Phenolic Antioxidants

Robin L. M. Cheung, BScH, MBA

IIntroductiion ntroduct on
Oxygen. Its a matter of life and death. Without oxygen most life on earth would cease to exist. But the same reactivity that makes it ideal for using sugars for energy can also backfire and damage the very cells that are using that energy. If allowed to run amok, oxygen can damage DNA, lipids, or proteins. Damage to DNA will either kill cells or turn cancerous. Damage to lipids causes damage to membranes. Damage to proteins inactivates enzymes. All of these effects result in serious disease. Over time, evolution has developed ways to help mitigate this problem. Antioxidants are compounds that help protect cells from oxidative damage. They function by blocking the reactive compounds caused by oxygen. A quick glance at a drug stores health food area will demonstrate the tremendous growth in popularity of antioxidants. Although the popular press provides a lot of recommendations and superficial second-hand information about antioxidants, many of these reports misinterpret original research findings. It is only by referring to primary research journals critically that we can find the truth about antioxidants. One particular class of antioxidants is the group of phenolic antioxidants. They are so called because they are based on phenol, an alcohol composed of a benzene ring and a hydroxyl group. These are particularly interesting because as recently as a few years ago, they were believed only to be important for flavour (Escarpa and Gonzales 2001).

Reactve Oxygen Speciies Reactve Oxygen Spec es

The Trutth Aboutt Oxygen The Tru h Abou Oxygen
Any high school chemistry student knows that molecular oxygen comprises two oxygen atoms joined by a double-bond. Empirical evidence, however, begs to differ. The length of the bond between the oxygen atoms has been shown to be shorter (and stronger) than a single bond, yet longer (and weaker) than a double bond (Levine and Kidd, 1985; Halliwell, 1984). In 1980, Demopoulos et al. (1980) showed that molecular oxygen is in fact a diradicalthat is, it has a resonance structure with one single bond and two unpaired electrons in different orbitals. Forman and Fisher (1980) further refined the model to illustrate that ground state oxygen contains two unpaired electrons with same spins in separate orbitals, and is thus paramagnetic:

Figure 1 Ground state oxygen. Source: Levine and Kidd, 1985. p. 21

Consequently, oxygen is much more reactive than would be expected by its high school definition.

The Cattch--22 The Ca ch 22

Given its tremendous reactivity, that life on earth evolved to use it is clearly a Catch-22most life requires oxygen to live; yet, the same oxygen can easily run amok and oxidize vital cellular structures. This fact has given rise to the free radical theory of aging, which states that almost all aging processes are directly related to damage by free radicals (Atkins, 2000; Perez-Campo, 1998). In most of these cases, the myriad species of free radicals can all be traced back to chain reactions initiated by reactive oxygen species.

The Cullpriitts The Cu pr s

While all forms of oxygen are very reactivehence the term oxidation for all losses of electronsthere are four forms of oxygen that are particularly reactive. These reactive oxygen species includein order of increasing reductionsinglet oxygen, superoxide radical, hydrogen peroxide, and hydroxyl radical. Once biomolecules are damaged, they can lead to aging, cancer, or even heart disease and stroke. Siingllett Oxygen S ng e Oxygen So-called because of its single peak on an Electron Spin Resonance graph (Halliwell, 1984), singlet oxygen can exist in two forms. One form is an energetic form of molecular oxygen wherein the two outer shell electrons share the same orbital. This form is therefore diamagnetic and not a radical. The other form has the two outer shell electrons in different orbitals, as in ground state oxygen; however, the electrons are of opposite spins. This form of singlet oxygen is therefore quasi-radical (Halliwell, 1984). It is capable of rapidly oxidizing many molecules, including membrane lipids. Halliwell and Gutteridge (1984) point out that single oxygen is commonly produced in human eye lenses. This species could therefore be important in the formation of cataracts and age-related hardening of the lens (presbyopia).

Figure 2 Singlet oxygen electron configuration. Source: Levine and Kidd, 1985

Superroxiide Radiicall Supe ox de Rad ca

O2 + e-

O2-

Adding a single electron to molecular oxygen produces superoxide radical (Halliwell, 1984), which is an anion due to its net negative charge. Although in aqueous solutions, it is only

moderately reactive, it is extremely reactive in organic solvents. It is largely responsible for causing ordinarily-unreactive carbon tetrachloride (CCl4) to become toxic by forming chlorine radicals. In aqueous solutions, this radical slowly combines with another superoxide radical to form one molecule of hydrogen peroxide (H2O2) (Halliwell, 1984).

Figure 3 Superoxide electron configuration. Source: Levine and Kidd, 1985

Superoxide radicals are cytotoxic, fragmenting DNA, causing inflammation, and damaging cellular structures. Halliwell and Gutteridge (1984) also point out that helpful cells, such as immune system cells, produce superoxide radical to bombard and destroy foreign invaders. Hydrrogen Perroxiide Hyd ogen Pe ox de

O2- + e- + 2H+

H2O2

Reduction of the superoxide radical yields hydrogen peroxide (Levine and Kidd, 1985). Although in vitro experiments have shown that hydrogen peroxide is less damaging than superoxide radical (Halliwell, 1985), the presence of catalase and glutathione peroxidase clearly show that hydrogen peroxide is an unwanted product. These two enzymes are responsible for degrading hydrogen peroxide (Diplock, 1981). While hydrogen peroxide may not be highly toxic in itself, it is easily reduced further to hydroxyl radical, which is extremely damaging.

Figure 4 Hydrogen Peroxide electron configuration. Source: Levine and Kidd, 1985

Hydrroxyll Radiicall Hyd oxy Rad ca

H2O2 + e- + H+

H2O + OH

When hydrogen peroxide comes into contact with metal ions, such as copper or iron, it is reduced to water and hydroxyl radical (OH) (Halliwell and Gutteridge, 1984). Hydroxyl radical, an fiercely reactive oxygen species, is not at all to be confused with hydroxyl ion, a common constituent of dissociated bases.

Figure 5 Hydroxyl radical electron configuration. Source: Levine and Kidd, 1985.

Perhaps even worse, heat or radiation can split hydrogen peroxides O-O bond, yielding two hydroxyl radicals (Halliwell and Gutteridge, 1984).

Sources of Free Radiicalls Sources of Free Rad ca s

Free radicals have a variety of origins. They are often produced spontaneously, are catalyzed by metal cofactors, or are by-products of cellular processes. Environmental pollution and synthetic drugsnow major sources of free radicalsare novel sources of free radicals that were unimportant even a few decades ago. The following are just a few of the common sources of free radicals:

Sponttaneouslly Produced Free Radiicalls Spon aneous y Produced Free Rad ca s

As Halliwell (1984) showed previously, singlet oxygen is easily produced from molecular oxygen with heat or radiation. Once singlet oxygen is produced, it can proceed down the cascade through superoxide, hydrogen peroxide, and hydroxyl radical, wreaking havoc along the way. Radiant energy can boost one electron of a pair from a lower to a higher orbital, thus creating an unfilled orbital. Because this electron is now farther away from the positively-charged nucleus, this electron is now much more easily stolen by an oxidizing agent.

Cellllullar Respiirattiion Ce u ar Resp ra on

Whereas mitochondria reduce most oxygen to water, Sacheck and Blumberg (2001) point out that between 2% and 5% of the oxygen used escapes the system as reactive oxygen species. During exercise, even more oxygen can escape as radicals. This accounts for why marathon runners and other endurance athletes often show signs of premature aging, such as wrinkles (Atkins, 2000).

Mettalls iin tthe Bllood Cattallyze Free Radiicall Producttiion Me a s n he B ood Ca a yze Free Rad ca Produc on
Levine and Kidd (1985) showed that Iron(II) salts rapidly catalyze the production of hydroxyl radical from hydrogen peroxide: Fe2+ + H2O2 Fe3+ + OH- + OH

Halliwell and Gutteridge (1984) showed that Copper(I) salts catalyze this reaction even faster than iron: Cu+ + H2O2 Cu2+ + OH- + OH

The presence of free copper or iron in the bloodstream is therefore critically dangerous. It is not surprising, therefore, that most iron in the body is sequestered in hemoglobin or other transport proteins, and copper is tightly bound to amino acids or small peptides (Halliwell and Gutteridge, 1984).

Ciigaretttte Smokiing C gare e Smok ng

The primary source of indoor free radicals also happens to be the most easily preventable. Cigarette smoke contains over 3,000 separately-identifiable compounds and delivers one hundred trillion (1x1014) biologically active molecules in an average puff (Levine and Kidd, 1985). Many of these compounds are potent mutagens and carcinogens. Notably, the list includes benzo[a]pyrene, the carcinogenic heterocyclic amine found in charred meats responsible for causing intestinal and stomach cancers. The following table illustrates some of the common harmful ingredients in cigarette smoke: Principal Ingredients of Cigarette Smoke (Levine and Kidd, 1985) tar pyrene nicotine benzo[a]pyrene pyrene benzo[e]pyrene Particulate Matter cadmium chrysene lead fluoroanthene carbon monoxide Ammonia acetone Formaldehyde Vapours polonium-210 (radioactive) Toluene nitrogen dioxide Hydrogen cyanide

Free Radiicall Chaiin Reactiions Free Rad ca Cha n React ons

Many reactions in nature occur through radical chain reactions. Gillespie et al. (1994) describe a chain reaction as A multi-step reaction with an initial step that produces a reactive intermediate, chainpropagation steps in which the product is formed and the reactive intermediate is regenerated, and one or more chain-termination steps, in which the reactive intermediate is removed.

Formattiion off HCll:: An Examplle off Radiicall Chaiin Reacttiions Forma on o HC An Examp e o Rad ca Cha n Reac ons
One common example of a radical chain reaction is the formation of HCl from H2 and Cl2 gases. As Gillespie et al. (1994) point out, chain reactions comprise three phases: initiation, propagation, and termination. The net chemical reaction is: H2 + Cl2 2 HCl, but as closer examination of the mechanism points out, this net reaction is not an accurate representation of what is actually happening.

IIniittiiattiion n a on Light with wavelength = 493 nm initially dissociates chlorine molecules into chlorine radicals: Cl2 2 Cl

Like all free atoms with unpaired electrons, the chlorine radicals are extremely reactive. This leads countless cycles of the propagation steps. Prropagattiion P opaga on One of the chlorine radicals attacks a hydrogen molecule, forming one molecule of hydrogen chloride and a hydrogen radical: Cl + H2 HCl + H

This new hydrogen radical reacts further with a subsequent chlorine molecule to form another molecule of HCl and yet another chlorine radical: H + Cl2 HCl + Cl

This new chlorine radical seeks out other reactants and the propagation cycle continues until one of the radicals encounters another radical. Then the termination step ensues. Terrmiinattiion Te m na on This cascade ends when one radical encounters another radical. Their unpaired electrons are thus paired without forming another radical: 2 Cl 2 H H + Cl Cl2 H2 HCl

The formation of a single chlorine radical by photolysis can create many thousands of HCl molecules before encountering another radical and terminating. The same type of reactions occur in animal systems. Rather than chlorine and hydrogen, however, the radicals are oxygen, lipids, or other vital cell components. It is obvious that damage caused by radical chain reactions can be catastrophic. Damage to DNA can cause cell deathor worsecancer. Damage to membrane lipids can cause skin wrinkles or hardening of membranes. Oxidized cholesterol becomes more sticky and contributes greatly to atherosclerosis. Control of radicals in living systems is therefore a prime factor in determining an animals lifespan.

Why Are Free Radiicalls So Bad? Why Are Free Rad ca s So Bad?
Liipiid Peroxiidattiion L p d Perox da on
Lipids are a vital part of every cell. Singer and Nicholson (1972) demonstrated that fluidity of cell membranes is a crucial characteristic. When the fatty acid chains in lipids become damaged by free radicals, they become cross-linked and damaged (Kong and Davison, 1980). Through a series of steps, lipids become lipid radicals and lipid peroxides, which then regenerate further radicals through chain reactions. Although any lipid can be damaged by radicals, polyunsaturated fatty acidsthe same type conventional medicine advises to be the most healthyare more susceptible to oxidation than saturated fats or cholesterol (Halliwell, 1984). According to Halliwell (1984), this is because polyunsaturated fatty acids (PUFA) are characterized by multiple double bonds. Double bonds have higher electron densities than single bonds. Thus, PUFAs, with their multiple double bonds are prime targets for free radical (FR) damage: FR + PUFA-H FR-H + PUFA

Free radicals, attracted by the high electron density of PUFA double-bonds, easily abstract a hydrogen atom, quenching the free radical, but turning the PUFA into a free radical, itself. The unpaired electron is then free to shift along the PUFA molecule, causing the PUFA to change shape (Halliwell, 1984). Obviously, a misshaped PUFA will no longer perform its structural function properly. Even worse, this oxidized PUFA radical can now continue the chain reaction, damaging other neighbouring PUFA molecules. This is easy, since the fatty acids are locked in place next to each other in a membrane.

Oxygenattiion tto a Peroxyll Radiicall Oxygena on o a Peroxy Rad ca

As if the PUFA radical alone were not bad enough, oxygen can react with the PUFA radical to form a peroxyl fatty acid radical (Levine and Kidd, 1985): PUFA + O2 PUFA-O-O

These lipid peroxide radicals can then attack other PUFA and regenerate the PUFA radical.

How Phenolliic Antiioxiidants Work How Pheno c Ant ox dants Work

It has long been known that phenolic compounds are effective antioxidants. Indeed, phenolic compounds have been used for decades as industrial antioxidants. Phenol comprises a benzene ring and an alcoholic hydroxyl group. Originally called carbolic acid, phenol was first used by Joseph Lister as an antiseptic in 1865 (Brown, 1995). Although phenol is an alcohol, it does not behave like typical alcohols, and is thus categorized in its own

family (Fox and Whitesell, 1994). Phenols are more acidic than alcohols; the pKa of the phenolic hydroxyl is 9.95 (Brown, 1995). The ability of phenolic compounds to quench free radicals arises because of both their acidity (ability to donate protons) and their delocalized -electrons (ability to transfer electrons while remaining relatively stable) characteristic of benzene rings (Brown, 1995). Electron delocalization and ease of ionization are probably responsible for their bright colours. Perhaps the most well-known physiological phenolic antioxidant is vitamin E. It is an apt antioxidant for membranes because its long aliphatic tail can embed itself in lipid membranes while exposing its phenolic head group to the aqueous environment (McKay and King, 1980). A free radical can easily abstract a hydrogen from phenols hydroxyl group, creating a radical of vitamin E. Spontaneous reactions only occur when they have a negative G (Rayner-Canham et al., 1989). The reaction of a vitamin E radical with another lipid to continue the chain reaction has a positive G and is therefore energetically unfavoured. The vitamin E radical therefore stabilizes itself by converting to a quinone (cyclohexanedienedione) (McKay and King, 1980). This conversion to a quinone terminates the chain reaction and protects surrounding lipids from oxidation. Vitamin E therefore acts as a bulletproof vest.

Hiisttory off Viittamiin E H s ory o V am n E

By 1922, researchers had established the existence of vitamins A, B, C, and D (Mason, 1980). Osborne and Mendel (1919) and Mattill and Conklin (1920) showed that diets sufficient in vitamins A, B, C, and D could support normal growth in rats; yet these semipurified diets would not allow the rats to reproduce. Some factor was clearly missing from their diets that was not necessary for normal growth but was crucial to reproduction. Evans and Bishop (1922) later discovered the existence of the unknown dietary factor that modulated reproductive ability. The last vitamin discovered at that time was vitamin D. Evans was quoted as saying, We have adopted the letter E as the next serial alphabetical designation, the antirachitic vitamin now being known as vitamin D (Evans, 1925). In 1936, Evans et al. reported the isolation of an alcohol from wheat germ oil that had demonstrable biological activity. This they named -tocopherol (C29H50O2). Two years after this discovery, they isolated two other tocopherols from oils of other vegetables. They named these tocopherols - and - tocopherols. Emerson et al.(1937) later noted that these tocopherols were of lesser biological activity than the form. The major question involving tocopherols thenand even nowis whether tocopherols have functions other than biological antioxidants. Also, despite the many reports that -tocopherol prevents or cures human ailments, its precise role in human health still remains elusive. Since the discovery of vitamin E, researchers have encountered numerous other compounds shown to have antioxidant activity. They share one characteristic in common with vitamin E they are all phenolic compounds, based on the alcohol of a benzene ring.

Where Do You Fiind Antiioxiidants? Where Do You F nd Ant ox dants?

Phenolliic Conttentt Pheno c Con en
Phytochemicals are bioactive non-nutrient compounds found in plants. They are often responsible for the bright reds and blues in berries and vegetables (Atkins, 2000; Escarpa and Gonzales, 2000). The most important class of phytochemicals in plant food sources is the group of phenolic compounds. There are thousands of phenolic phytochemicals, but they can be organized into two main groups: polyphenols and flavonoids (Escarpa and Gonzales, 2000). Pollyphenolls Po ypheno s A common constituent of tea, tannins are plant metabolites that are commonly found in tea, cereal grains, fruits, vegetables, and nuts (Nakamura et al., 2001). Polyphenols are so named because they have multiple phenolic constituents (Escarpa and Gonzales, 2001). They function as efficient free radical scavengers by donating their alcoholic hydrogen or one of their delocalized electrons (Madsen et al., 2001). Tea has been shown to contain about 3 grams of polyphenols per serving of tea leaves (Hibasami et al., 2000). Hibasami et al. (2000) found that, among Japanese and Chinese smokers, there was an abnormally low incidence of lung cancers, which they attributed to the high polyphenol content in tea. The average daily intake of tea in Asia is six or seven cupsthe minimum amount recommended for antioxidant protection. Although black tea is oxidized, it has still been shown to have protective effects, though less reliably than green (unoxidized) tea. Perhaps the most popular polyphenols today are the red wine polyphenols. Atkins (2000) recommends drinking one to two glasses of red wine per day. These polyphenols can protect against cancer by protecting DNA from radical damage or help prevent cardiovascular disease by preventing oxidation of lipids and cholesterol. Fllavonoiids F avono ds Flavonoids are actually a specific group of polyphenol. They are actually the most plentiful group of polyphenol compounds and so are given their own category (Madsen et al., 2000). Commonly found in apples, berries, and other brightly-coloured fruits and vegetables, flavonoids play a dual role for plants. The bright colours attract pollinating insects, while they protect lipids and vital cell components from damaging oxidation (Escarpa and Gonzalez, 2001). Flavonoids are potent free radical scavengers because they can donate their alcoholic hydrogen atom to free radicals (Madsen et al., 2000).

Fruiitts and Vegettablles Fru s and Vege ab es

Within the past two decades, scientists have started to recognize the value of fruits and vegetables beyond their contribution in helping prevent vitamin deficiencies. A significant number of phytochemicals have been found in fruits, vegetables and other plant foodstuffs, which is linked to the reduction of risk of disease. The anti cancer promotion of fruits and vegetables has highly been promoted recently. But fruits and vegetables not only help decrease chances of developing

cancer. They also decrease the risk of developing coronary heart disease and strokes. Compounds in fruits and vegetables have even been found to slow aging.

Wholle Graiins Who e Gra ns

The Canadian food guide recommends eating whole grains in order to prevent against chronic disease. Whole grains contain many antioxidants, including vitamins and phenolic acids. Grains are rich sources of phenolic acids in the bran layer of the grain. (Slavin et al 1999). Slavin et al. (1999) point out that whole grain phenolics probably work by activating detoxification systems and blocking activation of carcinogens.

IIndusttriiall Anttiioxiidantts ndus r a An ox dan s

Antioxidants are not only important to living systems. Many industrial processes, such as plastic and oil processing, involve high heat and pressures. These conditions are likely to cause oxidative damage to products. Knop and Scheib (1979) show that various phenolic substances are used to protect rubber, plastic, and other materials during processing.

Recommendatiions Recommendat ons

Although the decision to supplement with antioxidants should be closely supervised by a physician, there are several lifestyle changes that can increase natural dietary intake of common antioxidants and help extend healthy lives. Sttop Smokiing S op Smok ng Smoking is the primary controllable cause of cancer and heart disease. By cutting out smoking, each breath takes in 1014 fewer free radicals. Eatt Morre Frruiitts and Vegettablles Ea Mo e F u s and Vege ab es Because studies are still not clear about the benefits of supplementing individual antioxidants, it is likely that they must all work together for maximum benefit. The brightly-coloured fruits and vegetables contain the most phenolic antioxidants. Drriink Tea D nk Tea Studies have shown that drinking six to seven cups of tea per day, as the Asians do, reduces risk of cancer and heart disease. While these simple guidelines can help prolong healthy lives, it should be pointed out that much research must still be done. Many experiments are carried out in vitro, and many other experiments show that in vivo results may be different.