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Department of Pharmacology,
Navodaya Medical College,
Raichur.
Post Graduate Seminar
on
Antioxidants.
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Antioxidants.
Introduction.
The balance between oxidant and antioxidantlevels determines the health status and the risk of
chronic disease. Increased oxidative stress due togeneration of excessive amount of free radicalsderived from oxygen and nitrogen is linked withthe enhanced risk of chronic diseases such as
cardiac diseases, cancer, diabetes, andneurological diseases like Alzheimers diseaseand Parkinsons disease.
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Free Radical:
A free radical is a molecularspecies having an un
paired electron and thus is a highly reactive entity
being unstable.
Electrons in an atom occupy spaces known as
orbit. Each orbit can hold a maximum of 2
electrons spinning in opposite directions as is the
case with most of the biological molecules whichcan be termed as non-radicals.
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On the contrary, a free radical contains one or
more unpaired electrons in their orbit. A free
radical may donate one unpaired electron or
may accept one from another molecule or may
simply combine with a non-radical molecule.
In each case the non-radical is transformed to aradical to set up a chain reaction. The free
radical activity is terminated only when 2 free
radicals meet.
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Types of free radicals:
(A) Superoxide radical(B) Nitric oxide radical
(C) Hydroxyl radical
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(A) Superoxice radical:
It is an oxygen molecule deficient of one electron.
These are derived from inevitable leakage from
mitochondrial electron transport chain reactions.
These are also generated during metabolism of
various drugs. Eg: Paracetamol
Some enzymes catalyse the formation of superoxide
radical.
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(i)Biosynthesis:
(a) Enzymatic Sources:
It includes NADPH Oxidases located on the cellmembrane of polymorphonuclear cells, macrophages,and endothelial cells.
Cytochrome p-450 dependent oxygenases.
The proteolytic conversion of xanthine dehydrogenase
to xanthine oxidase.
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(b) Non-enzymatic source:
Superoxide radical is produced inside the
organelles such as mitochondria. The energy
needed to fuel the biological functions is
produced by mitochondria which is stored inthe form of ATP. The process in which ATP is
produced, called as oxidative phosphorylation,
involves the transport of hydrogen ions acrossthe inner mitochondrial membrane by means
of the electron transport chain.
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In the electron transport chain,electrons are passed through a series of
proteins via oxidation-reduction reactions. The
last destination for an electron along this chainis an oxygen molecule. Normally oxygen is
reduced to produce water , however
superoxide radical is produced when an
electron is directly transferred to oxygen.
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(ii) Mechanism of action:
Bcl-2 proteins present on the surface of mitochondria,detect the damage and activate a class of proteinscalled Bax, which punches holes in mitochondrialmembrane , causing cytochrome C to leak out. This
cytochrome C binds to Apaf-1 , which is free-floatingin cells cytoplasm. Using energy from ATPs , theApaf-1 & cytochrome C bind together to formapoptosomes. The apoptosomes binds and activates
caspase-9 which cleaves the proteins of mitochondrialmembrane causing it breakdown & starts a chainreaction of protein denaturation. If too much damageoccurs to its mitochondria, a cell undergoes apoptosis
or programmed cell death.
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Bcl-2 proteins
Activates
Baxleaks out
Cytochrome C
Binds
Apaf-1
ATP
Apoptosomes
Activates
Caspase-9
Apoptosis
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(B) Nitric oxide radicle: [NO]
It is a simple, unique yet multifunctional molecule andis reported to regulate a wide variety of physiological
functions.
Excess nitric oxide generation and its reactive products
with oxygen free radicles may be highly cytotoxic.
Combination of nitric oxide with superoxide radicleresults in formation of peroxynitrite (OONO) which is
highly toxic.
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(i) Biosynthesis:
NO synthase
L-arginine + O2 NO + citrullineCo-Factors
Isoforms of NO synthase:
(i) Neuronal (Type I) constitutive, present
(ii) Endothelial (Type III) under physiologicalcondition
(iii) Inducible (Type II) Expressed in pathological situations{inflammation, invasion by micro organism}
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(ii) Mechanism of action:
NO + Haem moiety of guanylate cyclase
Activation of guanylate cyclase
Increase in CGMP
Activates protein kinase
Phosphorylation of myosin
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t1/2 of NO is 5 seconds, direct measurement are difficult.
Studies on NOS provides the information about NO activity.
NADPH diaphorase is widely used marker for NOS & it has
been shown that NOS is distributed widely in different tissues.
The constitutive variety of NOS is present in the endothelium,
platelets, renal mesangial cells, osteoblasts/ osteoclasts, in CNS,
GIT, Respiratory tract, adrenal glands etc.
Inducible NOS is generated by macrophages, lymphocytes and
neutrophils during inflammation and immunological reactions.
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(iii) Physiological role:
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(iv) NO and disease:
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(v) NO and therapeutics:
Vasodilators like niroglycerine, isosorbide mononitrate, isosorbidedinitrate, nitroprusside promote vasodilation & inhibit platelet
aggregation by NO production.
Inhibitors of inducible NOS are effective in septic shock byreversing the hypotension.
Glucocorticoids also inhibits inducible NOS & are effective by
offering protection in septic states.
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NO is important for penile erection, so NO donors may be
useful in erectile impotence.
Inhaled NO (10-40 ppm) is useful in treatment of pulmonary
hypertension and Adult respiratory distress syndrome (ADRS). It
causes selective pulmonary vasodilatation with minimal systemic
cardiovascular effects. However higher concentrations(> 50-100ppm) may be toxic to lungs.
NOS type III could be potentially useful in preventing re
stenosis after angioplasty.
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(C) Hydroxyl radical:
It is the most reactive free radical which plays an important role
in tissue damage caused by radiation.
It causes DNA fragmentation and extensive chemical alteration
in purine and pyrimidine bases.
It is also known to induce lipid peroxidation of fatty acid side
chains of membrane phospholipids. Accumulation of lipid
peroxides in a biological membrane disrupts its integrity andfunction. In addition these lipid peroxides can break down to highly
cytotoxic end products.
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Promoters of free radicals:
Several transition metals like iron and copper havevariable oxidation numbers which accordingly can accept
a single electron or donate it to non-radicals. As a result
these metals serves as excellent promoters of free
radicals.
Eg: Iron and Copper ions can convert hydrogen peroxide
into a highly reactive free hydroxyl radical which canaccelerate lipid peroxidation and lead to accumulation of
highly cytotoxic end products.
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Reactive oxygen species(ROS):
Inorganic species like hydrogen peroxide,hypochlorus acid and nascient oxygen are not free
radicals but these are very powerful oxidising agents.
Such highly reactive species which are not free radicals
are called as ROS
Nascient oxygen is a transient atomic state of
oxygen which is highly reactive than oxygen and isimplicated in causation of several oxidative processes
and photosensitisation reactions.
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ROS Formation:
Reactive oxygen species are formed by several different
mechanisms:
The interaction of ionising radiation with biological
molecules.As an unavoidable byproduct of cellular respiration.Some electrons passing "down" the electron transportchain leak away from the main path and go directly to
reduce oxygen molecules to the superoxide anion. Synthesized by dedicated enzymes like NADPHoxidase and myeloperoxidase in phagocytic cells likeneutrophils and macrophages.
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ROS are Essential:
ROS performs important functions in the cell.
Examples:
The cells of the thyroid gland must make hydrogenperoxide in order to attach iodine atoms to
thyroglobulin in the synthesis of thyroxine
Macrophages and neutrophils must generate ROS in
order to kill some types of bacteria that they engulf by
phagocytosis.
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Neutrophils (but not macrophages) also kill off
engulfed pathogens by using the enzyme
myeloperoxidase which catalyzes the reaction of
hydrogen peroxide (made from superoxide anions)with chloride ions to produce the strongly antiseptic
hypochlorite ion.
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Chronic Granulomatous Disease (CGD):
This rare genetic disorder demonstrates theimportance of ROS in protecting us from many typeof bacterial infection. It is caused by a defective genefor one of the subunits of NADPH oxidase.
People with CGD have a difficult time riddingthemselves of bacterial infectionsespecially thosecaused by bacteria (e.g. staphylococci, Salmonella)and fungi (e.g., Aspergillus) that produce catalase to
protect themselves against the hydrogen peroxidegenerated by the macrophages and neutrophils thatengulf them. Often the result is the development of a
persisting nest of infected cellscalled agranuloma.
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The gene for one of the subunits of NADPH most
frequently mutated in CGD is on the X chromosome.
Thus males are principally affected.
In June 2005, two cases of successful gene therapy
for CGD were reported. Blood stem cells from the
patients were removed, and the active gene for theNADPH subunit inserted into them using a retroviral
vector. The transformed cells were returned to the
patients, took up residence in their bone marrow,
proliferated successfully, and improved their
symptoms.
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Evidences for the involvement of free radical indifferent diseases:
(A)Cardivascular diseases:
oxidized LDL-cholesterol
Formation of foam cells
Enhancing platelet adhesion & aggregation
Plaque formation
Thrombosis
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Oxidized LDL-cholesterol
Activates AP-2 & E2F
Vascular smooth muscle proliferation
Plaque formation
Thrombosis
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Free radicals
Endothelial cells
Impairs NOS pathway
Impairing NO production
Decreased coronary artery dilatation
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(B) Neurological diseases:
Parkinson disease:
Normal brain has highest concentration of unsaturatedfatty acids compared to other organs and these are
very susceptible to lipid peroxidation.Autopsy samples of substantia nigra from patients of
PD revealed increased oxidant levels and decreasedantioxidant levels.
Increased levels of free iron were also demonstratedin autopsy samples as well as in brains of living PD
patients by iron mediated contrast MRI.
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Alzheimers diseases:
Homogenates of frontal cortex from the patients ofAD obtained at autopsy revealed 22% higher
production of free radicals & in presence of iron 50%higher production of free radicals.
Increased levels of oxidized proteins are found inblood of AD patients.
Increased neuronal NOS expression in reactiveastrocytes corelated with apoptosis in hippacamalneurons of AD brains.
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Diabetes:
Several complications of diabetes likepolyneuropathy, proliferative retinopathy, cataract and
renal vascular diseases appears to be related to excess
production of free radicals.
Hyperglycemia is the primary factor responsible for
increased oxidative stress.
Use of multiple antioxidants have been shown to
prevent many biological changes induced by diabetes.
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AIDS:
Immune deficiency is the principal contributingfactor in the initiation and progress of AIDS.
Free radicals due to increased oxidative stress causesapoptosis of CD4+ T-lymphocytes resulting in low
CD4+ counts in AIDS patients.
The role of free radicals in AIDS is also supported
by the fact that antioxidant supplementation with vit
E, selenium, or B-carotene produced beneficial effects
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Antioxidants:
An antioxidant is a molecule capable of slowing orpreventing the oxidation of other molecules.
oxidation reaction can produce free radicals, which
starts chain reaction that damages cell. Antioxidants
terminate these chain reactions by removing free
radical intermediates, and inhibit other oxidation
reactions by being oxidized themselves.
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Classification:
(i) Natural antioxidants.Eg: Super oxide dismutase, Alpha tocopherol, Ascorbic acid
(ii) Synthetic antioxidants.
Eg: Ebselen, Xanthine oxidase inhibitors, desferrioxamine.
(iii) Drugs with additional antioxidant action:
Eg: NSAIDs, ACE inhibitors, calcium channel antagonists
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Antioxidants can also be classified as
(i) Hydrophilic Antioxidants:In general these antioxidants reacts with oxidants in the cell
cytosol and the blood plasma.
Eg: Ascorbic acid, Glutathione, lipoic acid,
(ii) Hydrophobic Antioxidants:
These antioxidants protects cell membranes from lipid
peroxidation.
Eg: Carotenes, alpha tocopherol, Ubiquinol.
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1) Superoxide dismutase [SOD]:
These are a class of closely related enzymes that catalyze thebreak down of superoxide radical in to hydrogen peroxide and
oxygen.
It contains metal ion co factors depending on which it has 3different isoforms
a) Copper / Zinc SOD present in cytosol.
b) Manganese SOD present in mitochondria.
c) Copper / Zinc SOD present in extracellular fluid.
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SOD has been shown to afford protection against
ischemic or reperfusion injury seen after myocardial
infarction or cerebral stroke. Maximal oxidative damage
occurs after re flow of blood following ischemia.
The large inflow of oxygen during reperfusion results
in generation superoxide & hydrogen peroxide which is
scavanged by SOD.
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2)Alpha tocopherol: [ Vitamin E ]
Its the most important lipid soluble antioxidant.
Its prolonged deficiency has been implicated in severe neurological damage,
haemolytic syndromes, atherosclerosis, thrombotic vascular diseases and
fibroplasia.Beneficial results have been reported it the above conditions.
It protects cell membranes from lipid peroxidation.
It has been shown to protect against LDL oxidation, raises HDL, lowers totalcholesterol and improves blood pressure.
Sources include Soya bean, mangoes, almonds, nuts and broccoli.
inhibit smooth-muscle cell growth.
inhibit platelet adhesion.
improves endothelial function.
Clin Cardiol 1993;16:I16-18
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CHAOS Study
(Cambridge Heart Antioxidant Study)
A prospective randomized trial of 2,002
patients with prior coronary disease
treated with vitamin E (400-800 IU / day) for3 years
- 77% reduction in nonfatal MI
no change in total mortality
Lancet 1996;347(9004):781-786
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3) Ascorbic acid: [ Vitamin C ]
It protects arteries against oxidative damage.
It neutralizes reactive oxygen species like hydrogen peroxides.
It is important in repair and maintenance of epithelial andconnective tissue.
It also acts as a substrate for antioxidant enzyme, ascorbate
peroxidase.
Sources include many fruits like oranges, vegetables.
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4) Carotene: [ vitamin A ]
contained in yellow and orange vegetables andfruits, and leafy green vegetables, carrots, sweet potatoes,
pumpkin and mangoes.
The Physician Health Study
In a subset of 333 subjects with preexisting coronary disease
beta-carotene was associated with a 44% reduction of
coronary events
Circ 1990;82s:202
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Health effects of antioxidants:
Disease treatment:
The brain is uniquely vulnerable to oxidative injury,
due to its high metabolic rate and elevated levels of
polyunsaturated lipids, the target of lipid peroxidation.
Antioxidants are commonly used as medications to
treat various forms of brain injury. Here, superoxide
dismutase mimetics,sodium thiopental and propofol areused to treat reperfusion injury and traumatic brain
injury,while the experimental drug NXY-059 and
ebselen are being applied in the treatment of stroke.
h d id i i
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These compounds appear to prevent oxidative stress in
neurons and prevent apoptosis and neurological
damage.
Antioxidants are also being investigated as possible
treatments for neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, andamyotrophic lateral sclerosis.
Di i
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Disease prevention:
Antioxidants can cancel out the cell-damaging
effects of free radicals.
people who eat fruits and vegetables, which are good
sources of antioxidants, have a lower risk of heart
disease and some neurological diseases, and there is
evidence that some types of vegetables, and fruits ingeneral, probably protect against a number of
cancers.These observations suggested that antioxidants
might help prevent these conditions.
There is some evidence that antioxidants might help
prevent diseases such as macular degeneration and
neurodegeneration.
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Many nutraceutical and health food companies now
sell formulations of antioxidants as dietary
supplements and these are widely used inindustrialized countries.
These supplements may include specific antioxidant
chemicals, like resveratrol (from grape seeds orknotweed roots), combinations of antioxidants, like the
"ACES" products that contain beta carotene
(provitamin A), vitamin C, vitamin Eand Selenium, or
herbs that contain antioxidants - such as green tea and
jiaogulan.
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Adverse effects of antioxidants:
Relatively strong reducing acids can have antinutrienteffects by binding to dietary minerals such as iron and
zinc in the gastrointestinal tract and preventing them
from being absorbed. Notable examples are oxalic
acid, tannins and phytic acid, which are high in plant-based diets.
Toxicity associated with high doses of water-solubleantioxidants such as ascorbic acid are less of a
concern, as these compounds can be excreted rapidly
in urine.
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The beta-Carotene and Retinol Efficacy Trial (CARET)
study of lung cancer patients found that smokers givensupplements containing beta-carotene and vitamin A
had increased rates of lung cancer. Subsequent studies
confirmed these adverse effects.
These harmful effects may also be seen in non-
smokers, as a recent meta-analysis including data from
approximately 230,000 patients showed that -carotene, vitamin A or vitamin E supplementation is
associated with increased mortality but saw no
significant effect from vitamin C.
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Uses of antioxidants in technology
A) Food preservativesAntioxidants are used as food additives to help guard
against food deterioration. Exposure to oxygen and
sunlight are the two main factors in the oxidation of
food.
These preservatives include ascorbic acid (AA,E300), propyl gallate (PG, E310), tocopherols
(E306), tertiary butylhydroquinone (TBHQ),butylated hydroxyanisole (BHA, E320) and butylate
hydroxytoluene (BHT, E321).
B) Industrial uses :
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B) Industrial uses:
Antioxidants are frequently added to industrial
products. A common use is as stabilizers in fuels andlubricants to prevent oxidation, and in gasolines to
prevent the polymerization that leads to the formationof engine-fouling residues.
They are widely used to prevent the oxidativedegradation of polymers such as rubbers, plastics andadhesives that causes a loss of strength and flexibilityin these materials.
Antioxidant preservatives are also added to fat-basedcosmetics such as lipstick and moisturizers to preventrancidity.
References:
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References:
1] HL Sharma and KK Sharma, Principles of
Pharmacology, 1stedition.
2] Kamlesh Kohli, Contemporary perspectives on clinical
pharmacotherapeutics, 1st
edition.
3] Pharmacology, Bhattacharya, 2ndedition.
4] Wikipedia. hank you !