carotene, beta-carotene and lutein by 7.2-, 15.3- and 5.1-fold respectively.
A full understanding of bioavailability implies a description of the delivery of the substance under investigation to target tissues. In the case of the carotenoids this is made more complex by their hydrophobicity, which ensures that they are transferred to the circulation with the chylomicrons, and transported as components of the plasma lipoproteins. About 80% of plasma beta-carotene and lycopene are transported by low density lipoproteins (LDL) but lutein and zeaxanthin also occur at significant levels in high density lipoproteins (HDL). Lipoprotein metabolism varies between individuals to an extent that can significantly modify the apparent concentrations of carotenoids in plasma. Because of this, Faulks and Southon have cautioned against the assessment of carotenoids bioavailability without also taking into account such between-subject differences (Faulks et al., 2005).
The glucosinolates are another complex group of biologically active compounds, which occur in cruciferous plants, and enter the human food chain in Brassica vegetables such as cabbages, broccoli and brussel sprouts, and in cruciferous salad vegetables including mustard greens, rocket and radishes (Mithen et al., 2000). All glucosinolates contain a common sulphur group, linked to a variable side chain, and a glucose molecule. They are stable, water soluble glycosides, sequestered within the plant tissue until acted upon by endogenous hydrolytic enzyme, myrosinase, which is released by mechanical disruption of the tissue. Hydrolysis liberates glucose and an unstable intermediate which undergoes further reactions to release a variety of products, the most important of which are the isothiocyanates. These pungent compounds impart flavour and aroma to cruciferous vegetables and herbs. They are released from raw plant tissue during food preparation, or by chewing and digestion, and they are absorbed passively across the intestinal surface. Like flavonoids, they are rapidly metabolised both in the gut epithelial cells and in the liver. Petri et al. (2003) used intraluminal tubes to infuse liquidised broccoli containing the isothiocyanate sulforaphane into the intact human intestine, and to recover the luminal contents for analysis. This study showed that most of the absorbed sulforaphane was metabolised to glucuronides and sulphates, and a large proportion was re-secreted into the gut. Nevertheless a much larger fraction of an ingested dose of isothiocyanates is absorbed and metabolised than is the case for the flavonoids, and low concentrations of intact isothiocyanates can be detected in human plasma (Verkerk et al., 2009). Interestingly, the concentration of sulforaphane metabolites in the urine of human volunteers after consumption of a test-meal of broccoli depends on their genetic status in relation to one of the major classes of Phase II enzymes, glutathione-S transferase (GST), which varies markedly in activity between individuals because of polymorphisms in the genes coding for the various components of its super-family (Gasper et al., 2005). Variations in the expression of the various sub-types of GST may influence the availability of isothiocyanate metabolites to the tissues, and seem to determine the degree to which humans benefit from the anticarcinogenic effects of Brassica vegetables (London et al., 2000).
The lectins, unlike the other main classes of phytochemicals reviewed here, are proteins, of diverse structure and high molecular weight. They occur in the human food chain mainly as plant constituents, but they are found in the animal kingdom as well. Their defining characteristic is their capacity to bind specifically to carbohydrates, and most notably to the carbohydrate moieties of glycoproteins or glycolipids that occur as constituents of cell membranes. It is this property that accounts for their frequent role in mechanisms involving specific bio-recognition phenomena, and for their laboratory use in cellular agglutination reactions. Plant lectins may also have evolved as natural pesticides; many act as anti-nutritional factors and can be toxic to humans (Vasconcelos et al., 2004).
Lectins are generally very resistant to digestion in the gut, and their high molecular weight makes them poor candidates for intestinal absorption. They do however frequently show a strong tendency to interact with glycoconjugation sites on the mucosal surfaces of the intestine, and this is thought to account for many of their well documented biological activities in the gastrointestinal tract. In animals, these effects include stimulation of intestinal epithelial cell proliferation to higher than normal levels, an effect which has been reported to occur in humans (Ryder et al., 1998). The lectin (phytohemagglutinin) derived from uncooked beans (Phaseolus vulgaris) causes aberrant growth and precocious maturation of the gastrointestinal tract in suckling rats. Linderoth et al. (2006) showed that the effects on the gut mucosa occurred when the lectin was given by direct introduction into the alimentary tract (enteral exposure), but not when it was given by sub-cutaneous injection. However subcutaneous exposure did lead to effects on systemic organs not seen after enteral exposure. These results suggest that this lectin is highly biologically active within the gut lumen but is unlikely to be absorbed and become available to other organs via the circulation. One other possible route of delivery of biologically active lectins to sub-epithelial tissues in the gut is via uptake and translocation by intestinal M cells, which are known to sample intraluminal proteins and present them to the lymphoid cells of the gastrointestinal immune system. Transport of the mistletoe lectin (Viscum album L, var. coloratum agglutinin) through this pathway has been demonstrated using an in vitro model of the intestinal mucosa (Lyu et al., 2008) but it is not clear whether this mechanism is of biological importance to human consuming lectins from conventional food sources.
The very strong evidence for protective effects of plant foods against cancer and cardiovascular disease that began to appear in the early 1990s prompted a very significant burst of research activity on the biological effects of phytochemicals in in vitro systems, animal models and humans, but in 2003 the authors of a report published by the International Agency for Research on Cancer (IARC, 2003) came to somewhat more cautious conclusions about the benefits of fruit and vegetable consumption than previous authors (Block et al., 1992), Their findings were that: ‘There is limited evidence for a cancer-preventive effect of consumption of fruit and of vegetables for cancers of the mouth and pharynx, oesophagus, stomach colon-rectum, larynx, lung, ovary (vegetables only), bladder (fruit only) and kidney. There is inadequate evidence for a cancer-preventive effect of consumption of fruit and vegetables for all other sites’. This general trend towards a more conservative assessment of the protective effects of fruits and vegetables against cancer has continued. The most recent report (Boffetta et al., 2010) on fruits and vegetables from the very large European Prospective Investigation into Cancer and Nutrition (EPIC), concluded that a statistically significant protective effect was detectable, but for men and women combined it amounted to only around 10% reduction in overall risk of cancer for the highest quintile of fruit and vegetable consumption (>647 g/day) compared to the lowest quintile (0-226 g/day).
The relationship between fruit and vegetable consumption and risk of coronary heart disease has been much less intensely studied than that for cancer, but recent research suggests a somewhat similar level of protection. For example, Dauchet et al. (2006) conducted a meta-analysis of nine cohort studies and calculated that across the entire population of 91 000 men and 130 000 women, the risk of coronary heart disease was decreased by 4% for each extra portion of fruits and vegetables consumed per day, and by about 7% for each additional portion of fruit. Against this background, some of the most important lines of evidence for the protective mechanisms of particular groups of phytochemicals against cancer and some forms of cardiovascular disease are discussed in the remainder of this section.
Free radicals are highly reactive, short-lived species generated by a variety of biological mechanisms, including inflammation (Hussain et al., 2003), or as a side effect of the reactions occurring during normal oxidative metabolism (Poulsen et al., 2000). During their short lifespan they readily interact with macromolecules, including lipids, proteins and nucleic acids, damaging their structures and often modifying their functionality. Mammalian cells have evolved a complex arsenal of antioxidant mechanisms to defend their constituent macromolecules from free-radical mediated damage but, even so, the steady-state level of oxidative DNA adduct formation caused by free radicals such as the hydroxyl radical (· OH) released from H2O2 in the presence of iron, nitric oxide (NO·) and peroxynitrite (ONOO) has been estimated to be about 66 000 adducts per cell (Helbock et al., 1998). The cumulative effects of such damage include mutations resulting from faulty DNA repair, and double strand-breaks (Bjelland et al., 2003). Free-radical reactions can also cause oxidative damage to proteins such as p53 that are involved in the regulation of cellular proliferation and apoptosis, and can thereby contribute directly to tumour promotion (Hofseth et al., 2003). High levels of free-radical production also occur in vascular tissues during the development of disease. Superoxide reacts with NO, forming peroxynitrite, and impairing NO-mediated processes essential to the maintenance of vascular health.
Fruits and vegetables are rich in both antioxidant nutrients such as ascorbate, which is a powerful electron donor, and which therefore acts as a reducing agent in a range of free-radical and other reactions. The donation of an electron by ascorbate, in reactions with O2- and OH., gives as a product the radical semidehydroascorbate, which is only weakly reactive. Vitamin E (α-tocopherol) is an important lipid-soluble antioxidant nutrient, that tends to accumulate in cell membranes, and which acts by reacting with lipid free-radicals, blocking peroxidation chain reactions, and thus protecting cell membranes from oxidative damage. Many naturally occurring polyphenols act as chain-breaking antioxidants in a similar way to vitamin E.
The fact that polyphenols and other secondary plant metabolites exhibit strong antioxidant activity in vitro led to the hypothesis that many of the putative protective effects of fruits and vegetables against cardiovascular disease and cancer are a direct consequence of strengthened antioxidant defences. Much of the experimental work underpinning this hypothesis was based on the use in vitro systems, but there have also been many attempts to demonstrate direct benefits of dietary antioxidant supplementation in human volunteers, using antioxidant activity in plasma or target tissues, or changes in the production of end-products of oxidative damage, as biomarkers. One widely used technique for the investigation of antioxidant effects in biological systems is the oxygen radical absorbance capacity assay (ORAC), which works by measuring the effect of some biological sample on a standard free-radical mediated reaction between R-phycoerythrin and a peroxyl radical generator, 2,2´-azobis(2-amidinopropane) dihydrochloride (AAPH). The synthetic, water-soluble antioxidant Trolox® is often used as a standard, so that the antioxidant activity of the biological system under investigation can be expressed in Trolox equivalents. Cao et al. (1998a) used the ORAC assay to explore the effects of fruit and vegetable consumption on the antioxidant capacity of plasma in a group of healthy non-smoking volunteers. At the outset of the study, the baseline antioxidant capacity of their plasma was positively correlated with their fruit and vegetable intake as estimated from a food-frequency questionnaire. The subjects then entered a metabolic laboratory, where they all consumed one or other of two controlled diets consisting of ten servings of fruit and vegetables per day for 15 days, or a similar diet with two additional servings of broccoli, with a washout period of six weeks between experiments. All subjects showed a significant increase in the antioxidant capacity of the plasma in response to both experimental diets. These effects were associated with an increase in α-tocopherol (vitamin E) in the plasma, but it was shown that the increased antioxidant capacity could not be accounted for by antioxidant nutrients alone. The authors therefore proposed that phytochemicals, including flavonoids, were the probable cause of the observed effects. In a separate study from the same laboratory the acute effects of strawberries, spinach, red wine and vitamin C were evaluated in elderly women (Cao et al., 1998b). As in the previous study, the theoretical effects of other sources of antioxidant nutrient activities were accounted for, and shown not to fully explain the observed increases in antioxidant activity. The authors concluded that much of the excess antioxidant capacity was due to absorption of food-borne polyphenolic phytochemicals, but this conclusion was not directly verified.
Other studies have confirmed that dietary intervention with flavonoid-rich berries and other fruits leads to a significant increase in the antioxidant activity in human plasma (Pedersen et al., 2000), but the causal relationship between this effect and reductions in the risk of disease remains largely hypothetical. Furthermore, the precise reasons for the observed changes in plasma antioxidant capacity in response to dietary intervention often remain ambiguous. Much of the work in this field has been based on the assumption that the antioxidant effects of fruits and vegetables can be ascribed largely to their phytochemical content, but the relevance of the antioxidant effects observed in vitro to clinical findings has been challenged by Lotito and colleagues, who argued that the rise in antioxidant capacity following fruit and vegetable consumption is often caused by an increase in plasma urate levels (Lotito et al., 2004). Uric acid accumulates in human plasma as an end-product of purine metabolism, and can reach concentrations close to 0.5 mM/L. Ames (1981) showed that uric acid is a powerful antioxidant and argued that it accounts for most of the antioxidant capacity of human plasma. Given the low bioavailability of flavonoids, which seldom reach concentrations in the micromolar range in human plasma, it seems highly unlikely that they can make a major contribution to antioxidant activity when consumed from conventional fruits and vegetables. Furthermore, it has been shown that a post-prandial increase in urate levels occurs in response to the metabolism of fructose via fructo-kinase mediated production of fructose 1-phosphate, which enables the rate of adenosine monophosphate degradation to urate to rise (Lotito et al., 2004). This transient rise in plasma urate levels may be the primary cause of increased postprandial antioxidant capacity after ingestion of apples and other fructose-rich foods.
Another approach to testing the antioxidant hypothesis is to conduct dietary interventions with foods rich in antioxidant phytochemicals and then to search for evidence of a reduction in free-radical mediated damage to macromolecules. Actual disease endpoints are extremely difficult to study under controlled experimental conditions, but biomarkers have been used in this way to study the effects of dietary intervention on oxidative damage in human trials. Several different markers of oxidative damage to lipids, proteins and DNA have been employed for this purpose. One very widely used measure of lipid peroxidation is the level of thiobarbituric acid-reactive substances (TBARS) present either in plasma or in low density lipoproteins obtained from the blood (Wade et al., 1989). In a small study with five subjects, Young et al. (1999) explored the effects of three daily doses of blackberry and apple juice (750, 1000 and 1500 ml) consumed for one week, on markers of lipid and protein peroxidation. Total plasma TBARS were reduced following the intervention with 1500 ml of juice but plasma 2-amino-adipic semialdehyde residues increased with time and dose, suggesting an unexpected pro-oxidant effect of the juice on plasma proteins. Bub et al. (2000) used a similar approach to measure changes in lipid peroxidation in 23 healthy male subjects after a period of dietary antioxidant depletion, and after intervention periods with 330 ml tomato juice, 330 ml carrot juice and finally with 10 g of spinach powder. Consumption of tomato juice reduced plasma TBARS by 12% (P < 0.05) and lipoprotein oxidisability as measured by an increased lag time by 18% (P < 0.05). However carrot juice and spinach powder had no effect on lipid peroxidation, and antioxidant status did not change during any of the study periods. In contrast van den Bergh et al. undertook a randomised placebo-controlled cross-over trial, lasting three weeks with a two-week washout period between treatments, in a group of 22 male smokers with a relatively low vegetable and fruit intake (van den Berg et al., 2001) . During the treatment phase the subjects consumed a vegetable burger and fruit drink, and showed increased plasma levels of vitamin C, carotenoids and total antioxidant capacity. However there were no effects on any marker of oxidative damage to lipids, proteins or DNA, or on other biomarkers of oxidative stress.
A group of Dutch and Scandinavian collaborators undertook a large and very thorough human intervention study to explore in some depth the effects of prolonged dietary supplementation with fruits and vegetables on antioxidant status and other aspects of metabolism in humans. For 25 days, a group of 43 healthy volunteers consumed either 600 g of fruits and vegetables per day, an equivalent quantity of vitamins and minerals or a placebo (Dragsted et al., 2004). The so-called ‘6-a-day’ study was designed to explore both the direct antioxidant effects of prolonged fruit and vegetable consumption, and the induction of enzymes involved in the metabolism, conjugation and excretion of potentially toxic substances. The use of a positive control group consisting of subjects receiving micronutrient supplements whilst consuming an essentially fruit- and vegetable-free control diet also enabled the researchers to deduce what proportion of any physiological response to the fruit and vegetables supplementation could be ascribed to phytochemicals. In practice however, despite the high levels of supplementation with fruits and vegetables and the assessment of a variety of sophisticated biomarkers, few important biological effects were observed. None of the markers of plasma antioxidant capacity that were measured showed any statistically significant response to dietary intervention. There was some evidence of an increased resistance of plasma lipoproteins to oxidation, but also an increase in protein carbonyl formation at lysine residues, which is indicative of increased protein oxidation. Interestingly, the latter effect was attributed to a pro-oxidant effect of ascorbate, which is known to occur under certain conditions. This finding emphasises the complexity of free-radical biology in living systems other than simple in vitro models. In another paper from the same study, it was reported that neither the prolonged period of fruit and vegetable depletion, nor the supplementation with either fruits and vegetables or micronutrients had any significant effects on the levels of oxidative damage to DNA (Moller et al., 2003). The authors concluded that the inherent antioxidant defence systems of these healthy human subjects were sufficient to protect their circulating mononuclear cells from oxidative damage.
The development of cancer is a prolonged, multi-stage process, involving a progressive series of molecular events, beginning with damage to DNA in a single dividing cell. Cells that have undergone the first step of initiation and continue to divide and multiply, are increasingly vulnerable to further mutations, leading to an increasingly abnormal phenotype that gradually acquires the ability to migrate to other tissues and establish secondary tumours. Some of the earliest studies on the ability of natural food-borne chemicals to inhibit the development of cancer were conducted by Wattenberg, who observed that anticarcinogenic chemicals could be defined as either blocking agents, which act immediately before or during the initiation of carcinogenesis by chemical carcinogens, or as suppressing agents, which act at later stages of promotion and progression (Wattenberg et al., 1985). Blocking agents are drugs or phytochemicals that prevent the initial damage to DNA by chemical carcinogens, either by inhibiting their activation from procarcinogens or by enhancing their detoxification and excretion. These effects occur primarily through changes in the activity of the Phase II metabolic enzymes mentioned earlier in the context of bioavailability. Phase II enzymes act downstream from Phase I metabolism, which is mainly due to the cytochrome p450 enzymes that orchestrate the oxidation, reduction and hydrolysis of environmental chemicals such as drugs, toxins and carcinogens. The products of Phase I metabolism are often highly reactive genotoxic intermediates that form substrates for Phase II enzymes such as glutathione S-transferase (GST), NAD:quinone reductase and γ-glutamylcysteine synthetase. Phase II catalyses the formation of less reactive, water-soluble conjugates that are readily excreted via the kidneys or in bile. Certain phytochemicals induce the transcription of genes expressing Phase I and II enzymes, and the most effective are those that selectively induce Phase II enzymes, without simultaneously inducing activation of carcinogens via increased Phase I activity (Prochaska et al., 1988). Several groups of phytochemicals have now been identified as potent inducers of Phase II enzymes; two of the most actively investigated are the flavanols, including epigallocatechin gallate (EGCG), which is the principal biologically active component of green tea (Chou et al., 2000), and the isothiocyanate sulforaphane derived from broccoli (Talalay et al., 2001).
A very substantial amount of research to elucidate the mechanisms of action of anticarcinogenic phytochemicals has been done using cultured tumour cells in vitro, but much of this work is also supported by studies with experimental animals. For example, the compound indole-3-carbinol obtained from Brassica vegetables (Morse et al., 1990) and the isothiocyanate phenethyl isothiocyanate (PEITC) from watercress (Hecht, 1996) have been shown to modify the metabolism of the tobacco smoke carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and inhibit the development of lung tumours in rats. In the case of NNK, the shunting of NNK metabolism away from the lung leads to increased metabolism in the liver, and higher urinary excretion of NNK metabolites. In some cases it has been possible to confirm the existence of such anticarcinogenic activity in studies with human volunteers. Thus smokers who consumed 170 g of watercress (Rorippa nasturtium-aquaticum) per day for three days showed increased urinary excretion of two NNK metabolites, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and (4-methylnitrosamino)-1-(3-pyridyl)but-1-yl)-beta-omega-D-glucosiduronic acid (Hecht, 1995). Overall, the evidence from both experimental and epidemiological studies (London et al., 2000) is consistent with the hypothesis that glucosinolate breakdown products modulate Phase II metabolism of tobacco smoke carcinogens in humans, and help prevent lung cancer, at least in some genetically distinct sub-groups in the population. The significance of these effects in relation to public health remain to be fully established, but the evidence has been strong enough to encourage the development of Brassica varieties rich in glucosinolates for human consumption (Mithen et al., 2003).
Prolonged exposure to carcinogens such as those present in cigarette smoke inevitably leads to an accumulation of genetic damage, and to further molecular events favouring the development of cancer. These include the appearance of further mutations and other genetic abnormalities that cause progressively abnormal gene expression. This so-called promotion stage of cancer development is characterised by poorly regulated cell proliferation and differentiation, and a reduced tendency for damaged cells to undergo programmed ‘suicide’ (apoptosis). Eventually the surviving cells acquire the full cancer phenotype, but there are several biologically plausible mechanisms whereby phytochemicals may delay or interrupt this process and thereby lead to tumour suppression. For example it is increasingly recognised that inflammation is a risk-factor for certain cancers (Balkwill et al., 2001) and there is strong evidence that prolonged use of aspirin and other anti-inflammatory drugs reduce the risk of cancers of the colon and other sites (Chan et al., 2005). These various lines of evidence have focused attention on the molecular mechanisms of inflammation, on the pathways through which they may promote cancer and on the phytochemicals that may be used to inhibit them.
One key factor in the activation of inflammatory processes in human disease is nuclear transcription factor κB (NF-κB). In its inactive form NF-κB resides in the cytoplasm as a complex with its main regulatory protein IκB. The activation pathway for NF-κB involves phosphorylation of IκB by the enzyme IκB kinase (IKK), which marks it for destruction by proteolytic enzymes. This step frees NF-κB to translocate to the nucleus, where it binds to a specialised sequence motif in the nuclear DNA, and functions as a transcription factor favouring the expression of at least 200 genes involved in the regulation of inflammation, cell proliferation, differentiation and apoptosis. There is strong evidence that the chronic, abnormal up-regulation of NF-κB is a key factor in the promotion and growth of many tumours (Karin et al., 2002). A variety of secondary plant metabolites (resveratrol, curcumin, limonene, glycyrrhizin, gingerol, indole-3-carbinol, genistein, apigenin) have been shown to inhibit NF-κB activity at various stages in its regulatory pathway. To take one example, curcumin, which is an established anticarcinogenic plant metabolite found in the spice cumin (Cuminum cyminum), suppresses TNF-induced activation of IKK (Singh et al., 1995). In contrast, caffeic acid phenethyl ester has been shown to prevent the binding of NF-κB to its target DNA sequence (Natarajan et al., 1996).
The enzyme cyclooxygenase (prostaglandin H synthase) exists as two distinct isoforms; COX-1, which is expressed, in normal healthy tissues, produces prostaglandins essential to platelet aggregation and gastric mucosal integrity, whereas COX-2 produces prostaglandins involved in inflammatory processes. The downstream effects of NF-κB include increased expression of COX-2, and so inhibition of NF-κB can inhibit inflammation by this route. Other phytochemicals also act as naturally occurring COX inhibitors, amongst which perhaps the earliest and best known example is salicylate, which was originally isolated from the willow tree (Salix alba). Both COX-1 and COX-2 are inhibited by aspirin, an acetylated derivative of salicylate. Salicylates have been shown to irreversibly inhibit the COX enzymes by selectively acetylating the hydroxyl group of a single serine residue, and also to suppress NF-κB, by inhibiting IKK kinase activity (Yin et al., 1998). Many flavonoids are also COX-2 enzyme inhibitors, and some (apigenin, chrysin and kaempferol) can suppress COX-2 transcription by mechanisms including activation of the peroxisome proliferator-activated receptor (PPAR) gamma transcription factor (Liang et al., 2001) and inhibition of NF-κB expression (Liang et al., 1999). As noted previously, flavonoids are extensively metabolised during and after absorption, but in vitro studies have established that COX-2 transcription is inhibited by flavonoid metabolites found in human plasma, including quercetin 3-glucuronide, quercetin 3’-sulphate and 3’ methylquercetin 3-glucuronide (O’Leary et al., 2004).
At a later stage in cancer promotion, anticarcinogenic phytochemicals may act directly on tumour growth by inhibiting cell proliferation (mitosis), or favouring cell death (apoptosis). The Wnt proteins are extracellular signalling molecules involved in the regulation of cell proliferation via the β-catenin signal pathway. They play an important role in gut formation during embryogenesis, and they contribute to the maintenance of normal gut morphology in the adult. About nineteen Wnt genes are known to code for cysteine-rich Wnt glycoproteins that are released into the extracellular environment. Their function is to regulate signalling by the cytoplsmic protein β-catenin in target cells, by interacting with the membrane receptors Frizzled and LRP. β-catenin regulates many aspects of cellular organisation, including cytoskeletal structure, cell proliferation and apoptosis (Wikramanayake et al., 2003), and it is itself tightly regulated by a sequence of interactions with other proteins. It is present in the cytoplasm as a complex with the adenomatous polyposis coli protein (APC) and the scaffolding protein Axin. This complex then associates with casein kinase I (CKI), which phosphorylates the N terminus of β-catenin, and glycogen synthase kinase 3β,an enzyme that phosphorylates other β-catenin residues. The phosphorylated β-catenin molecule is then marked for degradation, which tightly regulates the levels of β-catenin in the cytoplasm. In normal cells there is a relatively large and stable pool of inactive β-catenin associated with the cytoskeletal protein cadherin, and a small labile pool in the cytoplasm. However in cancer cells the degradation pathway is often suppressed and the balance is altered in favour of the labile cytoplasmic pool (Gregorieff et al., 2005). Active β-catenin then migrates to the nucleus, where it activates transcription factors regulating COX-2, and many other genes linked to cell proliferation.
It is well established that synthetic COX-2 inhibitors suppress β-catenin mediated gene transcription in colorectal carcinoma cells, and a number of phytochemicals, including quercetin (Park et al., 2005), also interact with the β-catenin pathway in vitro (Jaiswal et al., 2002). It has also been shown that both green tea, and its active flavanoid constituent epigallocatechin gallate (EGCG), suppressed nuclear β-catenin activity in kidney tumour cells in vitro (Dashwood et al., 2002). Furthermore, in the APCmin mouse, which is a widely used animal model of colorectal cancer, treatment with green tea and the COX-2 inhibitor sulindac both suppressed the growth of tumours (Orner et al., 2003).
One of the most important characteristics of a tumour cell is its ability to evade the induction of programmed cell death, which is a normal response to the many genetic abnormalities that typify cancer. In principle, the enhancement of apoptosis could eliminate genetically damaged cells from a tissue, or tip the balance of cell proliferation in a tumour towards regression rather than growth (Johnson, 2001). Several classes of phytochemicals, including organolsulphur compounds from garlic (Allium sativum) and isothiocyanates from cruciferous plants have been shown to induce apoptosis in vitro. It has been mentioned that glucosinolate breakdown products act as powerful inducers of Phase II enzymes and modulate carcinogen metabolism, both in vitro and in vivo, but this may not be their only mode of action. There is also much evidence that sulforaphane and other isothiocyanates can block mitosis and initiate apoptosis in a variety of epithelial cell lines and tissues. Using an animal model, Smith et al. showed that both oral administration of the pure glucosinolate sinigrin (Smith et al., 1998), which is the precursor of allyl isothiocyanate, and consumption of diets rich in raw brussel sprouts (Brassica oleracea var. gemmifera) rich in sinigrin (Smith et al., 2003), both caused an amplification of the apoptotic response induced in rat colorectal crypts 48 h after exposure to the chemical carcinogen 1, 2 dimethylhydrazine (DMH).
Like cancer, cardiovascular disease, which includes both coronary heart disease and stroke, is a major cause of both death and long-term morbidity in the developed world, and a similar amount of effort has been devoted toward understanding its causes at the cellular and molecular level. All the major and minor blood vessels, including the capillaries, are lined by squamous epithelial cells, which collectively comprise the endothelium. Endothelial cells play a crucial role in the maintenance of normal vascular physiology through their surface properties, their barrier functions and their importance in the regulation of vasodilation. Disruption of these physiological mechanisms, coupled with the onset of endothelial inflammation, is important in the development of cardiovascular disease, and there is much interest in the possible role of phytochemicals in their maintenance. Interest in the possibility that phytochemicals may help to prevent heart disease and stroke began with epidemiological data showing reduced risk of disease in heavy consumers of fruits and vegetables, but recently the attention of both scientists and food manufacturers has become more focussed on a few rich sources of dietary polyphenols, including grapes and wine, tea and cocoa products (Ghosh et al., 2009).
Endothelium-dependent vasodilation is regulated primarily through the signalling molecule, nitric oxide (NO), a short-lived diffusible gas that readily crosses cell membranes. The maintenance of optimal levels of NO within the vascular endothelial tissues is essential to vascular health because of its role as a smooth muscle relaxant and platelet aggregation antagonist, and its inhibitory activity against NF-κB dependent expression of cytokines and inflammatory factors that mediate the formation of atherosclerotic plaque. NO levels are controlled largely by the activity of endothelial nitric oxide synthase (eNOS), which catalyses oxidation of the guanidine group of L-arginine, releasing NO and L-citrulline. Interest in the role of phytochemicals as modulators of eNOS activity began with observations showing that treatment of vascular tissue with red wine or with polyphenols derived from red wine, caused NO-mediated dilation of isolated blood vessels in vitro (Fitzpatrick et al., 1993). These in vitro effects have since been shown to be due to the induction by red wine polyphenols of eNOS activity in endothelial cells, leading to a sustained increase in production of NO (Leikert et al., 2002).
The standard technique for the investigation of endothelial function in humans is the measurement of flow-mediated dilatation (FMD). FMD occurs when increased flow within vessels is detected by the endothelial cells, which respond by releasing dilator factors, the most important of which is probably NO. The effect can be measured non-invasively in humans by using an inflatable cuff to regulate the flow of blood into the vessels of the forearm. This technique has been widely used to study the effects of phytochemical supplements and other nutritional interventions. In one study, healthy male volunteers received a high-fat diet, which led as expected to a reduction in FMD, but the adverse effects were prevented by simultaneous daily consumption of 240 ml of red wine for 30 days (Cuevas et al., 2000). However in another study, red wine consumption failed to improve FMD in type II diabetics, although it did have the useful benefit of improving insulin sensitivity (Napoli et al., 2005).
Cocoa powder, which is prepared from pods of the cocoa tree Theobroma cacao, is amongst the most promising sources of biologically active flavonoids, principally oligomeric procyanidins, currently available to the food industry. In a double-blinded controlled intervention trial, Fisher et al. (2003) administered approximately 821 mg of flavanols/day, containing (-)-epicatechin and (+)-catechin, as well as oligomeric procyanidins, and measured changes in peripheral vasodilation. The cocoa supplementation induced significant vasodilation, which was reversed by infusion of a nitric oxide synthase inhibitor. In another controlled trial with chocolate, it was shown that consumption of moderate daily quantities (46 g) of dark chocolate, rich in flavonoids, led to a measurable increase in plasma concentrations of epicatechin, to more than 200 nM/L, compared to around 18 nM/L in the control group, and increased FMD significantly compared to controls (Engler et al., 2004). No reduction in blood pressure was observed by Engler et al., but reductions in blood pressure after supplementation with dark chocolate (Grassi et al., 2005) or with relatively high doses of cocoa (Davison et al., 2010) have been reported. It is interesting to note that although chocolate is a high calorie product containing relatively high levels of sucrose and fat, epidemiological evidence shows an inverse correlation between chocolate consumption and coronary heart disease in the USA, even after correction for other variables (Djousse et al., 2010). Cross-sectional population studies cannot establish causal mechanisms, but they do provide evidence in support of hypotheses derived from mechanistic studies.
Tea, in both its black and green forms, is a widely consumed beverage and one of the major sources of biologically active flavonoids in the human diet. It also has the advantage that tea drinking is not associated with any significant risk of over-consumption of either alcohol or energy. As with red wine and cocoa, epidemiological studies do suggest an inverse relationship between both black and green tea consumption and the risk of coronary heart disease (Hodgson et al., 2010), though as is usually the case it has often been difficult to completely separate the effects of tea from those of confounding factors. A recent dose-response study provides some evidence for effects of black tea consumption on blood pressure in humans, which if confirmed could prove to be of considerable significance for public health (Grassi et al., 2009).
Whilst it is probably true to say that the evidence for protective effects of diets rich in fruits and vegetables against chronic disease has tended to become less impressive with the passage of time, our understanding of the biological effects of their constituent phytochemical has grown at a near exponential rate. Clearly the so-called antioxidant hypothesis for the protective effects of fruits and vegetables remains, at best, unproven. There is little doubt that plant foods are rich in antioxidant constituents, but their poor bioavailability probably limits their effectiveness as regulators of antioxidant damage in humans. Even where there is evidence that consumption of high levels of fruits and vegetables modifies some biomarkers of antioxidant capacity and redox status, the active constituents of these dietary supplements may not be phytochemicals, and it is far from clear that the health of western consumers eating a normal diet is indeed compromised by a shortage of antioxidant nutrients. At present then, the lack of consistency of evidence across the field makes it difficult to reach a definitive conclusion about the real significance of antioxidant phytochemicals for human health. Nevertheless the last two decades have provided an abundance of new evidence for other potentially important protective mechanisms operating at the cellular and organ levels, and research on all aspects of phytochemicals and their physiological and biochemical effects continues apace. This growing evidence-base has stimulated interest in the broad concept of chemoprevention, focussed attention on particular fruits and vegetables rich in the most active compounds, and encouraged a more mechanistic approach to the epidemiology of diet and disease. It seems likely that new plant varieties and novel products based on these advances will emerge and become commercially viable in the very near future.
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