ANTIOXIDANTS in health and diseases

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ANTIOXIDANTSin health and diseases
MUHAMMAD IQBAL CHOUDHARY
Dr. Panjwani Center for Molecular Medicine and
Drug Research International Center for Chemical
and Biological Sciences University of Karachi,
Karachi-75270
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Oxidation and Human Health

• One of the paradoxes of life on this planet is
  that the molecule that sustain aerobic life,
  oxygen, is not only fundamentally essential for
  energy metabolism and respiration, but implicated
  in many diseases and degenerative conditions.
• Marx, Science, 235, 529-531 (1985).

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Learning Objectives

• Understanding the relationship between oxidative
  stress, and health and diseases
• What are oxidants or ROS, types, sources, and
  activities?
• Their role in normal physiological process
• Their detrimental role in the onset and
  progression of diseases, chemical basis of
  oxidative damage!
• Biomarkers of oxidative damage to the vital
  biomolecules and their analysis
• What are antioxidants, types, sources and
  activities?
• Perceived role of antioxidants in the
  preservation of health and prevention of diseases
• Anti-oxidant drug development- challenges and
  opportunities

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CONTENT

• What is oxidation?
• Oxidation in biological system
• What are free radicals?
• Sources of free radicals
• Harmful effects of free radicals
• Damage to proteins and associated diseases
• Damage to DNA and associated diseases
• Damage to lipids and associated diseases
• Damage to carbohydrates and associated diseases
• What are antioxidants?
• Natures antioxidants system
• Dietary sources of antioxidants
• Oxidative Stress- Imbalance between oxidation and
  anti-oxidation
• Types of antioxidants
• Mechanism of anti-oxidation
• Bioassays used to discover new antioxidants

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Why This Topic?

• If you search for Antioxidants
• Sci-Finder 305,081 Articles (April 2013)
• Pubmed 384,341 Hits (April 2013)
• Google Search Over 7,000,000 Web pages (Jan.
  2014)
• Chemical Abstracts 131,961 Publications
  (2003-2013)

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What is Oxidation?

• Combination of substrate with oxygen.
• Reaction in which the atoms in a compound lose
  electrons.
• Any compound, including oxygen, that can accept
  electrons is an oxidant or oxidizing agent
  (pro-oxidant), while a substance that donates
  electrons is a reductant or reducing agent
  (antioxidant).

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Oxidation in Biological System

• We live in an aerobic environment
• Oxygen is the life sustaining element
• We consume approximately 3.5 kilograms of oxygen
  every day
• 2.8 percent of the oxygen is not properly used
  and forms free radicals
• Several kilograms of peroxides (harmful oxidized
  lipids) are produced in our body every year

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What are Free Radicals?

• Free radicals (pro-oxidants) are any chemical
  species, capable of independent (although
  extremely short) existence with one or more
  unpaired electrons
• Highly unstable and reactive
• Looking for electrons from other sources to
  stabilize themselves. In this process they
  initiate a chain reaction of oxidation
• Most commons are Reactive Oxygen Species (ROS)
• Reactive Nitrogen Species (RNS) (NO., ONOO-, etc)
  or Reactive Sulfur Species (RSS)

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What are Reactive Oxygen Species (ROS)?

• ROS are
• oxygen derived radicals (O2.-, .OH, ROO., 1O2,
  RO.)
• oxygen-derived non radical species (O2, H2O2, O3,
  ROOH, HOCl)
• They are now considered as major players in
  biochemical reactions, cellular response, and
  clinical outcome

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Common Free Radicals
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
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Common 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
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What Free Radical does?

• Free radicals are cellular renegades they wreak
  havoc by damaging DNA, altering biochemical
  compounds, corroding cell membranes and killing
  cells outright. Such molecular mayhem, scientists
  increasingly believe, plays a major role in the
  development of ailments like cancer, heart or
  lung diseases and cataracts. Many researchers are
  convinced that the cumulative effects of free
  radicals also underlie the gradual deterioration
  that is the hallmark of aging in all individuals,
  healthy as well as sick.
• TIME, April 6, 1992 publications

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Internal Sources of Free Radicals

• Mitochondria
• Phagocytes (macrophages)
• Xanthine oxidase
• Reaction involving iron and other transition
  metals
• Arachidonate pathways
• Peroxisomes
• Exercise
• Inflammation
• Ischaemia/Reperfusion

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External Sources of Free Radicals

• Cigarette smoke
• Environmental pollutants
• Radiations
• Ultraviolet radiations
• Ozone
• Certain drugs, pesticides, anesthetics and
  industrial solvents

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Useful Functions of Free Radicals

• Necessary in the maturation processes of cellular
  structures
• Necessary in the antibacterial activity- White
  blood cells (phagocytes) releases free radicals
  to destroy invading pathogenic microbes as part
  of the bodys defense mechanism
• Necessary in the immune system
• Necessary in the prostaglandin biosynthesis
• Some of them play an important role in cell
  signaling

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History Harmful Effect of Free Radicals

• 1775 Priestly- Toxicity of oxygen to the organism
  similar to burning of candle
• 1954 Gilbert and Gersham- Free radicals are
  important player in biological environment and
  responsible for deleterious process in the cell
• 1969 Mc Cord and Fridovich- Superoxide theory of
  toxicity

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Harmful Effect of Free Radicals- Perception

• Free radicals can damage all cellular
  macromolecules including proteins, carbohydrates,
  lipids and nucleic acids
• Destructive effect play a role in the onset and
  progression of different diseases and in normal
  aging.

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Oxidative Damage to Organs

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Ageing is one of the major consequences of
oxidative damage
1956 Denham- Free Radical Theory of Ageing
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Human Ailments Associated with Oxidative Damage

• Neurological
• Alzheimers Disease
• Parkinsons Disease
• Endocrine
• Diabetes
• Gastrointestinal
• Acute Pancreatitis

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Human Ailments Associated with Oxidative Damage

• Others Conditions
• Obesity
• Loss of catalytic functions of proteins
• Toxicity
• Chronic Inflammation and arthritis

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Diseases Related to Oxidative Damage

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Exogenous v/s Endogenous Sources of Free Radicals

• Exogenous ROS are extremely high
• Exposure to endogenous oxidants is much more
  important and extensive, because it is a
  continued process during the entire life span
• Mitochondria play an extremely important role in
  endogenous ROS production
• Presence of metals (iron, copper, chromium,
  cobalt, vanadium) in un-complexed form
  significantly increase the level of oxidative
  stress.

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Damage to Lipids

• Lipids are highly prone to get oxidized.
• Polyunsaturated fatty acid (PUFA)- major part of
  the low-density lipoprotein (LDL) in blood.

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Damage to Lipids

• Lipid peroxidation, if not terminated rapidly,
  can cause damage to cell membranes.
• Removal of lipid peroxides is essential for
  mammalian life (Glutathione peroxidase IV
  knock-out mouse doesnt survive beyond embryonic
  state).
• Malondialdehyde (MDA) is an important biomarker
  of oxidative stress. It reacts with DNA bases to
  for DNA-adduct

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End Products of Lipid Peroxidation
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-o
ctdecdienoic acid 4-HNE Hydroxynonenal
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Damage to Lipids-Associated Diseases

• Alterations in the structures of lipid molecules
  lead to change in their physical properties, such
  as permeability, surface adhesion, etc.
• It cause damage to the cell membrane, made up of
  mainly lipids.
• Risk of cardiovascular diseases (CVD), including
  atherosclerosis.

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Damage to Lipids-Associated Diseases

• End products of lipid peroxidation can also cause
  mutagenesis and carcinogenesis. For example
  malondialdehyde reacts with deoxyadenosine and
  deoxyguanosine in DNA to form a variety of DNA
  adducts.
• Body has evolved a range of molecules, such as
  Vitamin E, and enzymes such as SOD, catalase, and
  peroxidase to control lipid peroxidation.
• Knockout animals (which can not produce
  anti-oxidant enzymes), generally do not survive.

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Atherosclerosis and Oxidative Stress

• Compelling evidence points oxidative stress as an
  important trigger in the complex chain of events
  leading to atherosclerosis.
• It involves accumulation of macrophages in the
  arterial wall. Which then promptly incorporate
  oxidized LDL to form foam cells.
• ROS can lead to platelet activation and
  thrombosis formation.
• Probucol has shown reduced progression of carotid
  atherosclerosis in clinical trials.

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Oxidative Damage to Proteins

• .OH and RO. and cause damage to proteins
• Direct damage include peroxidation, damage to
  specific amino acid residues, change in tertiary
  structures, degradation and fragmentation
• No efficient mechanism of repair of protein
  damage exists
• Proteolytic enzymes play an important role in the
  removal of damaged proteins

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Protein Oxidation Products

• Aldehydes, keto and other carbonyl compounds
• 3-Nitrotyrosine, produced by interaction of
  tyrosine and ONOO-, is a useful biomarker of
  oxidative protein damage
• Ortho- and meta-tyrosines from phenylalanine.
• Other damaged products include hydroxyproline,
  glutamyl semialdehyde, etc
• Crossed linked proteins

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Damage to Proteins- Associated Problems

• Modified oxidized proteins are susceptible to
  many changes in their functions
• This include chemical fragmentation, inactivation
  and increased proteolytic degradation
• Oxidative changes in the structures of catalytic
  proteins lead to loss of enzyme activity
• Altered cellular functions such as energy
  production, interference with the creation of
  membrane potential and change in the type and
  level of cellular proteins
• Non- enzymatic glycation of proteins lead to
  multiple poteopathic disorders
• Serum protein carbonyl concentration is directly
  related to muscle dysfunctions.

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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.
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Oxidative Damage to DNA

• DNA is stable, well protected molecules
• ROS, specially .OH, can interact with it and
  cause several types of damage
• Including modification of DNA bases, single- and
  double helical breaks, loss of purines, damage to
  deoxyribose sugar, DNA protein cross linkage and
  DNA repair system
• Out of four bases, guanine is the most easily
  oxidizable nucleic acid base.

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Oxidative Damage to DNA

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Oxidative Damage to DNA

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

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Oxidative Damage to DNA

• ROS in the cells lead to DNA damage, cause stable
  DNA lesions which are mutagenic, if un-repaired
• Damaged DNA provide the wrong genetic code
  leading to unregulated protein synthesis and/or
  cell growth which results in cancer.
• Presence of 8-oxo-2-deoxyguanosine (oxo8dG) in
  DNA is an important indicator of oxidative damage
  to DNA
• Oxidative damage to DNA accumulate with ageing,
  increasing the possibilities of cancers and other
  disorders

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Damage to DNA- Associated Problems

• Number of oxidative hits to DNA per cell per day
  is about 100,000 in the rat and about 10,000 in
  the human (Reason????)
• There is an inherent mechanism (specific repair
  glycosylases, etc.) to repair most of the DNA
  damage caused by ROS
• Oxidative lesions in DNA accumulate with age and
  eventually lead to serious health challenges
  (well established relationship between onset of
  cancers and age)

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Oxidative Stress Markers Oxidative stress end
products detection

• Lipoperoxidation markers
• malondialdehyde (MDA), conjugated dienes,
  isoprostanes
• Oxidative damage to protein markers
• protein hydroperoxides
• Oxidative damage to DNA
• modified nucleosides

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Potentialities of oxidative/nitrosative
stress-related biomarkers
Acta Medica Okayama, 61 (4), 181-189, 2007
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What are Antioxidants?

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

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

• Antioxdiants produced during normal metabolism
  include glutathione, ubiquinol and uric acid
• Antioxidant enzymes include glutathione
  peroxidases, superoxide dismutases and catalase
• Antioxidants from dietary sources such as
  Vitamins E and C and carotenoids
• Antioxidants from non-dietary sources include
  phenolic or polyphenolic compounds as well as
  selenium

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What Antioxidant Do???
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WHY ARE ANTIOXIDANTS IMPORTANT ?

• 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.

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Sources of Antioxidants

• More than 4,000 antioxidants are known
• Endogenous- Antioxidant enzymes include
  glutatione peroxidases, superoxide dismutases and
  catalase
• Antioxidants from dietary sources, such as
  Vitamin E, Vitamin C and carotenoids
• Antioxidants from non-dietary sources include
  phenolic or polyphenolic compounds

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Antioxidant Enzymes

• Glutathione peroxidases (Seleno proteins)
  catalyze the reduction of lipid hydroperoxides to
  their corresponding alcohols
• Superoxide dismutases, a family of
  metal-containing enzymes (Mn, Fe, Zn, Cu),
  catalyze the dismutation of superoxide into
  oxygen and hydrogen peroxide
• Catalases catalyze the decomposition of hydrogen
  peroxide to water and oxygen

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Major Antioxidants- Vitamin E

• Vitamin E, fat-soluble vitamin which exists in
  eight different forms. a-Tocopherol is the most
  active form in humans.
• Vitamin E protect cells through its ability to
  limit production of free radicals
• Dietary sources include wheat, almonds, sunflower
  seeds, etc.

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Major Antioxidants- Vitamin C

• Vitamin C, or L-ascorbate is an essential
  nutrient, water-soluble
• Vitamin C scavenge aqueous peroxyl radicals

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Major Antioxidants- Carotenoids

• Organic pigments naturally occur in plants
• 600 known carotenoids, and tetraterpenoids exist
  in nature
• Most common are lycopene and vitamin A precursor
  b-carotene
• They quench singlet oxygen primarily by a
  physical mechanism in which the excess energy of
  the singlet oxygen is transferred to the
  carotenoids electron rich structure

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Lycopene
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Major Antioxidants- Plant Phenolics

• A large number of plant phenolics, such as
  flavanoids, and catechins act as free radical
  scavengers
• Tea is the richest source of plant phenolics
• French paradox Flavanoids in red wine

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Major Antioxidants- Plant Phenolics

• Apigenin

• Apigenin
  Catechin

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Antioxidants at a glance

• Nutrient RDI Dietary Sources Evidence
• Vitamin E 30 IU Vegetable oils (soy, corn,
  olive, 100-800 IU may lower cotton-seed,
  safflower, sunflower), heart disease risk
  by nuts, sunflower seed, wheat germ 30-40
• Vitamin C 60 mg Citrus, strawberries,
  tomatoes, no evidence that RDI
  cantaloupe, broccoli, asparagus, supplement
  form can peppers, spinach, potatoes prevent
  CHD or cancer
• ß-Carotene NA Dark green, yellow, and orange may
  protect against vegetables spinach, collard
  green CHD and macular broccoli, carrots,
  peppers, sweet degeneration potatoes yellow
  fruits peaches
• Selenium 70 ug Egg yolks, tuna, seafood,
  chicken, 150-200 ug may lower
• 55 ug liver, whole grains, plant grown
  in prostate cancer risk selenium-rich soil.

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Balance Between Oxidation and Antioxidation- Key
to Health

• 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.

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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
  organisms 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, Alzheimers disease, arthritis, etc.
• Difficult to measure.

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Oxidative Stress Markers

• Free radical detection
• Very difficult to analyze because of chemical and
  physical properties e.g. short half life
• Oxidative stress end products detection (foot
  print measurement)
• more simple, a wide range of techniques available
  

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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)

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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.

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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!!!!

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Antioxidant Drug Development- Challenges and
Opportunities

• Clinical trials nightmare
• Non-conclusive results
• Requires new type of clinical studies than
  conventional double blind placebo trials.
• Preventive v/s therapeutic
• Raise questions to old believes?

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THANK YOU VERY MUCH