Antioxidant Properties

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As part of normal metabolism, radicals are generated. Radicals, as discussed in Chapter 2, are compounds with free (i.e. unpaired) electrons. Radicals are very reactive, and, when left unchecked, can cause oxidative damage to the molecules in a cell, and hence have negative impacts on the cellular metabolism. An excess of radicals can cause oxidative stress.

Compounds that can scavenge radicals are also referred to as antioxidants. The best known anti-oxidants are vitamin C and vitamin E. Vitamin C is L-ascorbate (7.8), a good reducing agent that prevents oxidation of other molecules. The oxidized form of L-ascorbate is L-dehydroascorbic acid (7.9). Vitamin E is a mixture of a-, P-, y-, and S-tocopherol (7.10a-d). Of these four compounds, a-tocopherol is the most effective. Vitamin E is lipid-soluble and has the ability to disrupt the chain reaction during lipid peroxidation (see Chapter 2, Section 1.9).


A lack of vitamin C in the diet results in the disease scurvy. Scurvy's symptoms include purple lesions on the skin, rotten gums, and, as a consequence, loss of teeth. This disease was common among 16th and 17th century sailors who relied on preserved foods and an overall unbalanced diet on their long journeys. The intake of fresh fruits and vegetables, which are rich in vitamin C, can effectively prevent scurvy. The biochemical basis of scurvy is the reduced activity of the enzyme prolyl hydroxylase (E.C., probably because the iron atom that is part of the enzyme cannot be maintained in its active, ferrous state due to the lack of vitamin C. This reduced enzyme activity then results in insufficient hydroxylation of collagen, a structural protein that gives elasticity to the skin and blood vessels. The lack of elasticity causes the lesions in the skin and the rupture of blood vessels (Stryer, 1988).

In the current era scurvy is a rare disease, but many other diseases can arise from low levels of these vitamins. For example, low plasma levels of a-tocopherol and L-ascorbate correlate with an increased incidence of myocardial infarction and of some forms of cancer (Gey et al., 1987). In fact, many diseases are thought to be associated with higher levels of radicals in the cell (Halliwell, 1991). An example includes rheumatoid arthritis (RA), whereby joint tissues have an excess of activated neutrophiles that secrete radicals, such as O2-. Under normal circumstances the radicals are used to kill pathogenic microorganisms, but in the case of RA, the excess of activated neutrophiles contribute to the inflammation and swelling, and hence aggravate the disease symptoms. Other diseases in which reactive oxygen species are implicated include atherosclerosis, adult respiratory distress syndrome (ARDS), myocardial infarction and some forms of cancer. Thus, the ability to scavenge radicals can prevent the onset of a disease, slow down the progress of the disease, or alleviate its symptoms.

Aside from vitamins C and E many other compounds present in fruits and vegetables have been shown to have anti-oxidant properties. Among these compounds there are several classes of phenolic compounds. Aside from preventing scurvy these compounds have a positive influence on cardiovascular health.

An interesting case is the prevention of cardio-vascular diseases as a result of the consumption of wine. Like most fruits grapes are rich in polyphenols, and the process of wine making results in the concentration of polyphenols. Wine polyphenols are considered to have beneficial effects on coronary heart disease and atherosclerosis. The presence of polyphenols in wine are thought to be the reason for the 'French paradox': France was shown to have a coronary mortality rate close to that of China and Japan in spite of the high amount of saturated fat and cholesterol levels in the French diet. The consumption of red wine in France, however, is considerably higher than in either China or Japan (Staggs, 1996).

Wollin and Jones (2001) investigated the effects and mechanisms of action of consumption of red wine compared to other alcoholic beverages on the risk of cardiovascular disease. Of particular interest was the form and quantity of alcohol consumed. The relationship between alcohol consumption and mortality is supported by epidemiologic studies suggesting that different forms of alcohol alter the relative risk for mortality. Evidence from various epidemiologic and experimental studies suggests a protective effect against the development of cardiovascular disease by moderate consumption of red wine. They point out that components of red wine that are thought to be responsible for the protective effects include various phenolic compounds as well as alcohol content.

The effects of wine and its polyphenol constituents on early indicators of coronary heart disease such as elevated levels of plasma lipids, platelets and serum antioxidant activity were discussed in a review by Cooper et al. (2004). This review also addressed whether the polyphenols or alcohol are responsible for the beneficial effects of wine on cardio-vascular health. The authors conclude that red wine polyphenols have little effect on plasma lipid concentrations, but that wine consumption reduces the susceptibility of low-density lipoprotein (LDL) cholesterol to oxidation and increase serum antioxidant capacity. These effects, however, do depend on the amount of wine that is consumed and the period of supplementation. It was suggested that specific polyphenols appear to have endothelium-dependent vaso-relaxing abilities. Red wine phenolics also have an inhibitory effect on platelet aggregation. Evidence suggests that alcohol has a positive synergistic effect with wine polyphenols on some atherosclerosis risk factors. Thus, evidence that wine drinking is beneficial for cardiac health appears positive.

Flavonoids may benefit health in cardiovascular disease by adjusting adhesion of monocytes (large mononuclear leukocytes in the blood) in the inflammatory process of atherosclerosis. Most in vitro studies have used types of flavonoids present in food rather than those that appear in plasma after food ingestion. Koga and Meydani (2001) tested the effects of plasma metabolites of the flavonoids (+)-catechin (1.39) and quercetin (1.43) on the alteration of monocyte adhesion to human aortic endothelial cells and on the production of reactive oxygen species. Plasma extracts of flavonoid metabolites were prepared after administration of pure compounds to rats. The plasma preparations contained sulfate or glucuronide conjugates or both, as well as methylated forms. Adhesion of U937 monocyte cells to human aortic endothelial cells was measured, and the production of reactive oxygen in the endothelial cells was monitored, when the cells were pretreated with either pure compounds or plasma extracts from control or treated rats. Adhesion assays were performed with endothelial cells stimulated with interleukin or cells activated with phorbol myristylacetate. Reactive oxygen species were measured after challenging the human aortic endothelial cells with interleukin-1b (IL-1b) or hydrogen peroxide.

Pretreatment of endothelial cells with (+)-catechin (1.39) metabolites inhibited U937 cell adhesion to interleukin IL-1b-stimulated endothelial cells, whereas pretreatment with intact (+)-catechin had no effect. Generation of reactive oxygen species in hydrogen peroxide-stimulated cells was inhibited by (+)-catechin, its metabolites, and control plasma extract, whereas reactive oxygen species generation in IL-1b-stimulated cells was inhibited by (+)-catechin metabolites only. In contrast, quercetin inhibited U937 cell adhesion to interleukin IL-1b-stimulated cells, whereas its metabolites were not effective. The authors concluded that metabolic conversion of flavonoids such as (+)-catechin and quercetin modifies the biological activity of the flavonoids. It was suggested that metabolites of flavonoids, rather than their intact forms, contribute to the effects of flavonoids on reducing the risk of cardiovascular disease.

The seeds of fenugreek (Trigonella spp.) are rich in phenolic compounds. Kaviarasan et al. (2004) evaluated fenugreek seeds for their potential to protect erythrocytes from oxidation induced by hydrogen peroxide (H2O2). Human erythrocytes from diabetic and non-diabetic subjects were incubated with increasing amounts of fenugreek seed extract and challenged with H2O2. They were then analyzed for hemolysis (release of hemoglobin) and lipid peroxidation. Erythrocytes from diabetic subjects were more susceptible to hemolysis and lipid peroxidation than those from non-diabetic subjects. It was significant that incubation of the cells with the polyphenol-rich seed extract significantly reduced the oxidative modifications in both cell groups. The inhibition of lipid peroxidation was concentration-dependent. The extract contained 0.75 mM gallic acid-equivalents (1.41) of phenolic compounds. The findings demonstrated the potent antioxidant properties of the phenol-rich fenugreek seeds.

Palmerini et al. (2005) reported that phenols in the Mediterranean diet are free radical scavengers and have antioxidant properties. Yet the mechanisms of their effects are not fully understood. Palmerini and co-workers hypothesized an effect on the concentration of Ca2+, which plays an important role in intracellular signaling and regulates various processes. To test this hypothesis they incubated human lymphomonocytes with two phenolic compounds isolated from olive oil: 3,4-dihydroxyphenyl-ethanol (7.11) and p-hydroxyphenyl-ethanol (7.12). They showed that both of these compounds increased the intracellular concentration of Ca2+ in a dose-dependent manner, both in the presence and in the absence of calcium in the extracellular medium. This effect was antagonized by the drug nifedipine (7.13), a calcium channel blocker administered as a muscle relaxing agent to patients suffering from chest pain.





The olives themselves contain many phenolic compounds with antioxidant properties. Bouaziz et al. (2005) investigated the olive cultivar "Chemlali" from Tunisia. Oleuropein (7.14), a bitter glycoside esterified with a phenolic acid, was the major compound present. Phenolic monomers and twelve flavonoids were also identified. The antioxidant activity of the extract was evaluated. Acid hydrolysis of the extract enhanced its antioxidant activity. p-Hydroxyphenyl-ethanol (7.12) and quercetin (1.43) showed antioxidant activities similar to that of 2,6-di-tert-butyl-4-methyl phenol (7.15), a reference compound with known antioxidant properties. It was suggested that a hydroxyl group at the ortho-position on the flavonoid B ring could contribute to the antioxidant activity of the flavonoids.


In addition to the contribution of olives and olive oil to antioxidant activity in the Mediterranean diet, spices and dressings have also been shown to have health-promoting activity. In a study by Ninfali et al. (2005), twenty-seven vegetables, fifteen aromatic herbs and some spices consumed in Central Italy were studied to determine total phenolic flavonoid content as well as their antioxidant capacity measured by the oxygen radical absorbance capacity method. A comparison of antioxidant capacity was made between different salads, as well as between salads to which aromatic herbs had been added. Lemon balm and marjoram at a concentration of 1.5% (w/w) increased the antioxidant capacity of a salad by 150% and 200%, respectively. A 200-gram portion of a salad enriched with marjoram corresponded to an intake of 200 mg phenolics and 4000 oxygen radical absorbance capacity units. Olive oils, wine and apple vinegars were salad dressings that provided the highest increase in antioxidant capacity. Of the spices tested, cumin and ginger made the most significant contribution to the antioxidant capacity.

Both intact compounds and their metabolites - formed either in human tissues or meabolized in the colon by microflora - might explain the effects on health of dietary polyphenols. In order to assess the importance and biological activities of microbial metabolites in vivo, Gonthier et al. (2003) measured the microbial metabolites formed in rats fed a diet supplemented with three levels of catechin (1.43) or red wine powder containing proanthocyanidins, phenolic acids, flavanols, anthocyanins and flavonols. This was compared to rats fed an unsupplemented diet. Aromatic acid metabolites in urine were assayed by an HPLC-electrospray ionization-mass spectrometry method. The primary metabolites formed from the catechin diet were 3-hydroxyphenylpropionic acid, 3-hydroxybenzoic acid and 3-hydroxyhippuric acid. Their total urinary excretion accounted for 4.7% (w/w) of the catechin ingested, and that of intact catechins for 45.3% (w/w). When the diet was supplemented with red wine powder, the same metabolites observed with the catechin diet were identified in urine, along with p-hydroxybenzoic (1.4), 3-hydroxyphenylacetic (see 1.11 for a comparable structure), p-coumaric (1.13), vanillic (1.8), and hippuric (N-benzoylglycine) acids. These aromatic acids accounted for 9.2% (w/w) of the total wine polyphenols ingested, whereas intact catechins accounted for only 1.2% (w/w). It was suggested that the higher excretion of aromatic acids by rats fed wine polyphenols was due to their poor absorption in the proximal part of the gut. Some of the microbial metabolites still contained a reducing phenolic group and should also prevent oxidative stress in inner tissues. The authors suggested that attention be given in the future to these microbial metabolites and their biological properties to help explain the effects of polyphenols that are not easily absorbed through the gut.

Whole grains provide another source of phenolic antioxidants, but whole grains contain many other compounds that have a positive effect on human health. They have high concentrations of dietary fiber, starch, and oligosaccharides, and contain phytate, phyto-oestrogens such as lignans, plant stanols and sterols, vitamins and minerals. Epidemiological studies have shown that whole-grain intake is protective against cancer, cardiovascular disease, diabetes, and obesity (Slavin et al., 2004). Despite recommendations to consume three servings of whole grains daily, usual intake in Western countries is only about one serving. Feeding studies show that consumption of whole grains improves biomarkers such as weight loss, blood-lipid levels, and the concentration of antioxidants. Although it is difficult to separate the protective properties of whole grains from dietary fiber and other components, the disease protection seen from whole grains in prospective epidemiological studies far exceeds the protection from isolated nutrients and phytochemicals in whole grains

An interesting question is whether the health-promoting properties of phenolic compounds is consistent, or whether there are effects of the culture practice during crop production, the location of the field where the crop is grown, and the specific cultivar that was selected. Emmons and Peterson (2001) investigated whether cultivar and location had an effect on phenolic contents and antioxidant activities of alcohol-soluble extracts from groats (i.e. the edible part of the grain) of oat (Avena sativa L.). Antioxidant activities (AOA) and concentrations of eight phenolic compounds having AOA were measured in three cultivars grown at seven locations in Wisconsin during 1998. The phenolic compounds included p-coumaric acid (1.13), ferulic acid (1.15) and avenanthramides (7.16a-d). Avenanthramides are phytoalexins found in oats. There are several different compounds -avenanthramide A, B, D and G - that differ in the substitution pattern of the two aromatic rings, as is shown below. Avenanthramide L (7.17) contains an additional carbon in the chain linking the two aromatic rings (Okazaki et al., 2004).

There were significant differences among cultivars for AO A concentrations of all of the phenolic compounds measured, except p-coumaric and ferulic acids, and for total free phenolic contents (FPC). Location significantly affected the concentrations of five of the phenolics and total FPC, but did not affect AOA. There were significant cultivar x location interactions for the concentrations of avenanthramides and for total FPC. The presence of this interaction means that the cultivar with the highest FPC level in one location, does not produce the highest FPC levels in another location. The unexpectedly high concentrations of avenanthramides from the Sturgeon Bay location were confirmed by analysis of groats from 1999 and 2000. Based on these observations it should be possible to improve the AOA and phenolic concentrations of oat as quantitative traits in a cultivar development program (see Chapter 3, Section 3.4), but significant location effects may slow down progress.

Similarly, Tarozzi et al. (2004) assessed the impact of cultivation practices, commercial processing, and storage of fruits and vegetables on phenolic antioxidants. They investigated the influence of commercial cold-storage periods on antioxidant properties of apples grown by organic or

integrated systems. Regardless of the production method, total phenolics and total antioxidant activity decreased in apples with the peel intact only in the first three months of storage. It was suggested that cold storage rapidly depletes antioxidant properties in apple skin but not in the pulp. Antioxidant activity was assessed in vitro in terms of intracellular antioxidant, cytoprotective, and anti-proliferative activities in human colon carcinoma (Caco-2) cells. Time-related decreases in antioxidant activity after six months cold storage were found regardless of the cultivation method. These data suggest that cold storage should be taken into account when evaluating the cancer-preventive benefits of fruits and vegetables. Furthermore, the authors concluded that organic production methods of apples do not provide health benefits. This latter conclusion is in contrast with a study by Halweil (2003), who concluded that organic produce was richer in health-promoting phenolic compounds.

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The Mediterranean Diet Meltdown

The Mediterranean Diet Meltdown

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