Antioxidants: Role on Health and Prevention

T Srdic-Rajic, Institute for Oncology and Radiology of Serbia, Belgrade, Serbia

A Konic Ristic, Institute for Medical Research University of Belgrade, Belgrade, Serbia
2016 Elsevier Ltd. All rights reserved.

Oxygen is essential element for life. Human cells, as well as

those of other aerobic organisms, use oxygen to break down
nutrients and provide energy. In the mitochondrial energygenerating system, oxygen is reduced to water, and energy is
stored inside ATP molecules. However, this process is a natural
phenomenon, as it is essential for life and can be harmful to
our body at the same time. Different chemical entities that
contain partially reduced oxygen are called reactive oxygen
species (ROS). They are continuously produced in the mitochondrial respiratory chain and some other biochemical reactions and have very important signaling role in various cellular
processes. Most of them are very reactive, with great affinity to
vital molecules of human cells proteins, lipids, and deoxyribonucleic acids (DNA). Oxidative damage of macromolecules
can lead to the disturbance of their function and the development of various diseases. However, in normal conditions, the
levels of ROS are low, and human cells protect themselves from
their action with efficient antioxidant machinery. Surprisingly,
the human body can even use the deleterious action of ROS to
help the immune system, destroy foreign substances, and combat infectious diseases. Undoubtedly, the delicate balance
between physiological effects of ROS, as signaling molecules
and efficient component of immune cells, and their pathological effects is under the control of complex system of antioxidant defense and represents a key aspect of life. However, in
pathological states, production of ROS can overcome the capacity of antioxidants. This state is called oxidative stress. The
precise definition given by Sies in the early 1990s describes
oxidative stress as an imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage.
So, it is not just about the increase in ROS (if it induces more
efficient antioxidant defense) or about the decrease in antioxidants (if the level of ROS are low); it is a disturbance in their
balance that could end with the damage of our body.
ROS is a term that encompasses all highly reactive, oxygencontaining molecules, including free radicals. ROS includes
superoxide (O2
), hydroxyl (OH), peroxyl (ROO),
lipid peroxyl (LOO), alkoxyl (RO) radicals. Reactive nitrogen species (RNS) include nitric oxide (NO) and nitrogen
dioxide (NO2). Oxygen and nitrogen free radicals can be
readily converted to other nonradical reactive species that are
also dangerous for health. Hydrogen peroxide (H2O2), ozone
(O3), singlet oxygen (1O2), hypochlorous acid (HOCl), nitrous
acid (HNO2), peroxynitrite (ONOO
), dinitrogen trioxide
(N2O3), and lipid peroxides (LOOH) are not free radicals but
generally named oxidants and can easily lead to free radical
reactions in living organisms.
All ROS and RNS are capable of reacting with membrane
lipids, nucleic acids, proteins and enzymes, and various small
molecules, resulting in cellular damage.

Encyclopedia of Food and Health

Mechanisms of ROS Generation

ROS can be produced from both endogenous and exogenous
sources. Production of ROS in the body is continuous and a
normal part of our physiology.
Endogenous sources of ROS include the following:
Mitochondrial respiratory chain (MRC). It is the main source
of ROS, particularly O

2 , the most crucial one as it can induce
formation of several other reactive oxygen intermediates. O

2 is
formed by reduction of molecular oxygen with electron
leaked from the MRC.
Respiratory burst and NADPH oxidase. Respiratory burst is the
process by which phagocytic cells consume large amounts of
oxygen during phagocytosis, mainly via activation of NADPH
oxidase and O

2 release into the extracellular space or phagosomes. NADPH oxidase is an enzyme present in the plasma
membrane and phagosomes of phagocytes such as monocytes,
macrophages, neutrophils, and eosinophils. Relocation of the
cytosolic components of NADPH oxidase to the cell membrane
induces its activation. Enzyme is normally latent in phagocytes
but is activated in the membrane before respiratory burst.
There are six homologues of NADPH oxidase, collectively
called the NOX family of NADPH oxidases.
Xanthine oxidase (XO). It is found on the outer surface of the
plasma membrane and in the cytoplasm. It catalyzes oxidation
of hypoxanthine to xanthine and, further, to uric acid as part of


purine catabolism. Both reactions generate O

2 . However, O2
has a short half-life and is readily reduced to H2O2, so is not
considered as highly reactive. Additionally, due to charged
moiety of O

2 , it cannot pass through lipid membranes. The
production of xanthine and XO is greatly enhanced during
ischemia, accompanied with the loss of antioxidant enzymes.
O2 is an electron acceptor and cofactor for XO, thus generating
O

2 and H2O2, a major ROS in ischemia, causing damage to
ischemic cells and tissues of different origins.
Lipoxygenases (LOX). They are nonheme iron enzymes that
catalyze dioxygenation of polyunsaturated fatty acids and
formation of hydroperoxyl derivatives that can be subjected
to reduction and give corresponding hydroxyl derivatives,
including leukotrienes and lipoxins. In humans, oxidation of
arachidonic acid by LOX generates ROS. Five LOX enzymes
identified in humans, named based on position of oxygenated
residues, catalyze four different reactions that produce fatty
acid hydroperoxides. LOX are deeply involved in the process
of atherogenesis.
Myeloperoxidase (MPO). It is a heme enzyme localized in
lysosomes of neutrophils, macrophages, and monocytes. It
catalyzes the reaction of H2O2 to highly reactive HOCl and
an oxidation of thiocyanate (SCN
) to hypothiocyanite
(OSCN
).

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Antioxidants: Role on Health and Prevention

Nitric oxide synthase (NOS). It is a heme-containing monooxygenase that generates NO. Three different isozymes of NOS
have been identified: constitutively expressed neuronal NOS
(nNOS or NOS I), endothelial NOS (eNOS or NOS III), and
endotoxin- or cytotoxin-inducible NOS (iNOS or NOS II). All
types of NOS catalyze the oxidation of L-arginine to an intermediate, N-hydroxy-L-arginine, followed by generation of
L-citrulline and NO. NOS can also generate H2O2 within a

reaction with O

2 at low L-arginine levels. NO is a weak oxi

dant, but in reaction with O2 generates OONO
. NO and
OONO
can further form very stable nitrite (NO

2 ) and nitrate
(NO

3 ) ions, which accumulate in cells, and form reactive
intermediates: NO2, N2O3, and NO. They are capable to
nitrate and nitrosate important biological macromolecules
such as DNA, RNA, proteins, and lipids and disrupt their
function. 8-Nitroguanine, a nitration product of DNA and
RNA, is a potent prooxidant and mutagen.
Cyclooxygenase (COX). It is a bifunctional enzyme with both
COX and peroxidase activities. COX releases arachidonic acid
(AA) from membrane phospholipids and catalyzes further
conversion of AA to prostanoids. There are two isoforms of
COX, COX-1 and COX-2. COX catalyzes oxidation of AA to
unstable cyclic hydroperoxide (PGG2) and further its reduction
of PGG2 endoperoxide due to its peroxidase activity (PGH2).
PGH2 is converted to stable prostanoids such as PGE2, prostacyclins, and thromboxane A2. The peroxidase activity of
COX generates NAD
and NADP
radicals, which can further
generate O

2 .
Transition metals. Transition metal ions such as iron (Fe2)
and copper (Cu) are involved in the Fenton reaction that
generates HO and OH from H2O2, and they are oxidized to
Fe3 and Cu2, respectively. The generation of HO  through
this pathway accelerates lipid peroxidation.
Oxidative stress can also be triggered by external factors
acting as direct or indirect sources of ROS:
Radiation and chemotherapy. Ionizing radiation can produce
HO by radiolysis of water or other ROS via secondary reactions. Particularly, susceptible systems are the cerebrovascular,
gastrointestinal (GI), and hematopoietic systems. Cancer chemotherapy induces generation of ROS and decrease in vitamin
E and beta-carotene levels that often cause toxic side effects.
Cigarette smoke. It is a significant contributor to oxidative
stress as source of a large number of free radicals and other
oxidative and aromatic agents acting as direct or indirect ROS
generators.
Xenobiotics including drugs, food, and alcohol. Food can contain different ROS or compounds that can generate ROS within
a human body (iron, copper, trans fatty acid, and acrylamide).
Thermally treated lipids and alcohol can be significant contributors to oxidative stress. Increase in oxidative stress has been
observed in postprandial states after high-fat and/or high-sugar
meals. Many drugs (glucocorticoids, anesthetics, and nonsteroidal anti-inflammatory agents) and xenobiotics generate
free radicals. This is also an important feature of a great number of anticancer agents, which often act as therapeutic through
generation of ROS.
Mental stress. It can trigger the production of free radicals as
toxic by-product of intensive metabolism. In addition, hormones that mediate stress reaction in the body (cortisol and
catecholamine) decompose into destructive free radicals.

Pollutants. Air pollutants (asbestos, benzene, carbon monoxide, chlorine, formaldehyde, ozone, and toluene), chemical
solvents (cleaning products, glue, paints, paint thinners, perfumes, and pesticides), and water pollutants (chloroform and
other trihalomethanes) are potent generator of free radicals.
Burning of organic matter during cooking, forest fires, and
volcanic activities also can generate free radicals.

Antioxidant Defense System

Nature has endowed each cell of our body with adequate
antioxidant mechanisms for protection against any harmful
effects of ROS generated within the body or those that entered
our body from the environment. Endogenous antioxidant
defense system (ADS) comprises enzymatic and nonenzymatic
antioxidants. Besides endogenous antioxidants, different
substances or agents that can scavenge reactive oxygen metabolites, block their generation, or enhance capacity of endogenous antioxidants act as exogenous antioxidants. Dietary
compounds, including vitamins, minerals, polyphenols, isothiocyanates, and carotenoids, are the most important exogenous antioxidants. The fact that they are safe, cheap, and orally
bioavailable resulted in their most often use as antioxidant
supplements in the prevention or therapy of stress-related
diseases. Antioxidants act as radical scavengers, hydrogen
donors, electron donors, peroxide decomposers, singlet oxygen
quenchers, enzyme inhibitors, enzyme inducers, synergists, or
metal-chelating agents.

Endogenous Enzymatic Antioxidants

The major antioxidant enzymes in human cells are superoxide
dismutases, glutathione peroxidase, glutathione reductase, and
catalase. SOD and catalase provide major antioxidant defenses
against ROS.
Superoxide dismutases (SOD) are enzymes that catalyze dismutation of O

2 into O2 and H2O2. There are three isoforms of
SOD in humans: cytosolic copper- and zinc-containing SOD
(Cu-Zn-SOD), manganese-containing mitochondrial SOD
(Mn-SOD), and extracellular copper- and zinc-containing
SOD (EC-SOD). Mn-SOD is essential for life.
Glutathione peroxidase (GPX) converts glutathione into oxidized glutathione (glutathione disulfide, GSSG) and simultaneously reduces H2O2 to H2O and lipid hydroperoxides
(ROOH) to corresponding stable alcohols. This reaction is
coupled to the reaction catalyzed with glutathione reductase
(GSSG-R), the enzyme responsible for maintaining optimal
reduced glutathione (GSH) levels. Neurons are characterized
by very low levels of GSH and thus are particularly vulnerable
to free radical damage. GPX has very important role in protecting
cells from the harmful effects of peroxide decomposition. There
are eight isotypes of GPX in humans, and most of them have
selenocysteine residues at their active site, crucial for their action.
O

2 formed in the mitochondria is converted to H2O2 by
the action of Cu-Zn-SOD of the mitochondrial intermembranous space and Mn-SOD of the mitochondrial matrix. Formed
H2O2 is reduced by GPX present in the mitochondrial matrix.
Uncharged H2O2 passes the mitochondrial membranes and

Antioxidants: Role on Health and Prevention

can be scavenged in the cytosol by cytosolic Cu-Zn-SOD or

catalase.
Catalase is a heme enzyme located mainly in peroxisomes.
Liver and kidney cells, as well as erythrocytes, are rich in
catalase. It converts H2O2 to H2O and O2.
Glutathione reductase (GR or GSR) is a homodimeric flavoprotein disulfide oxidoreductase. It reduces oxidized glutathione (GSSG) to GSH. GSH is an important antioxidant, and
thus, GRs main role is to protect red blood cells, hemoglobin,
and cell membranes from oxidative stress.
Heme oxygenase (HO) catalyzes degradation of heme and
formation of CO, biliverdin, and iron. It does not have a direct
antioxidant enzymatic function, but indirectly, via its products,
it can protect cells against oxidative stress. It exists in two isoforms, inducible HO-1 and constitutively expressed HO-2.
HO-1 expression is induced by heat shock, UV radiation, or
ischemia/reperfusion injury.

Endogenous Nonenzymatic Antioxidants

Glutathione (GSH), a tripeptide consisting of glutamate, cysteine, and glycine, is found in all eukaryotic cells and represents
one of the key nonenzymatic antioxidants in the body. It is
mainly present in its reduced form, GSH. It is accompanied by
three enzymes involved in its homeostasis: GR, GPX, and
glutathione S-transferases (GST). They form the glutathione
system. In the gut mucosa, the GSH system serves as an antioxidative barrier.
Thioredoxin system is composed of thioredoxin (Trx) and
thioredoxin reductases (TrxR). Trx is disulfide-containing oxidoreductase that modulates activity of redox-sensitive transcription factors. In its dithiol form, it scavenges ROS and
recovers from the formed oxidized Trx state (Trx-S-S) via reduction catalyzed by a flavoenzyme TrxR and NADPH.
Melatonin is a hormone of the pineal gland but can be
found also in the retina, lymphocytes, GI tract, and bone
marrow. It neutralizes HO and peroxyl radicals, CO

3 , NO2,
O

2 , and HOCl. The characteristic of this antioxidant is that it
is irreversibly oxidized, so it is often called a suicidal or terminal antioxidant. It is a valuable protector of the mitochondria
against oxidative stress, as it can pass through the mitochondrial membrane, and thus, it is an important protector of the
liver from oxidative stress induced by alcohol consumption.

Exogenous Antioxidants
Vitamin C or ascorbic acid is the primary antioxidant in the
plasma and cells. It is a water-soluble vitamin found in all body
fluids. As an essential nutrient, it needs to be taken from foods,
mainly fresh fruits and vegetables. It donates two electrons from
C-2 and C-3 double-bond carbons, which results in the formation of an intermediate free radical, semidehydroascorbic
acid E. The resulting ascorbate free radicals reduce to a neutral
ascorbate molecule. It can react with various ROS, RNS, sulfur
radicals, O3, nitrosating compounds, and HOCl.
Vitamin E and a-tocopherol, as the most active form of this
vitamin, protect cells lipids from peroxidation by scavenging
ROS, but they can also act as prooxidants and reduce transition
metals. The mode of action depends on the level of
a-tocopherol.

229

Carotenoids and vitamin A are thought to be antioxidants,

but they can act as either prooxidant or oxidant depending on
the level of O2 and carotenoids.
Minerals (zinc (Zn), copper (Cu), manganese (Mn), iron
(Fe), and selenium (Se)) are antioxidant micronutrients as they
are cofactors of important antioxidant enzymes. Zn, Mn, and
Cu are cofactors of superoxide dismutase (Cu/Zn-SOD), and
Fe is a component of catalase. Se is a major antioxidant as it is a
component of selenoproteins. Se deficiency can result in toxicity through increased O

2 , NO, and lipid peroxidation.
Bioactive plant polyphenols are secondary plant metabolites,
widely distributed in fruits and vegetables. Plant polyphenols
are important dietary antioxidants, and dietary intake of these
compounds can be up to 1200 mg day
1. Main polyphenol
classes are flavonoids, phenolic acids, and procyanidins, and
they all posses strong antioxidant potential confirmed in
chemically based assays. However, their direct antioxidant
potential is compromised by their low bioavailability and
extensive metabolism, resulting in very low levels of the parent
compounds (in aglycone from) and metabolites. They can also
be susceptible to the metabolic transformation by intestinal
microbiota resulting in a wide variety of simple compounds,
which bioactivity still needs to be confirmed. Polyphenols are
shown to be potent inhibitors of XO, COX, LOX, GST, microsomal monooxygenases, and NADH oxidase and are capable to
chelate transition metals and indirectly suppress ROS production. Isothiocyanates are bioactives of cruciferous vegetables,
present in the form of their precursors, glucosinolates. They do
not exert direct antioxidant activity, but are potent inducers of
antioxidant enzymes, such as GST, and induce increase in
cellular glutathione levels. They can also be cytotoxic depending on their concentration and the side chain. Less lipophilic
isothiocyanates enter the cell in a time-dependant manner and
react with GSH, and the decrease in GSH levels is a signaling
factor for GST induction, resulting in net increase of cellular
GSH levels. More lipophilic ones enter the cell very fast resulting in immediate deprivation of GSH levels and induction of
cytotoxic effects. Dietary levels of isothiocyanates cannot exert
toxic effects on normal cells.

Levels of Antioxidant Action

An antioxidant is a molecule stable enough to donate an
electron to a rampaging free radical and neutralize it, thus
reducing its capacity to damage. These antioxidants delay or
inhibit cellular damage mainly through their free radicalscavenging property. Antioxidants interact with free radicals
and terminate chain reactions by removing free radical
intermediates and inhibit other oxidation reactions by being
oxidized themselves. A large number of antioxidants, acting
this way, including glutathione, ubiquinol, and uric acid, are
produced in the body during normal metabolism. Other antioxidants are obtained from a diet.
The ADS assumes several different acting levels such as
prevention, radical scavenging, repair and de novo synthesis,
and adaptation as the lines of defense.
The first line of defense comprises preventive antioxidants,
able to suppress the formation of free radicals. GPX,
GST, phospholipid hydroperoxide glutathione peroxidase
(PHGPX), and peroxidase are known to decompose lipid

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Antioxidants: Role on Health and Prevention

hydroperoxides to corresponding alcohols. PHGPX is unique

as it can reduce hydroperoxides of phospholipids integrated
into biomembranes. Glutathione peroxidase and catalase
reduce hydrogen peroxide to water.
The second line of defense comprises antioxidants that scavenge the active radicals to suppress chain initiation and/or
break the chain propagation reactions. The examples include
both hydrophilic (vitamin C, uric acid, bilirubin, albumin, and
thiols) and lipophilic (vitamin E and ubiquinol).
The third line of defense is the repair and de novo synthesis of
antioxidants. The proteolytic enzymes, proteinases, proteases,
and peptidases, present in the cytosol and in the mitochondria
of mammalian cells, recognize, degrade, and remove oxidatively modified proteins and prevent their accumulation. The
DNA repair systems also play an important role in the total
defense system against oxidative damage. Various kinds of
enzymes such as glycosylases and nucleases, which repair the
damaged DNA, are known.
The fourth line of defense is another adaptation mechanism,
where the signal for the production and action of reactive
species induces formation and transport of the appropriate
antioxidant to the right site of action.

Antioxidants in Human Diet

Vitamin C cannot be stored in the body and should be included
in regular diet. Important sources include citrus fruits, green
peppers, broccoli, green leafy vegetables, strawberries, raw cabbage, and potatoes.
Vitamin E (tocopherols and tocotrienols) is a fat-soluble vitamin that can be stored in the liver and other tissues. It is often
indicated for a range of states, from aging delay to sunburn
healing. Important dietary sources of vitamin E are wheat
germ, nuts, and seeds.
Beta-carotene, as the most studied of more than 600 different carotenoids that have been discovered, protects dark green,
yellow, and orange vegetables and fruits from solar radiation
damage. It is hypothesized that it plays a similar role in the
body. Carrots, squash, broccoli, sweet potatoes, tomatoes,
kale, collards, cantaloupe, peaches, and apricots are particularly rich sources of beta-carotene.
Selenium is an important component of antioxidant
defense. Selenium should be taken from foods, as large doses
that can be present in supplements can exert toxic effects. Good
food sources include fish, shellfish, red meat, grains, eggs,
chicken, and garlic. Vegetables can also be a good source if
grown in selenium-rich soils. Cereals contain selenomethionine, a naturally occurring amino acid that is the most important nutritional source of Se.
Plant bioactives. In addition to many vitamins and
minerals as nutrients, plants are rich sources of nonnutritive
dietary compounds and plant secondary metabolites
phytochemicals and many of them are bioactive, influencing
different processes of the human body. One of the most investigated activities is their antioxidant potential, confirmed in
numerous chemically based assays. However, very often, their
metabolites, which are the form of bioactives that can be found
in the circulation, lose the antioxidant potential of the parent
compounds. Still, they have been shown to affect redox states
in the cell by acting as signaling molecules. Depending on the

particular class or subclass of polyphenols, main dietary

sources of these bioactive compounds are numerous. Rich
sources of phenolic acids are berry fruits, kiwi, cherries, aubergine, chicory, artichoke, potatoes, corn flour, ciders and coffee.
Berry fruits are also the main dietary source of anthocyanins
(subclass of flavonoids) that are also present in substantial
quantities in grapes, plums, and wine. Bioactives of other
flavonoid subclasses can be found in onion, kale, tomato,
cherry, broccoli, berry fruits, apricots, apples, green and black
tea, and beans (flavonols); parsley, celery, and capsicum pepper (flavones); citrus fruits and juices (flavanones), soy and soy
products (isoflavones); and chocolate, beans, apricots, grapes,
peach, apple, green and black tea, and wine (monomeric flavanols). Isothiocyanates are the main bioactives of cruciferous
vegetables including broccoli, cauliflower, cabbage, kale,
watercress, garden salad, turnip, and horseradish.
Glutathione (GSH) protects cells from toxins including free
radicals. The human body produces GSH from the synthesis of
three key amino acids: cysteine, glycine, and glutamic acid.
Food sources with the highest amounts of naturally occurring
GSH include asparagus, avocado, grapefruit, squash, potato,
cantaloupe, peach, zucchini, spinach, broccoli, watermelon,
and strawberries. Fish, meat, and foods that yield sulfurcontaining amino acids (e.g., eggs) are the preferred sources
for maintaining and increasing bodily GSH levels.
Peroxidase is an enzyme occurring especially in plants, milk,
and leukocytes and consisting of a protein complex with hematin groups that catalyzes the oxidation of various substances.
Food sources of peroxidase are horseradish root, soybean,
mango fruit, and turnip.
Cysteine is an important antioxidant in cellular systems.
Cysteine is incorporated in the cellular glutathione, which
works along with vitamin E to protect cells against free radical
oxidant damage. Cysteine is a nonessential amino acid, synthesized in the liver from methionine. Animal proteins are
better sources of sulfur amino acids compared to proteins
from vegetables. Therefore, a balanced diet that includes both
animal and vegetable proteins (from grains and beans) would
provide adequate cysteine intake. Excessive intake of cysteine
can result in liver damage, kidney stone formation, or even
some forms of neurological disturbances.

Role on Health and Prevention

Oxidative Stress and Disease
In physiological conditions, the human body is in a steady
state with ROS being continuously generated and quenched. In
pathological conditions, in the state of oxidative stress,
increased levels of ROS can cause long-term damage that has
been implicated in numerous degenerative diseases. Oxidative
stress has been demonstrated by depressed levels of antioxidant substances (e.g., carotenoids, vitamin C, glutathione,
vitamin E, and uric acid); change in overall antioxidant capacity of plasma using different chemically based assays, including
total reactive antioxidant potential, the Trolox-equivalent antioxidant capacity, the ferric reducing/antioxidant power, the
oxygen radical absorbance capacity, or the ferrous oxidationxylenol orange assays; low levels of enzymes that are important
parts of the ADS or their activity (SOD, GPX, and CAT); and

Antioxidants: Role on Health and Prevention

increased levels of oxidation products (e.g., malondialdehyde,

8-hydroxy-2-deoxy-guanosin, oxi-LDL, and protein carbonyls)
or markers of macromolecular damage (comet assay for DNA
degradation and skin markers for protein degradation). Oxidative stress, as a result of either excessive generation of reactive
species or disturbed antioxidant defense, has been implicated
as underlying cause of various diseases. Most of previous
research was targeted to the elucidation of its role on the
pathogenesis of cardiovascular diseases, cancer, and neurodegenerative diseases. However, the putative list of diseases and
pathological conditions considered to be associated with oxidative stress should include atherosclerosis, coronary heart
disease, an impaired immune system, and increased risk of
infectious disease; diabetes (both noninsulin-dependent and
insulin-dependent diabetes); autoimmune conditions including rheumatoid arthritis; various respiratory diseases; and eye
diseases including cataracts and retinal damage leading to agerelated macular degeneration.
The effects of antioxidants on the prevention of these
pathologies were supported by the results of large prospective
studies (Nurses Health Study (NHS), Health Professionals
Follow-Up Study (HPFS), the European Prospective Investigation into Cancer and Nutrition (EPIC), etc.), suggesting an
inverse association between diet rich in fruits, vegetables, and
whole grains and the incidence of major chronic diseases,
including cancer and cardiovascular diseases. Evaluation of
obtained data by focusing on particular class of antioxidants
or particular disease or risk factors provides more precise information. However, initiated by epidemiological studies, a great
number of intervention trials were conducted in the past aiming to investigate the effects of antioxidants in the prevention
(primary, secondary, or tertiary) or therapy of chronic diseases,
in healthy subjects or participants with the diseases. And the
results of recent meta-analyses vary between supportive,
inconclusive, and even disappointing, depending on the type
of antioxidant and the targeted population. However, there are
many gaps that still need to be filled before the final conclusion about the relevance of antioxidant activity (both direct
and indirect) as a mechanism of action of dietary compounds
in the prevention and therapy of various diseases. Based on
their expertise and experience drawn from up-to-date
evaluations, European Food Safety Authoritys (EFSAs) Panel
on Dietetic Products, Nutrition and Allergies (NDA), responsible for verifying the scientific substantiation of the health
claims, published a guidance document on the scientific
requirements for health claims related to antioxidants, oxidative damage, and cardiovascular health. Regarding the effects of
antioxidants, major concerns were related to the causal correlation between certain biomarkers and health benefit (biomarkers of plasma antioxidant status), their specificity
(Comet assay and plasma markers of protein oxidation), and
applied methodology (ELISA). Still, numerous studies using
valid biomarkers show promising results. However, metaanalyses of studies on markers of diseases instead on markers
of disease risks were less supportive. Some authors share opinion that disappointing results obtained with exogenous antioxidants are not a proof of their inefficiency but rather indicate
a misleading approach with the whole body antioxidant
defense as a target instead of focusing on disease relevant
sites, where the disturbance has been shown and correlates

231

with the pathogenesis. Accordingly, a strategy that will include

an approach similar to that in pharmaceutical research could
lead to a discovery of new, advanced, and more efficient antioxidant nutraceuticals.

Antioxidants and Cardiovascular Disease

Animal and human data implicate increased levels of ROS
associated with oxidative stress in the pathogenesis of cardiovascular disease. However, in numerous clinical trials, at least
with small-sized antioxidants, supplementation failed to
reduce cardiovascular morbidity or mortality. Even the results
from German EPIC study on the associations between vitamin/
mineral supplementation and cancer, cardiovascular, and allcause mortality were unsupportive or at least inconclusive. On
the contrary, recent data from the Cancer Prevention Study II
Nutrition Cohort indicated an association of flavonoid consumption with lower risk of death from CVD. Surprisingly,
associations were strongest with intermediate intakes, suggesting that even relatively small amounts of flavonoid-rich foods
may be beneficial. Another prospective study showed inverse
association between anthocyanin intake and myocardial
infarction risk. This was confirmed in large number of intervention trials, showing beneficial effects of polyphenols intake
on variety of CVD risk factors and biomarkers. As shown in
numerous intervention studies, polyphenols in general exert
pleiotropic effects on cardiovascular health. It is still questionable if these effects were a result of direct or indirect antioxidant effects or more specific interactions with relevant
molecular and cellular targets. Cruciferous vegetable consumption is associated with a reduced risk of total and cardiovascular disease mortality. Results from intervention trials were
promising as well, suggesting relatively new area of isothiocyanate research.

Antioxidants and Cancer

Cancer is uncontrolled cell growth, in which cells lose their
natural function and spread throughout the blood in the entire
body. Oxidative stress is involved in the process of the development of cancer and tumors, due to the damage of the macromolecules by generated ROS. Beyond direct effect on DNA,
ROS-induced oxidation products of lipids (malondialdehyde)
can cause mutations or breaks in the DNA double chain or
modifications in guanine and thymine bases and sister chromatid exchanges, which can affect the activities of signal transduction, transcription factors, and gene tumor suppressors.
Oxidative damage or genetic defects in enzymes that repair
mutations favor age-dependent cancer. Importantly, some chemotherapeutics or radiation therapy increases ROS and
decreases antioxidant content, producing a state of severe oxidative stress, causing side effects. However, it was shown that if
generation of ROS is deeply involved in the mechanism of
action of anticancer agents, supplementation with antioxidants
could compromise the final outcome of the therapy.
Based on Cochrane reviews, there is no scientific evidence
for the effects of antioxidant supplementation in the prevention of GI cancers, lung cancer, or skin cancer. On the contrary,
some antioxidant supplements seem to increase overall mortality in general population and cancer patients.

232

Antioxidants: Role on Health and Prevention

Natural compounds such as polyphenols, in particular (
)epigallocatechin gallate and resveratrol, were shown to have a
promising future as antioxidants and anticarcinogenesis
agents. However, at this moment, the evidence for polyphenol
intake associations with cancer incidence is mostly limited to
the cancers of gastrointestinal tract. In substantial number of
prospective studies, intake of cruciferous vegetables was positively associated with lower risk of breast, colon (but not
rectal), prostate, and lung cancers.
Generally, the discrepancies between epidemiological data
and clinical trials with supplements (mainly vitamins and
minerals) might be due to the synergistic effects of various
bioactives in the whole food (antioxidants, vitamins, and minerals) compared to the effects of isolated compounds.

Antioxidants and Immune Function

The protective function against external pathogens carried out
by the immune system is by itself a source of ROS, since
activated neutrophils produce free radicals to a significant
extent. Frequent claims suggest that antioxidants benefit the
immune system, but recent meta-analyses indicated that the
results are uncertain even for vitamin C and its beneficial
effects on common cold, despite long history of use, and null
for vitamins and minerals in general, as supportive therapy in
pneumonia or asthma.

Antioxidants and Gastrointestinal Diseases

The GI tract is an important source of ROS. This is a putative
mechanism of various GI diseases including ulcers, malignancies, and inflammatory bowel disease. Dietary antioxidants,
mainly polyphenols, have been shown to exert beneficial
effects on oxidative stress in GI tract. They provide support to
the protective function of epithelial barrier against deleterious
effects of prooxidants and proinflammatory agents in the gut
lumen through their prebiotic properties on GI microbiota,
anti-inflammatory effects on GI epithelial cells, and direct
interaction with their lipid bilayer. In addition to the beneficial
effect of plant bioactives on the prevention of GI cancers,
several studies postulated an effect on other diseases of GI tract.

Antioxidants and Macular Degeneration

Some positive messages were expected from studies of particular antioxidants in macular degeneration, the major cause of
blindness in elderly people. The proposed mechanism is based
on the fact that ROS, produced in retina during light absorption, are deeply involved in the progression of the disease.
Some (but not all) studies initially suggested that specific
antioxidant supplements help in the protection against further
degeneration. The final conclusion drawn in the most recent
Cochrane review was supporting, with the limitation that most
of the data were taken from one randomize trial. The results for
age-related cataract were negative, showing no effects of antioxidants (beta-carotene, vitamin E, and vitamin C) in the
prevention and progression delay. There are also some evidences of benefits from vegetables and fruits rich in lutein
and zeaxanthin as antioxidants. Egg yolk is also a good source
of these compounds. A recent and extensive review reports no
benefits of vitamin E, beta-carotene, or any antioxidant supplements for preventing age-related macular degeneration.

Antioxidants and Neurodegenerative Diseases

Oxidative processes have been implicated in the pathogenesis
of neurodegenerative diseases including Alzheimers and Parkinsons diseases. The suggestion that they are triggered, at least
in part, by oxidative and nitrosative stress and sustained by
inflammatory cytokine production rationalizes the protection
of the central nervous system against these damaging mechanisms with antioxidants as a useful prophylactic and therapeutic approach. The results of both the Rotterdam Study and the
Cache County Study supported the hypothesis that high dietary intake of vitamin C and vitamin E may lower the risk of
Alzheimers disease, but not cognitive decline and dementia in
general. Additionally, based on the systematic review of the
literature, other antioxidants that could be effective in the
prevention of Alzheimers disease include garlic extract, curcumin, melatonin, resveratrol, Ginkgo biloba extract, and green
tea. Generally, the Kame project confirmed a hypothesis that
polyphenol-rich fruit and vegetable juice intake may exert
beneficial effects in delaying the onset of Alzheimers disease,
particularly among susceptible individuals. Recent studies support the use of antioxidants specifically targeted to the mitochondria as promising therapy of Parkinsons disease.

Antioxidants and Aging

For many people, the greatest interest is in antioxidants antiaging potential. Since the bodys production of its own antioxidants decreases with age, few doubt the potential value of
dietary sources. However, there is no evidence that supplementation with antioxidants will stop hair graying, prevent
wrinkles, or provide a fountain of youth. However, promising
results were obtained in a study investigating the effects
supplementation with vitamins and mineral antioxidants
(SU.VI.MAX) on cognitive performance. A direct correlation
was also shown for cognitive performance and total flavonoid
intake in a prospective Nurses Health Study. In general, cognitive function is considered as a very promising target for the
action of polyphenols and is relevant, considering the rise in
the percentage of aged population in developed countries.

Further Reading
Angelo G, Drake VJ, and Frei B (2014) Efficacy of multivitamin/mineral supplementation
to reduce chronic disease risk: a critical review of the evidence from observational
studies and randomized controlled trials. Critical Reviews in Food Science and
Nutrition. http://dx.doi.org/10.1080/10408398.2014.912199.
Cassidy A, Mukamal KJ, Liu L, Franz M, Eliassen AH, and Rimm EB (2013) High
anthocyanin intake is associated with a reduced risk of myocardial infarction in
young and middle-aged women. Circulation 127: 188196.
Chandel NS and Tuveson DA (2014) The promise and perils of antioxidants for cancer
patients. New England Journal of Medicine 371: 177178.
Day BJ (2014) Antioxidant therapeutics: Pandoras box. Free Radical Biology &
Medicine 66: 5864.
Dinkova-Kostova AT and Kostov RV (2012) Glucosinolates and isothiocyanates in
health and disease. Trends in Molecular Medicine 18: 337347.
Droge W (2002) Free radicals in the physiological control of cell function. Physiological
Reviews 82: 4795.

Antioxidants: Role on Health and Prevention

Engelhart MJ, Geerlings MI, Ruitenberg A, et al. (2002) Dietary intake of

antioxidants and risk of Alzheimer disease. Journal of the American Medical
Association 287: 32233229.
Hooper L, Kroon PA, Rimm EB, et al. (2008) Flavonoids, flavonoid-rich foods, and
cardiovascular risk: a meta-analysis of randomized controlled trials. The American
Journal of Clinical Nutrition 88: 3850.
Jin H, Kanthasamy A, Ghosh A, Anantharam V, Kalyanaraman B, and Kanthasamy AG
(2014) Mitochondria-targeted antioxidants for treatment of Parkinsons disease:
preclinical and clinical outcomes. Biochimica et Biophysica Acta
1842: 12821294.
Kesse-Guyot E, Fezeu L, Jeandel C, et al. (2011) French adults cognitive performance
after daily supplementation with antioxidant vitamins and minerals at nutritional
doses: a post hoc analysis of the supplementation in vitamins and mineral
antioxidants (SU.VI.MAX) trial. The American Journal of Clinical Nutrition
94: 892899.
Landete JM (2013) Dietary intake of natural antioxidants: vitamins and polyphenols.
Critical Reviews in Food Science and Nutrition 53: 706721.

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McCullough ML, Peterson JJ, Patel R, Jacques PF, Shah R, and Dwyer JT (2012)
Flavonoid intake and cardiovascular disease mortality in a prospective cohort of US
adults. American Journal of Clinical Nutrition 95: 454464.
Murphy MP (2014) Antioxidants as therapies: can we improve on nature? Free Radical
Biology & Medicine 66: 2023.
Sorice A, Guerriero E, Capone F, Colonna G, Castello G, and Costantini S (2014)
Ascorbic acid: its role in immune system and chronic inflammation diseases. MiniReviews in Medicinal Chemistry 14: 444452.
Visioli F and Davalos A (2011) Polyphenols and human health: a prospectus. Critical
Reviews in Food Science and Nutrition 51: 524546.

Relevant Websites
http://www.cochranelibrary.com/ Cochrane.
http://ebasis.eurofir.org/ EuroFIR AISB.
http://www.efsa.europa.eu/ European Food Safety Authority.