Clinica Chimica Acta 436 (2014) 332–347

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Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Invited critical review

Antioxidants and human diseases

Peramaiyan Rajendran a, Natarajan Nandakumar b, Thamaraiselvan Rengarajan a, Rajendran Palaniswami d,
Edwinoliver Nesamony Gnanadhas e, Uppalapati Lakshminarasaiah f, Jacob Gopas b,c, Ikuo Nishigaki a,⁎
a
NPO-International Laboratory of Biochemistry, 1-166, Uchide, Nakagawa-ku, Nagoya 454-0926, Japan
b
Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Israel
c
Oncology Department Soroka University Medical Center, Be'er-Sheva 84105, Israel
d
Department of Applied Zoology and Biotechnology, Vivekananda College (A Gurukula Institute of Life Training), Affiliated to Madurai Kamaraj University, Thiruvedakam West, Madurai 625234, India
e
Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
f
Department of Clinical Biochemistry and Pharmacology, Soroka University Medical Center, Ben-Gurion University of the Negev, Be'er-Sheva 84105, Israel

a r t i c l e i n f o a b s t r a c t

Article history: Oxidative stress plays a pivotal role in the development of human diseases. Reactive oxygen species (ROS) that
Received 4 April 2014 includes hydrogen peroxide, hyphochlorus acid, superoxide anion, singlet oxygen, lipid peroxides, hypochlorite
Received in revised form 4 June 2014 and hydroxyl radical are involved in growth, differentiation, progression and death of the cell. They can react with
Accepted 5 June 2014
membrane lipids, nucleic acids, proteins, enzymes and other small molecules. Low concentrations of ROS has an
Available online 13 June 2014
indispensable role in intracellular signalling and defence against pathogens, while, higher amounts of ROS play a
Keywords:
role in number of human diseases, including arthritis, cancer, diabetes, atherosclerosis, ischemia, failures in im-
Antioxidant munity and endocrine functions. Antioxidants presumably act as safeguard against the accumulation of ROS
Oxidative stress and their elimination from the system. The aim of this review is to highlight advances in understanding of the
Diabetes ROS and also to summarize the detailed impact and involvement of antioxidants in selected human diseases.
Cancer © 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
2. Measuring oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
2.1. Pro-oxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
3. Potential antioxidant biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.1. Superoxide dismutase (SOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.2. Catalase (CAT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.3. Glutathione peroxidase (GPx) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.4. Thiol index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.5. Xanthine oxidase (XO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.6. NADPH Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
3.7. A biomarker of cardiovascular damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
4. Mechanism of antioxidant action and nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
5. Antioxidants vs human diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
5.1. Antioxidant vs arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
5.2. Anti oxidant vs cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
5.3. Antioxidant vs diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
5.4. Antioxidant vs arthrosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
5.5. Anti oxidant vs neurodegerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

⁎ Corresponding author.
E-mail address: nishigaki@se.starcat.ne.jp (I. Nishigaki).

http://dx.doi.org/10.1016/j.cca.2014.06.004
0009-8981/© 2014 Elsevier B.V. All rights reserved.
 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347
1. Introduction
The beneficial effect of antioxidants on the maintenance of health in
human has become an important subject that has engaged many
scientists across the world over the last decade. In the last few years, an-
tioxidants have become the indispensable nutrients of the nutritional
world. Antioxidants are important in terms of their ability to protect
against oxidative cell damage that can lead to conditions, such as
Alzheimer's disease, cancer, heart disease and also linked with chronic
inflammation [1]. It is defined as the substances which at low concen-
tration significantly inhibits or delay the oxidative process, while often
being oxidized themselves. Recent reports suggest that several endoge-
nous and exogenous antioxidants are used to neutralize free radicals
and protect the body from free radicals by maintaining redox balance
[2,3]. Singh et al. (2010) [4] quoted that antioxidants have gone from
“Miracle Molecules” to “Marvellous Molecules” and finally to “Physio-
logical Molecules” that they play a vital role in metabolic pathways
and protect cells. However recent conflicting evidence has forced the
scientists to dig deeper in order to explore the role of antioxidants and
pro-oxidants, since free radical reactions have been implicated in
every human pathological condition which includes neurodegenerative
disorders like Alzheimer's disease, Parkinson's disease, multiple
sclerosis, amyotrophic lateral sclerosis, memory loss, depression and
cardiovascular diseases such as atherosclerosis, ischemic heart disease,
cardiac hypertrophy, hypertension, shock and trauma. Further, it also
implicated in pulmonary disorders which include inflammatory lung
diseases such as asthma and chronic obstructive pulmonary disease
and additionally diseases associated with premature infants such as
bronchopulmonary dysplasia, periventricular leukomalacia, intraven-
tricular hemorrhage, retinopathy of prematurity and necrotizing en-
terocolitis and in some autoimmune diseases like rheumatoid arthritis
and also in several renal disorders such as glomerulonephritis,
tubulointerstitial nephritis, chronic renal failure, proteinuria, uremia
and finally gastrointestinal diseases like peptic ulcer, inflammatory
bowel disease and colitis, diabetes, tumors and cancers [5].
2. Measuring oxidative stress
As oxidative stress is indicative of an inequity between oxidants and
antioxidants, methods for quantifying oxidative stress mostly include
straight or indirect measurement of oxidants and antioxidants [6,7]. In
the following segment, some principles and commonly used methods
for the measurement of oxidative stress and damage will be briefly
outlined.
2.1. Pro-oxidants
The most abundant free radicals in biological systems are the
oxygen-centered free radicals and their metabolites, usually referred
to as ROS [7]. ROS are formed continuously as normal by-products of
cellular metabolism; and, in low concentrations, they are essential for
several physiological processes, including protein phosphorylation,
transcription factor activation, cell differentiation, apoptosis, oocyte
maturation, steroidogenesis, cell immunity, and cellular defense against
microorganisms [7]. However, when produced in excess, ROS can dam-
age cell functionality as they can harm cellular lipids, proteins, and DNA
[7,8]. The plasma level of ROMs is considered an indicator of free-radical
production [7]. ROMs is a collective term that includes not only oxygen-
centered free radicals such as the superoxide anion and hydroxyl
radical, but also some non-radical derivatives of oxygen, such as hydro-
gen peroxide (H2O2) and hypochlorous acid [9]. A ROMs kit has been
developed to assess oxidant levels in plasma and other biological fluids;
and the ROMs test has been validated by electron spin resonance [10],
which is considered the “gold standard” for measuring total oxidative
status. However, electron spin resonance is not suitable for routine anal-
ysis, as the method is complex and requires specific technical assistance
333
not available in most laboratories. The utility of the ROMs assay in
monitoring oxidative stress in goats [11], sheep [12], and dairy cows
[13] has been reported.
The concentrations of individual oxidant components can be
measured separately in the laboratory; but such measurements are
time-consuming, labor intensive, and costly. Free-radical analytical
system 4 technology has been shown to offer a quick, simple, precise,
and reliable method for assessing the oxidative status in dairy cows
[14] and in horses [15]. Such technology is particularly useful in the
field, where it is not always practical or possible to get samples to a
laboratory immediately. The possibility of assessing oxidative stress
directly in blood samples provides veterinarians with a simple and
reliable method for measuring oxidative stress in clinical situations
such as the monitoring of therapy and in the antioxidant supplementa-
tion of domestic animals. However, given the lack of reference values
for ROMs in ruminants, it is difficult to establish if and when these ani-
mals are actually experiencing oxidative stress. Therefore, it is important
to calculate the specific referral ranges; because a correct biochemical
evaluation of oxidative status is an essential premise to prevent and
eventually to treat the effects of oxidative stress in ruminant medicine.
Advanced oxidation protein products (AOPPs) are terminal products of
proteins exposed to free radicals and arise from the reaction between
plasma proteins and chlorinated oxidants mediated by myeloperoxidase,
a neutrophil enzyme [16,17]. In humans, AOPPs have been linked to
several diseases, such as chronic renal failure [18], diabetes mellitus
[19], diabetic nephropathy [20], coronary artery diseases [21], and
obesity [22]. Chronic accumulation of AOPPs has been demonstrated to
promote inflammatory processes in the diabetic kidney [20] and in
chronic renal failure [18], indicating that these products might be a by-
product of neutrophil activation during infections. Studies in ruminants
have shown higher levels of AOPPs than normal in lambs [23] and
dairy cows [24] supplemented with Yerba Mate (Ilex paraguanensis).
More information about the role of protein oxidation in ruminants'
health and about oxidated proteins could be obtained by a comparison
of AOPPs with other indicators of protein oxidation, such as advanced
glycation end products (AGE). However, a correlation between AGE
and inflammatory parameters is usually not found or is only weak, and
the induction of proinflammatory activities caused by AOPPs seems to
be more intense [25,26], suggesting that oxidative stress is more closely
linked to inflammation and acute-phase reactions than to the advanced
glycation process and its end products. AOPPs could thus better describe
acute inflammation, whereas AGE might serve more as a marker of
chronic long-lasting damage [25]. These observations are highly relevant,
as increased levels of AOPPs could indicate the presence of inflammatory
processes that can potentially compromise the correct embryonic
development in dairy cows [14,27]. Lipids, in particular those that are
polyunsaturated, are prone to oxidation. Lipids are one of the most
susceptible substrates to damage by free radicals, and biomarkers of
lipid peroxidation are considered the best indicators of oxidative stress
[28]. Malondialdehyde (MDA) is one of several low-molecular-weight
end products formed during radical-induced decomposition of polyun-
saturated fatty acids [29]. MDA readily reacts with thiobarbituric acid,
producing a red pigment that can be easily measured by spectrophotom-
etry in the form of thiobarbituric acid-reactive substances (TBARS, [29]).
It is worth noting that the MDA assay has been criticized for its low
specificity and for artifact formation, since only a fraction of the MDA
measured is actually generated in vivo. Furthermore, the TBARS assay,
a common method used to measure MDA, is considered inaccurate,
and returns results that differ according to the assay conditions used
[30]. For example, studies on dairy cows have yielded contrasting results,
with some reports failing to show any significant changes in plasma
MDA concentrations during the peripartum period [31]; whereas in
other studies MDA or TBARS concentrations were shown to increase
around calving [8,16,32]. This apparent discrepancy could also have
been mainly due to the great variation in individual MDA concentrations
measured in the studies by Castillo et al. [31]. Similarly, studies on
334 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347

transported cattle have failed to report a consistent change in their MDA
concentration. It seems that this discrepancy is partly due to the different
methodologies employed to assess MDA levels. TBARS represent a wide
range of lipid peroxidation products, and the thiobarbituric acid reaction
is rather non-specific for MDA [33]. High-pressure liquid chromatogra-
phy would be expected to be highly specific, and perhaps, more accurate
than the spectrophotometric procedures [34]. More recently, an ELISA-
based method for the detection of isoprostane, which is considered to
be the most reliable marker of lipid oxidation [35], has become commer-
cially available and might be able to shed more light on the role of lipid
peroxidation during the peripartum period in ruminants.
non spontaneous decomposition of hydrogen peroxide, by the method
described by Aebi [44].
3.3. Glutathione peroxidase (GPx)
The biochemical function of glutathione peroxidase is to reduce lipid
hydroperoxides to their corresponding alcohols and to reduce free H2O2
to water. In contrast to catalase, peroxidase possesses a high affinity for
and can remove H2O2 even when it is present in a low concentration
[52]. Its activity can be estimated by the method described by Flohe
and Gunzler [45].

3. Potential antioxidant biomarkers

3.4. Thiol index

Epidemiologic literature that focuses on antioxidant status and

The GSH/GSSG index is a parameter of the intracellular redox status.
chronic disease risk has in the past relied primarily upon biomarkers
GSH is the major endogenous antioxidant produced by cells; and it
of exposure to antioxidant nutrients. However, this approach is in
participates directly in the neutralization of free radicals and reactive-
essence using exposure estimates to a select group of nutrients as a sur-
oxygen compounds, as well as in maintaining exogenous antioxidants
rogate for estimating actual oxidative defense or oxidative stress status.
such as vitamins C and E in their reduced forms [53]. GSH and GSSG
Emphasis is now being placed on developing functional biomarkers of
levels can be measured in erythrocytes by the method described by
oxidative stress status, that is, biomarkers that integrate the effect of
Hissin and Hilf [46].
exposure to oxidants coupled with the full range of antioxidant
protectivemechanisms in vivo. Many of such biomarkers are being
3.5. Xanthine oxidase (XO)
studied including various measures of oxidation products of lipid,
DNA, and protein (Table 1). Some of these biomarkers are now being
XO is a key enzyme involved in the formation of reactive oxygen
applied in research of pathologies related to oxidative stress.
species, and it plays a major role in cell oxidative stress. This enzyme
catalyzes the oxidation of hypoxanthine to xanthine and can further
3.1. Superoxide dismutase (SOD)
catalyze the oxidation of xanthine to uric acid [54]. Xanthine oxidase
can be estimated by the method of Haining and Legan [47].
The term superoxide dismutase characterizes a family of enzymatic
proteins differing in their structure and cofactors, among them being
Mn-SOD and Cu–Zn-SOD. SOD activity enhances the spontaneous 3.6. NADPH Oxidase
dismutation of superoxide radicals to H2O2 [52]. SOD can be measured
by utilizing the technique of Misra and Fridovich [43], which is based The NADPH-oxidase complex utilizes electrons to produce superoxide
on inhibition of the formation of nicotine amide adenine dinucleotide, radicals from the oxygen molecule. NADPH oxidase may be measured by
phenazine methosulfate, and amino blue tetrazolium formazan. a chemiluminescence [55] or electrochemical method among others.
Approaches to quantitate oxygen consumption, extracellular release of
3.2. Catalase (CAT) O∙2 or H2O2, and intracellular O∙2 production provide reliable assessment
of NADPH oxidase activity in a given population of cells [56].
The end product of the dismutation reaction, i.e., H2O2, can be re-
moved by the activity of the enzyme catalase. CAT is an enzyme with 3.7. A biomarker of cardiovascular damage
a very high KM for its substrate and can remove H2O2 present in high
concentrations [52]. The activity of CAT can be measured through the Endothelial microparticles (EMPs) are nuclear fragments of cellular
membrane shed from oxidatively stressed or damaged cells. Typically
defined as having a diameter of 0.1 to 1.0 mm, these microparticles con-
Table 1 tain surface proteins and cytoplasmic material of the parental cells [57].
Biomarkers of oxidative stress.
Many biomonitoring studies have investigated the role of antioxi-
Biomarkers Location Reference dants in reducing oxidatively generated DNA damage in urine and
Carbonyls Extracellular Mohanty et al. [36] white blood cells. A collective interpretation is difficult because many
Lipid peroxidation studies lack sufficient control and have unreasonably high baseline
•Malondialdehyde Extracellular Ohkawa et al. [37] levels of oxidatively damaged DNA. to investigating antioxidant effects
•F2-isoprostane Extracellular Collins et al. [38]
has been the use of biomarkers of oxidatively damaged DNA. This is
•4-Hydroxynonenal Extracellular Halliwell and Gutteridge [39]
Ferric reducing ability of plasma Extracellular Benzie and Strain [40] based on the mechanistic rationale that dietary antioxidants inhibit
Plasma vitamins the oxidation of DNA. With the possibility of detecting 8-oxo-7,8-
•Vitamin C Extracellular Roe and Kuether [41] dihydro-2′-deoxyguanosine (8-oxodG), the first of many antioxidant
•Vitamin E Extracellular Teissier [42] supplementation studies with focus on this lesion in WBC appeared in
Antioxidant enzymes
the beginning of the 1990s. At the same time, reliable detection of uri-
•Superoxide dismutase Intracellular Misra and Fridovich [43]
•Catalase Intracellular Aebi and Bergmeyer [44] nary 8-oxodG excretion was achieved, and this was soon followed by
•Glutathione peroxidase Intracellular Floh'e andG¨unzler [45] antioxidant supplementation studies using urinary 8-oxodG excretion
•GSH/GSSG ratio in erythrocyte Intracellular Hissin and Hilf [46] as key biomarker. However, by far the most popular method in antiox-
Prooxidant enzymes
idant intervention trials has been the comet assay that detects DNA
•Xanthine oxidase Intracellular Haining and Legan [47]
•NADPH oxidase Intracellular Nauseef [48] strand breaks (SB). An enzyme-modified version of the comet assay
Others has been developed to detect oxidatively altered nucleobases by
•Endothelial microparticles Extracellular Burger and Touyz [49] including a DNA digestion step with DNA glycosylase or endonuclease
•Endothelial progenitor cells Extracellular Touyz and Schiffrin [50] enzymes [58]. The characteristics of the intervention studies are
•Ischemia modified albumin Extracellular Sinha et al. [51]
outlined in Table 2.
 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347 335

Table 2
Administrations of dietary antioxidant with assessment of oxidatively DNA damage in white blood cells and urine.

Supplement per day Subjects Age Effect References

(years)

β-carotene (20 mg) for 14 weeks 122 M (S) 39 (NR) No effect on urinary excretion of 8-oxodG/24-h (HPLC) [59]
β-carotene (30 mg) for 1 months 14 M (NS) 19–22 No effect on urinary excretion of 8-oxodG/24-h (HPLC) [60]
Carotenoidsa for 3 weeks 32 MF(NS) 32 ± 11 No effect versus baseline, but decreased urinary excretion of 8-oxodG ELISA) [61]
in active group post-supplementation
Vitamin C (500 mg as plain or slow release formulations) 48 M (S) 39 ± 12 Decreased ENDOIII and FPG sites (Comet) in the group that ingested tablets [62]
and vitamin E (182 mg) for 4 weeks with the slowrelease vitamin C formulation
Multi-vitamin tablet (100 mg vitamin C, 280 mg vitamin 100 M (50S) 50–59 Decreased ENDOIII sites (Comet) after 20 weeks in WBC [63]
E, and 25 mg b-carotene) for 20 weeks
Vegetable consumption (3.6 vs 12 servings) for 64 F (NR) 23–81 Lower 8-oxodG in WBC (HPLC) among those subjects eating 12 servings per day [64]
2 weeks
Kiwi fruit (1–3 pieces) for 3 weeks 14 MF (NS) 26–54 Decreased ENDOIII and FPG (Comet) sites [65]
Cranberry juice (750 ml) for 2 weeks 20 F (2S) 18–40 No effect on ENDOIII (Comet) in WBC [66]
Blackcurrant juice or anthocyanine drink 57 MF (6S) 19–52 No effect on ENDOIII and FPG sites (Comet) in WBC [67]
(475–1000 ml) for 3 weeks
Green tea or black tea (four cups) for 1–4 months 120 MF(S) 18–79 Decreased excretion of 8-oxodG (ELISA) in spot urine samples after 4 mo [68,69]
in green tea group, but not before
Two interventions of green tea with 300 ml for 7 days 68 MF (13S) 18–45 Decreased 12-h urinary excretion of 8-oxodG (HPLC) [70]
or 32 oz for 7 days
Green tea extract b for 3 weeks 16 M (8S) 20–31 No effect related to supplementation on 24-h urinary excretion of 8-oxodG [71]
(HPLC) but a decreased excretion during the study in all groups
Green tea (500 or 1000 mg) among high-risk subjects 124 NR Lower 8-oxodG/24-h (HPLC) after 3 months supplementation but not after [72]
of liver cancer for 3 months MF(NR) 1 month
Soya-hypocotyl tea (N1000 ml) for 1 months 38 F (NR) NR Decreased excretion of 8-oxodG (ELISA) in active group (statistical [73]
test not reported)
Soy milk, rice milk, or cow milk (1 l) for 4 weeks 10 M (NS) 20–50 Decreased ENDOIII sites (Comet) for soy milk [74]
Polyphenol-rich olive oils (25 ml) for 40 days 12 M (NS) 20–22 Decreased excretion of 8-oxodG (HPLC) in spot urinary samples following [75]
supplementation in a dose-dependent manner
Olive oil (25 ml) with three different content of phenolic 182 M (NS) 20–60 Decreased 8-oxodG/24-h urinary excretion, but no difference in urinary excretion [76]
compounds for 3 weeks of 8-oxoGua/24-h. No difference in the effect of the different olive oils
Antioxidant rich diet or tabletsc for 5 weeks 55 MF (NR) 71 ± 6 No effect versus baseline, but decreased 24-h urinary excretion of 8-oxodG [77]
(ELISA) in active group post-supplementation

Number of subjects indicated as males (M) and females (F). Smokers (S) and non-smokers (NS) are indicated in brackets. Lacking information is indicated as NR (not reported) Age is
shown as range or mean ± standard deviation. aSupplement constitute b-carotene (6 mg), a-carotene (1.4 mg), lycopene (4.5 mg), bixin (11.7 mg), lutein (4.4 mg), and paprika carot-
enoids (2.2 mg), b Consisted of 1000 mg extract/kg bodyweight in meat patties (total phenolics were 23.5 mg/10 MJ), cConsisted of tablets with vitamin antioxidants (400 mg vitamin C,
150 mg vitamin E, 4 mg b-carotene), capsules (90 mg vitamin C, 18 IU vitamin E, 2.4 mg b-carotene, and powder or extract of fruits and berries), or a carotenoid-rich diet.

4. Mechanism of antioxidant action and nutrients
It is reported that antioxidants can execute protective role against
free radicals by a variety of different mechanism including the catalytic
systems to neutralize or divert ROS, binding or inactivation of metal ions
prevents generation of ROS by Haber–Weiss reaction, suicidal and chain
breaking antioxidants scavenge and destroy ROS or absorb energy,
electron and quenching of ROS. In 21st century, demand for intake of
antioxidant food or dietary antioxidant increasing with the hope to
keep body healthy and free from diseases [78,79] and the potential
beneficial effects of antioxidants in protecting against disease have
been well established. It is increasingly thought that nutrition may
play a vital role in helping to defend against oxidative stress and damage
induced by free radicals. Therefore, certain nutrients and dietary com-
ponents with antioxidant properties are important for the protection
against oxidative stress injury of the body. Food consumption is a
major source of exogenous antioxidants and has been estimated that a
typical diet provides more than 25,000 bioactive food constituents as
nutrients and many of this may modify a multitude of processes that
are related to different diseases. Generally, antioxidants are abundant
in vegetables and fruits and are also found in grain cereals, peas,
legumes, nuts and other food products. At this juncture, a systematic
survey has also identified more than 3100 antioxidants in foods, like
beverages, spices, herbs and supplements which are regularly
consumed by different cultures [80]. It has been speculated that the de-
crease in the intake of nutritional and antioxidants rich food may

Table 3
Effects of the health associated to the intake of Antioxidants.

Antioxidants Effect of health References

Vitamin C Protects against cancers, Protects from heart disease, Improvement of the health of
cartilage, joints and skin, Maintaining a healthy immune system, Improvement in the
antibody production, Increase in the absorption of nutrients, Increases protection
against H2O2-induced DNA strand breaks
Vitamin E Prevents coronary heart disease, Prevents the formation of blood clots Decreases
incidence of breast and prostate cancers, Brain protection, Reduces long-term risk
of dementia Decreases risk of Parkinson's disease
Polyphenol Inhibit oxidation of LDL, Inhibit platelet aggregation, Improve endothelial dysfunction
Lower risk of myocardial infarction, Effect anticarcinogenic Prevent neurodegenerative
diseases, Protect against neurotoxic drugs, treatment of diabetes, treatment to prevent
osteoporosis, Inhibit non-heme iron absorption
Cu, Zn, Mn, Se Cofactors of antioxidant enzymes SOD-Cu/Zn, Mn-SOD and GSH-Pox
Other carotenoids Protection against oxidation of lipids, LDL, proteins and DNA. Abduction and free
(lycopene) radical scavenging
Barry [85], Liu et al. [86], Wang et al. [87], Wintergerst et al. [88],
Woo et al. [89], Thankachan et al. [90] and Riso et al. [91].
Pryor [92], Traber et al. [93], Weinstein et al. [94], Muller et al. [95],
Devore et al. [96] and Miyake et al. [97].
Manach et al. [98], Russo et al. [99], Sch¨achinger et al. [100],
Corder et al. [101], Yang et al. [102] Halliwell [103], Pan et al. [104],
Zunino et al. [105], Atmaca et al. [106], Hurrell et al. [107].
Visioli F et al. [108]
Visioli F et al. [108]
336 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347
increase the chances of oxidative stress which may lead to cell damage,
therefore intake of such natural antioxidants may give protective effect
against free radical induced diseases [80,81]. In view of the importance
of antioxidants, Suntres [82] reported that antioxidant liposomes will
hold an important role in future research on antioxidants, and also
reports that it facilitates the delivery of antioxidants to specific sites as
well as achieving prophylactic and therapeutic action. In addition,
Bouayed and Bohn [83] have suggested that the balance between oxida-
tion and anti-oxidation is critical in maintaining a healthy biologic sys-
tem and low doses of antioxidants may be favorable to this system,
but high quantities may disrupt the balance. It is true that antioxidants
are beneficial and display a useful role in the maintenance of the ho-
meostasis in human ROS system, but so are pro-oxidants, therefore
the scientific community should search deeper into the kinetics and
in vivo mechanisms of antioxidants to uncover the optimal concentra-
tions or desired functions in order to push forward against cancer,
neurodegenerative and cardiovascular diseases [84] (Table 3). Various
studies related to free radicals, oxidative stress and antioxidant activity
of food reveals the prominent beneficial role of antioxidant and its spe-
cific role against different diseases individually. However the collective
and concise data on the role of antioxidants in human diseases will be
better and most appropriate one in order to know the exact role of an-
tioxidants in all kinds of human diseases (Fig. 1). Hence, an attempt
has been made in this review, to summarize the detailed role and
impact of antioxidants in certain selected dreadful human diseases.
Fig. 1. Antioxidants/oxidative stress and health diseases.
5. Antioxidants vs human diseases
5.1. Antioxidant vs arthritis
Chronic autoimmune inflammation, which is commonly observed in
rheumatoid arthritis (RA), disrupts the delicate balance between bone
resorption and calcification causing the destruction of the bone and
joints. Multiple aetiologies are suspected to contribute to the formation
of RA, including defective articular cartilage structure and biosynthesis,
joint trauma, joint instability, congenital and developmental abnormal-
ities, and inflammatory conditions [109,110]. Oxidative stress induced
damage to essential cell components caused by oxygen free radicals is
a mechanism in the patho-biology of degenerative rheumatoid arthritis
[109]. Hydroxyl radicals cause degradation of isolated proteoglycans,
and hypochlorous acid (HOCl) fragments of collagen. Hydrogen perox-
ide, which is highly diffusible, readily inhibits cartilage proteoglycan
synthesis, by interfering with ATP synthesis, in part by inhibiting the
glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase in
chondrocytes, aggravating the effects of proteolytic and free-radical-
mediated cartilage degradation. Peroxynitrite and HOCl may facilitate
cartilage damages by inactivating TIMPs. TIMP-1 inhibits stromelysins,
collagenases and gelatinasesand this ability is lost after nitrus
oxide (NO− 3 ) or HOCl treatment. HOCl can also activate latent forms of
neutrophil collagenases and gelatinase with obvious consequences.
Hypochlorous acid, NO− −
3 and O2 react with ascorbate, which is essential
Table 4
Clinical trials (variables observed: vitamins C, E, selenium, and β-carotene, ORAC).

Study Sample size and Cancer agents Dosage and mode Study design Results Strengths/weaknesses
demographics

Hematologic malignancies
Clemens et al. [155] Subjects: n = 19 CY, MEL, VP16, TBI α-Toc 9 mg (TPN) Cohort study Subjects (TBI+): serum α-Toc and β-Car 2 Significant differences between two groups at
(baseline v day +12); TBI-α-Toc over time baseline; nonrandomized, small cohort, no
(P b .001), no significant changes in β-Car controls; intervention assessed the effects of
RDA only
Clemens et al. [156] Subjects: (TBI+) CY, VP16, BU, TBI TBI+: α-Toc 825 mg, β-Car Nonrandomized, ↓ Hepatotoxicity; no significant differences in Study groups received different TPN regimens;
N = 16, mean age: 45 mg, AA 450 mg, (PO); TBI+: nonblinded, controlled rates of relapse significant differences were not observed at
30.5 y, Controls: (TBI+) AA 530 mg, α-Toc 9 mg (TPN) trial each time point; P values NR; small cohort, dif-
n = 10, mean age: ferent anticancer regimens; TBI+ also received
37.5 y 130 mg AA and 13.1 mg α-Toc by TPN

P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347

Hunnisett et al.,[157] Controls: n = 19, mean High-dose chemotx, VC 25 mg; VE 1000 IU (TPN) 20 sequential patients Acute 2 in VC and VE (P b. .001; P b .001); no Nonrandomized, small cohort; cancer
age: 33 y; Subjects: ±TBI significant differences after day 0 diagnosis and agents NR
n = 10, mean age: 37 y
Lloyd et al. [158] Controls: n = 26, age NR AA 1 g X 4 days (mode NR) Cohort study Prior to intervention: mean platelet and plasma Inquired about dietary intake/Cancer therapy
range: 18–28 y; subjects: ↓AA In CML controls (P b .001; P b .05), mean NR; time of blood withdrawal, length of
n = 16, intervention: platelet AA↓ in CML v controls (P b .05); after observation, and timing of intervention NR;
n=6 intervention: ↑ in platelet and plasma AA in AL methodology NR
& CML (P b .025) ↑ plasma AA in CLL (P b .05)

Gynecologic cancer
Sundstrom et al. [159] Subjects: n = 56; DXR, CY, CDDP, MELPH Four arms: 1) VE 300 mg, Se Controlled trial ↑ in serum Se in Se group 1 and group 2 v Placebo controlled trial/multiple gynecologic
controls: n = 44, mean 96 μg; 2) Se 96 μg; 3) VE controls (P b .001; P b .001) cancers, stages, and regimens
age: 58.7 y 300 mg; 4) placebo (PO)

Lung cancer
Jaakola et al. [160] Subjects: n = 18, age CY, ADR, VP16, VCR, MV β-Car 10,000–20,000 U, Nonrandomized Study group had increased survival rates Variable doses administered depending on
range: 51.8–69.6 y DXR, EPI CBPT, RT α-Toc 300–800 U, AA 2000– controlled trial compared to historical controls (P value NR); “individual” micronutrient analysis
5000 U, Se 856 μg (PO) plasma Se, α-Toc NS, plasma AA, β-Car NR (methodology NR); multiple stages combined;
one subject received no cancer therapy

Breast cancer
Lockwood et al. [161] Subjects: n = 32, age Chemotx, RT, Surgery VC 2850 mg, VE 2500 IU, β-Car Nonrandomized, Whole blood β-Car, VE, and Se ↑ after 3 and Whole blood VC NR, no controls, chemother-
range: 32–81 y 32,500 U, Se 387 μg (PO) noncontrolled trial 12 months (P b .01), P b .05), P b .01); no apy agents NR; subjects also administered MV
deaths, expected four; no subject had weight containing Co-Q10 and essential fatty acids; P
loss, ↓ use of pain killers, ↑ QOL values not consistently reported

Abbreviations: ORAC, Oxygen radical absorbance capacity; CY, cyclophosphamide; MELPH, melphalan; VP16, etoposide; 2, decrease; 1, increase; TBI, total body irradiation; α-Toc, alpha tocopherol; TPN, total parenteral nutrition; β-Car, beta carotene;
RDA, recommended dietary allowances; y, years; BU, busulfan: PO, orally; AA, ascorbic acid; NR, not reported; VC, vitamin C; VE, vitamin E; DZ, diaziquone; TP, thiotepa; Ara-C, cytosine arabinoside; k-Cal, total caloric intake; CML, chronic myelocytic
leukemia; AL, acute leukemia; CLL, chronic lymphocytic leukemia; VCR, vincristine; DXR, doxorubicin; EPI, epirubicin;
CBPT, carboplatin; RT, radiation therapy; MV, multivitamin; NS, nonsignificant; CDDP, cisplatinum diammine dichloride; Se, selenium; chemotx, chemotherapy; QOL, quality of life; Co-Q10, coenzyme Q 10; y-TOC, gamma tocopherol (Modified from
Elena J. Ladas et al., 2004).

337
338 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347
for cartilage function, leading to low levels of ascorbate in synovial fluid.
Low concentrations of hydrogen peroxide (H2O2), O.− 2 or both, acceler-
ate bone resorption by osteoclasts, whereas nitric oxide (NO.) inhibits it
and promotes chondrocyte apoptosis, thereby inhibits proteoglycan
synthesis and activates latent metalloproteinases and cyclooxygenase.
ROS, produced by activated phagocytes, could alter the antigenic behav-
ior of immunoglobulin G, producing fluorescent protein aggregates that
can further activate phagocytic cells. Radical-exposed IgG is able to bind
rheumatoid factor and results in the generation of C3a. This reaction
may be self-perpetuating within the rheumatoid joint, suggesting that
free radicals play a role in the chronicity of the inflammatory reaction
which is a key question regarding to which extent free radicals contrib-
ute to the consequences of inflammation, such as the cartilage and
bone destruction. Reactive oxygen intermediates can also function as
signaling messengers to activate transcription factors, like NF-κB
Change all to NF-κBand activator protein 1(AP-1), and induce gene
expression [111]. All this knowledge might serve to apply a rational
selection of antioxidants for possible therapeutic purposes, of the
inflammatory joint disease.
Many raw foods contains natural antioxidants, including enzymes
such as superoxide dismutase, glutathione peroxidase and catalase,
which are usually inactivated during food processing, and non-
enzymic antioxidants such as carotenoids (e.g. canthaxanthin and
astaxanthin in some farmed fish), β-carotene, lutein, lycopene, tocoph-
erols (in oils) and other compounds in plants. The latter include other
carotenoids and ascorbate (vitamin C). The plasma concentrations of
these are largely determined by dietary intake and it is possible that
two or more antioxidants can act together synergistically [112]. In
view of the high antioxidant content of the French diet which is rich
in fruit, vegetables and red wine [113,114], it is intriguing to note the
relatively low incidence of arthritis [115]. More work is required on tis-
sue distributions and bioavailability of antioxidant molecules within
joints since lipophilic antioxidant molecules, such as vitamin E or
β-carotene, may not have the same access to tissues as hydrophilic an-
tioxidants, such as vitamin C. Perhaps, the differences in their effects in
disease processes may depend on the hydrophilicity of the antioxidant
molecules concerned and the resulting pattern of tissue distribution in
different tissue areas. One major problem is that there is no assay
currently available to measure oxidant activity within joints and possi-
bly the activity could occur from an alternative mechanism.
5.2. Anti oxidant vs cancer
Reactive Oxygen Species is a hallmark of human cancer. ROS and
their functions with respect to the cancer initiation and signalling in
cancer cells is a prime concern of cancer research. Tobacco smoke also
plays a very important role in increasing the risk for inflammation and
cancer due to its high carcinogenic potential and the synergistic effects
with other respirable particulate to generate ROS and catalyse redox re-
actions in the cells of humans, leading to oxidative stress and increased
production of mediators of inflammation [116–118]. Inflammatory cells
are particularly effective in generating most of the reactive oxygen
species. The activation of the redox metabolism of the inflammatory
cells generates a highly oxidative environment within an organ of aero-
bic organisms. Much of the oxygen biochemistry, through the activation
of plasma membrane NADPH oxidase, of macrophages and neutrophils
is directed towards the release of superoxide anion, hydrogen peroxide
and hydroxyl radicals [119–121]. Inflammation acts through the forma-
tion of ROS and reactive nitro species (RNS) which cause oxidative
damage to cellular components. Many pro-inflammatory mediators,
especially cytokines, chemokines and prostagladins, turn on the angio-
genesis switches mainly controlled by vascular endothelial growth
factors [122]. Cancer-associated inflammation is also linked with
immune-suppression that allows cancer cells to evade detection by
the immune system. Inflammation is a critical component of tumor pro-
gression. Many cancers arise from sites of infection, chronic irritation
and inflammation. It is now becoming clear that the tumor microenvi-
ronment, which is largely orchestrated by inflammatory cells, is an
indispensable participant in the neoplastic process, fostering prolifera-
tion, survival and migration. In addition, tumor cells have co-opted
some of the signalling molecules of the innate immune system such as
selectins, chemokines and their receptors for invasion, migration and
metastasis. These insights are fostering new anti-inflammatory thera-
peutic approaches to cancer development [123]. Pathological angiogen-
esis is a hallmark of cancer and various ischaemic and inflammatory
diseases. Chronic inflammation is associated with angiogenesis, a
process that helps cancer cells to grow. Angiogenesis is necessary for
expansion of tumor mass and macrophage, platelets, fibroblasts and
tumor cells are a major source of angiogenic factors (vascular endothe-
lial growth factors, chemokines, NO, etc.) [124,125]. The inflammation
in the tumor microenvironment is characterized by leukocyte infiltra-
tion, ranging in size, distribution and composition, as: tumor-
associated macrophages (TAM), mast cells, dendritic cells, natural killer
(NK) cells, neutrophils, eosinophils and lymphocytes. These cells produce
a variety of cytotoxic mediators such as ROS and RNS, serine and cysteine
proteases, membrane perforating agents, matrix metalloproteinase
(MMP), tumor necrosis factor α (TNFα), interleukins (IL-1, IL-6, IL-8),
interferons (IFNs) and enzymes, likecyclooxygenase-2 (COX-2),
lipooxygenase-5 (LOX-5) and phospholipase A2 (PLA2). Other re-
searchers discovered that tumor-associated macrophages are key regula-
tors of the link between inflammation and carcinogenesis [126] and
experimental studies showed that, tobacco smoke produce ROS that
have been associated with activation of mitogen-activated protein kinase
(MAPK) family members and activation of transcription factors such as
NF-κβ and AP-1. These signaling pathways have been implicated in pro-
cesses of inflammation, apoptosis, proliferation, transformation and dif-
ferentiation [127]. The activation protein-1 (AP-1) contributes to basal
gene expression in biological systems. ROS can activate AP-1 through
several mechanisms and the effect of AP-1 activation is an increased cell
proliferation due to increased expression of growth stimulatory genes
such as cyclin D1 and suppression of the protein p21waf. Studies showed
that AP-1 and NF-kB, inducible by tumor promoters of oxidative stimuli,
show differential protein levels or activation in response to tumor pro-
moters in JB6 cells. Researchers suggest that as long as oxidative events
regulate AP-1 and NF-kβ transactivation, these oxidative events can be
important molecular targets for cancer prevention [128,129]. NF-kB is
expressed and participates in a wide range of biological processes, such
as cell survival, differentiation, inflammation and growth [130]. NF-kB ac-
tivation has been linked to a wide spectrum of extracellular stimuli and
oxidants and subsequent involvement in the carcinogenic process
through promotion of angiogenesis and tumor cell invasion and metasta-
sis [131,132].
Dietary factors like chlorogenic acid have also been found to protect
against environmental carcinogen induced carcinogenesis by their up-
regulation of phase II conjugating enzymes and suppression of ROS-
mediated correct, AP-1, and MAPK activation [133]. Cellular defence
system is comprised of several Phase II detoxification enzymes such as
glutathione-S-transferases (GSTs), NADP (H) quinoneoxidoreductase
(NQO1), Glutathione peroxidases (GPx), Catalase, superoxide
dismutases (SODs), epoxide hydrolase, hemeoxygenase (HO-1), UDP-
glucuronosyltransferases (UGTs), gammaglutamylcysteinesynthetase
and many others. Expression of these proteins protects cells from oxida-
tive damage and can prevent mutagenesis and cancer [134–137]. To
counterbalance the oxidative damage from ROS, aerobic organisms
have created a variety of antioxidant mechanisms to maintain their
genomic stability. These mechanisms include phase II detoxification
and other anti-oxidation enzymes that act in cellular defence such as
catalase and SOD, GPx, GST, other constitutive and inducible antioxi-
dants, DNA repair enzymes, and other cellular mechanisms of genomic
surveillance, such as cell cycle checkpoint control systems [133]. More-
over, several growth factors such as serum, insulin like growth factor I,
or fibroblast growth factor 2 generates ROS in MIA PaCa-2 and PANC-1
 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347
cells, which are human pancreatic adenocarcinoma cells that promotes
cell survival. Inhibiting ROS generation with the antioxidants or NADPH
oxidase (Nox4) antisense, or MnSODover expression would stimulate
apoptosis in PaCa-2 and PANC-1 cells. This mechanism might play an
important role in pancreatic cancer resistance for treatment and thus
represent a novel therapeutic target. In gastrointestinal and colon
cancer, oxidative pentose pathway (OPP) and the glutathione (GSH) an-
tioxidant defense system play an important role in the regulation of cell
growth and apoptosis. The OPP modulate intracellular redox status and
provides NADPH for the synthesis of GSH, which is responsible for the
inactivation of intracellular ROS that induce apoptosis and cell injury.
Depletion of GSH increases the sensitivity of cells to ROS. Therapeutic
inhibition of the OPP and/or the GSH defense system might increase
the sensitivity of gastric and colon cancer cells to anti-cancer therapy
[138]. Recent studies have shown that the enzymatic product of thymi-
dine phosphorylase (TP) generated ROS within cancer cells that help in
maintaining the growth of colon cancer cells thus may provide im-
proved therapeutic results as well as a preventative effect on carcino-
genesis of the colorectum [124]. Emerging evidence also suggests that
supranutritional doses of selenite could induce typical apoptosis in
colorectal cancer cells in vitro and in xenografttumors by inducing
ROS and it indicates that antioxidants can activate the apoptotic
machinery through redox-dependent activation and could be useful in
cancer therapy [139].
The MAPK signalling cascade is activated by a wide variety of recep-
tors involved in growth and differentiation including receptor tyrosine
kinases (RTKs), integrins and ion channels. This pathway has been re-
ported to be activated in over 50% of acute myelogenous leukemia,
acute lymphocytic leukemia and are frequently activated in breast,
prostate cancers and other cancer types [140,141]. Under optimal
growth conditions, transiently elevated ROS levels confer a growth ad-
vantage to tumor cells. However, exposure of these cells to anticancer
agents induces a prolonged increase in ROS levels resulting in potentiat-
ing of apoptosis. Thus ROS modulates the ability of stress kinases to
stimulate cell growth or cell death and it depends on signal intensity
and signal duration [142]. This idea has been nurtured to explain the
complex nature of ERK signaling in cell cycle regulation and in stress ac-
tivated protein kinase signalling (SAPK). Transient, reduced activity of
SAPK promotes cell proliferation, whereas persistent, increased activity
results in cell killing [143]. Conversely, in drug-resistant tumors, phase II
enzymes or other antioxidant defences are often up regulated, shielding
cells from apoptosis. ASK1, an upstream regulator of SAPKs, is inhibited
in normal cells through its association with thioredoxin, which is an
antioxidant capable of metabolizing ROS. Increased levels of ROS lead
to the dissociation of this complex and thereby facilitate the activation
of ASK1 and subsequently SAPKs [144]. A similar redox control has
been identified for JNK where increased level of ROS triggers the detach-
ment of JNK associated glutathione-S-transferase-π (GSTp) and thereby
facilitating JNK activation. Moreover, ROS-dependent activation of JNK
may also lead to knock down of a JNK phosphatase via an unknown
mechanism linking ROS and stress induced kinases an important player
in cancer cell growth [145]. Growth inhibition of pancreatic cells is de-
pendent on the efficiency of scavenging ROS as well as effective inhibi-
tion of ERK1/2 signaling pathway activation as demonstrated by using
the free radical scavenger N-Acetyl Cysteine (NAC) or the MEK/ERK1/
2 inhibitor (PD98059). Moreover, ERK1/2 induction is dependent on
ROS production as demonstrated by a complete removal of ERK1/2
phosphorylation by NAC. Thus ROS act as pro-survival, anti-apoptotic
factor in pancreatic cancer cells [52]. Moreover, treatment of SW620
(colon cancer cells) with berberine, which is a major constituents of
Coptidisthizoma, resulted in activation of apoptosis by phosphorylation
of JNK and p38 MAPK, as well as generation of the ROS [146]. Further-
more, proline oxidase (POX), that often considered as a ‘housekeeping
enzyme’, induce apoptosis through both intrinsic and extrinsic
pathways and is involved in nuclear factor of activated T cells (NFAT)
signaling and regulation of the MEK/ERK pathway in colon cancer
339
cells. It has been suggested that, as a nutrition factor, POX may modulate
apoptosis signals induced by p53 or other anti-cancer agents and
enhance apoptosis in stress situations [147].
Components of the cell signaling network, especially those which
converge on the ubiquitous eukaryotic redox-sensitive transcription
factor nuclear factor-kappa B (NF-κB), have been implicated in patho-
genesis of many inflammation-associated disorders. Under normal
physiologic conditions, NF-κB is sequestered in the cytoplasm by bind-
ing to the inhibitory protein called IκBa. Phosphorylation and subse-
quent ubiquitination results in degradation of IκBa by proteasomes.
Phosphorylation of IκBa is mainly mediated by the IκB kinase (IKK)
complex. Phosphorylation of specific serine residues of p65 subunit of
NF-κB has been considered to facilitate the translocation of NF-κB to
nucleus and interaction with the coactivator CBP/p300. Induction of
κphase-2 detoxifying or antioxidant genes represents an important cel-
lular defense in response to oxidative and electrophilic insults. Nuclear
transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2)
plays a crucial role in regulating phase-2 detoxifying/antioxidant gene
induction. Like NF-κB, Nrf2 is present in the cytoplasm as an inactive
complex with the inhibitory protein subunit, in this case Keap1. Dissoci-
ation of Nrf2 from Keap1 is prerequisite for nuclear translocation and
subsequent transactivational activity. Once translocated into nucleus,
Nrf2 interacts with a small Maf protein, forming a heterodimer that
binds to antioxidant responsive elements (ARE) or electrophile respon-
sive elements (EpRE) present in the promoter/enhancer regions of
genes encoding many antioxidant and detoxifying enzymes. Keap1 con-
tains several cysteine residues that function as sensors of redox changes.
Oxidation or covalent modification of these critical cysteine thiols
diminishes the affinity of Nrf2 for Keap1, releasing Nrf2 for nuclear
translocation. Dissociation of the Nrf2-Keap1 complex is also assumed
to be stimulated through the phosphorylation of Nrf2 by distinct up-
stream kinases such as MAPKs, PKC, PI3K, etc. As in the case of NF-κB,
phosphorylation of Nrf2 is also considered to facilitate the interaction
of this redox-sensitive transcription factor with CBP/p300.NRF2 is
a known regulator of the antioxidant response [148,149]. NRF2-
regulated phase II enzymes protect against the development of cancer
by catalyzing reactions that convert highly reactive, carcinogenic
chemicals to less reactive products [150,151]. Singh et al. (2012) [152]
recently demonstrated that vitamin C and BHA provide protection
against E2-mediated oxidative DNA damage but the mechanism is not
well understood. Many antioxidants derived from dietary and medicinal
plants have been found to modulate Nrf2-Keap1 signaling, thereby
potentiating cellular antioxidant capacity or facilitating detoxification
of carcinogens and other toxicants [153,154]. Table 4 describes 7 obser-
vational studies reporting changes in serum, plasma, or whole blood
levels of vitamins Cand E, selenium,β-carotene, and total radical antiox-
idant parameter (TRAP), which represents total body antioxidant status,
in patients undergoing anticancer therapy. Information is grouped by
malignancy and includes subject demographics, cancer data, cancer
treatment, end points evaluated, results, and comments. No specific
treatment was consistently associated with changes in the individual
antioxidants (Table 4) [162].
5.3. Antioxidant vs diabetes
Glucose overload may damage the cells through oxidative stress.
This is currently the basis of the “unifying hypothesis” that
hyperglycemia-induced oxidative stress may account for the pathogen-
esis of all diabetic complications [163,164]. Increasing evidence in both
experimental and clinical studies suggests that oxidative stress plays a
major role in the pathogenesis of both types of diabetes mellitus. Free
radicals are formed disproportionately in diabetes by glucose oxidation,
non-enzymaticglycation of proteins, and the subsequent oxidative
degradation of glycatedproteins [165]. This increased superoxide
production causes the activation of 5 major pathways involved in
the pathogenesis of complications: polyol pathway flux, increased
340 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347
Fig. 2. Shows the involvement of cellular antioxidant enzymes in cardiovascular diseases [216–227].
formation of AGEs (advanced glycation end products), increased ex-
pression of the receptor for AGEs and its activating ligands, activation
of protein kinase C isoforms, and overactivity of the hexosamine path-
way. It also directly inactivates 2 critical anti-atherosclerotic enzymes,
endothelial nitric oxide synthase and prostacyclin synthase. Through
these pathways, increased intracellular reactive oxygen species cause
defective angiogenesis in response to ischemia, activate a number of
pro-inflammatory pathways, and cause long-lasting epigenetic changes
that drive persistent expression of proinflammatory genes after glyce-
mia is normalized (“hyperglycemic memory”). Atherosclerosis and car-
diomyopathy in diabetes are caused in part by pathway-selective
insulin resistance, which increases mitochondrial ROS production
from free fatty acids and by inactivation of anti-atherosclerosis enzymes
by ROS. Over expression of antioxidant enzymes in diabetic patients
prevents diabetic retinopathy, nephropathy, and cardiomyopathy
[166]. Antioxidants have proven to play a minimal if any role in the
treatment of diabetic complications in humans [167]. Decreases in ex-
pression, and in some instances the activity of antioxidant enzymes,
has been previously reported in diabetic microvascular disease [168].
Indeed, the over expression of Cu+Zn2+ superoxide dismutase (SOD)
protects against end organ damage in models of type II diabetic ne-
phropathy [169]. Other studies in mice with genetic deletions of various
antioxidant enzymes have also provided insight into the specific relative
contributions of Mn2+SOD [170] to the development of diabetes com-
plications. Mn2+SOD mimetics such as MnTBAP have also shown effica-
cy in preventing ROS-induced injury in vitro, although the utility in vivo
of such drugs may be limited [171,172]. Further strengthening a poten-
tial role for the antioxidant Mn2+SOD, specific polymorphisms of the
Mn2+SOD gene are associated with the development of diabetic com-
plications [173]. Interestingly, GPx-1-deficient mice have no increased
risk for microvascular disease, in particular diabetic complications
[174], most likely because of redundancy with respect to GPx isoforms.
Over-expression of catalase in experimental models of type 2 diabetic
appears to be protective the diabetic complications [175]. In contrast
to Mn2+SOD, however, studies in humans have indicated no relation-
ship between catalase gene polymorphisms that interfere with its
cellular expression and the incidence of type 2 diabetic patients [176].
Kunisaki et al. [177] investigated the effects of treatments with clas-
sical antioxidants such as vitamin E,vitamin C and lipoic acid. Specifical-
ly, vitamin E normalizes retinal blood flow and protein kinase C (PKC)
activity in the vascular tissue of diabetic rats. One short-term experi-
mental study showed that high doses of vitamin C can improve some as-
pects of endothelial dysfunction in diabetes. Another experimental
study has demonstrated that intra-peritoneal administration of α-
lipoic acid to streptozotocin (STZ) diabetic Wistar rats normalises
TBARS levels in plasma, and the retina, liver, and pancreas [178]. Fur-
thermore, it has been reported that α-lipoic acid leads to a decrease in
the severity of diabetic neuropathy by maintaining GSH levels and/or
by its direct antioxidant properties [179]. However, lipoic acid adminis-
tration improved endothelial function in subjects with metabolic syn-
drome. In another study to determine the effects of vitamin E on
oxidative stress and cell membrane fluidity in the brains of diabetes-
induced experimental rats, Hong et al. [180] reported that vitamin E
was found to be effective for strengthening the antioxidant defense sys-
tem. They noted a reduction of the accumulation of ROS such as super-
oxide radicals, a decrease in the generation of oxidative damaging
substances such as the carbonyl value, a significantly improved lipid
composition, and maintenance of membrane fluidity in the brains of
the rats. Coleman suggested that triple antioxidant therapy (vitamin E,
lipoic acid and vitamin C) in diabetic volunteers attenuates the
 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347
experimental oxidative stress of met-hemoglobin formation in vitro and
reduces haemoglobin glycation in vivo [181]. Panjwani et al. [182]
suggested that vitamin C administered alone or in combination with
vitamin E reduced the fall in ulnar nerve conduction velocity. Sivan
and Reece [183] investigated whether dietary supplementation with
vitamin E would reduce the incidence of diabetic embryopathy in an
in vivo rat model. Fifty pregnant rats were designated as the control
group and received a normal diet while the diabetic group received vita-
min E supplements (400 mg/day). This experimental study found that
supplementation with vitamin E reduces the incidence of neural tube
defects by more than 75% [184]. These findings suggest that vitamin E re-
duces this oxidative load, confers a protective effect against diabetic
embryopathy, and thus may potentially serve as a dietary prophylaxis in
the future. These results are in line with the reports of Chang et al. [184]
which showed that diabetic embryopathy of rat or mouse embryos is
prevented by vitamin C, vitamin E, SOD, N-acetyl-cysteine, or glutathione
ethyl ester. Another study by Otero et al. [185] showed the effects of
supplementation with vitamin E to diabetic mice. The beneficial effect
of vitamin E observed in this model was shown to retard coronary
atherosclerosis accelerated by DM, and was demonstrated to be due to a
reduction in oxidative stress, and not secondary to a decrease in plasma
glucose or cholesterol since their respective plasma concentrations
remained unchanged in the diabetic mice supplemented with vitamin E
[186]. Furthermore, it has been recently reported that macrophages
from diabetic mice are under excess oxidative stress, and that the antiox-
idant vitamin E can attenuate macrophage oxidative stress which exists in
DM and leads to accelerated atherosclerosis development. In a placebo-
controlled, randomized trial of diabetic patients, Reaven investigated
the effect of 1600 IU of RRR-α-tocopherol supplementation daily for
10 weeks on hyperglycemia-induced LDL modifications. The result of
the study pointed to a reduction of approximately 60% of plasma LDL
oxidation in diabetic patients, which was statistically significant when
compared to healthy controls [187]. These results are supported and con-
firmed by other similar clinical data. Salonen et al. [188] found that doses
equal to or higher than 450 IU are sufficient to significantly ameliorate
the susceptibility of LDL to oxidation, indicating that relatively
high doses of RRR-α-tocopherol for supplementation are needed.
Furthermore, Bellomo and others have shown that the effects of RRR-
α-tocopherolsupplementation on LDL oxidation are accompanied by a
concomitant reduction in autoantibody levels againsthyperglycemia-
induced LDL modifications. Moreover, clinical studies have shown
an inhibitory effect of RRR-α-tocopherol supplementation on
thehyperglycemia-induced LDL modifications inT1DM and T2DM dia-
betic patients. Based on nutrition recommendations and interventions
for diabetes, there is no clear evidence of benefits that can be derived
from vitamin or mineral supplementation in people with diabetes.
Routine supplementation with antioxidants, such as vitamins E and C
and carotene, is not advised because of lack of evidence of efficacy, and
concern related to long term safety, and therefore cannot be recom-
mended [189]. The majority of studies included in this review support
a possible role of antioxidant supplementation in reducing the risk of
diabetic complications
5.4. Antioxidant vs arthrosclerosis
The mechanisms of oxidative stress and antioxidants are in patients
with atherosclerosis have been evaluated in extensive animal experi-
ments and clinical research. There is a relationship between atheroscle-
rotic risk factors (ARF) and increased vascular production of ROS; the
most important ARFs are heredity, age, hypertension, dyslipidemia,
diabetes and smoking [190]. It has been reported that in in-vitro studies
an increased vascular production of ROS, particularly superoxide causes
deleterious effects in cell death [191,192]. ROS and reactive nitrogen
species (RNS) are closely linked to the disease process. Incomplete scav-
enging of ROS and RNS influences the mitochondrial lipid cardiolipin,
stimulates the release of mitochondrial cytochrome c and finally
341
activates the intrinsic death pathway [193]. Local generation of RNS
may contribute to vascular tissue injury. Therefore, ROS and RNS partic-
ipate as signalling molecules that regulate diverse pathophysiological
signalling pathways. High levels of ROS are potent inducers of the intrin-
sic apoptotic pathway and tissue injury in pathophysiological condi-
tions as an integral part of atherosclerotic plaque stabilization. The
most important sources of ROS production associated with CVD pathol-
ogy are the mitochondrial respiratory chain, nicotinamide adenine
dinucleotide phosphate oxidases (NADP oxidase). The pathogenesis of
atherosclerosis is further related to inflammation, immune response
and the proliferative process. Endothelial denuding injury leads to
platelet aggregation and releases platelet-derived growth factor,
which triggers the proliferation of smooth muscle cells forming the
nidus of the atherosclerotic plaque in the arterial intima, implicating in-
flammatory changes in the development of the disease [194,195].
Supplementing treatment with antioxidants might be helpful to pa-
tients. Vitamin E reduces the consequences of lipid peroxide content
in mouse peritoneal macrophages (MPMs) [196]. E-selectin has been
proposed as an important factor in the development of the inflammato-
ry process underlying atherothrombosis. The expression of E-selectin is
decreased by vitamin E in the diabetic rats [197]. Vitamin C has also
been investigated in studies on the prevention of atherosclerosis. The
following proposals have been put forward regarding the mechanisms
of vitamin C in preventing atherosclerosis: First, vitamin C has been
shown to prevent apoptosis caused by cytokines in cultured endothelial
cells [198]. It also decreases the release of micro-particles derived from
endothelial cells and suppresses pro-apoptotic activity in congestive
heart failure patients in vivo [199]. Second, vitamin C stimulates all
types of collagen synthesis by specific hydroxylase enzymes [200].
Endothelial cell proliferation is, in part, associated with the synthesis
of type IV collagen [201]. Thus, the lack of vitamin C prevents the
generation of type IV collagen in cultured endothelial cells [202].
Third, vitamin C protects the vascular endothelium by enhancing endo-
thelial NO synthase. Endothelial NO synthase activity is inhibited by ROS
that oxidize and deplete the co-factor tetrahydrobiopterin [203]. There-
fore, vitamin C prevents the loss of NO synthase activity by maintaining
tetrahydrobiopterin [204]. Glutathione monoethyl ester, but not ascor-
bic acid, exerted protective effects against ischemia–reperfusion injury.
Interestingly, the protective effects of glutathione monoethyl ester are
enhanced by co-administration with vitamin C in rat hearts subjected
to ischemia and reperfusion [205]. The oxidized LDL leads to increased
platelet-endothelial cell adhesion, which can be prevented by superox-
ide dismutase (SOD) and catalase [206]. In fact, the vascular extracellu-
lar expression of SOD is stimulated by NO [207]. Narang et al. [208]
reported that dietary palmolein oil, which is rich in monounsaturated
fatty acid and antioxidant vitamins, “protected rat heart from oxidative
stress associated with ischemiareperfusion injury”. Das et al. [209]
found “a role for c-Src [a family of proto-oncogenic tyrosine kinases]
in post ischemic cardiac injury and dysfunction and demonstrate direct
cardio protective effects of [the tocotrienol-rich fraction of palm oil
(TRF)]. The cardio protectiveproperties of TRF appear to be due to
inhibition of c-Src activation and proteasome stabilization”. Carlson
et al. [210] using a rat model, reported: “Antioxidant vitamin therapy
[vitamin C, vitamin E, vitamin A and zinc] abrogated myocardial inflam-
matory cytokine signaling and attenuated sepsis-related contractile
dysfunction, suggesting that antioxidant vitamin therapy may be a
potential approach to treat injury and disease states characterized by
myocardial dysfunction”. Hypertension is directly regulated by the
kidneys and cardiovascular system and adversely affects these organs.
Tian et al. [211] found that in salt sensitive rat model of hypertension,
vitamin C and vitamin E treatments “decreased renal inflammatory
cytokines and chemokines, renal immune cells, NF-κB, and arterial pres-
sure and improved renal function and damage”. As noted above, chronic
zidovudine administration promotes cardiovascular damage, and vita-
min C has been found to combat this effect in rats [212]. Diabetes
mellitus may initiate increased myocardial vulnerability to ischemia–
342 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347
reperfusion injury and pro/antioxidant imbalance. Resistance to
ischemia-induced ventricular arrhythmias and levels of endogenous an-
tioxidants (α-tocopherol) has been found to be increased in diabetic rat
myocardium [213]. The currently available evidence needs to be con-
firmed in clinical trials. Consumption of fruit and vegetable diets rich
in antioxidant nutrients can be recommended for all individuals, and
there is some indication that such a diet can be beneficial in the effects
of cardiovascular events. Fig. 2 shows the involvement of cellular anti-
oxidant enzymes in cardiovascular diseases.
5.5. Anti oxidant vs neurodegerative diseases
Neurodegenerative disorders are a heterogeneous group of diseases
of the nervous system, including the brain, spinal cord, and peripheral
nerves that have much different aetiology. ROS are particularly active
in the brain and neuronal tissue as the excitatory amino acids and
neurotransmitters, whose metabolism is factory of ROS, which are
unique to the brain and serve as sources of oxidative stress. ROS attack
glial cells and neurons, which are post-mitotic cells and therefore,
they are particularly sensitive to free radicals, leading to neuronal dam-
age [226]. It has been reported that deleterious effects of ROS on human
cells may end in oxidative injury leading to programmed cell death i.e.
apoptosis [227]. A multitude of genes involved in redox status, anti-
inflammation and detoxification are transcribed by Nrf2–ARE pathway
activation. These genes are known to be involved in cytoprotection
from various oxidative insult and cellular injuries in numerous different
tissues and organs including brain. Intracellular peroxidases are cleared
by a group of enzymes that are transcribed by Nrf2–ARE pathway. The
peroxisomal CAT catalyses the conversion of H2O2 to water and molec-
ular oxygen. However, the specific activity of CAT is much lower in brain
than peripheral tissue [228]. Glutathione-s-peroxidase (Gpx) is another
enzyme that metabolizes H2O2 and depends on reduced glutathione
(GSH). The oxidized GSH (GS-SG) is recycled to GSH by glutathione
reductase (GR). Glutathione (GSH), a tripeptide γ-glutamyl-cysteinyl-
glycine, is the most abundant low molecular weight thiol expressed
ubiquitously. It is widely recognized as an endogenous non-enzymatic
antioxidant and an oxyradical scavenger, and is thereby critical to main-
taining a reducing environment in the cell and protect against oxidative
damage by ROS [229–233]. GSH has been implicated in a wide range of
metabolic processes, including cell division, DNA repair, regulation
of enzyme activity, and activation of transcription factors, modulation
of anion and cation homeostasis, and protection against oxidative
damage [234].
GSTs are key detoxification enzymes that catalyze the conjugation of
various electrophiles, reactive alkenals, and numerous other xenobiotic
to GSH. These GSH-S-conjugates are removed from cells by the multi-
drug resistant protein-1 (MRP-1), an ATP binding cassette (ABC) family
protein [235,236]. MRP-1 is an integral plasma membrane protein that
exports glutathione-S conjugates out of the cell in an ATP-dependent
manner [237,238]. Studies have shown reduced GST activity in brain
and ventricular fluids in AD [239]. Increased expression of GST leads
to increased resistance towards oxidative stress in neuroblastoma cells
and provides protection against HNE-mediated toxicity in neuronal
cell culture [239]. Growing evidence demonstrates that the AD brain is
under tremendous oxidative stress. A significantly increased HO-1 ex-
pression was reported in post-mortem AD temporal cortex and hippo-
campus compared to aged-matched control [240]. Additionally, an
increased Nqo1 activity and expression was found in astrocytes and
neurons of AD brain [229] and Nrf2 was predominantly localized in
cytoplasm in AD hippocampal neurons [241]. Furthermore, there is in-
creased protein oxidation [242,243] and lipid peroxidation [244–246]
in AD brain when compared to aged matched controls. Recent studies
in aged APP/PS1 AD mouse models showed reduced Nrf2, Nqo1, GCL
catalytic subunit (GCLC) and GCL modifier subunit (GCLM) mRNA and
Nrf2 protein levels [247]. Additionally, in a triple transgenic AD
mouse, the GSH/GSSG ratio was reported to be reduced [248].
The hallmark of PD is a severe reduction of dopamine in all compo-
nents of the basal ganglia. Dopamine and its metabolites are depleted
in the caudate nucleus, putamen, globuspallidus, nucleus accumbens,
the ventral tegmental area, and the substantianigra pars compacta and
reticulata. Moderate losses of dopamine are found in the lateral hypo-
thalamus, medial olfactory region and amygdaloid nucleus [249]. In
early parkinsonism, there appears to be a compensatory increase in
dopamine receptors to accommodate the initial loss of dopamine
neurons [250]. As the disease progresses, the number of dopamine re-
ceptors are decreases, apparently due to the concomitant degeneration
of dopamine target sites on striatal neurons. In the remaining neurons in
patients with PD, dopamine turnover seems greatly increased, judging
from the concentrations of homovannilic acid [HVA] in the nerve termi-
nals in the striatum and the cell bodies and dendrites in the substantia
nigra [251] and the ROS production may very well increase in conse-
quence. This hypothesis is strengthened by a study showing that the
concentrations of GSH decrease when dopamine turnover increase
after reserpine treatment in rats, indicating increased activity of the
peroxide scavenging enzyme GSH-Px [252]. If the increase in ROS
production due to increased dopamine turnover is not buffered by the
scavenging enzymes (SOD, catalase, and GSH-Px), the compensatory
hyperactivity of the dopaminergic neurons may become self-
destructive. Chronic administration of L-DOPA would then only exacer-
bate the production of destructive ROS [253]. The administration of
L-DOPA itself has been postulated to enhance the accumulation of ROS
[254]. Another index of oxidative stress in PD might be the evidence
of a robust increase of NF-κB in the nuclei of dopaminergic neurons in
the substantia nigra of PD patients [255]. This clinical finding is consis-
tent with in vitro data showing that oxidative stress induced by C2-
ceramide treatment causes nuclear translocation of NF-κB in cultured
mesencephalic neurons [256]. More recently, it has been shown that
the neurotoxin 6-OHDA activates NF-κB in PC12 cells by enhancing in-
tracellular ROS levels [257]. Interestingly, in this experimental model,
NF-κB seems to sustain cell survival by stimulating the expression of
the anti-apoptotic proteins Bcl-2 and Bfl-1144. Moreover, as already
mentioned, the potent green tea polyphenol antioxidant EGCG exerts
a neuroprotective effect in a MPTP mouse model of PD [258]. However,
there are still few and controversial epidemiological data on this impor-
tant point, which might be partly due to the intrinsic difficulties in
performing epidemiological surveys regarding the dietary habits of
large populations. Nevertheless, it is desirable that future studies
aimed at investigating the relationship between dietary antioxidant
intake and the relative risk for neurodegenerative disorders such as
AD, PD, and ALS will throw more light on this very important aspect of
public health.
6. Conclusion
In summary the reactive oxygen species and oxidative stress play an
important role in the aetiology and progression of major human degen-
erative diseases has triggered enormous and worldwide interest in
endogenous and exogenous antioxidants. There is now abundant evi-
dence that substances in fruit and vegetables are potent preventives of
various diseases, especially arthritis, cancer, heart disease, diabetes
and neurodegenerative diseases. With these recent developments in
scientific knowledge which firmly establish the links between food
factors and the prevention of disease. The present review reveals a
vast number of relevant studies related to oxidative stress and selected
human diseases. The research field actually presents a variety of
approaches that have been produced suggestive evidence that antioxi-
dants might have an impact against many diseases. However, more
systematic research in this line is imperative in order to reveal the rela-
tionship and involvement of antioxidants with other diseases. The large
diversity in the molecular mechanisms and the methodology of antiox-
idants discussed here are factors probably responsible for the consistent
findings of their prevention of diseases. This shows that it will be very
 P. Rajendran et al. / Clinica Chimica Acta 436 (2014) 332–347
useful in controlling many oxidative stress mediated diseases as
discussed above. In comparison with the effects of the antioxidants
analysed in the previous study we can expect that many plant derived
compounds which have excellent antioxidant property and were suc-
cessfully investigated here and validates or corroborates the antioxidant
hypothesis of many diseases [165,259–263]. Current review highlights
the perspectives and possibilities of unrevealing the uncapped poten-
tials of antioxidants in herbal plants for disease cure and warrants the
need for systematic research in the field to clear the insights in this
very complex matter. However, recent research work with antioxidant
therapy suggests another promising area i.e., combining potential anti-
oxidants and using them during the early onset of the disease as a
prophylactic measure to prevent the disease that may eventually
prove to be more effective in treating several devastating diseases.
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