14] antioxidants

8/12/2019 14] Antioxidants

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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 !

14] antioxidants

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