Chapter 1 Introduction to Flavonoids

CHAPTER 1

INTRODUCTION

CHAPTER 1 INTRODUCTION

11 Free radicals and disease

A free radical can be defined as any molecular species capable of independent

existence which contains an unpaired electron in an orbital such as CH3 or Cl the

dot representing a single (unpaired) electron (Halliwell and Gutteridge 1989 Kaul et

al 1993) This electronic configuration makes free radicals highly unstable and

chemically very reactive Gomberg (1900) was the first scientist to demonstrate the

existence of organic free radicals in the form of the triphenylmethyl radical In 1954

Gerschman and co-workers (Gerschman et al 1954) demonstrated the presence of

oxygen-containing free radicals in a biological system by showing that compounds

that protected animals against radiolysis also protected them against oxygen

challenge This result contributed to the free radical theory of ageing which was

proposed by Harman in 1956 (Harman 1956)

Free radicals are weakly attracted to a magnetic field and are described as

paramagnetic Electrons are more stable when paired together in an orbital the two

electrons in a pair have different directions of spin Hence radicals are generally

less stable than non-radicals although their activity can vary considerably Once

radicals are formed they can either react with another radical to form a non-radical

species or with another molecule by various interactions (Cadogan 1973) As noted

before most radicals are highly reactive and thus are short lived They are therefore

difficult to measure directly and their activity is often determined by indirect

measurements of tissue damage products or ldquofootprintsrdquo (Halliwell and

Chirico 1993) One direct technique for the detection and study of free radicals

themselves is electron spin resonance (ESR) or electron paramagnetic resonance

(EPR) spectroscopy This technique exploits the absorption of microwave radiation

of particular frequencies when the radical is located in a strong magnetic field (Kaur

and Perkins 1991)

The rate and selectivity of reactions undergone by radical species depends on the

radical concentration the extent to which delocalisation of the single electron of the

radical occurs and on the absence of weak bonds in any other molecules present with

which the radical could interact (Bensasson et al 1993)

The most important free radicals in many disease states are oxygen derivatives due to

the ubiquitous presence of oxygen in higher species and diatomic oxygens ability to

readily accept electrons Oxygen plays an essential role in aerobic life including the

production of energy and the synthesis of a variety of important compounds In

many of these reactions iron copper and other transition metals play an essential

role usually bound in specific complexes within proteins Any major disruption of

these complex oxidative reactions rapidly leads to death (Olson 1995) Molecular

oxygen normally contains three unpaired electrons and in this state is known as

triplet oxygen When triplet oxygen reacts with transition metals and other

compounds partly reduced and highly active forms of oxygen may be produced of

which the hydroxyl radical (OH) is one of the most reactive (Cheeseman and Slater

1993 Halliwell and Gutteridge 1995) When the intermediates of oxygen reduction

ie reactive oxygen species (ROS) are not strictly regulated they are potentially

problematic as they are more reactive than ground state molecular oxygen A variety

of adverse consequences may occur including lipid carbohydrate and nucleic acid

oxidation protein inactivation disruption of membrane function and activation of

pro-carcinogens and other xenobiotics (Thomas 1994)

111 Sources of free radicals

There are several endogenous sources of free radicals These are produced during

normal metabolism in mitochondria during the process of oxidative phosphorylation

as electrons are passed along the electron transport chain at the mitrochondrial inner

membrane (Kehrer and Smith 1994) During normal aerobic metabolism

mitochondria consume molecular oxygen and reduce it sequentially to produce

water The inevitable by-products of this reaction are superoxide hydrogen peroxide

and the hydroxyl radical It has been calculated that over 2 kg of superoxide are

produced in the human body every year (Halliwell 1996)

Peroxisomes which contain fatty acyl CoA oxidase dopamine -hydroxylase urate

oxidase and other oxidative enzymes produce hydrogen peroxide during

metabolism which is then degraded by catalase Some hydrogen peroxide escapes

degradation and leaks into other cellular compartments and increases oxidative

damage

Other enzyme reactions are also important endogenous sources of radical production

The cytochrome P-450 mixed function oxidase system constitutes a primary defence

against various xenobiotics and endogenous substances and enhances production of

free radicals Some superoxide is produced deliberately for example by NADPH

oxidase in phagocytic cells to aid the destruction of bacteria or virus-infected cells

with an oxidative burst of superoxide hydrogen peroxide hypochlorite and nitric

oxide There are also important exogenous drivers of free radical production such as

a high intake of iron and copper cigarette smoke inhaled atmospheric pollutants

radiation (Kubow 1993) and lipid peroxidation products in foods

112 Reactive oxygen species (ROS)

ROS include all oxygen containing free radicals and related non-radical oxygen

containing species and are constantly formed in human body by various

physiological processes and insults as noted previously (Figure 11)

Purposeful directed useful but potentiallydamaging if excessive prolonged oruncontrolled

eg superoxide and hypocholorous acid productionduring phaocytic lsquorespiratory burstrsquo endothelial cellrelease of nitric oxide for maintenance of normalvascular tone

Sources of ROS in vivo

Physiological (lsquonormalrsquo)production of ROS

Accidental potentially harmful

eg leakage of electrons from cytochrome in themitochondrial electron transport chain exercise-induced local ischaemia lsquoautoxidationrsquo ofcatecholamines and other endogenous compounds

Pathological (lsquoabnormalrsquo)production of ROS

Serving no purpose always potentially damaging

eg in food cigarette smoke pollutants ozoneradiation-induced water splitting radical forms of toxinsand drugs from peroxisomal and cytochrome P-450metabolism as a consequence of reactions betweenperoxides and free iron or copper during ischaemia andpostischaemia reperfusion

Figure 11 Sources of reactive oxygen species (adapted from Strain and Benzie 1998)

Many of them serve useful physiological functions but they can be toxic when

generated in excess or in inappropriate environments and this toxicity is usually

aggravated by the presence of ions of such transition metals as iron or copper

Table 11 Reactive oxygen species (ROS) of physiological interest

Radical Name Typical biological target

O2macrbull Superoxide Enzymes

H2O2 Hydrogen peroxide Unsaturated fatty acids

OHbull Hydroxyl All biomolecules

Rbull R-yl Oxygen

RObull R-oxyl Unsaturated fatty acids

ROObull R-dioxyl (R-peroxyl) Unsaturated fatty acids

ROOH Hydroperoxide Unsaturated fatty acids1O2 Singlet molecular oxygen H2O

NObull Nitroxyl Several

(Muggli and Hoffmann 1993)

ROS can be divided into two main groups these being oxygen containing free

radicals such as the superoxide anion hydroxyl radical peroxyl and alkoxyl

radicals and non-radical ROS such as hydrogen peroxide singlet oxygen

hydroperoxides and hypochlorite

The most important free radicals in many disease conditions are oxygen derivatives

specifically the superoxide anion (O2macr bull) and the hydroxyl radical (OHbull) Superoxide

(O2macr bull) is produced in vivo by adding a single electron to the diatomic oxygen

molecule

O2 + emacr O2macr bull

In biological systems the relatively short half-life of O2macr bull limits its diffusion away

from its site of production Part of the O2macr bull formed in vivo is a chemical accident

(Halliwell 1991) As an example when mitochondria are functioning some of the

electrons passing through the respiratory chain leak from the electron carriers and

pass directly onto oxygen forming O2macr bull (Fridovich 1974) Other molecules such as

adrenaline several sugars including glucose can also oxidize in vivo to produce

oxygen radicals Superoxide is both a reducing and an oxidizing agent In aqueous

solutions it can oxidise ascorbic acid It can also reduce certain iron complexes such

as cytochrome-C and ferric-ethylenediamine-tetraacetic acid (Fe-EDTA)

Superoxide dimutase (SOD) removes O2macr bull by catalysing a dismutation reaction

converting it to hydrogen peroxide (H2O2) and oxygen (O2) (Halliwell and

Gutteridge 1989)

2O2macr bull + 2H+ H2O2 + O2

Although some of the O2macr bull production in vivo may be accidental much is functional

as O2macr bull production is important in allowing phagocytes to kill some of the bacterial

strains which they can engulf Also in the presence of the enzyme myeloperoxidase

the H2O2 produced by dismutation of O2macr bull oxidizes chloride ions to hypochlorous

acid (HOCl) which is powerful antibacterial agent usually found in household

bleaches

H2O2 + Clmacr HOCl + OHmacr

A further example of the usefulness of O2macr bull may be provided by the endothelial cells

that line blood vessels It is thought that endothelial cells produce nitric oxide (NO)

a free radical that is identical to endothelium-derived relaxing factor (EDRF)

Vascular endothelium also appears to produce small amounts of O2macr bull which can

inactivate NO combining to give a non-radical product the nitrate ion (NO3macr)

NO + O2macr bull NO3macr

Thus variations in the production of NO and O2macr bull by endothelium may provide one

mechanism for the regulation of vascular tone (Halliwell 1989)

Hydrogen peroxide (H2O2) has a relatively long half-life is membrane permeable

and may traverse considerable distances causing damage at sites distant from its

origin (Kaul et al 1993) Although H2O2 is poorly reactive at low levels at higher

levels it can attack several cellular energy producing systems For instance it can

inactivate the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase

Hydrogen peroxide can sometimes be toxic to cells because it can give rise to OHbull

which is highly reactive Thus H2O2 acts as a conduit to transmit ROS induced

damage across cell compartments and between cells (Young and Woodside 2001)

Stohs and Bagchi (1995) suggested that there are several mechanisms by which OHbull

may be produced but the most important mechanism is likely to be the transition

metal ion catalysed decomposition of O2macr bull and H2O2 The most important transition

metals in terms of OHbull generation are iron and copper Fenton (1894) was the first

scientist to describe the involvement of transition metals in the formation of hydroxyl

radicals

Fe (II) + H2O2 Fe (III) + OHbull + OHmacr

Biochemical studies of free radicals indicate that O2macr bull is the main source of

hydrogen peroxide in vivo In the presence of transition metal ions such as iron or

copper the reaction of O2macr bull and H2O2 together can directly form a hydroxyl radical

at rapid speed

Fe (III) + O2macr Fe (II) + O2

This can be simplified as the Haber-Weiss reaction (Haber and Weiss 1932)

O2macr + H2O2 OHmacr + OHbull + O2

The hydroxyl radical (OHbull) is a highly reactive oxygen-centred radical that attacks all

molecules in the human body and is likely to be the final mediator of most free

radical induced tissue damage (Loyd et al 1997) It is the most reactive radical

known with an extremely short half-life and a very limited diffusion capacity It can

attack almost every molecule found in living cells and because it is a radical it can

leave behind a legacy in the cell in the form of free-radical chain reactions Thus if

OHbull attacks DNA free-radical chain reactions occur within the DNA and lead to

chemical alteration of the deoxyribose purines and pyrimidines which in turn can

lead to mutations and DNA strand breakage Imperfect repair of DNA damage can

result in oncogene and carcinogenesis Probably the most common biological

damage caused by OHbull is its ability to promote lipid peroxidation which occurs when

OHbull is generated adjacent to the fatty acid side-chains of membrane lipids Lipid

hydroperoxides and their aldehyde decomposition products are toxic and are

responsible for the rancidity of peroxidized food material (Halliwell 1991)

The actual reaction in the body may be more complicated than the described

reactions in that they may involve the formation of the ferryl or perferryl radical

The propensity of transition metal ions to drive OHbull formation means that it is

important for these metals to be sequestered in forms that are not available to

catalyse the final reaction (Lloyd et al 1997)

The peroxyl (RO2bull) and alkoxyl radicals (RObull) are organic radicals typically

encountered as intermediates during lipid peroxidation which will be discussed in

more detail later

Singlet oxygen (1O2) is an electronically excited non-radical form of dioxygen and a

highly reactive oxidizing agent The major source of 1O2 is photosensitizing

reactions although small amounts are produced by self-reaction of peroxyl radicals

during lipid peroxidation (Muggli and Hoffmann 1993)

113 Consequences of excessive production of free radicals

Although free radicals are critical for the maintenance of normal physiological

function overabundance or uncontrolled chain reactions initiated as well as

propagated by free radicals are potentially lethal for the cell (Kaul et al 1993)

Excess generation of ROS within tissues can damage DNA lipids proteins and

carbohydrate Which is the most important target of damage depends on the cell type

subjected to the oxidative stress and how the stress is imposed Biological systems

respond to oxidative stress in a poorly understood fashion with a cascade of

interacting reactions at the molecular cellular and organ level (Figure 12 Muggli

and Hoffmann 1993) Free radicals have been implicated in more than one hundred

disease conditions in humans including atherosclerosis arthritis ischemia and

reperfusion injury of many tissues central nervous system injury gastritis tumour

promotion and carcinogenesis and AIDS (Pitot and Dragan 1991 Kehrer 1993)

OXIDATIVE STRESS HYPOTHESIS

Oxidative stress

1O2 O2macr H2O2 OH

Reactive oxygen species (ROS)

Lipids Proteins Receptors Chromosomes

Molecular reaction with biological substances

Enzymes Membranes Nucleic acids Carbohydrates

Cellular disturbances

Tissue injuries

DISEASE

Figure 12 Cascade of multiple derangements of cell metabolism by oxidative stress (from Muggli and Hoffmann 1993)

Peroxidation of lipids is most likely the most extensively investigated of all the

radical-mediated mechanisms

12 Lipid oxidation and disease

Lipid oxidation may be defined as the process of oxidative deterioration of

polyunsaturated fatty acids and is initiated and propagated by free radicals (Henning

and Chow 1988) In cellular systems lipid oxidation is of absolute importance

particularly in membranes where most of the oxygenndashactivating enzymes are found

and there is typically a relatively high concentration of polyunsaturated fatty acids

which are more susceptible to oxidation Lipid oxidation in cell membranes can

disintegrate the membrane structure and cause loss in the function of the cell

organelles (Kappus 1985)

Neurological tissue is particularly susceptible to oxidation because it consumes a

high rate of oxygen and has relatively low levels of antioxidant defences In

addition there is a high content of polyunsaturated fatty acids and transition metals

ions There are some examples of neurodegenerative disorders in which lipid

peroxidation is likely to be important including Parkinsonrsquos and Alzheimerrsquos

diseases

Myoglobin the major heme protein in muscle tissues involved in transport and

storage of oxygen has been identified as a powerful catalyst able to initiate lipid

oxidation In its higher oxidation states it has been shown to oxidise a wide range of

biological substrates including proteins nicotinamide adenine dinucleotide (reduced

form) (NADPH) ascorbate carotenoids and plant polyphenols (Kanner and Harel

1985) Under physiological conditions deoxymyoglobin has been found not to be

pro-oxidative (Kanner and Harel 1985) On the other hand pro-oxidative activity

has been identified under acidic conditions such as at inflammation and ischemic

sites (Fantone et al 1989) The catalytic activity of myoglobin in biological systems

is expected to originate from its interaction with lipid peroxides and particularly with

hydrogen peroxide (Kanner and Harel 1985) Table 12 (from Jadhav et al 1995)

outlines range of diseases which can be caused by lipid oxidation in vivo

Table 12 Lipid peroxidation induced diseases and effects

Disease Remarks

1 Haemochromatosis Organ damage due to Fe overload leading to increased lipid oxidation

2 Keshan disease Selenium deficiency causes a decrease in glutathione peroxidase activity leading to increased lipid peroxidation

3 Rheumatoid arthritis Due to Fe-induced lipid oxidation

4 Atherosclerosis Lipid peroxides and reaction products of lipid oxidation such as hydroxyalkenals alter low density lipoproteins which is important in atherosclerotic lesions

5 Ischaemia Occurs during reperfusion injury of heart and brain Also results in lipid peroxidation probably by transformation of xanthine dehydrogenase to xanthine oxidase and by the production of reactive oxygen species

6 Ageing May be due to lipid peroxidation but has been confirmed in erthyrocytes

7 Carcinogensis Wide speculation about the involvement of lipid peroxidation in carcionogensis This is due to genotoxic effects of lipid peroxides

(Jadhav et al 1995)

121 Mechanism of lipid peroxidation

Lipid peroxidation has been extensively reviewed by a number of authors including

Labuza (1971) Burton and Ingold (1984) Halliwell and Chirico (1993) and

Morrissey et al (1994) The process of lipid peroxidation includes three main steps -

initiation propagation and termination

Initiation Xbull + RH Rbull + XH (a)

Rbull + O2 ROObull (b)

Propagation ROObull + RH ROOH + Rbull (c)

2ROOH RObull + ROObull + H2O (d)

Termination Rbull RObull ROObull stable non-propagating species (e)

Figure 13 Outline of the reactions involved in the autoxidation of unsaturated fatty acids (Coultate 2002)

The initiation reactions result in the production of a small number of highly reactive

fatty acids molecules with unpaired electrons the free radicals denoted by Rbull

(Figure 13a) During the propagation reactions atmospheric oxygen reacts with

these radicals to form peroxy radicals ROObull (Figure 13b) which are also highly

reactive and further react with other unsaturated fatty acids to generate

hydroperoxides ROOH and another free radical (Figure 13c) The free radical can

go round and repeat this process thereby forming a chain reaction (Figure 13b) but

the hydroperoxide can break down to give other free radicals (Figure 13d) which can

also behave much as ROObull did Consequently the ever increasing number of free

radicals accumulate in the lipid which absorbs considerable quantities of oxygen

from the air Eventually the free radical concentration reaches a point when they

start to react with each other to produce stable end-products and these are known as

the termination reactions (Figure 13e)

122 Lipoprotein oxidation

Within the human body the oxidation of lipoporotein is a key early stage in the

development of atherosclerosis (Young and McEneney 2001)

1221 Lipoproteins

Lipids such as triglycerides and cholesterol are essential to the body and serve a

number of causes The triglycerides are required within the body as a source of

energy being composed of fatty acids and glycerol Cholesterol is a structural

component of cell membranes and nerve sheaths being required for the synthesis of

steroid and adrenocortical hormones and bile acids As these lipids are insoluble in

water they are transported in the blood bound to proteins (apoproteins and

apolipoproteins) Thus they become water-soluble complexes and are termed

lipoproteins

The lipoprotein complexes undergo metabolism by the body during which the

initially large lipid-filled low-density complexes which transport either diet derived

or endogenously synthesised lipids undergo gradual conversion to smaller denser

particles that are rich in protein and phospholipid prior to their utilisation or

excretion (Thomas 2001) The lipoproteins are therefore classified by density due

to their change in lipid content during metabolism (Thomas 2001)

12211 Chylomicrons

These are the form in which lipids that are consumed in the diet are absorbed from

the gastrointestinal tract During circulation round the body their triglyceride is

gradually removed by skeletal muscle cells and adipose tissue under the influence of

lipoprotein lipase The remnants of the chylomicron are taken up by the liver and

reassembled into new lipoproteins

12212 Very low-density lipoproteins (VLDL)

VLDL particles are constructed by the liver from chylomicron remnants and are

comprised mainly of triglyceride The VLDL maintain a supply of triglyceride for

energy production to body tissues in the fasting state

12213 Intermediate-density lipoproteins (IDL)

IDL are derived from partially degraded VLDL and are short-lived intermediates

12214 Low-density lipoproteins (LDL)

LDL particles are derived from VLDL As the triglyceride is removed by the body

cells from VLDL the remaining cholesterol is concentrated within LDL for transfer

to the peripheral tissues Approximately 60 of total circulating cholesterol is

contained within LDL

12215 High-density lipoproteins (HDL)

HDL are small dense particles derived from the breakdown of the chylomicrons and

are composed of protein cholesterol and phospholipid HDL have an important role

transporting cholesterol from cells back to the liver

1222 Oxidation of VLDL

VLDL is susceptible to oxidation because it contains high levels of triglycerides

cholesteryl esters and phospholipids that harbour unsaturated fatty acids such as

linoleic acid and arachidonic acid (Arai et al 1999) Apolipoprotein (apoE) a

component of VLDL acts as a ligand for the LDL receptor and heparan sulfate

proteoglycan on the cell surface and plays an important role in regulating lipoprotein

metabolism (Weisgraber 1994) In addition apoE contributes to restoration and

regeneration of nerve tissue (Ignatius et al 1986) McEneny et al (1996) found that

oxidation of VLDL in vitro increases macrophage uptake and promotes foam cell

formation Recent studies suggest that both oxidised VLDL and HDL may play a

role with LDL in the pathogenesis of atherosclerosis Much work has been done on

the oxidative modification of LDL as will be discussed However a much smaller

number of studies have been performed investigating the properties of oxidised

VLDL and HDL Mohr and Stocker 1994) reported that radical-mediated lipid

peroxidation proceeded via a similar mechanism in isolated human LDL and VLDL

A difference in the compositional structure of VLDL compared to other lipoproteins

was reported by Bailey and Southon (1998) in that C181 (oleic acid) is more

prominent in VLDL In all sub-fractions of LDL and HDL C182 (linoleic acid) is

the most abundant long chain fatty acid with C160 (palmitic acid) being the next

most abundant This may be important in relation to lipoprotein oxidation as

monounsaturated fatty acids resist oxidation due to an absence of diene sites at which

free radical initiation can take place and hence conjugation (Yamamoto 1990)

1223 Oxidation of LDL

Increasing evidence indicates that oxidative modification of LDL is a crucial factor

in the process of atherogenesis (Witzum and Steinberg 1991 Esterbauer et al 1992

Parthasarathy and Rankin 1992) The initial step in the development of

atherosclerosis is believed to be injury to the endothelium or lining of the artery

Where damage has occurred macrophages ingest LDL and other atherogenic

particles to form foam cells Upon death of the macrophages the lipid remains in the

arterial wall accumulating over time to form a lipid core or pool The formation of

this fatty streak is the first step in plague development which eventually leads to

narrowing of the artery and inadequate supply of oxygen to the heart muscle and

consequently coronary heart disease (Thomas 2001) It is believed that modification

of LDL within the arterial wall particularly by oxidation as noted previously is

crucial to the cellular uptake of LDL in the first stages of plaque development

(Young and McEneny 2001)

Oxidation of LDL is initiated by free radicals or by one of several enzymes such as

lipoxygenase and myeloperoxidase within the walls of arteries (Heinecke 1997) It

appears that following the initial attack by a free radical on the polyunsaturated fatty

acids in the LDL particles lipid peroxidation (as described previously) occurs The

lipid hydroperoxides fragment in the presence of catalytically active iron or copper to

form a wide variety of aldehydes which then combine covalently with the lysl and

other amino acid residues of the protein moiety of LDL apolipoprotin B-100 (Leake

1995) The modified protein then becomes recognised by the scavenger receptors of

macrophages The bound LDL may then be internalised rapidly by the cells and

deposit its cholesterol within them thereby giving rise to the foam cells that are

characteristic of atherosclerotic lesions (Figure 14) Once initiated oxidation of

LDL is a free-radical-driven lipid peroxidation chain reaction

Figure 14 Pathogenesis of atherosclerosis

(a) The initiating stages of lesion development are characterised by endothelial dysfunction when adhesion molecules are upregulated resulting in the attachment of leukocytes The endothelium becomes more permeable facilitating the transmigration of cells and intimal accumulation of plasma lipoproteins

(b) A fatty streak develops which consists of lipid loaded monocytes and macrophages (foam cells) This occurs in conjunction with smooth muscle cell migration and proliferation

(c) Fatty streak progresses to an advanced lesion following the development of a fibrous cap A necrotic core is formed as a result of cell apoptosis and necrosis the proteolytic degradation of the extracelluar matrix plaque calcification and the accumulation of extracellular lipid

(Adapted from Ross 1999)

Lipoprotein-like particles with oxidative damage have been isolated from

atherosclerotic lesions (Witzum and Steinberg 1991) and lipid oxidation products

such as malondialdehyde have been immunohistochemically detected in human and

animal atherosclerotic lesions (Esterbauer et al 1992) The main reason it is

unlikely that oxidation of LDL occurs in the plasma is due to the presence of high

antioxidant concentrations and proteins that chelate metal ions Thus oxidation of

LDL is likely to occur in the intima of large arteries as noted previously Oxidation

may be mediated by lipoxygenases and myeloperoxidase

Lipoxygenases are cytosolic enzymes present in macrophages endothelial cells and

smooth muscle cells and they directly oxidise polyunsaturated fatty acids

(Yamamoto 1992) particularly those bound to phospholipids within LDL Amounts

of 12-lipoxygenase increase in cholesterol-loaded macrophages (Mather et al 1985)

Heinecke (1997) reported that 15-lipoxygenase may oxidise LDL in early stages of

atherosclerotic lesions

Phagocytes secrete hydrogen peroxide and the heme protein myeloperoxidase which

interact to generate antimicrobial toxins (Klebanoff 1980) This enzyme uses

hydrogen peroxidase to convert chloride to hypochlorous acid (Harrison and Schultz

1976) and to convert L-tyrosine to the tyrosyl radical Myeloperoxidase with its

ability to generate a range of reactive species may play a role in lipoprotein

oxidation throughout atherosclerotic lesion development and may therefore

represent an important link between chronic inflammation and development of

atherosclerotic plaques in the human artery wall (Daugherty et al 1994)

1224 Oxidation of HDL

Plasma HDL is a powerful negative risk factor for atherosclerotic cardiovascular

disease (Rifkind 1990) as it has been found to promotes reverse transport of

cholesterol from peripheral cells to the liver In addition HDL protects LDL against

oxidation an effect mediated mainly by HDL associated enzymes such as

paraoxonase platelet activating factor acetylhydrolase (PAF-AH) and lecithin-

cholesterol acyltranferase (L-CAT) (Schnell et al 2001) These enzymes have been

shown to inhibit LDL oxidation in vitro and to convert bioactive lipid peroxidation

products into inactive compounds Oxidative modification of HDL results in

deterioration in several biological functions critical to its role in reverse cholesterol

transport (Schnell et al 2001) Sakai et al (1992) found that oxidative modification

of HDL impairs its binding to cells thereby reducing its effectiveness in promoting

cellular lipid efflux Recent studies (Chait et al 2004) have indicated that HDL is

much more susceptible to oxidation than LDL

1225 Factors influencing lipoprotein oxidation

The oxidation of lipoprotein in vivo is influenced by the composition of the

lipoprotein (intrinsic factors) and the microenvironment in which it is found

(extrinsic factors) Among the intrinsic factors the fatty acid composition of LDL is

of primary importance as a high concentration of polyunsaturated fatty acids

(PUFAs) will make the LDL significantly more susceptible to oxidation

Conversely a high concentration of monounsaturated fatty acids such as oleic acid

(C181) can protect against oxidation Also of importance is the concentration of

endogenous antioxidants in lipoproteins such as -tocopherol ubiquinol-10 and

carotenoids since the propagation phase of LDL oxidation begins after these have

been consumed (Young and Woodside 2001)

In terms of extrinsic factors susceptibility of LDL to oxidation in vivo is likely to be

influenced by its microenvironment including transition metal availability pH the

presence of specific enzyme systems and local antioxidant concentration the latter of

which is now receiving significant attention

13 Antioxidant defence against disease

Nature has developed a variety of sophisticated defences against undesirable side

effects of oxygen To offset the undesirable effects a complex interactive network of

antioxidants has evolved (Krinsky 1992 Olson 1995) The term antioxidant has

two parts anti which means against and oxidant or an oxidising agent which means

any substance which accepts electrons and causes another reactant to be oxidised

A reductant or a reducing agent is a substance that donates electrons and thereby

causes another reactant to be reduced Reductant and oxidant are chemical terms

whereas antioxidant and pro-oxidant have meanings in the context of biological

systems (Prior and Cao 1999) Antioxidants may be defined as any substance which

when found at low concentrations compared with those of an oxidizable substrate

significantly prevents or delays a pro-oxidant initiated oxidation of the substrate An

antioxidant is a reductant but a reductant is not necessarily an antioxidant A pro-

oxidant is a toxic substance that can cause oxidative damage to lipids proteins and

nucleic acids resulting in various pathological events or diseases

Intracellular extracellular lipophilic and aqueous antioxidant mechanisms are found

throughout the body and work together to

(i) prevent generation of ROS (preventative antioxidants)

(ii) destroy or inactivate ROS which are formed (scavenging antioxidants)

(iii) terminate chains of ROS-initiated peroxidation (chain-breaking antioxidants)

(Strain and Benzie 1998)

Many endogenous and exogenous constituents of biological fluids have been shown

to exhibit antioxidant activity in vitro However for an antioxidant to have a

physiological role there are certain criteria that must be met

(i) the proposed antioxidant must be able to react with ROS found at sites in the

body where the proposed antioxidant if found

(ii) upon reacting with the ROS the proposed antioxidant must not be changed

into a more reactive species than the original ROS

(iii) the proposed antioxidant must be found in sufficient quantity at the site of its

presumed action in vivo for it to make an appreciable contribution to

antioxidant defence if its concentration is very low there must be some

recycling or replenishing mechanism in vivo

Some of these antioxidant mechanisms are highly integrated enzymatic systems

including endogenous antioxidants such as catalase superoxide dimutase

glutathione peroxidase and reductase Exogenous antioxidants encompass a

comprehensive group of free radical scavengers including lipophilic antioxidants that

protect membranes and lipoproteins This type of antioxidant includes vitamin E

vitamin A and the carotenoids The hydrophilic antioxidants that protect against free

radicals in the aqueous phase include ascorbate and uric acid The antioxidants can

be conveniently divided into three groups these being

(i) antioxidant enzymes (ii) transition metal binding proteins and (iii) chain

breaking antioxidants

131 Antioxidant enzymes

1311 Catalase

Catalases help prevent the accumulation of HO2 within cells and catalyse the

following reaction

2HO2 2H2O + O2

They are located in animal and plant tissues in sub-cellular organelles bound by a

single membrane and known as peroxisomes (Halliwell and Gutteridge 1989

Robinson 1991) These organelles also contain some of the cellular H2O2 generating

enzymes such as glycollate oxidase urate oxidase and flavoprotein dehydrogenases

involved in the -oxidation of fatty acids

Catalases were initially found in aerobic cells whereas most anaerobic organisms do

not contain the enzyme activity In animals catalase is present in all major body

organs being specifically accumulated in the liver and erythrocytes (Halliwell and

Gutteridge 1989) The brain heart and skeletal muscle contain only low amounts

although the activity does vary between muscles and even among different regions of

the same muscle

Halliwell and Gutteridge (1989) reviewed the mechanism of dismutation of H2O2 into

oxygen and water by catalase The reaction proceeds in two steps

Catalase-Fe (III) + H2O2 compound (k1)

Compound I + H2O2 catalase-Fe (III) + H2O +O2 (k2)

The second order rate constants k1 and k2 for rate liver catalase have values of

17x107M-1 and 26x107M-1 respectively

1312 Glutathione peroxidases (GSH-Px) and glutathione reductase

Mills (1957) was the first scientist who discovered glutathione peroxidases (GSH-

Px) in animal tissues as an enzyme which protects red blood cells against

haemoglobin oxidation and haemolysis GSH-Px uses a simple tripeptide (Glu-Cys-

Gly glutathione) as a co-factor abbreviated to GSH in its reduced form The

oxidised form GSSG consists of two GSH molecules joined by a disulphide bridge

GSH is the predominant form of free glutathione in vivo The enzyme GSH-Px

catalyses the oxidation of GSH to GSSH at the expense of H2O2 as in the following

reaction

H2O2 + 2GSH GSSG + 2H2O

The enzyme mechanism of GSH-Px is concluded in 3 main steps The first step

includes the oxidation of the co-factor by a peroxidase substrate and produces the

corresponding alcohol This is followed by two successive additions of GSH and

release of GSSG (Flohe and Gunzler 1974)

The ratios of GSH to GSSG in normal cells generally are kept high In order to

maintain this GSSG must be reduced back to GSH a reaction catalysed by

glutathione reductase

GSSG + NADPH + H+ 2GSH + NADP+

Glutathione reductase can also catalyse the reduction of certain mixed disulfides such

as that between GSH and coenzyme-A (Halliwell and Gutteridge 1989) NADPH is

required for these reactions and is available in animal tissues via the oxidative

pentose phosphate pathway Glutathione reductase contains two protein sub-units

each of them having flavin FAD as its active site During the catalytic cycle the

NADPH reduces the FAD which then passes its electrons on to a disulfide (-S-S-)

between two cysteine residues in the protein The twondashSH groups so formed then

interact with GSSG and reduce it to 2 GSH therefore re-forming the protein

disulfide

1313 Superoxide dimutase (SOD)

Mann and Keilin (1938) discovered superoxide dimutase (SOD) initially thinking

that it played an important role in copper storage In 1960 McCord and Fridovich

(1960) demonstrated that the protein previously discovered catalyses the dismutation

of superoxide radicals (O2macrbull) to hydrogen peroxide (H2O2) as described previously

SOD is found widely in all oxygen-consuming organisms (McCord et al 1971) All

SODs are metalloproteins containing copper iron or manganese at their active sites

There are three forms of SOD in mammalian tissues each of which has a specific

sub-cellular location and different tissue distribution Copper zinc superoxide

dimutase (CuZn-SOD) is found in animals plants and yeasts Mammalian CuZn-

SODs are highly thermostable resistant to protease attack and tolerant to organic

solvents

Manganese superoxide dimutase (Mn-SOD) is found in the mitochondria of all cells

(Weisiger and Fridovich 1973) These are more susceptible to denaturation by heat

or chemicals such as detergents (Halliwell and Gutteridge 1989) although they are

not affected by H2O2

Iron superoxide dimutases are generally dimeric proteins with each sub-unit having

a molecular mass of about 22000 kDa and containing a functional iron ion Fe-SOD

exhibits a very high degree of sequence homology with Mn-SODs They have

helical proteins each monomer consisting of 6 basic helical segments and three

strands of anti-parallel -sheet Halliwell and Gutteridge (1989) claimed that the

iron in the resting state is Fe(III) and that it probably oscillates between Fe(III) and

Fe(II) states during the catalytic cycle

132 Transition metal binding antioxidants

Transition metals bind proteins that is ferritin transferrin lactoferrin and

caeruloplasmin act as a crucial component of the antioxidant defence system by

sequestering iron and copper so that they are not available to drive the formation of

the hydroxyl radical

The principle copper-binding protein caeruloplasmin may also function as an

antioxidant enzyme that can catalyse the oxidation of divalent iron as follows

4Fe(II) + O2 + 4H+ 4Fe(III) + 2H2O

Fe(II) is the form of iron that drive the Fenton reaction and the rapid oxidation of

Fe(II) to the less reactive Fe(III) for is therefore an antioxidant effect (Young and

Woodside 2001)

133 Chain-breaking antioxidants

Chain-breaking antioxidants may be defined as small molecules that can receive an

electron from a radical or donate an electron to a radical with the formation of stable

by-products (Halliwell 1995) Generally the charge associated with the presence of

an unpaired electron becomes dissociated over the scavenger and the resulting

product will not readily accept an electron from or donate an electron to another

molecule thereby preventing further propagation of the chain reaction (Young and

Woodside 2001) Such chain breaking antioxidants can be divided into aqueous

phase and lipid phase antioxidants

1331 Aqueous phase chain-breaking antioxidants

13311 Vitamin C

Vitamin C or ascorbic acid (ascorbate) is a water-soluble vitamin known as anti-

haemorrhagic agent when its discovery was associated with curing scurvy It is an

essential nutrient with a number of specific functions as a nutrient such as collagen

formation and it acts as an essential co-factor for several enzymes catalysing

hydroxylation reactions Fresh fruits and vegetables are the main sources of vitamin

C particularly citrus fruit parsley peppers broccoli spinach and tomatoes It can

be synthesised by most mammals though not by humans primates guinea pigs and

fruit bats It is a non-thermostable vitamin and is affected by cooking

Ascorbate is a chain-breaking antioxidant and is considered the most important

antioxidant in extracellular fluids It can efficiently scavenge superoxide hydrogen

peroxide the hydroxyl radical hypochlorous acid aqueous peroxyl radicals and

singlet oxygen (Sies et al 1992) Frei (1991) reported that ascorbic acid is

sufficiently reactive to intercept oxidants in the aqueous phase before they can attack

and cause detectable oxidative damage to lipids

The principal pathway of oxidation and turnover of ascorbic acid is thought to

involve the successive removal of two electrons yielding firstly the ascorbate free

radical (AFR) and then dehydroascorbate (Thurnham et al 2000) Two molecules

of AFR may react together to form one molecule of ascorbate or one of

dehydroascorbate Alternatively AFR may be reduced by a microsomal NADH-

dependent enzyme mono-dehydro-L-ascorbate reductase It has been shown that

AFR is less reactive than many other free radicals (Bielski et al 1975) thus the

reason for its protective role as a free-radical chain terminator In addition as

ascorbate is readily soluble in aqueous fluids it is easily dispersed through the

tissues and in contact with probably all biological membranes where vitamin E is the

principal antioxidant (Thurnham et al 2000)

Vitamin C is capable of restoring oxidised vitamin E back to its original form In

other words the unique ability of low concentrations of vitamin E to act as an

efficient antioxidant in biological systems is due to its ability to be reduced from its

chromanoxyl radical form to its native state by intracellular reductants such as

ascorbate (Packer and Kagan 1993)

Although ascorbate can play a key role in protecting cells against oxidative damage

in the presence of the transition metals Fe(III) or Cu(II) it can promote generation of

the same ROS it is known to destroy (Stadtman 1991) This ability to act as a pro-

oxidant is due to the ability of ascorbate to reduce Fe(III) and Cu(II) to Fe(II) and

Cu(I) respectively and to reduce oxygen to the superoxide ion and hydrogen

peroxide Thus ascorbate can play a dual role as a pro-oxidant and an antioxidant

13312 Uric acid

Uric acid is formed in the body by the breakdown of purines and by direct synthesis

from 5-phosphoribosyl pyrophosphate (5-PRPP) and glutamine In humans uric

acid is normally excreted in the urine while in other mammals it is further oxidized

to allantoin prior to excretion Uric acid can scavenge free radicals being converted

in the process to allantoin It is particularly important in protecting against certain

oxidising agents such as ozone (Cross et al 1992) Ames et al (1981) suggested

that the increase in the life-span that has existed during human evolution could be

partly explained by the protective effect of uric acid in human plasma Urate can

directly quench free radicals and contribute to the formation of stable non-reactive

complexes with iron (Frei et al 1988)

13313 Albumin

Albumin is also an efficient radical scavenger when bound to bilirubin (Frei et al

1988) Copinathan et al (1994) suggested that albumin may play an important role

in protecting the neonate from oxidative damage because of lack of other chain-

breaking antioxidants in the neonates Albumin is the predominant protein in plasma

and makes the major contribution to plasma sulphydryl groups although it also has

several other antioxidant properties It contains 17 disulphide bridges and has a

single remaining cysteine residue and this residue is responsible for antioxidant

properties (Stocker and Frei 1991) Albumin also has the capacity to bind copper

ions and will inhibit copper-dependent lipid peroxidation and hydroxyl radical

formation (Young and Woodside 2001) It has also been shown to act as a powerful

scavenger of hypocholorous acid and provides the main plasma defence against this

oxidant (Hu et al 1993)

13314 Reduced glutathione (GSH)

Glutathione (Glu-Cys-Gly) or GSH is the only significant electron donor substrate

for the metabolite reactions involving glutathione peroxidase (GSH-Px) although the

latter can use a variety of hydroperoxide acceptor substrates GSH is also a

scavenger of hydroxyl radicals and singlet oxygen Since it is present at high

concentrations in many cells it may help protect against these radical species

(Deshpande et al 1995)

1332 Lipid phase chain-breaking antioxidants

13321 Vitamin E

Vitamin E is the principal component of the secondary defence mechanism against

free radical-mediated cellular injuries It is the only natural physiological lipid-

soluble antioxidant that can inhibit lipid peroxidation in cell membranes

In 1922 Evans and Bishop discovered vitamin E as a substance in lettuce which

prevented sterility in animals (Evans and Bishop 1922) It was later isolated as a

closely related family of compounds designated as tocopherols -Tocopherol was

thus known as an anti-sterility factor the name being derived from the Greek words

tokos and pherein which mean to bring forth children In 1936 the vitamin was

isolated from wheatgerm oil (Evans et al 1936) Vitamin E is a fat-soluble vitamin

a deficiency of which can lead to a group of symptoms in animals and poultry such

as embryonic degeneration liver necrosis encephalomalacia and testicular

degeneration although deficiency in humans is rare Vegetable oils nuts and plant

sources are good sources of the vitamin while animal sources such as lard and butter

are less important Vitamin E is absorbed from the gut with the aid of bile salts and

the vitamin is not esterfied as is the case with retinol It is transported to the blood

stream via chylomicrons and distributed to the various tissues via lipoproteins

Vitamin E refers to two groups of compounds the tocopherols and the tocotrienols

and includes eight different forms which differ greatly in their degree of biological

activity The tocopherols include and -tocopherol and have a chromanol

ring and a phytyl tail and differ in the number and position of the methyl groups on

the ring The tocotrienols and are structurally similar but have

unsaturated tails Both groups of compounds have antioxidant properties (Horwitt

Burton et al (1983) reported that probably vitamin E is the most important lipid

antioxidant -Tocopherol (TOH) is quantitatively the most important antioxidant in

plasma and LDL because it is present in concentrations at least 10-15 times higher

than any of other lipid soluble antioxidants (Burton et al 1983) It is an essential

component of biological membranes as it has membrane-stabilising properties the

hydrophobic tail being the means by which TOH inserts into lipoproteins or anchors

into membranes next to unsaturated fatty acids (Diplock 1985) The chromanol

nucleus lies at the surface of a lipoprotein or at the surface of membranes and it is

the phenolic hydroxyl group that quenches free radical (Packer and Kagan 1993)

TOH is only active during the propagation phase of lipid peroxidation that is the

consumption of this lipid soluble antioxidant is associated with the formation of lipid

hydroperoxides The hydrogen atom of the phenolic hydroxyl group on the

chromanol ring is very easy to remove It therefore reacts readily with lipid peroxy

and alkoxy radicals by donating the labile hydrogen to them thereby terminating the

chain reaction of peroxidation by scavenging chain-propagating radicals (Deshpande

et al 1995)

When the chromanol phenolic group of TOH encounters a peroxy radical (ROO bull) it

forms a hydroperoxide (ROOH) and in the process a tocopheroxyl radical (TO bull) is

formed

TOH + ROObull ROOH + TObull

The tocopheroxyl radical (TObull) generated is poorly reactive and is therefore unable to

attack adjacent fatty acid side chains TOH might be regenerated by reaction at the

aqueous interface with ascorbate as noted previously or another aqueous phase chain

breaking antioxidants such as glutathione or urate On the other hand two

tocopheroxyl radicals might combine to form a stable dimer or the radical may be

completely oxidised to form tocopherol quinone (Young and Woodside 2001)

13322 Ubiquinol-10

Ubiquinol-10 the reduced form of coenzyme-Q has long been known to be a

component of the mitochondrial respiratory chain (Crane et al 1993) During the

last three decades it has also been shown that in its reduced form it can serve as a

potent antioxidant (Ernster and Forsmark 1993) and protect membrane

phospholipids (Takayanagi et al 1980) and LDL cholesterol (Stocker and Frei

1991) It has been pointed out (Ernster and Forsmark 1993) that ubiquinol is the

only known lipid soluble antioxidant that is synthesised de novo in animal cells and

for which there exists enzymic mechanisms by which it can be maintained in the

reduced form The reduced form of coenzyme QH2 is also effective as a lipid

soluble chain breaking antioxidant (Shi et al 1999)

Ubiquinol-10 is found in lower concentrations than vitamin E but it can trap lipid

peroxyl radicals more efficiently than vitamin E or -carotene and can also restore

membrane bound vitamin E from the tocopheroxyl radical (Lass and Sohal 1998)

Thomas et al (1996) reported that ubiquinol-10 is the first antioxidant consumed

during oxidation of LDL cholesterol and this may suggest that it is of particular

importance in preventing the propagation of lipid oxidation Scientific work in this

area has been hampered by the instability of ubinquinol-10 during sample handling

or analysis

13323 Carotenoids and vitamin A

The carotenoids are a group of lipid soluble antioxidants based around an isoprenoid

carbon skeleton the most important being -carotene Recently there has been

considerable interest in the antioxidant activity of carotenoids and their potential role

in the pathogenesis of several diseases There are particularly efficient scavengers of

singlet oxygen (Fukuzawa et al 1998) although they can also trap peroxy radicals

at low oxygen pressure with an efficiency at least as great as that of TOH Thus the

carotenoids may play a role in preventing in vivo lipid peroxidation The other

important role of certain carotenoids is as precursors of vitamin A (retinol) the latter

also having antioxidant properties (Keys and Zimmerman 1999) As the carotenoids

are part of the phytochemical group of compounds their protective effect in vivo

will be discussed in more detail later in the chapter

14 Phytochemicals

Fruits and vegetables contain a wide variety of low-molecular weight secondary

metabolites which can (i) protect the plant against attack by predators and pathogens

(ii) function with physical barriers and some defensive enzymes to provide a system

of defence against physiological stress and attack by other organisms (iii) provide

organoleptic or characteristics such as flavour compounds and colour pigments and

(iv) the reduction of characteristics with adverse acceptability properties such as

tannins (Rhodes 1996) Such potentially protective plant compounds are termed

phytochemicals Many of these phyotnutrients are ubiquitous throughout the plant

kingdom and are therefore present in the human diet To date approximately 30000

phytochemicals have been identified of which 5000 to 10000 are present in the

food consumed in the human diet (Cassidy and Dalais 2003)

Although they might not be considered as essential nutrients interest in these

phytonutrients as they are otherwise known as bioactive components is currently of

major interest due to increasing epidemiological evidence that they are involved in

the maintenance of healthy tissues and organs in the body For example there is

evidence that diets rich in fruit and vegetables could have a protective effect against

a number of cancers (Block et al 1992) and other chronic health conditions such as

cardiovascular disease (John et al 2002) According to Rice-Evans et al (1997)

many dietary polyphenolic compounds derived from plants are more effective

antioxidants in vitro than vitamins E or C and therefore might contribute to the

protective effects in vivo It is though that these dietary phenolic compounds might

make a contribution to total antioxidant capacity

Phytochemicals may be classified as carotenoids phytosterols and sulphur

containing compounds (sulfides and glucosinolates) and phenolic phytochemicals

(tannins stilbenes and lignans and the flavonoids)

141 Carotenoids

Carotenoids (Figure 15) are natural pigments present in plants and responsible for

colours such as red orange and yellow in various fruits and vegetables such as

carrots tomatoes spinach oranges and peaches More than 600 different

compounds have been identified in various organisms Carotenoids containing only

carbon and hydrogen atoms are collectively referred to as carotenes (-carotene

-carotene and lycopene) However most natural carotenoids contain at least one

oxygen function such as keto (violerythrin) hydroxy (lutein) or epoxy groups

(violaxanthin) and these are collectively referred to as xanthophylls or

oxocarotenoids (Stahl and Sies 1999)

Beta-carotene -carotene and -cryptoxanthin have important nutritional roles in

the body as they act as pro-vitamins for vitamin A The non pro-vitamin A

compounds with implications for human health are lutein zeaxanthin and lycopene

These together make up major dietary carotenoids the major dietary sources being

vegetables such as broccoli carrots greens parsley green and orange peppers

spinach tomatoes and various fruits including apricots grapefruit and oranges

(Mangels et al 1993) They can also be found in various seafoods such as lobster

and salmon (Liaaen-Jensen 1990) On a commercial basis the carotenoid

astaxanthin is used as a feed additive to obtain the typical colour of salmon flesh

(Bjerkeng 1992) Carotenoids such as crocetin and bixin are becoming increasingly

popular as food colourants with many now officially sanctioned for use

Although many hundreds of carotenoids have been identified only a small number

have been investigated for their effects on human health (Ottaway 2001)

Increasingly there is evidence that a diet rich in carotenoids is associated with a

reduction in risk for certain cancers and cardiovascular disease Although early

epidemiological studies suggested a protective effect of -carotene in the 1990s it

was reported that large doses of supplemental -carotene could increase the risk of

lung cancer in heavy smokers and asbestos workers (ATBC Study Group 1994

Albanes et al 1996) There is evidence to suggest that lutein and zeaxanthin the

predominant carotenoids in human macula lutea may protect against age-related

macular degeneration (AMD) a leading cause of irreversible blindness among the

65+ age group (Seddon et al 1994)

-carotene

Lycopene

Lutein

Figure 15 Chemical structure of carotenoids

Lycopene the major carotenoid in tomatoes is one of the most effective scavengers

of singlet oxygen free radicals Research has suggested that eating tomatoes every

day will reduce the risk of developing a cancer in the upper aero-digestive tract

stomach lungs or respiratory tract However observations suggest that it is not

lycopene alone that is responsible for the protective effect of tomatoes but possibly

other compounds present in the tomatoes such as -carotene vitamins C and E

folates phenolics and lignans A number of studies have suggested a relationship

between lycopene and a risk reduction for prostate cancer particularly in aggressive

cases (Giovannucci et al 1995) A protective effect of lycopene against pancreatic

cancer has also been suggested (Burney et al 1989)

142 Phytosterols

Phytosterols (Figure 16) have been reported in measurable amounts in over 250

plants (Cassidy and Dalais 2003) These compounds have a similar structure to

cholesterol but with some modifications to the side chain including the addition of a

double bond andor methyl or ethyl group

The most common dietary phytosterols are sitosterol campesterol and stigmasterol

representing lt50 of the total intake of sterols in the Western diet the remainder

being cholesterol (Subbiah 1971) The major dietary sources include legumes nuts

seeds soyabeans and unrefined plant oils (Pennington 2002) The daily diet in the

UK contains an average of 200-400 mg plant sterols although vegetarians may eat

around 800 mg daily (Flora 2003)

Interest in phytosterols has centred on their cholesterol lowering properties thereby

offering protection from cardiovascular disease (Law 2000) Numerous studies have

shown that plant sterols and stanols derived from wood pulp and vegetable oils lower

total and LDL cholesterol by inhibiting cholesterol absorption from the intestine in

humans As plant stanols are virtually unabsorbable they are more ideal

hypocholesterolaemic agents than plant sterols (Nguyen 1999)

Sitosterol

Stigmasterol

Campesterol

Figure 16 Chemical structure of phytosterols

Esterification of plant stanols has allowed their incorporation into various food

products such as margarine without having an adverse effect on the taste or texture of

such foods It has been shown that plant stanol esters at a level of 2-3 g per day can

reduce LDL cholesterol by 10-15 However it has been shown that plant sterols

may also reduce the level of carotenoids although these reductions are well within

the variation observed in dietary intake of carotenoids between winter and summer

and are therefore not biologically significant (Weststrate and Meijer 1998) Recent

studies have shown that dietary advice to consume an additional daily serving of a

high-carotenoid fruit or vegetable when consuming spreads containing esterified

plant sterols or stanols will maintain plasma carotenoid concentrations and at the

same time significantly lower LDL cholesterol concentrations (Noakes et al 2002)

143 Sulphur-containing compounds

1431 Sulfides

Naturally occurring sulphur-containing compounds (the allium family) are found in

large quantities in bulbous plants such as garlic onions and leeks Such vegetables

contain a range of S compounds which are derivatives of L-cysteine sulphoxides

(CSO) and a potential source of protective compounds in the diet against

degenerative disease (Rhodes 1996)

Figure 17 Chemical structure of allicin

The 1-propenyl derivative is particularly associated with onions and the allyl

derivative with garlic the compounds being odourless in undamaged plant tissues

Only when the garlic is crushed or the onion sliced does the enzyme allinase gain

access to its substrate and the flavour becomes obvious In garlic dimerisation of the

allyl sulfenic acid leads to the formation of allicin which is well known for its

antibacterial properties (Coultate 2001) There has been a long history of the

medicinal properties of garlic and allicin although results in clinical trials with

human subjects have been variable Studies have shown that consumption of garlic

may have benefits with regard to cholesterol blood pressure platelet aggregration

and coagulation time (Lawson et al 1992)

1432 Glucosinolates

The glucosinolates (Figure 18) are a large group of sulfur-containing compounds

that occur principally in the family Cruciferae including the brassicas (cabbage

Brussels sprouts broccoli) and many plants renowned for their pungency when raw

namely horse-radish black and white mustards and radish Over 100 such

compounds have been described thus far (Rhodes 1996)

R mdash CS mdash D-glucose

N mdash OSO3-

Figure 18 Structure of glucosinolates

Total glucosinolates are normally present at levels of around 2 gkg fresh weight with

the highest amounts being found in the most pungent species or varieties (Coultate

2001) The glucosinolates are not themselves pungent but when the plant tissue is

disrupted by chewing chopping in the kitchen or suffers insect or other damage in

the field the glucosinolates are hydrolysed by the enzyme myrosinase to form

isothiocyantes or nitriles The volatile isothiocyanates are the most likely products to

the formed (collectively referred to as mustard oils) and it is these that have the

pungent taste With regard to health benefits of consumption of fruits and

vegetables it is recognised that at least some of the beneficial effects especially in

relation to cancers of the colon rectum and thyroid can be related to the

consumption of glucosinolates in cruciferous vegetables Various experiments

carried out with laboratory animals and tissue cultures of human cells have shown

that allylisothyiocyanate from sinigrin and sulforophane from glucoraphanin at

realistic concentrations have been shown to kill cancerous cells (Zhang and Talalay

1994) It appears that sulforophane causes cell to increase production of glutathione

transfereases a group of enzymes involved in the neutralisation of carcinogens

144 Phenolic phytochemicals

This group of phytochemicals includes a broad range of compounds that are

widespread in commonly consumed fruits vegetables and beverages derived from

plants such as tea The term phenolic encompasses a variety of plant compounds

containing an aromatic ring with one or more hydroxyl groups They are partly

responsible for the colour taste and smell of many foods and are influenced by

factors such as growing conditions cultivar ripeness processing and storage

(Cassidy and Dalais 2003) Research has highlighted the potential role of these

phytochemicals as important contributing factors to the antioxidant activity of the

diet and hence protective effects for human health (Rice-Evans and Millar 1996

Rice-Evans et al 1997) The flavonoids form the most important group of phenolic

phytochemicals found in foods and beverages

15 Flavonoids

To date more than 4000 chemically unique flavonoids have been identified in plant

material being found in the upper epidermal layer of leaves flowers stems roots

seeds and fruits They are derived biosynthetically from phenylalanine the general

structure being two benzene groups connected by a three-carbon bridge (C6-C3-C6

structure) with phenolic OH groups (Figure 19) Three moles of malonyl-coenzyme

A (CoA) from glucose metabolism condense to form ring A the reaction being

catalysed by chalcone synthetase Rings B and C also come from glucose

metabolism but via the shikimate pathway through phenylalanine which is

converted to cinnamic acid and then to coumaric acid Subsequently coumaric acid

CoA and three malonyl CoAs are condensed in a single enzymatic step to form

naringenin chalcone The C-ring closes and becomes hydrated to form 3-

hydroxyflavonoids (eg catechins) 34-dio flavonoids (eg quercetin) and

procyanidins (Merken and Beecher 2000) In plant foods the flavonoids are present

mainly as glycosides in which phenolic hydrogen or hydrogens are substituted to the

sugar moiety (Terao 1999) Glucose is the most commonly encountered sugar with

galactose rhamnose xylose and arabinose not uncommon and mannose fructose

glucuronic and galacturonic acids being rare (Robards and Antolovich 1997)

Figure 19 Basic structure of flavonoids

From a dietary point of view the most important classes of flavonoids are the

flavones and flavonols the catechins and related tannins based on the flavan-3-ol

structure and the anthocyanins these being the pigments responsible for the red and

blue colours of many fruits and vegetables (Rhodes 1996) Furthermore the

isoflavones are related to these compounds and have activity as phytoestrogens

although this group of compounds are largely limited to the pea family whereas the

flavonoids are found in nearly all fruits and vegetables

151 Flavonols flavones and flavanols

The three major sub-classes of flavonoids are the flavonols flavones and flavanols

Common flavonols include quercetin kamepferol myricetin and isohamnetin

(Figure 110) Glycosylation occurs preferentially at the 3-hydroyl group

Quercetin

Kaempferol

Myricetin

Figure 110 Chemical structure of flavonols

The two most common flavones of dietary importance are luteolin and apigenin

(Figure 111) They are structurally different to the flavonols in that they lack a

hydroxyl group on the 3-position of the C ring Other flavones include nobiletin and

sinensetin found in sweet orange peel and taneretin found in tangerine oil (Robards et

al 1997)

Apigenin

Luteolin

Figure 111 Chemical structure of flavones

Flavanols (or flavan-3-ols) differ from the flavonols and flavones as they lack an

oxygen molecule at the 4 position of the C ring The principal flavanols found in the

plant materials include catechin epicatechin (Figure 111) and gallocatechin

Figure 112 Chemical structure of epicatechin

The flavanols are also called tea flavonoids are these compounds are only rich in tea

leaves where catechins may constitute up to 25 of dry leaf weight One of the most

concentrated dietary sources of catechins is green tea (Camellia sinensis) where

epicatechin epigallocatechin and their gallate esters are commonly found During

the preparation of black tea oxidative polymerization of flavanols occurs with

theaflavin theaflavingallates thearubigins and epitheaflavic acid being formed

(Figure 112)

(a) (b) (c)

Compound R R1 Compound R R1 Compound R1

Thearubigin H H Theaflavin H H Epitheaflavic acid H

Thearubigin-3-gallate galloyl H Theaflavin-3-gallate galloyl H Epitheaflavic gallate gallate

Thearubigin-3-gallate H galloyl Theaflavin-3-gallate H galloyl

Thearubigin-33-digallate

galloyl galloyl Theaflavin-33-digallate

galloyl galloyl

Figure 113 Chemical structures of thearubigin (a) theaflavin (b) epitheaflavic (c) and related compounds

The flavanols are also important constituents of fruits in oligomeric or polymeric

forms as proanthocyanidins or condensed tannins (Figure 114) They are widely

distributed in foods such as apples grapes strawberries plums grape seeds and

barley

Dimer TrimerProcyanidin B3 (R3 = H)Prodelphinidin B3 (R3 = OH)

Figure 114 Chemical structure of proanthocyanidins

152 Flavanones

Flavanones occur in small amounts compared with other flavonoids but are the

predominant flavonoid in citrus fruits (Robards et al 1997) The most commonly

found flavanones in citrus include naringenin (Figure 115) naringin hesperidin and

hesperetin In the case of sweet oranges mandarins lemons and citrons the

predominant flavanone is hesperidin while the major flavanone in grapefruit is

naringin

Figure 115 Chemical structure of naringenin

153 Anthocyanins

The anthocyanins are natural plant pigments occurring in nature as glycosides with

the aglycones being commonly known as anthocyanidins It is these compounds that

give the pink red mauve violet and blue colours of flowers fruit and vegetables

They are particularly rich in berries and the red grape

Anthocyanidins (R3 = R5 = H) Anthocyanin (R3 R5 = glycosides)

Compound R3 R5 Compound R3 R5

Delphinidin (De) OH OH De-3-glucosides glucose H

Cyanidin (Cy) OH H De-35-diglucosides glucose glucose

Pelargonidin (Pe) H H

Peonin (Pn) OCH3 H

Malvidin (M) OCH3 OCH3

Petunidin (Pt) OCH3 OH

Figure 116 Chemical structures of anthocyanins and related glycosides

In total six anthocyanidins (Figure 116) occur in nature although the diversity of the

patterns of glycosylation means there are innumerable different anthocyanins

Cyanidin is the most common followed by delphinidin peonin pelargonidin

petunidin and malvidin Of the six anthocyanidins only pelargonidin is not found in

grapes It is also interesting that there is much more variety in the patterns of

glycosylation and acetylation than in most plants (Coultate 2001) With regard to

vegetables cyanidin is the anthocyanidin of red cabbage while pelargonidin and

delphinidin occur in radishes and aubergines respectively

154 Hydroxycinnamates

Closely related to the flavonoids are some derivatives of cinnamic acid such as

caffeic -coumaric and ferrulic acids (Figure 117) also known as

hydroxycinnamates Such acids may acylate with the sugar moieties of the glycoside

and are effective antioxidants Caffeic acid ferulic acid and -coumaric acid are

found in fruits and vegetables such as asparagus cabbage olives tomatoes and white

grapes while chlorogenic acid is present in apple cherry pear tomato and peach

(Rice-Evans et al 1997)

Compound R1

Caffeic acid OH

p-Coumaric H

Ferulic acid OCH3

Figure 117 Chemical structure of cinnamic acid and derivatives

155 Isoflavones (Phyoestrogens)

Isoflavonids are a class of flavonoids also known as phytoestrogens due to the fact

that their structure (Figure 118) is similar to that to the mammalian hormone

oestrogen They are found principally in legumes with soy being the main dietary

source although they can be derived from other legumes In soya beans the

isoflavones are present as glycosides Following digestion these conjugated

isoflavones are hydrolysed by glycosidases and bioactive aglycones are formed The

major dietary aglycones are daidzein glycetin and genistein their glycosides being

glycitin daidzin and genistin The isoflavone content of soybean varieties ranges

from 12 - 38 mgg seed and depends on the variety growing conditions and

planting season Phytoestrogens produce both agonisitic and antagonistic activity as

estrogen receptrors depending upon the level of circulating endogenouse estrogen

and the type of estrogen receptor (Alpro 2003)

Daidzein Genistein

Figure 118 Chemical structure of isoflavones

16 Flavonoids and Health

Interest in plant-based diets is increasing because as noted previously this type of

diet may offer protection against some of the chronic diseases common in Western

countries

161 The Mediterranean diet

The well known term the Mediterranean Diet was first popularised in the 1970s

when studies showed that Mediterranean countries have diets associated with low

incidences of cardiovascular disease with later studies showing that these countries

also enjoy a low incidence of cancers of the colon and breast There is now little

doubt that the Mediterranean countries enjoy a low risk of many of the diet-related

diseases of affluence (Hill 1995) The Mediterranean diet is characterised by

consumption of fruit vegetables olive oil and dietary fibre and low intakes of meat

and saturated fats It not only produces favourable effects on blood lipids but also

protects against oxidative stress the latter being thought to represent one of the

mechanisms leading to chronic diseases such as atherosclerosis and cancer

Many studies suggest that a link exists between fruit and vegetables or the amounts

of plasma antioxidant vitamins and risk of death from cancer or coronary heart

disease (Ghiselli et al 1997) The preference for fresh fruit and vegetables typical

of Mediterranean populations results in a higher consumption of raw foods

involving a reduced production of cooking-related antioxidants with a consequent

decreased waste of nutritional and endogenous antioxidants It is thought that the

health benefits of the Mediterranean diet are mainly due to its high antioxidant

potential due to the abundance of flavonoids and other phytochemicals which may be

additionally or complimentarily involved in the reported protective effects of this diet

(Peluso and Vineis 2000)

162 The French Paradox

The low incidence of coronary heart disease (CHD) in France especially in southern

France despite having a diet high in fat and a high incidence of smoking has been

termed the French Paradox The incidence of CHD is also low in countries such as

Italy Greece and Spain and so maybe the phenomenon should be known as the

Mediterranean Paradox Mortality from ischaemic heart disease in France is about a

quarter of that in Britain although the risk factors are similar Undercertification of

ischaemic heart disease in France could account for about 20 of the difference It

has been postulated however that the high consumption of red wine by the French

could act as a protective factor particularly as it has been shown that red wine could

inhibit the oxidation of low density lipoprotein (LDL) (Kondo et al 1993)

It was postulated by Frankel et al (1993) that it is the phenolic substances extracted

from red wine that could potentially inhibit LDL oxidation the active antioxidants

being flavonoids The flavonoid in the greatest amount is quercetin which

comprises 60 of all the flavonoids in wine Other flavonoids in red wine such as

caffeic acid have greater antioxidant potential but are present in small amounts The

flavonoids resveratrol and epicatechin are also found in red wines but not in white

wines As red wine is fermented with the grape skin red wine contains procyanidins

(tannins) the absorption of which is facilitated by the 5-11 alcohol Frankel et al

(1993) showed that quercetin and phenolic substances isolated from red wine

effectively impaired copper ion-catalyzed oxidation of LDL while -tocopherol

exhibited only 60 of the potency of wine phenolics or quercetin

According to Renaud and deLorgeril (1992) moderate alcohol intake does not

prevent CHD through an effect on atherosclerosis but rather through a haemostatic

mechanism Data has shown that blood platelet aggregation which is related to

CHD is inhibited significantly by alcohol at levels of intake associated with reduced

risk of CHD This inhibition of platelet activity by wine (alcohol) may be one

explanation for protection from CHD in France since studies showed that the

tendency of platelets to aggregate is considerably less lower in subjects from the

south of France than in Scotland Most flavonoids decrease eicosanoid synthesis

Red wine has been shown to decrease concentration (inhibits synthesis) of

thromboxane B2 Overall the decrease in eicosanoid metabolism decreases

thrombaxane and increases prostacyclin Most flavonoids increase the formation of

endothelium-dependent relaxation factor maybe by increasing the formation of nitric

oxide (NO) (Fitzpatrick et al 1993) This effect of red wine if it occurs in vivo

could help to prevent vasospasm of coronary arteries or to inhibit platelet

aggregation as NO inhibits platelet aggregation The vasorelaxation effect of red

wine seems to be mediated by an increase in cyclic GMP resulting in vascular

smooth muscle relaxation

Other possibilities to explain the French Paradox are those eluded to earlier when

referring to the Mediterranean diet such as the high intake of monounsaturated fatty

acids mainly in the form of olive oil which also contains antioxidants The higher

temperatures of the Mediterranean countries compared to Northern Europe may be

another possible contributory factor (Leake 1995)

It is there thought that the cardiovascular benefits of red wine are a result of its

alcohol and flavonoid content Alcohol alone in doses of 20 to 30 g per day has been

shown to decrease CHD by 40 Red wine also contains a large amount of folic

acid other vitamins and potassium and magnesium which may also have beneficial

cardiovascular effects

The health benefits of phytochemicals such as the flavonoids from fruits and

vegetables are thus becoming more evident and have increasing importance in

nutritional science

163 Flavonoid intake and cardiovascular disease

Epidemiological studies have demonstrated that dietary flavonoid intake is inversely

associated with mortality from coronary heart disease (CHD) and incidence of stroke

(Hertog et al 1993 Knekt et al 1996)

Studies were undertaken by Hertog et al (1995) to determine whether flavonoid

intake could explain differences in mortality rates from chronic diseases between

populations The workers determined the mean intake of flavonoids at baseline

(1960) in the 16 participant cohorts of the Seven Countries Study by chemical

analysis of equivalent food composites and related it to 25 year mortality from CHD

cancer at various sites and all causes It was found that the mean flavonoid intake of

the cohorts varied from 26 mg per day in West Finland to 682 mg per day in

Ushibuka Japan with quercetin representing 100 (eg West Finland) to 39

(Zutphen the Netherlands) of total flavonoid intake Hertog and co-workers (1995)

showed that flavonoid and quercetin intake was highly correlated (r = 092) and the

relationhips between quercetin intake and disease mortality similar to those observed

for total flavonoid intake Results showed that the average flavonoid intake was

inversely related to CHD mortality with variance in flavonoid intake explaining

about 25 of the total variance in CHD mortality across the 16 cohorts Using

multivariate analysis it was shown that intake of saturated fat (73 p = 0001)

flavonoid intake (8 p = 001) and percentage of smokers per cohort (9 p = 003)

explained together independent of alcohol and antioxidant vitamins 90 of the

variance in CHD rates Adjustment for intake of vitamin E ascorbic acid -

carotene or alcohol did not increase the total explained variance

In the same studies by Hertog et al (1995) the average flavonoid intake was not

associated with lung or colorectal cancer mortality There was a strong and positive

correlation of flavonoid intake with stomach cancer mortality However using

multivariate analysis after additional adjustment for intake of ascorbic acid the

association was no longer statistically significant Average flavonoid intake was not

related to all-cause cancer mortality and adjustment for fat intake and percentage of

smokers did not change the association

Hertog et al (1995) concluded that average flavonoid intake may contribute to

differences in mortality from CHD across populations but not to differences in

cancer mortality The results obtained for this cross-cultural comparison by Hertog

and co-workers (1995) were in agreement to those of the prospective cohort Zutphen

Elderly Study (Hertog et al 1993 1994) that involved a cohort of 806 elderly men

(aged 65 - 84 years) in the Netherlands at baseline in 1985 In that particular study

flavonoid intake was inversely associated with CHD mortality but not with mortality

from cancer at all sites It was hypothesised by Hertog et al (1995) that diets rich in

flavonoids protect LDL against oxidation by free radicals The relative incidence of

heart disease among men from the Zutphen study who had the highest intake of

flavonoids was only on third of those who had the lowest intake of flavonoids The

result was the same even when adjustments were made for age body fat smoking

cholesterol blood pressure physical activity coffee consumption and the intake of

calories vitamin C vitamin E -carotene and dietary fibre Their findings also

supported the suggestion noted previously that flavonoids present in red wine could

be partly responsible for low CHD mortality in red wine drinkers

A Finnish cohort study undertaken to study the effect of flavonoid intake and

coronary mortality (Knekt et al 1996) used a large sample size of 2748 men and

2385 women The study was based on data collected at the Finnish mobile clinic

health examination survey from 1967-72 and followed up until 1992 and took into

account 30 communities from different parts of Finland In line with the Zutphen

Elderly Study the Finnish study indicated an inverse association between intake of

flavonoids and coronary mortality It was found that there was an inverse

relationship between flavonoid intake and total mortality both in men and women

when the major cardiovascular risk factors of smoking serum cholesterol

concentration blood pressure and body mass index were adjusted for It was found

that the intake of the antioxidant vitamins C and E and carotenoids were low in

Finland during the baseline study thereby suggesting the possibility that even smaller

amounts of dietary flavonoids are important in circumstances where the intake of

other dietary antioxidant agents is low For women coronary mortality was

significantly inversely associated with flavonoid intake but for men the inverse

association was not significant Further adjustment for intake of energy fatty acids

fibre and antioxidant vitamins weakened the associations in women The results are

in line with previous findings of Knekt et al (1994) which showed antioxidants are

of particular benefit to women It was therefore concluded by Knekt et al (1996)

that people with very low intakes of flavonoids have risks of coronary disease

A study carried out by Rimm et al (1996) investigated the association between

intake of flavonols and flavones and CHD in 34789 men enrolled in the US Health

Professionals Follow-up Study and followed prospectively for six years Results of

the study showed that no individual flavonoids were associated with an appreciable

reduction in risk from CHD and controlling for intake of dietary fibre saturated fat

or cholesterol did not alter the results The data did not therefore support a strong

inverse association between flavonoid intake and total CHD but did not exclude the

possibility that flavonoids have a protective effect in men with established CHD

Hertog et al (1997a) also investigated whether flavonol intake predicted a lower rate

of ischemic heart disease (IHD) in 1900 Welsh men aged between 45 and 59 years

who were followed up for 14 years (The Caerphilly Study) In this case flavonol

intake mainly from tea to which milk was normally added was not related to IHD

incidence but was weakly positively related to IHD and cancer mortality and strongly

related to total mortality Hertog and co-workers (1997a) concluded that the intake

of antioxidant flavonols the major food source of which is black tea is not inversely

associated with IHD risk in the UK These results contrasted strongly with the

findings of the Zuthphen Elderly Study (Hertog et al 1993) There was a possibility

that the flavonols from tea to which milk is added are not absorbed experimental

evidence suggesting that adding milk to tea abolishes the plasma antioxidant-raising

capacity of tea

Using data from the Zutphen study Arts et al (2001) evaluated the association

between catechin intake and the incidence of and mortality from IHD and stroke

They calculated the mean catechin intake in 1985 to be approximately 72 mg mainly

from black tea apples and chocolate It was found that catechin intake was

inversely associated with IHD disease mortality which is in contrast to the findings

of Hertog et al (1997a) noted previously for the Caerphilly Study However Arts et

al (2001) found no association between intake and stroke incidence or mortality

Work carried out by Arai et al (2000) calculated the intake of flavonols flavones

and isoflavones by 115 Japanese women aged between 29 and 78 years The total

mean intakes of flavonoids (sum of flavonols and flavones) and isoflavones were

167 and 472 mg per day respectively It was found that the total intake of

isoflavones exceeded that of other dietary antioxidants such as flavonoids

carotenoids and vitamin E and was approximately half that of the vitamin C intake

After adjustment for age body mass index and total energy intake it was

demonstrated that the total intake of flavonoids was inversely correlated with the

plasma total cholesterol concentration (TC) and plasma LDL cholesterol (LDL-C)

Quercetin as a single component was also inversely correlated with both TC and

LDL-C The results of the work carried out by Arai et al (2000) suggested that a

high consumption of both flavonoids and isoflavones by Japanese women may

contribute to their low incidence of CHD compared with women in other countries

As part of the Womens Health Study Sesso et al (2003) examined whether

flavonoids are associated with incident cardiovascular disease (CVD) in a large

cohort of female health professionals (38445) in the US They evaluated the

association between individual selected flavonols and flavones including quercetin

kaempferol myricetin apigenin and luteolin plus specific food sources of

flavonoids and the risk of CVD The data obtained from the large cohort of middle-

aged and older US women indicated that higher flavonoid intake was not associated

with a reduced risk of CVD after adjustment for lifestyle and dietary factors No

individual flavonol or flavone was associated with CVD

Knekt et al (2002) studied the association between flavonoid intake and risk of

several chronic diseases in Finland The total dietary intakes of 10054 men and

women were determined during the year preceding the baseline examination The

flavonoid intakes were estimated based mainly on the flavonoid concentrations in

Finnish foods and the incident cases of the diseases considered were identified from

different national public health registers Overall it was found that people with a

higher total flavonoid intake tended to have a lower total mortality Results from the

study suggested the presence of an inverse association between flavonoid intake and

subsequent occurrence of IHD cerebrovascular disease lung and prostate cancer

type 2 diabetes and asthma The potential benefits of the flavonoids were mainly

attributed to quercetin known to be the most potent antioxidant but also in some

cases to kaempferol myricetin hesperitin and naringenin The lower risk found for

IHD mortality was found to be mainly in those people with the higher intakes of

apples and onions and accordingly to the flavonols quercetin and kaempferol

However the authors did note that although their findings were independent of the

intake of antioxidant vitamins the potential importance of other biologically active

compounds in fruit and vegetables could not be ruled out They did however

conclude that the risk of some chronic diseases may be lower at higher dietary

flavonoid intakes

Recent work published by Mennen et al (2004) evaluated the relationship between

consumption of foods rich in flavonoids and estimated cardiovascular risk in a large

French population A cross-sectional analysis was carried out on 1286 women and

1005 men from the SUVIMAX (SUpplementation en VItamines et Mieacutenraux

AntioXidants) Study an 8 year trial evaluating the effect of antioxidant vitamin (C E

and -carotene) and mineral (selenium and zinc) supplementation on the incidence of

major chronic diseases (cancers of all sites and ischemic heart disease) in France In

women flavonoid-rich food consumption was inversely related to systolic blood

pressure No such relationship was obtained for men There was little difference

between genders with respect to intake of flavonoid-rich foods except for the

consumption of wine which was higher in men and the consumption of tea which

was higher in women Overall it was concluded that a diet rich in flavonoid-rich

foods such as apples tea chocolate onions citrus fruit and red fruit may be useful

in the prevention of CVD in women

The body of evidence available suggests that dietary flavonoids are beneficial to

health as they have a wide range of different biological activities including

antibacterial antithrombotic vasodilatory antiinflammatory and anticarcinogenic

effects mediated by different mechanisms (Knekt et al 2002 Middleton et al 2000

Nijveldt et al 2001) However it should be noted that the results obtained with

regard to an association between flavonoids and CHD are mixed In vitro studies

have indicated that there are considerable differences in the antioxidative potential of

different flavonoid sub-groups depending on their chemical structure (Rice-Evan et

al 1997) As knowledge on the bioavailability absorption and metabolism of the

polyphenols in human is limited it is likely that different groups of flavonoids have

different effects on human health

164 Mode of action of flavonoids in relation to coronary heart disease

Flavonoids can function as

metal chelators and reducing agents

scavengers of ROS (reactive oxygen species)

chain-breaking antioxidants

quenchers of the formation of singlet oxygen and

protectors of ascorbic acid or conversely ascorbic acid can protect flavonoids

against oxidative degradiation

(Middleton et al 2000)

In many of the cases reported it is not certain whether flavonoids inhibit the

formation of ROS or scavenge them Nonetheless flavonoids have been shown to be

react with the hydroxyl radical and therefore can act as highly important chain-

breaking antioxidants ROS that can be scavenged or whose formation can be

prevented by flavonoids have been presented previously in Table 13

Table 13 Reactive oxygen species that can be scavenged or whose formation can be inhibited by flavonoids

O2-middot (Superoxide ion) One-electron reduction product of O2 Produced by

phagocytes formed in autooxidation reactions (flavoproteins redox cycling) and generated by oxidaseses (heme proteins)

HO2-middot (Perhydroxy radical) Protonated form of O2

-middot

H2O2 (Hydrogen peroxide) Two-electron reduction product of O2 formed from O2

-middot (HO2-middot ) by dismutation or directly from O2

-middot Reactivity of O2

-middot and H2O2 is amplified in the presence of heme proteins

OH (Hydroxyl radical) Three-electron reduction product of O2 generated by Fenton reaction transition metal (iron copper)-catalyzed Haber-Weiss reaction also formed by decomposition of peroxynitrite produced by the reaction of O2

-middot with NO (nitric oxide radical)

ROmiddot (Alkoxyl radical) Example Lipid radical (LO)

ROOmiddot (Peroxyl radical) Example Lipid peroxy radical (LOO) produced from organic hydroperoxide (eg lipid hydroperoxide LOOH) ROOH by hydrogen abstraction

1O2 Singlet oxygen

(from Middleton et al 2000)

1641 Inhibition of lipid peroxidation

It has been reported that several flavonoids can lipid peroxidation by scavenging

OH The chain breaking antioxidant action of the flavonoids (F) can be represented

as follows (Middleton et al 2000)

LOO + FLminusOH LOOH + FLminusO

where FLminusOH represents the flavonoid

Termination of lipid radical (L) lipid peroxyl radical (LOO) and alkoxyl radical

(LO) by phenolic antioxidants is as follows

LOO L LO + AminusOH rarr LOOH LH LOH + AOmiddot

where AminusOH represents phenolics (eg -tocopherols flavonoids) and AO

represents the phenoxyl radical

There has also been a proposal that flavonoids react with lipid peroxyl radicals

(LOO) leading to termination of radical chain reactions (Takahama 1983)

Differences in the modes of action of the flavonoids as to whether they act as free

radical scavengers chain-breaking antioxidants or act by terminating radical chain

reactions indicate that different constituents are important for different biological

activities as noted previously

The characteristics of flavonoid structure for most effective radical-scavenging

activity are presented in Table 14 [taken from Middleton et al (2000)] It appears

that quercetin is an extremely efficient radical scavenger with myricetin being even

more active due to the presence of the third (pyrogallol) OH group on the B ring

Kaempferol has been shown to be a very good scavenger despite the fact that is has

only one OH group on the B ring (4 -OH) possibly because of the combination of the

other characteristics (C2-C3 double bond 3-OH group and 4-oxo group on ring C)

Although catechin has the catechol group on ring B and the 3-OH group on ring C it

is a weak scavenger as it lacks the C2-C3 double bond and the 4-oxo group on ring C

Table 14 Characteristics of flavonoid structure for most effective radical-scavenging activity

The catechol (O-dihydroxy) group in the B ring confers great scavenging ability with exceptions such as those described by Ratty and Das (1983) who thought it did not contribute towards lipid peroxidation in rat brain mitochondria

A pyrogallol (trihydroxy) group in B ring of a catechol as in myricetin produces even higher activity The C2-C3 double bond of the C ring appears to increase scavenger activity because it confers stability to the phenoxy radicals produced

The 4-oxo (keto double bond at position 4 of the C ring) especially in association with the C2-C3 double bond increases scavenger activity by delocalizing electrons from the B ring

The 3-OH group on the C ring generates an extremely active scavenger in fact the combination of C2-C3 double bond and 4-oxo group appears to be the best combination on top of the catechol group

The 5-OH and 7-OH groups may also add scavenging

Thus there appears to be a hierachy of flavonoid and isoflavonoid antioxidant

activities that is dependent on structure and defines the relative abilities of the

compounds to scavenge free radicals (Rice-Evans et al 1997) The antioxidant

activities relative to Trolox the water-soluble vitamin E analogue are consistent

with their structure (Rice-Evan et al 1997) The half-peak reduction (Ep2) has also

been ascribed as a suitable parameter for representing the scavenging activity of the

flavonoids (Van Acker et al 1996) A flavonoid with a low value for half-peak

reduction potential (ie lt02) is a good scavenger The half-peak reduction

potentials can be compared with the total antioxidant activity measured as the

Trolox Equivalent Antioxidant Capacity (TEAC) From Table 15 (Rice-Evans et al

1997) it can be seen that there is broad agreement between the TEAC value (pH 74)

and the Ep2 value (same pH) in that flavonoids with efficient scavenging properties

have a TEAC value of 19 mM and Ep2 values of 02 mV the exception being

kaempferol

Table 15 Hierarchy of flavonoid antioxidant activities and the relationship with reduction potentials

Flavonoid Antioxidant activitya (mM)

Half-peak reduction potentialb (mV)

Quercetin 47 003

Rutin 24 018

Catechin 24 016

Luteolin 21 018

Taxifolin 19 015

Apigenin 15 gt1

Naringenin 15 06

Hesperetin 14 04

Kaempferol 13 012

aMeasured as the TEAC (Trolox equivalent antioxidant activity)- the concentration of Trolox with the equivalent antioxidant activity of 1mM concentration of the experimental substancebDesignated Ep2 An Ep2 of lt02 indicates a chemical that is readily oxidizes and therefore an efficient free radical scavenger

(from Rice-Evans et al 1997)

1642 Decrease in LDL oxidation by flavonoids

As oxidation of LDL is implicated in the pathogenesis of atherosclerosis the

enhancement of the resistance of LDL to oxidation is one of the models used by

researchers to investigate the efficacy of dietary phytochemicals as antioxidants

against radicals generated in the lipophilic phase (Rice-Evans et al 1997)

DeWhalley et al (1990) reported that flavonoids inhibited cell-free oxidation of LDL

oxidation mediated by CuSO4 They appeared to act by protecting LDL against

oxidation caused by macrophages as they inhibited the generation of lipid

hydroperoxides and protected -tocopherol from being consumed by oxidation in the

LDL Consequently the flavonoids protected -tocopherol and possibly other

endogenous antioxidants in LDL from oxidation maintained their levels for longer

periods of time and delayed the onset of lipid peroxidation

Thus there is now good experimental evidence that flavonoids are potent inhibitors

of LDL oxidation although the mechanism by which they do so are not certain The

following possibilities however have been put forward (Middleton et al 2000)

Firstly the flavonoids may reduce the generation or release of free radicals in the

macrophages or may protect the -tocopherol in LDL from oxidation by being

oxidized by free radicals themselves

Secondly the flavonoids could regenerate active -tocopherol by donating a

hydrogen atom to the -tocopheryl radical The latter radical is formed when it

transfers its own OH hydrogen atom to a lipid peroxyl radical to terminate the chain

reaction of lipid peroxidation as noted previously

Thirdly the flavonoids may sequest metal ions such as iron and copper thereby

diminishing the endangered free radicals in the medium

Investigations by Negre-Salvayre et al (1991) have shown that the cytotoxicity of

oxidized LDL could be prevented by flavonoids (quercetin rutin and catechin) either

by inhibiting the lipid peroxidation of LDL (induced by UV radiation) or by blocking

at the cellular level the cytotoxicity of previously oxidized LDL Later work carried

out by Negre-Salvayre et al (1995) showed that LDL mildly oxidized by copper ions

or UV radiation exhibited a cytotoxic effect on cultured endothelial cells that could

be inhibited by rutin ascorbic acid and -tocopherol It appeared that these

antioxidants acted to inhibit oxidation of LDL and increase the resistance of the cells

to the cytotoxic effects of oxidized LDL the mixture of the three compounds having

a supra-additive effect

It has also been suggested that flavonoids may be protective again CHD by

influencing several other processes such as (i) an increase in HDL levels (ii) a

reduction of cardiac mast cell mediator release or by (iii) decreasing cardiovascular

inflammation (Middleton et al 2000)

165 Bioavailability of flavonoids

Demonstration of the fact that flavonoids are bioavailable after they are consumed

from fruits vegetables and beverages is essential in order to support the belief that

the flavonoids derived from these sources are of benefit to health Indirect evidence

of their absorption through the gut barrier is the increase in the antioxidant capacity

of the plasma after the consumption of flavonoid-rich foods (Hollman et al 1997)

More direct evidence on the bioavailability of a few flavonoids has been obtained by

measuring their concentrations in plasma and urine after the ingestion of either pure

compounds or of foodstuffs with a known content of the compound of interest

(Scalbert and Williamson 2000) The chemical structure of the flavonoids will

determine their rate and extent of absorption through the small intestine and the

nature of the metabolites circulating in the plasma

Limited information is available on the metabolism of flavonoids in humans Ring

scission occurs under the influence of intestinal bacteria which also accounts for the

subsequent demethylation and dehydroxylation of the resulting phenolic acids

(cinnamic acid derivatives and simple phenols) Intestinal microorganisms also

possess glycosidases capable of cleaving sugar residues from flavonoid residues

The flavonoids can also undergo oxidation and reduction reactions as well as

methylation glucuronidation and sulfation in animal species (Middleton et al

2000) The metabolism of quercetin is proposed to be via the splitting of the

flavonoid molecule to form hydroxyaromatic acids with a two-carbon side chain a

OH group is on the 3-carbon of the pyrone ring When a OH group is lacking in this

position as in hesperidin hyroxyaromatic acids with 3-carbon side chains are formed

Gugler et al (1975) studied the metabolism of quercetin in six subjects (four males

and two females) aged between 21 and 32 years After oral administration of a

single dose of 4 g no measurable concentrations of quercetin or its derivatives were

detected in plasma or urine However it was found that approximately 53 of the

oral dose were recovered unchanged in the faeces and it was calculated that only 1

of the original 4 g dose of quercetin or approximately 40 mg was absorbed through

the gastrointestinal tract

Work by Hollman et al (1997) investigated the relative bioavailabiltiy of quercetin

from onions and apples The participants in this study (five women and four men

mean age 248 years) followed a quercetin-free diet during three experimental

periods each of five days these periods being separated by nine days without

treatment or without a prescribed diet On day four of each experimental period the

subjects were provided with one out of three different quercetin-containing

supplements in random order namely fried onions apples and rutinoside (a

glycoside of quercetin) These supplements were given at breakfast with blood

samples being collected periodically over the next 36 hours and urine continuously

for 24 hours Results showed that the quercetin was found in the circulation after

consumption of the major dietary sources of quercetin However it was found that

the bioavailability and absorption kinetics varied markedly between sources a major

difference being the type of glycosides Most rapidly absorbed was the quercetin

from onions which contains only glucosides Bioavailability from apples which

contain a variety of glycosides and pure quercetin-3-rutinoside the major species in

tea was 30 relative to onions Peak levels were achieved less than 07 hours after

ingestion of the onions 25 hours for apples and 9 hours after ingestion of the

rutinoside The half-life of elimination was 28 hours for onions and was found to be

23 hours for apples The workers concluded that the experimental results pointed to

a predominant role of the sugar moiety in the bioavailability and absoprtion of

dietary quercetin in the human body They could not rule out however that

differences between apples and onions in cell wall structures location of glyosides in

cells or their binding to cell constituents also effect the liberation of quercetin from

these foods in the digestive tract

Olthof et al (2000) tested whether the position of the glucose moiety affected the

bioavailability of quercetin glucosides in humans by comparing quercetin-3-

glucoside with quercetin-4-glucoside the former being produced by splitting of the

rhamnose molecule from the quercetin-3-rutinoside The quercetin-3-glucoside

differs only from the highly bioavailable quercetin-4-glucoside in the position of the

glucose moiety on the quercetin aglycone The workers fed five healthy men and

four healthy women (19 - 57 years) with a single dose of each compound and

followed the plasma quercetin glucosides The bioavailability was found to be the

same for both quercetin glucosides and it was thus concluded that irrespective of the

position of the glucose moiety the quercetin glucosides are rapidly absorbed in

humans

Table 16 taken from the review by Scalbert and Williamson (2000) provides a good

summary of the bioavailability in humans of polyphenols consumed alone or in

foods As noted by these authors bioavailability is particularly low for quercetin and

rutin (03-14) but reaches higher values for catechins in green tea isoflavones in

soy flavanones in citrus fruits or anthocyanidins in red wine (3-26)

Interindividual variations have also been observed eg 5-57 of the naringin

consumed with grapefruit juice is found in urine according to the individual (Fuhr

and Kummert 1995)

According to the work reviewed by Scalbert and Williamson (2000) the

concentration of intact parent polyphenols in the plasma are often low (Table 16)

the maximum rarely exceeding 1 M after the consumption of 10-100 mg of a single

phenolic compound It has also been found that they do not account on their own for

the increase in antioxidant capacity in the plasma which is probably higher due to the

presence of metabolites formed in the bodys tissues or by the colonic microflora

These metabolites are still for the main unknown and not accounted for Work

carried out by Donovan et al (1999) demonstrated that catechin was found almost

exclusively as metabolites after the consumption of red wine being present both as a

sulfate conjugate and a conjugate containing both glucuronide and sulfate residues

They concluded that if flavonoids are protective nutrients the active forms are likely

to be metabolites which are far more abundant than the forms that exist in foods

Table 16 Bioavailability in humans of polyphenols consumed alone or in foods1

Polyphenol Source

Quantity of polyphenol

ingested

Maximum concentration

in plasmaExcretion in urine Reference

mg microM

Caffeic acid 1000 27 Jacobson et al 1983Quercetin Onion 68 074 139 Hollman et al 1997Quercetin Apple 98 030 044 Hollman et al 1997Quercetin-4-O-rhamnoglucoside

Pure compound 202 030 035 Hollman et al 1997

Quercetin-4-O-glucoside

Pure compound 144 32 Hollman et al 1999

Quercetin Onion 139 134 08 Aziz et al 1998Quercetin Mixed black currant and apple

juice 1000 mld for 7 d64 05 Young et al 1999

Epigallocatechin gallate

Green tea infusion 12 g 88 033 ND Lee et al 1995

Epigallocatechin 82 067 36Epicatechin gallate 33 ND NDEpicatechin 32 027 62Epigallocatechin gallate

Green tea infusion 5 g 105 013ndash031 Unno et al 1996

Green tea infusion 6 g 50 Maiani et al 1997

Green tea extract 525 44 Nakagawa et al 1997

Catechin Red wine 120 ml 34 0072 Donovan et al 1999Catechin Pure compound 500 20 045 Balant et al 1979Genistein Soy milk 19 074 198 Xu et al 1994Daidzein 25 079 53Genistein Soy proteins 60 gd for 1 mo 20 92 Cassidy et al 1994Daidzein 25 25Genistein Soy proteins 60 gd for 28 d 80 050 Gooderham et al

1996Daidzein 36 091Genistein Soy proteins 20 gd for 9 d 23 87 Karr et al 1997Daidzein 13 26Naringin Grapefruit juice 120 ml 43 lt4 88 Fuhr and Kummert

1995Naringin Grapefruit and orange juice

1250 ml each689 68 Ameer et al 1996

Hesperidin 89 244Naringin Pure compounds 500 49 Ameer et al 1996Hesperidin 500 30Anthocyanins Red wine 300 ml 218 10ndash67 Lapidot et al 1998

1 Polyphenols principally in the form of conjugated metabolites as sulfate esters or glucuronides in plasma and urine were hydrolyzed by acid or enzymes before chromatrographic or colorimetric analysis ND not detected

(from Scalbert and Williamson 2000)

17 Previous studies

As noted previously evidence from epidemiological and in vitro studies suggest

dietary flavonoids may be able to protect against CHD A collaborative study

between the Department of Food Science and the Department of Clinical

Biochemistry (Queenrsquos University-Belfast) ldquoDietary Flavonoids and their Possible

Role as Antioxidants in Preventing Atherosclerosis (McAnlis 1998) was

investigated both the in vitro and in vivo effects of the main dietary flavonoids The

work of McAnlis (1998) found that the flavonoids quercetin myricetin kaempferol

and apigenin were found at high concentrations in onions lettuce tomatoes cabbage

greens and black tea A series of in vivo studies investigated the ability of these

dietary flavonoids to protect LDL against oxidation All the flavonoids with the

exception of apigenin inhibited the oxidation of LDL in vitro in a dose dependent

manner The results from these series of initial studies suggested that the

consumption of flavonoid rich foods may be able to protect LDL against oxidation

thereby slowing the progress of atherosclerosis

The in vivo effects of the main dietary flavonoids were consequently investigated by

McAnlis and co-workers (McAnlis et al 1997 McAnlis 1998 McAnlis et al

1999) The studies carried out indicated that flavonoids can be absorbed from

dietary sources thereby increasing the total antioxidant capacity of the plasma but not

reducing the susceptibility of LDL to oxidation Further investigation revealed that

the flavonoids become bound to plasma proteins thus they were not present in the

LDL particle to protect against lipid oxidation It was therefore concluded that the

ingestion of flavonoid-rich foods does not directly protect the LDL against oxidation

but may protect against CHD by another mechanism

18 Objectives of the present investigations

The studies carried out in the present investigations followed on from those of

McAnlis and co-workers as summarised in Section 17 The first main objective of

the present studies was to determine flavonoid content in a range of fruits and

vegetables selected for analysis based on those listed in a food frequency

questionnaire currently being used as part of the EUREYE Study the aim of which is

to determine the effect of diet on eye disease in the over-sixties age group in Europe

The concentration of flavonoids in the raw fresh fruit and vegetables was to be

analysed and subsequently a range of fruits and vegetables subjected to the cooking

processes of baking boiling and frying

The second objective of the work was to use the values obtained from analysis of the

raw fruits and vegetables to estimate the flavonoid intake both profile and content

of the Northern Ireland cohort of the EUREYE Study participants

The third main objective of the work was to carry out a series of in vitro studies to

study the ability of the selected flavonoids to protect VLDL (very low density

lipoprotein) LDL (low density lipoprotein) and HDL (high density lipoprotein)

against oxidation a process of significant importance in the pathogenesis of

atherosclerosis

CHAPTER 1

INTRODUCTION





14 Phytochemicals

As noted previously evidence from epidemiological and in vitro studies suggest dietary flavonoids may be able to protect against CHD A collaborative study between the Department of Food Science and the Department of Clinical Biochemistry (Queenrsquos University-Belfast) ldquoDietary Flavonoids and their Possible Role as Antioxidants in Preventing Atherosclerosis (McAnlis 1998) was investigated both the in vitro and in vivo effects of the main dietary flavonoids The work of McAnlis (1998) found that the flavonoids quercetin myricetin kaempferol and apigenin were found at high concentrations in onions lettuce tomatoes cabbage greens and black tea A series of in vivo studies investigated the ability of these dietary flavonoids to protect LDL against oxidation All the flavonoids with the exception of apigenin inhibited the oxidation of LDL in vitro in a dose dependent manner The results from these series of initial studies suggested that the consumption of flavonoid rich foods may be able to protect LDL against oxidation thereby slowing the progress of atherosclerosis

The in vivo effects of the main dietary flavonoids were consequently investigated by McAnlis and co-workers (McAnlis et al 1997 McAnlis 1998 McAnlis et al 1999) The studies carried out indicated that flavonoids can be absorbed from dietary sources thereby increasing the total antioxidant capacity of the plasma but not reducing the susceptibility of LDL to oxidation Further investigation revealed that the flavonoids become bound to plasma proteins thus they were not present in the LDL particle to protect against lipid oxidation It was therefore concluded that the ingestion of flavonoid-rich foods does not directly protect the LDL against oxidation but may protect against CHD by another mechanism


The studies carried out in the present investigations followed on from those of McAnlis and co-workers as summarised in Section 17 The first main objective of the present studies was to determine flavonoid content in a range of fruits and vegetables selected for analysis based on those listed in a food frequency questionnaire currently being used as part of the EUREYE Study the aim of which is to determine the effect of diet on eye disease in the over-sixties age group in Europe The concentration of flavonoids in the raw fresh fruit and vegetables was to be analysed and subsequently a range of fruits and vegetables subjected to the cooking processes of baking boiling and frying

The second objective of the work was to use the values obtained from analysis of the raw fruits and vegetables to estimate the flavonoid intake both profile and content of the Northern Ireland cohort of the EUREYE Study participants