Download - ANTIOXIDANTS in health and diseases
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 248,724 Articles (April 248,724 Articles (April 2012)2012)
• PubmedPubmed 340,354 Hits (April 340,354 Hits (April 2012)2012)
• Google SearchGoogle Search 30,500,00030,500,000 Web Web pages (April 2012)pages (April 2012)
• Chemical AbstractsChemical Abstracts 14,609 Publications 14,609 Publications (2011 alone)(2011 alone)
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• Malondialdehyde (MDA) is a Malondialdehyde (MDA) is a
biomarker of oxidative stress.biomarker of oxidative stress.
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.
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), do not produce anti-oxidant enzymes), do not survive beyond embryonic stage.survive beyond embryonic stage.
Atherosclerosis and Atherosclerosis and Oxidative Stress Oxidative Stress
• Compelling evidence points oxidative stress Compelling evidence points oxidative stress as an important trigger in the complex chain as an important trigger in the complex chain of events leading to atherosclerosis.of events leading to atherosclerosis.
• It involve accumulation of macrophages in It involve accumulation of macrophages in the arterial wall. Which then avidly the arterial wall. Which then avidly incorporate oxidized LDL to form foam cells.incorporate 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 reduced progression of carotid Probucol has reduced progression of carotid atherosclerosis in clinical trials.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 Modified oxidized proteins are susceptible to many changes in their susceptible to many changes in their functionsfunctions
• 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 catalytic proteins lead to loss of enzyme activityenzyme activity
• Altered cellular functions such as Altered cellular functions such as energy production, interference with energy production, interference with the creation of membrane potential the creation of membrane potential and change in the type and level of and change in the type and level of cellular proteinscellular proteins
• Glycation of proteinsGlycation of proteins
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
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 sourcesAntioxidants from dietary sources such as Vitamin E, Vitamin C and such as Vitamin E, Vitamin C and carotenoidscarotenoids
• Antioxidants from non-dietary Antioxidants from non-dietary sourcessources include phenolic or include phenolic or polyphenolic compoundspolyphenolic compounds
Antioxidant Antioxidant EnzymesEnzymes
• Glutathione peroxidases catalyze Glutathione peroxidases catalyze the reduction of lipid the reduction of lipid hydroperoxides to their hydroperoxides to their corresponding alcohols corresponding alcohols
• Superoxide dismutases, a family Superoxide dismutases, a family of metal-containing enzymes of metal-containing enzymes (Mn, Fe, Zn, Cu), catalyze the (Mn, Fe, Zn, Cu), catalyze the dismutation of superoxide into dismutation of superoxide into oxygen and hydrogen peroxide oxygen and hydrogen peroxide
• Catalases catalyze the Catalases catalyze the decomposition of hydrogen decomposition of hydrogen peroxide to water and oxygenperoxide to water and oxygen
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 may lower100-800 IU may 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 may 150-200 ug may 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
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 DifficultDifficult
THANK YOU VERY MUCH