ANTIOXIDANTSANTIOXIDANTSin health and diseasesin health and diseases

Dr. Panjwani Center for Molecular Medicine and Drug ResearchInternational Center for Chemical and Biological Sciences

University of Karachi, Karachi-75270

MUHAMMAD IQBAL CHOUDHARYMUHAMMAD IQBAL CHOUDHARY

Oxidation and Human Oxidation and Human HealthHealth

One of the paradoxes of life on this One of the paradoxes of life on this planet is that the molecule that planet is that the molecule that sustain aerobic life, oxygen, is not sustain aerobic life, oxygen, is not only fundamentally essential for only fundamentally essential for energy metabolism and respiration, energy metabolism and respiration, but implicated in many diseases but implicated in many diseases and degenerative conditions.and degenerative conditions.

Marx, Marx, ScienceScience, , 235235, 529-531 , 529-531 (1985). (1985).

Learning Learning ObjectivesObjectives

Understanding the relationship Understanding the relationship between oxidative stress, and between oxidative stress, and

health and diseases…health and diseases…

• What are oxidants or ROS, types, sources, and What are oxidants or ROS, types, sources, and activities?activities?

• Their role in normal physiological processTheir role in normal physiological process• Their detrimental role in the onset and Their detrimental role in the onset and

progression of diseases, chemical basis of progression of diseases, chemical basis of oxidative damage!oxidative damage!

• Biomarkers of oxidative damage to the vital Biomarkers of oxidative damage to the vital biomolecules and their analysisbiomolecules and their analysis

• What are antioxidants, types, sources and What are antioxidants, types, sources and activities?activities?

• Perceived role of antioxidants in the Perceived role of antioxidants in the preservation of health and prevention of preservation of health and prevention of diseasesdiseases

• Anti-oxidant drug development- challenges and Anti-oxidant drug development- challenges and opportunitiesopportunities

CONTENTCONTENT• What is oxidation?What is oxidation?• Oxidation in biological systemOxidation in biological system• What are free radicals?What are free radicals?• Sources of free radicalsSources of free radicals• Harmful effects of free radicalsHarmful effects of free radicals• Damage to proteins and associated diseasesDamage to proteins and associated diseases• Damage to DNA and associated diseasesDamage to DNA and associated diseases• Damage to lipids and associated diseasesDamage to lipids and associated diseases• Damage to carbohydrates and associated diseasesDamage to carbohydrates and associated diseases• What are antioxidants?What are antioxidants?• Nature’s antioxidants systemNature’s antioxidants system• Dietary sources of antioxidantsDietary sources of antioxidants• Oxidative Stress- Imbalance between oxidation and Oxidative Stress- Imbalance between oxidation and

anti-oxidationanti-oxidation• Types of antioxidantsTypes of antioxidants• Mechanism of anti-oxidation Mechanism of anti-oxidation • Bioassays used to discover new antioxidantsBioassays used to discover new antioxidants

Why This Topic?Why This Topic?

If you search for If you search for Antioxidants…Antioxidants…

• Sci-FinderSci-Finder 305,081 Articles (April 305,081 Articles (April 2013)2013)

• PubmedPubmed 384,341 Hits (April 384,341 Hits (April 2013)2013)

• Google SearchGoogle Search Over 7,000,000Over 7,000,000 Web Web pages (Jan. pages (Jan. 2014)2014)

• Chemical AbstractsChemical Abstracts 131,961 131,961 Publications (2003-2013)Publications (2003-2013)

What is Oxidation?What is Oxidation?

• Combination of substrate with Combination of substrate with oxygen.oxygen.

• Reaction in which the atoms in a Reaction in which the atoms in a compound lose electrons. compound lose electrons.

• Any compound, including oxygen, Any compound, including oxygen, that can accept electrons is an that can accept electrons is an oxidant or oxidizing agent (pro-oxidant or oxidizing agent (pro-oxidant), while a substance that oxidant), while a substance that donates electrons is a reductant donates electrons is a reductant or reducing agent (antioxidant).or reducing agent (antioxidant).

Oxidation in Biological Oxidation in Biological SystemSystem

• We live in an aerobic environmentWe live in an aerobic environment• Oxygen is the life sustaining Oxygen is the life sustaining

elementelement• We consume approximately 3.5 We consume approximately 3.5

kilograms of oxygen every day kilograms of oxygen every day • 2.8 percent of the oxygen is not 2.8 percent of the oxygen is not

properly used and forms properly used and forms free free radicalsradicals

• Several kilograms of peroxides Several kilograms of peroxides (harmful oxidized lipids) are (harmful oxidized lipids) are produced in our body every year produced in our body every year

What are Free What are Free Radicals?Radicals?

• Free radicals (pro-oxidants) are any Free radicals (pro-oxidants) are any chemical species, capable of chemical species, capable of independent (although extremely independent (although extremely short) existence with one or more short) existence with one or more unpaired electronsunpaired electrons

• Highly unstable and reactiveHighly unstable and reactive• Looking for electrons from other Looking for electrons from other

sources to stabilize themselves. In this sources to stabilize themselves. In this process they initiate a chain reaction process they initiate a chain reaction of oxidationof oxidation

• Most commons are Most commons are Reactive Oxygen Reactive Oxygen SpeciesSpecies (ROS) (ROS)

• Reactive Nitrogen Species (RNS) (NOReactive Nitrogen Species (RNS) (NO.., , ONOOONOO--, etc) or Reactive Sulfur Species , etc) or Reactive Sulfur Species (RSS)(RSS)

What are Reactive Oxygen What are Reactive Oxygen Species (ROS)?Species (ROS)?

• ROS are: ROS are: a.a. oxygen derived radicals (Ooxygen derived radicals (O22

.-.-, , ..OH, OH, ROOROO.., , 11OO22, RO, RO..) )

b.b. oxygen-derived non radical species oxygen-derived non radical species (O(O22, H, H22OO22, O, O33, ROOH, HOCl), ROOH, HOCl)

• They are now considered as major They are now considered as major players in biochemical reactions, players in biochemical reactions, cellular response, and clinical cellular response, and clinical outcomeoutcome

Triplet Oxygen(ground state)

Singlet oxygen

Superoxide

Perhydroxyl Radical

Hydrogen Peroxide

Hydroxyl Radical

Hydroxyl Ion

Water

O O

O O

O O H

O OH H

O O

OH

OH

OH H

O O :

O O

O O : O O:

H : O-O : H

H : O + H : O : H

H : O : H

+22 +7.6

-21.7

-88

-53.7

FREE ENERGYKcal / mol

Oxidant Description

•O2-, superoxide anion

One-electron reduced state of O2, formed in many autoxidation reactions and by the electron transport chain. Rather unreactive but can release Fe2+ from iron-sulfur proteins and ferritin. Undergoes dismutation to form H2O2 spontaneously or by enzymatic catalysis and is a precursor for metal-catalyzed •OH formation.

H2O2, hydrogen peroxide

Two-electron reduction state, formed by dismutation of •O2- or by direct reduction of O2. Lipid soluble and thus able to diffuse across membranes.

•OH, hydroxyl radical

Three-electron reduction state, formed by Fenton reaction and decomposition of peroxynitrite. Extremely reactive, will attack most cellular components

Common Free RadicalsCommon Free Radicals

Oxidant Description

ROOH, organic hydroperoxide

Formed by radical reactions with cellular components such as lipids and nucleobases.

RO•, alkoxy and ROO•, peroxy radicals

Oxygen centered organic radicals. Lipid forms participate in lipid peroxidation reactions. Produced in the presence of oxygen by radical addition to double bonds or hydrogen abstraction.

ONOO-, peroxynitrite

Formed in a rapid reaction between •O2- and NO•. Lipid soluble and similar in reactivity to hypochlorous acid. Protonation forms peroxynitrous acid, which can undergo homolytic cleavage to form hydroxyl radical and nitrogen dioxide. Source= Wikipedia

Common Free RadicalsCommon Free Radicals

What Free Radical What Free Radical does?does?

Free radicals are cellular renegades; Free radicals are cellular renegades; they wreak havoc by damaging DNA, they wreak havoc by damaging DNA, altering biochemical compounds, altering biochemical compounds, corroding cell membranes and killing corroding cell membranes and killing cells outright. Such molecular mayhem, cells outright. Such molecular mayhem, scientists increasingly believe, plays a scientists increasingly believe, plays a major role in the development of major role in the development of ailments like cancer, heart or lung ailments like cancer, heart or lung diseases and cataracts. Many diseases and cataracts. Many researchers are convinced that the researchers are convinced that the cumulative effects of free radicals also cumulative effects of free radicals also underlie the gradual deterioration that underlie the gradual deterioration that is the hallmark of aging in all is the hallmark of aging in all individuals, healthy as well as sick. individuals, healthy as well as sick.

TIME, April 6, 1992 publicationsTIME, April 6, 1992 publications

Internal Sources of Free Internal Sources of Free RadicalsRadicals

• MitochondriaMitochondria• Phagocytes (macrophages) Phagocytes (macrophages) • Xanthine oxidaseXanthine oxidase• Reaction involving iron and other Reaction involving iron and other

transition metalstransition metals• Arachidonate pathwaysArachidonate pathways• PeroxisomesPeroxisomes• ExerciseExercise• InflammationInflammation• Ischaemia/ReperfusionIschaemia/Reperfusion

External Sources of Free External Sources of Free RadicalsRadicals

• Cigarette smokeCigarette smoke• Environmental pollutantsEnvironmental pollutants• RadiationsRadiations• Ultraviolet radiationsUltraviolet radiations• OzoneOzone• Certain drugs, pesticides, Certain drugs, pesticides,

anesthetics and industrial anesthetics and industrial solventssolvents

Useful Functions of Free Useful Functions of Free RadicalsRadicals

• Necessary in the maturation processes Necessary in the maturation processes of cellular structuresof cellular structures

• Necessary in the antibacterial activity- Necessary in the antibacterial activity- White blood cells (phagocytes) White blood cells (phagocytes) releases free radicals to destroy releases free radicals to destroy invading pathogenic microbes as part invading pathogenic microbes as part of the body’s defense mechanism of the body’s defense mechanism

• Necessary in the immune systemNecessary in the immune system• Necessary in the prostaglandin Necessary in the prostaglandin

biosynthesisbiosynthesis• Some of them play an important role Some of them play an important role

in cell signalingin cell signaling

History: History: Harmful Harmful Effect of Free RadicalsEffect of Free Radicals

• 1775 Priestly- 1775 Priestly- Toxicity of oxygen Toxicity of oxygen to the organism similar to to the organism similar to burning of candleburning of candle

• 1954 Gilbert and Gersham- 1954 Gilbert and Gersham- Free Free radicals are important player in radicals are important player in biological environment and biological environment and responsible for deleterious responsible for deleterious process in the cellprocess in the cell

• 1969 Mc Cord and Fridovich-1969 Mc Cord and Fridovich- Superoxide theory of toxicitySuperoxide theory of toxicity

Harmful Effect of Harmful Effect of Free Radicals- Free Radicals-

PerceptionPerception• Free radicals can damage all Free radicals can damage all

cellular macromolecules cellular macromolecules including proteins, including proteins, carbohydrates, lipids and nucleic carbohydrates, lipids and nucleic acidsacids

• Destructive effect play a role in Destructive effect play a role in the onset and progression of the onset and progression of different diseases and in normal different diseases and in normal aging.aging.

Oxidative Damage to Oxidative Damage to OrgansOrgans

Ageing is one of the major Ageing is one of the major consequences of oxidative consequences of oxidative

damagedamage

1956 Denham- Free Radical Theory of Ageing

Human Ailments Human Ailments Associated Associated with Oxidative with Oxidative DamageDamage

NeurologicalNeurological

AlzheimerAlzheimer’’s Diseases Disease

Parkinson‘s DiseaseParkinson‘s Disease

EndocrineEndocrine

DiabetesDiabetes

GastrointestinalGastrointestinal

Acute PancreatitisAcute Pancreatitis

Others Others CConditionsonditions

ObesityObesity

Loss of catalytic functions of Loss of catalytic functions of proteinsproteins

ToxicityToxicity

Chronic Chronic InflammationInflammation and arthritis and arthritis

Human Ailments Human Ailments Associated Associated with Oxidative with Oxidative DamageDamage

Diseases Related to Oxidative Diseases Related to Oxidative DamageDamage

Exogenous Exogenous v/sv/s Endogenous Sources Endogenous Sources

of Free Radicalsof Free Radicals• Exogenous ROS are extremely highExogenous ROS are extremely high• Exposure to endogenous oxidants is Exposure to endogenous oxidants is

much more important and extensive, much more important and extensive, because it is a continued process because it is a continued process during the entire life spanduring the entire life span

• Mitochondria play an extremely Mitochondria play an extremely important role in endogenous ROS important role in endogenous ROS production production

• Presence of metals (iron, copper, Presence of metals (iron, copper, chromium, cobalt, vanadium) in un-chromium, cobalt, vanadium) in un-complexed form significantly increase complexed form significantly increase the level of oxidative stress.the level of oxidative stress.

Damage to LipidsDamage to Lipids• Lipids are highly prone to get Lipids are highly prone to get

oxidized.oxidized.• Polyunsaturated fatty acid Polyunsaturated fatty acid

(PUFA)- major part of the low-(PUFA)- major part of the low-density lipoprotein (LDL) in density lipoprotein (LDL) in blood.blood.

Damage to LipidsDamage to Lipids• Lipid peroxidation, if not terminated Lipid peroxidation, if not terminated

rapidly, can cause damage to cell rapidly, can cause damage to cell membranes.membranes.

• Removal of lipid peroxides is essential for Removal of lipid peroxides is essential for mammalian life (Glutathione peroxidase mammalian life (Glutathione peroxidase IV knock-out mouse doesn’t survive IV knock-out mouse doesn’t survive beyond embryonic state).beyond embryonic state).

• Malondialdehyde (MDA) is an important Malondialdehyde (MDA) is an important biomarker of oxidative stress. It reacts biomarker of oxidative stress. It reacts with DNA bases to for DNA-adduct with DNA bases to for DNA-adduct

End Products of Lipid End Products of Lipid PeroxidationPeroxidation

These end products are the markers for lipid peroxidation determination. For example malondialdehyde (MDA) is detected in TBARS (Thiobarb-ituric Acid Reactive Substance) assay, specific for lipid peroxidation determination.

13-HPODE= 13- Hydroperxy-9Z,11E-octdecdienoic acid

4-HNE= Hydroxynonenal

Damage to Lipids-Damage to Lipids-Associated DiseasesAssociated Diseases

• Alterations in the structures of lipid Alterations in the structures of lipid molecules lead to change in their molecules lead to change in their physical properties, such as physical properties, such as permeability, surface adhesion, etc.permeability, surface adhesion, etc.

• It cause damage to the cell membrane, It cause damage to the cell membrane, made up of mainly lipids.made up of mainly lipids.

• Risk of cardiovascular diseases (CVD), Risk of cardiovascular diseases (CVD),

including atherosclerosis. including atherosclerosis.

Damage to Lipids-Damage to Lipids-Associated DiseasesAssociated Diseases

• End products of lipid peroxidation can End products of lipid peroxidation can also cause mutagenesis and also cause mutagenesis and carcinogenesis. For example carcinogenesis. For example malondialdehyde reacts with malondialdehyde reacts with deoxyadenosine and deoxyguanosine deoxyadenosine and deoxyguanosine in DNA to form a variety of DNA in DNA to form a variety of DNA adducts.adducts.

• Body has evolved a range of molecules, Body has evolved a range of molecules, such as Vitamin E, and enzymes such such as Vitamin E, and enzymes such as SOD, catalase, and peroxidase to as SOD, catalase, and peroxidase to control lipid peroxidation. control lipid peroxidation.

• Knockout animals (which can not Knockout animals (which can not produce anti-oxidant enzymes), produce anti-oxidant enzymes), generally do not survive.generally do not survive.

Atherosclerosis and Atherosclerosis and Oxidative Stress Oxidative Stress

• Compelling evidence points oxidative stress as Compelling evidence points oxidative stress as an important trigger in the complex chain of an important trigger in the complex chain of events leading to atherosclerosis.events leading to atherosclerosis.

• It involves accumulation of macrophages in the It involves accumulation of macrophages in the arterial wall. Which then promptly incorporate arterial wall. Which then promptly incorporate oxidized LDL to form foam cells.oxidized LDL to form foam cells.

• ROS can lead to platelet activation and ROS can lead to platelet activation and thrombosis formation.thrombosis formation.

• Probucol has shown reduced progression of Probucol has shown reduced progression of carotid atherosclerosis in clinical trials.carotid atherosclerosis in clinical trials.

Oxidative Damage to Oxidative Damage to ProteinsProteins

• ..OH and ROOH and RO.. and cause damage to and cause damage to proteinsproteins

• Direct damage include Direct damage include peroxidation, damage to specific peroxidation, damage to specific amino acid residues, change in amino acid residues, change in tertiary structures, degradation tertiary structures, degradation and fragmentationand fragmentation

• No efficient mechanism of repair No efficient mechanism of repair of protein damage existsof protein damage exists

• Proteolytic enzymes play an Proteolytic enzymes play an important role in the removal of important role in the removal of damaged proteinsdamaged proteins

Protein Oxidation Protein Oxidation ProductsProducts

• Aldehydes, keto and other carbonyl Aldehydes, keto and other carbonyl compoundscompounds

• 3-Nitrotyrosine, produced by 3-Nitrotyrosine, produced by interaction of tyrosine and ONOOinteraction of tyrosine and ONOO--, is a , is a useful biomarker of oxidative protein useful biomarker of oxidative protein damagedamage

• OrthoOrtho- and - and metameta-tyrosines from -tyrosines from phenylalanine.phenylalanine.

• Other damaged products include Other damaged products include hydroxyproline, glutamyl hydroxyproline, glutamyl semialdehyde, etcsemialdehyde, etc

• Crossed linked proteinsCrossed linked proteins

Damage to Proteins- Damage to Proteins- Associated ProblemsAssociated Problems

• Modified oxidized proteins are susceptible Modified oxidized proteins are susceptible to many changes in their functionsto many changes in their functions

• This include chemical fragmentation, This include chemical fragmentation, inactivation and increased proteolytic inactivation and increased proteolytic degradationdegradation

• Oxidative changes in the structures of Oxidative changes in the structures of catalytic proteins lead to loss of enzyme catalytic proteins lead to loss of enzyme activityactivity

• Altered cellular functions such as energy Altered cellular functions such as energy production, interference with the creation production, interference with the creation of membrane potential and change in the of membrane potential and change in the type and level of cellular proteinstype and level of cellular proteins

• Non- enzymatic glycation of proteins lead Non- enzymatic glycation of proteins lead to multiple poteopathic disordersto multiple poteopathic disorders

• Serum protein carbonyl concentration is Serum protein carbonyl concentration is directly related to muscle dysfunctions.directly related to muscle dysfunctions.

Mechanism of Glycation of Protein- Role of ROS

Catalyzed by transition metals (M) and the superoxide radical generated are converted to the hydroxyl radical via the Fenton reaction.

M nO2

M (n -1)M nO2 M (n -1)O2

OH H2O2

Glucose + Protein Schiff base

Amadori product

AdvancedGlycationEndproducts

Protein Enediol

Protein dicabonyl

Oxidative Damage to Oxidative Damage to DNADNA

• DNA is stable, well protected DNA is stable, well protected moleculesmolecules

• ROS, specially ROS, specially ..OH, can interact OH, can interact with it and cause several types of with it and cause several types of damagedamage

• Including modification of DNA Including modification of DNA bases, single- and double helical bases, single- and double helical breaks, loss of purines, damage breaks, loss of purines, damage to deoxyribose sugar, DNA to deoxyribose sugar, DNA protein cross linkage and DNA protein cross linkage and DNA repair systemrepair system

• Out of four bases, guanine is the Out of four bases, guanine is the most easily oxidizable nucleic most easily oxidizable nucleic acid base. acid base.

Oxidative Damage to Oxidative Damage to DNADNA

•

Oxidative Damage to Oxidative Damage to DNADNA

• Oxidative products of guanosine Oxidative products of guanosine serve as biomarkers of damage to serve as biomarkers of damage to DNA molecule. DNA molecule.

Oxidative Damage to Oxidative Damage to DNADNA

• ROS in the cells lead to DNA damage, ROS in the cells lead to DNA damage, cause stable DNA lesions which are cause stable DNA lesions which are mutagenic, if un-repairedmutagenic, if un-repaired

• Damaged DNA provide the wrong Damaged DNA provide the wrong genetic code leading to unregulated genetic code leading to unregulated protein synthesis and/or cell growth protein synthesis and/or cell growth which results in cancer.which results in cancer.

• Presence of 8-oxo-2-deoxyguanosine Presence of 8-oxo-2-deoxyguanosine (oxo8dG) in DNA is an important (oxo8dG) in DNA is an important indicator of oxidative damage to DNAindicator of oxidative damage to DNA

• Oxidative damage to DNA accumulate Oxidative damage to DNA accumulate with ageing, increasing the with ageing, increasing the possibilities of cancers and other possibilities of cancers and other disordersdisorders

Damage to DNA- Damage to DNA- Associated Associated ProblemsProblems

• Number of oxidative hits to DNA per Number of oxidative hits to DNA per cell per day is about 100,000 in the rat cell per day is about 100,000 in the rat and about 10,000 in the human and about 10,000 in the human (Reason????)(Reason????)

• There is an inherent mechanism There is an inherent mechanism (specific repair glycosylases, etc.) to (specific repair glycosylases, etc.) to repair most of the DNA damage repair most of the DNA damage caused by ROScaused by ROS

• Oxidative lesions in DNA accumulate Oxidative lesions in DNA accumulate with age and eventually lead to with age and eventually lead to serious health challenges (well serious health challenges (well established relationship between onset established relationship between onset of cancers and age) of cancers and age)

Oxidative Oxidative SStress tress MMarkersarkers

Oxidative stress Oxidative stress end end products products detectiondetection

Lipoperoxidation markers:Lipoperoxidation markers:

malondialdehyde (MDA), conjugated malondialdehyde (MDA), conjugated diendienees,s, isoprostanesisoprostanes

Oxidative damage to protein markers :Oxidative damage to protein markers :

protein hydroperoxidesprotein hydroperoxides

Oxidative damage to DNA :Oxidative damage to DNA :

modified nucleosidesmodified nucleosides

Potentialities of Potentialities of oxidative/nitrosative stress-oxidative/nitrosative stress-

related biomarkersrelated biomarkers

Acta Medica Okayama, 61 (4), 181-189, 2007

What are What are Antioxidants?Antioxidants?

• Antioxidants (reductants or Antioxidants (reductants or reducing agents) are compounds reducing agents) are compounds capable of preventing the pro-capable of preventing the pro-oxidation process or biological oxidation process or biological oxidative damage by scavenging oxidative damage by scavenging or stabilizing reactive oxidative or stabilizing reactive oxidative species.species.

An antioxidant is a molecule stable An antioxidant is a molecule stable enough to donate an electron to a enough to donate an electron to a rampaging free radical and neutralize rampaging free radical and neutralize it, thus reducing its capacity to it, thus reducing its capacity to damage.damage.

What are What are Antioxidants?Antioxidants?

• Antioxdiants produced during normal Antioxdiants produced during normal metabolism include glutathione, metabolism include glutathione, ubiquinol and uric acidubiquinol and uric acid

• Antioxidant enzymes include Antioxidant enzymes include glutathione peroxidases, superoxide glutathione peroxidases, superoxide dismutases and catalasedismutases and catalase

• Antioxidants from dietary sources Antioxidants from dietary sources such as Vitamins E and C and such as Vitamins E and C and carotenoidscarotenoids

• Antioxidants from non-dietary sources Antioxidants from non-dietary sources include phenolic or polyphenolic include phenolic or polyphenolic compounds as well as seleniumcompounds as well as selenium

What Antioxidant Do???What Antioxidant Do???

They inhibit the conversion of nitrites to

nitrosamines (which are tumor

promoters) and enhance the immune

response. Vitamins E, and C, ubiquinones, etc.

remove free radicals from the epidermis

of the skin and counteract their

potentially damaging effect. They terminate free radical- induced

cellular

damage and functional degeneration

(aging). They trap and neutralize free radicals

and protect our body tissues from

environmental pollutants.

WHY ARE ANTIOXIDANTS IMPORTANT ?

Sources of Sources of AntioxidantsAntioxidants

• More than 4,000 antioxidants are More than 4,000 antioxidants are knownknown

• Endogenous- AntioxidantEndogenous- Antioxidant enzymes include glutatione enzymes include glutatione peroxidases, superoxide peroxidases, superoxide dismutases and catalasedismutases and catalase

• Antioxidants from dietary Antioxidants from dietary sources,sources, such as Vitamin E, such as Vitamin E, Vitamin C and carotenoidsVitamin C and carotenoids

• Antioxidants from non-dietary Antioxidants from non-dietary sourcessources include phenolic or include phenolic or polyphenolic compoundspolyphenolic compounds

Antioxidant Antioxidant EnzymesEnzymes

• Glutathione peroxidases (Seleno Glutathione peroxidases (Seleno proteins) catalyze the reduction of proteins) catalyze the reduction of lipid hydroperoxides to their lipid hydroperoxides to their corresponding alcohols corresponding alcohols

• Superoxide dismutases, a family of Superoxide dismutases, a family of metal-containing enzymes (Mn, Fe, Zn, metal-containing enzymes (Mn, Fe, Zn, Cu), catalyze the dismutation of Cu), catalyze the dismutation of superoxide into oxygen and hydrogen superoxide into oxygen and hydrogen peroxide peroxide

• Catalases catalyze the decomposition Catalases catalyze the decomposition of hydrogen peroxide to water and of hydrogen peroxide to water and oxygenoxygen

Major Major Antioxidants- Antioxidants-

Vitamin EVitamin E• Vitamin E, fat-soluble vitamin Vitamin E, fat-soluble vitamin

which exists in eight different which exists in eight different forms. forms. -Tocopherol is the most -Tocopherol is the most active form in humans. active form in humans.

• Vitamin E protect cells through Vitamin E protect cells through its ability to limit production of its ability to limit production of free radicalsfree radicals

• Dietary sources include wheat, Dietary sources include wheat, almonds, sunflower seeds, etc.almonds, sunflower seeds, etc.

Major Antioxidants- Major Antioxidants- Vitamin CVitamin C

• Vitamin C, or L-ascorbate is an Vitamin C, or L-ascorbate is an essential nutrient, water-soluble essential nutrient, water-soluble

• Vitamin C scavenge aqueous Vitamin C scavenge aqueous peroxyl radicalsperoxyl radicals

Major Antioxidants- Major Antioxidants- CarotenoidsCarotenoids

• Organic pigments naturally occur in Organic pigments naturally occur in plantsplants

• 600 known carotenoids, and 600 known carotenoids, and tetraterpenoids exist in naturetetraterpenoids exist in nature

• Most common are lycopene and vitamin A Most common are lycopene and vitamin A precursor precursor -carotene-carotene

• They quench singlet oxygen primarily by a They quench singlet oxygen primarily by a physical mechanism in which the excess physical mechanism in which the excess energy of the singlet oxygen is transferred energy of the singlet oxygen is transferred to the carotenoids electron rich structureto the carotenoids electron rich structure

Lycopene

Major Antioxidants- Major Antioxidants- Plant PhenolicsPlant Phenolics

• A large number of plant A large number of plant phenolics, such as flavanoids, phenolics, such as flavanoids, and catechins act as free radical and catechins act as free radical scavengersscavengers

• Tea is the richest source of plant Tea is the richest source of plant phenolicsphenolics

• French paradox – Flavanoids in French paradox – Flavanoids in red winered wine

Major Antioxidants- Major Antioxidants- Plant PhenolicsPlant Phenolics

ApigeninApigenin

Apigenin Apigenin

CatechinCatechin

Antioxidants at a glanceAntioxidants at a glance

NutrientNutrient RDIRDI Dietary SourcesDietary Sources EvidenceEvidence

Vitamin EVitamin E 30 IU30 IU Vegetable oils (soy, corn, olive,Vegetable oils (soy, corn, olive, 100-800 IU 100-800 IU maymay lower lowercotton-seed, safflower, sunflower),cotton-seed, safflower, sunflower), heart disease risk byheart disease risk bynuts, sunflower seed, wheat germnuts, sunflower seed, wheat germ 30%-40%30%-40%

Vitamin CVitamin C 60 mg60 mg Citrus, strawberries, tomatoes,Citrus, strawberries, tomatoes, no evidence no evidence that RDI that RDI cantaloupe, broccoli, asparagus,cantaloupe, broccoli, asparagus,

supplement form cansupplement form canpeppers, spinach, potatoespeppers, spinach, potatoes prevent CHD or prevent CHD or

cancercancer

ß-Caroteneß-Carotene NANA Dark green, yellow, and orangeDark green, yellow, and orange may protect againstmay protect againstvegetables: spinach, collard greenvegetables: spinach, collard green CHD and macularCHD and macularbroccoli, carrots, peppers, sweetbroccoli, carrots, peppers, sweet degenerationdegenerationpotatoes; yellow fruits: peachespotatoes; yellow fruits: peaches

SeleniumSelenium 70 ug70 ug Egg yolks, tuna, seafood, chicken,Egg yolks, tuna, seafood, chicken, 150-200 ug 150-200 ug maymay lowerlower

55 ug55 ug liver, whole grains, plant grown inliver, whole grains, plant grown in prostate cancer riskprostate cancer riskselenium-rich soil.selenium-rich soil.

• Balance between pro-oxidants and oxidants is tightly

regulated and extremely important for maintaining

vital cellular and biological functions

• The objective of the antioxidant therapy is not to

eliminate all the ROS, but to strike a healthy balance.

Balance Between Oxidation and Antioxidation- Key to

Health

Oxidative and reductive stress

Oxidative stress Reductive stress

Oxidant Reductant

ReductantOxidant

What is Oxidative Stress?

• If there are too many free radicals produced and too few anti-oxidants, a condition of “oxidative stress” develop which may cause chronic diseases.

• An imbalance between the production of various reactive species and the ability of the organism’s natural protective mechanism to cope with these reactive compounds and prevent adverse effects.

• It has been proven to be related to degenerative diseases such as cancers, diabetes, premature ageing, Alzheimer’s disease, arthritis, etc.

• Difficult to measure.

Oxidative Oxidative SStress tress MMarkersarkers

Free radical detectionFree radical detection• VVery difficultery difficult to analyze to analyze because of because of

chemchemical and ical and physphysicalical ppropertiesroperties e.g. short e.g. short half lifehalf life

Oxidative stress Oxidative stress end end products detectionproducts detection (foot print measurement)(foot print measurement)

• more simple, a wide range of techniquesmore simple, a wide range of techniques availableavailable

Some Common In Vitro Antioxidant Assays

• NO· ( Nitric Oxide) Radical Inhibition• Assay for DPPH Radical Scavenging Activity• Assay for xanthine oxidase inhibition Activity• Assay for superoxide anion scavenging Activity

a. Enzymatic generation of superoxide anion (through xanthine oxidase)

b. Non-Enzymatic generation of superoxide anion (through NADH/Phenazine methosulphate system)

Some Common In Vivo Antioxidant Assays

• In vivo CCl4 (Carbon tetrachloride) hepatoxicity assay.

• Antioxidant testing kits are now available to detects free radical activity in body.

DILEMA OF REDOX IN LIVING SYSTEM

• Difficult to quantify the oxidizing stress• Difficult to extrapolate in vitro and in

vivo situations• Bioavailability is an issue• Interaction with other molecules• Whether antioxidants do help in the

prevention of diseases?• More questions than answers!!!!

Antioxidant Drug Antioxidant Drug Development- Challenges Development- Challenges

and Opportunitiesand Opportunities Clinical trials nightmareClinical trials nightmare Non-conclusive resultsNon-conclusive results Requires new type of clinical studies Requires new type of clinical studies

than conventional double blind than conventional double blind placebo trials.placebo trials.

Preventive v/s therapeutic Preventive v/s therapeutic Raise questions to old believes? Raise questions to old believes?

THANK YOU VERY MUCH