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antioxidants defense mechanism

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DOI: 10.13140/RG.2.2.33502.48966

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ANTIOXIDATIVE DEFENSE MECHANISMS

Prepare by
Hayder Mzedawee
 An antioxidant is a molecule capable of inhibiting
the oxidation of other molecules. Oxidation is a
chemical reaction that transfers electrons or
hydrogen from a substance to an oxidizing agent.

Oxidation reactions can produce free radicals. In

turn, these radicals can start chain reactions.
Antioxidant Defence Mechanism

 The atoms or molecules having unpaired electrons are known as free

radicals and are considered as a major source of ageing.

 These free radicals are stabilized by special category of substances known as

antioxidants which prevent and protect the cell from the damage caused by
free radicals.

 Few examples of such substances include beta-carotene; lycopene; vitamins

C, E and A; catalase; etc. which help in sustaining the rate of oxidation
reactions in a cell (Sies 1997).
 The process of transfer of electrons from one substance to another with low
reduction potential is termed as oxidation, and these oxidation reactions are
responsible for the generation of free radicals, which can cause injury to the
cells by an order of chain reactions.

 In order to prevent damage by oxidation, plants and animals have a

complex systems of antioxidants like catalase, superoxide dismutase,
glutathione peroxidises, vitamin C; etc.
 Antioxidant Defence Mechanism

Enzymatic
Antioxidants Non-Enzymatic
Antioxidants
SOD
ASH
CAT GSH
APX Phenolic compounds
GR alkaloids
MDHAR flavonoids
DHAR carotenoids
non-protein
GPX amino acids
GOPX a-tocopherols (vit E)
GST Ascorbic acid (vit C)
 Types of Antioxidants

Primary or natural Secondary or synthetic

antioxidants antioxidants.

Antioxidant enzymes (NDGA

Antioxidant vitamins (BHA)
(TBHQ)
Antioxidant minerals (BHT)
Phytochemicals (PG)
(EDTA)
(flavonoids)
Primary Antioxidants
Primary antioxidants are also referred to as type 1 or chain-breaking
antioxidants.

Because of the chemical nature of these molecules, they can act as free radical
acceptors/scavengers and delay or inhibit the initiation step or interrupt the
propagation step of autoxidation.

Secondary Antioxidants
Secondary antioxidants are also classified as preventive or class II
antioxidants.
They offer their antioxidant activity through various mechanisms to slow the
rate of oxidation reactions.
The main difference with primary antioxidants is that the secondary
antioxidants do not convert free radicals into stable molecules.

ROOH: hydro peroxide

ROO : peroxy radical

Figure : Possible interactions of primary and secondary antioxidants with

lipid oxidation pathway in foods.
Natural Antioxidants
Antioxidant Enzymes

 All cells in the body contain powerful antioxidant enzymes.

 The three major classes of antioxidant enzymes are the superoxide

dismutases, catalases and glutathione (GSH) peroxidases.

 In addition, there are numerous specialized antioxidant enzymes reacting

with and detoxifying oxidants
 1- Superoxide dismutases (SODs)

They are a class of closely related enzymes that catalyse the breakdown of the
superoxide anion into oxygen and hydrogen peroxide
SOD
O2 .– + O2 .– + 2H H2O2 + O2

They are present in almost all aerobic cells and in the extracellular fluids.

 There are three distinct types of SOD classified on the basis of the metal
cofactor:
 the copper/zinc (Cu/Zn - SOD) in cytoplasm , nucleus and plasma
 the manganese (Mn-SOD) in mitochondria
 and the iron (Fe-SOD) isozymes in chloroplasts
In the absence of superoxide dismutase, this reaction occurs non-enzymatically
but at a very slow rate

Hydrogen peroxide(H2O2) is mainly produced by enzymatic reactions. These

enzymes are located in microsomes, peroxysomes and mitochondria.

In plant and animal cells, superoxide dismutase is able to produce H2O2 by

dismutation of O2˙¯, thus contributing to the lowering of oxidative reactions.

The natural combination of dismutase and catalase contributes to remove

H2O2 and thus has a true cellular antioxidant activity.

H2O2 is also able to diffuse easily through cellular membranes

Since SOD is present in all aerobic organisms and most (if not all) subcellular
compartments that generate activated oxygen, it has been assumed that SOD
has a central role in the defense against oxidative stress (Scandalias, 1993).

Elimination of superoxide
 Catalase
Catalase (CAT) is a heme-containing enzyme that catalyses the dismutation of hydrogen
peroxide into water and oxygen.

The peroxisomes are major sites of Hydrogen peroxide production.

Three type of this enzyme : Cat-1 and Cat-2 are localized in peroxisomes and the cytosol,
whereas cat-3 is mitochondrial.

Catalase
2H2O2 2H2O + O2

Elimination of H2O2
Three isoforms of CAT found in maize (corn yellow) (present on different
chromosomes and independently regulated).
 Glutathione peroxidase

 Glutathione peroxidase (GPx, 1.11.1.9) is a selenium-dependent enzyme, which

decomposes H2O2 and various hydro- and lipid peroxides (Kinnula et al., 1995).

 The classical form of GPx is cellular and dispersed throughout the cytoplasm,

 but GPx activity is also found in mitochondria (Buettner, 1998), and has been detected in
several human tissues, including lung (Chu et al., 1992).

 Selenium is essential for the protein synthesis and enzymatic activity of GPx. Severe
selenium deficiency may cause liver necrosis and degenerative heart disease (Buettner,
1998).

 GPx requires reduced glutathione as a co-substrate, and the role of glutathione reductase
is to restore glutathione from an oxidized to a reduced form (Kinnula et al., 1995).
 During this reaction hydrogen peroxide (H2O2) is reduced to water and glutathione (GSH)
is oxidized to glutathione disulfide (GSSG).

Glutathione Peroxidase
• H2O2 + 2GSH H2O + GSSG

contains selenocystein, reduces H2O2 and hydroperoxides of phospholipids (ROOH) .

2 G-SH + H-O-O-H  G-S-S-G + 2 H2O
2 G-SH + R-O-O-H  G-S-S-G + R-OH + H2O
SOD, catalase, and glutathione peroxidase are three primary enzymes,
involved in direct elimination of active oxygen species (hydroxyl radical,
superoxide radical, hydrogen peroxide)

whereas glutathione reductase, glucose-6-phosphate dehydrogenase, and

cytosolic GST are secondary enzymes, which help in the detoxification of ROS
by decreasing peroxide levels or maintaining a steady supply of metabolic
intermediates like glutathione and NADPH necessary for optimum functioning
of the primary antioxidant enzymes.
 Non Enzymatic Antioxidant
Ascorbic acid

L-ascorbic acid (vitamin C) is an important vitamin in the human diet and is abundant in
plant tissues. Green leaves have the same amount of ascorbate as chlorophyll.

Ascorbate functions as a reductant for many free radicals, thereby minimizing the damage
caused by oxidative stress.

Ascorbate can directly scavenge oxygen free radicals with and without enzyme catalysts and
can indirectly scavenge them by recycling tocopherol to the reduced form.
Antioxidant effects of vitamins C and E on lipid peroxidation (LPO). The LPO chain reaction can be initiated by many
radical species (indicated by R•) and converts LH into LOO•, which attacks another LH generating L •

Ascorbic acid may scavenge the initiating radical species R• and reduce the tocopheroxyl radical, generating the ascorbyl
radical, which can be reduced by glutathione-dependent enzymes.

Key to reaction steps:

1, initiating event;
2, radical propagation reaction;
3, termination of the radical reaction by tocopherol (TocH);
4, dismutation of ascorbyl radicals (Asc•–);
5, reduction of dehydroascorbate (DHAsc) by GSH-dependent dehydroascorbate reductase;
6, GSH peroxidase (GPx);
7, further oxygenation and nonenzymatic cleavage of carbon–carbon bonds yields 4-hydroperoxy-2(E)-nonenal (HPNE);
8, reduction yields 4-hydroxy-2(E)-nonenal (HNE) (Fig. ).
 Glutathione

Glutathione (GSH) is a tripeptide (Glu-Cys-Gly) whose antioxidant function is facilitated by

the sulphydryl group of cysteine (Fig. ).

Fig. structure of glutathione

On oxidation, the sulphur forms a thiyl radical that reacts with a second oxidized
glutathione forming a disulphide bond (GSSG).

GSH is found in most tissues, cells and subcellular compartments of higher plants. GSH
concentration is highest in the chloroplast .

GSH can function as an antioxidant in many ways. It can react chemically with singlet
oxygen, superoxide and hydroxyl radicals and therefore function directly as a free radical
scavenger.

GSH may stabilize membrane structure by removing acyl peroxides formed by lipid
peroxidation reactions (Price et al., 1990). GSH is the reducing agent that recycles ascorbic
acid from its oxidized to its reduced form by the enzyme dehydroascorbate reductase
(Loewus, 1988).
 Overall Glutathione (GSH)
NH2 H O

 tripeptide N
HOOC  N COOH

 γ-glutamylcysteinylglycine O CH2 H
SH
 made in all cells
 reducing agent (-SH)
 reduces H2O2 and ROOH (glutathione peroxidase)
 reduces many ROS
 regenerates -SH groups of proteins and coenzyme A
 regenerates tocopherol and ascorbate
25
 Tocopherol (vitamin E)

 Lipophilic antioxidant of cell membranes and lipoproteins

 Reduces peroxyl radicals of phospholipids to hydroperoxides which are further reduced by GSH,
tocopherol is oxidized to stable radical Toc-O·

PUFA-O-O· + Toc-OH PUFA-O-O-H + Toc-O·

 Toc-O· is partially reduced to Toc-OH by ascorbate or GSH

CH3 CH3
HO O

R R
H3C O H3C O
CH3 CH3
CH3 CH3
Natural vitamins Es are known as α, β, γ, and δ according to the methyl or proton groups
that are bound to their Benzene rings and the most common and biologically active form is
alpha-tocopherol (Fig. 6) (Brigelius-Flohe and Traber,1990) .

The principal reserve of natural vitamin E is vegetable oil where its function is to protect
tissue from oxidative damage.
It is a liposoluble molecule, and, therefore, after dietary intake, vitamin E is not only
absorbed easily from the intestinal lumen but is also dispersed between lipids and proteins
in cell membranes.
Vitamin E molecules can interrupt free radical chain reactions by capturing the free radical.
This imparts to them their antioxidant properties.
The free hydroxyl group on the aromatic ring is responsible for the antioxidant properties.
The hydrogen from this group is donated to the free radical, resulting in a relatively stable
free radical form of vitamin E (Fig. (Sies and Murphy, 1991).

Fig. Antioxidant effect of vitamin E. α-Tocopherol reacts with a lipid hydroperoxyl (LOO•) radical. The resultant
tocopheryl radical is resonance-stabilized and does not react with oxygen (unlike L• radicals) and it can be converted
back to α-tocopherol by ascorbate
Fig. 8. Antioxidant mechanism of tocopherols. LH: Lipid molecule, LOOH: Lipid peroxide, LOO+: Lipid Peroxide radical, a-Toc-
OH: α-Tocopherol, a-Toc-O+: α-Tocopherol radical, GSH: Glutathione, Vitamin C+: Vitamin C radical, GS+: Glutathione radical,
GSSG: Oxidized glutathione, NADPH: Reduced nicotinamide adenine dinucleotide phosphate, NADP+: Oxidized nicotinamide
adenine dinucleotide phosphate
Asada-Halliwell pathway (hydrogen peroxide sacvenging and ascorbic acid regeneration).
 The most widely studied dietary antioxidants are vitamin C, vitamin E, and beta-carotene.
 Vitamin C is considered the most important water-soluble antioxidant in extracellular
fluids, as it is capable of neutralizing ROS in the aqueous phase before lipid peroxidation
is initiated.
 Vitamin E is a major lipid-soluble antioxidant, and is the most effective chain-breaking
antioxidant within the cell membrane where it protects membrane fatty acids from lipid
peroxidation. Beta-carotene and other carotenoids also provide antioxidant protection to
lipid rich tissues.
 fruits and vegetables are major sources of vitamin C and carotenoids
 while whole grains, i.e., cereals and high quality vegetable oils are major sources of
vitamin E.
 Carotenoids

 polyisoprenoid hydrocarbons (tetraterpens)

 eliminate peroxyl radicals

 they can quench singlet oxygen

 food sources: green leafy vegetables, yellow, orange, red vegetables and fruits

 very potent antioxidant is lycopene (tomatoes, more available from cooked tomatoes,
ketchup etc.)

Based on their composition, carotenoids are divided in two classes, carotenes containing only
carbon and hydrogen atoms, and oxocarotenoids (xanthophylls) which carry at least one
oxygen atom.
Most carotenoids contain an extended system of conjugated double bonds, which
is responsible for their antioxidant activity (Fig. )

Fig. Structures of β-Carotene and Lycopene

 References
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 Ahmed M Kabel .2014 Free Radicals and Antioxidants: Role of Enzymes and Nutrition . World Journal of Nutrition and Health, Vol.
2, No. 3, 35-38

 Hossam S. EL-Beltagi, Heba I.Mohamed , 2013. Reactive Oxygen Species, Lipid Peroxidation and Antioxidative Defense Mechanism .
Not Bot Horti Agrobo, 41(1):44-57

 Neeti Mehla, Vinita Sindhi, Deepti Josula, Pooja Bisht, and Shabir H. Wani, 2017. An Introduction to Antioxidants and Their Roles in
Plant Stress Tolerance . Springer Nature Singapore Pte Ltd.

 Mohammed Amr El-Missiry, 2012. Antioxidant Enzyme.

 Antonis Z. Renata M, 2015. Antioxidants Antioxidants in Health and Disease . by Taylor & Francis Group, LLC .

 ZORICA J . ANTIOXIDATIVE DEFENSE MECHANISMS IN THE AGING BRAIN, 2014 . Arch. Biol. Sci., Belgrade, 66 (1), 245-252,
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 Maysa M. Hatata E. Adel Abdel-Aal , 2008. Oxidative Stress and Antioxidant Defense Mechanisms in Response to Cadmium
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