Antioxidants

●​ Definition: Antioxidants are compounds that protect biological systems from harmful
effects of excessive oxidation, as defined by the United States Department of
Agriculture.
●​ Role: Maintain a balance between prooxidants (promote oxygen release for energy) and
antioxidants to prevent cellular damage from free radicals.
●​ Free Radicals: Chemical species with unpaired electrons, making them unstable and
highly reactive. They can:
○​ Lose an electron (oxidized).
○​ Gain an electron (reduced).
●​ Impact of Free Radicals: Cause cumulative tissue injury, contributing to diseases like
cancer, atherosclerosis, radiation damage, and accelerated aging.
●​ Redox Reactions: Most frequent mechanism in biological systems, involving electron
transfer (reduction: electron addition; oxidation: electron removal), producing Reactive
Oxygen Species (ROS) and non-oxygen free radicals.

2. Free Radicals of Oxygen

Types of Free Radicals

●​ Superoxide Radical: Formed when molecular oxygen accepts one electron.

○​ Can accept a proton (H⁺) to form perhydroxy radical (HOO), which is fat-soluble
and highly toxic to lipid-rich cell membranes.
●​ Peroxide and Hydrogen Peroxide:
○​ Superoxide takes another electron to form peroxide, which combines with H⁺ to
form hydrogen peroxide (H₂O₂).
○​ H₂O₂ is more stable, less toxic, but can travel far, causing damage.
○​ Not a free radical but induces free radical formation.
●​ Hydroxyl Radical:
○​ Formed via Fenton’s Reaction in cells rich in transition metals (iron, copper),
splitting H₂O₂ into hydroxyl radical and ion.
○​ Highly toxic but short-lived, unable to diffuse across membranes.
●​ Singlet Oxygen: Molecular oxygen with a lost electron, classified as a ROS.

Reactive Oxygen Species (ROS): Include superoxide, hydrogen peroxide, hydroxyl radicals,
and singlet oxygen.
3. Sources of Free Radicals and ROS

Physiological Sources

●​ Respiration: Small amounts of ROS formed in cells, especially red blood cells (high
oxygen and iron content), and inactivated naturally.
●​ Respiratory Burst:
○​ Triggered by foreign bodies (bacteria, pollutants, tobacco smoke, dust).
○​ Neutrophils engulf and digest foreign matter, producing excessive ROS and
hypochlorite (toxic radical) in mucosal surfaces (GI and respiratory tracts).
○​ Beneficial effect: Free radicals kill bacteria during respiratory burst.

Environmental and Other Factors

●​ Physiological Factors:
○​ Energy metabolism, diabetes, exercise, acute illness, immune response, injury,
obesity, other diseases, trace minerals (iron, copper).
●​ Environmental Factors:
○​ Air pollution, asbestos, high vitamin C levels, high oxygen levels, radioactive
emissions (e.g., radon gas), herbicides, tobacco smoke, ultraviolet light,
xenobiotics (drugs/additives).
●​ Detoxification:
○​ Cyt P450-mediated mixed function oxidases in liver detoxify drugs (antibiotics,
anticancer drugs, analgesics), pesticides, and chemicals (benzene, hexane),
producing large amounts of ROS, damaging liver cells.
●​ Ischemic Conditions: Oxygen inadequacy generates excessive ROS.
●​ Autooxidation: Small molecules (adrenaline, ferredoxin, glucose) produce free radicals
when in excess.
●​ Nitrate Metabolism:
○​ Nitrate in water/foods reduced to nitrite in the stomach’s acidic environment.
○​ Nitrite reacts with protein amines/amides to form N-nitros derivatives, free
radical generators found in cured/smoked meat, fish, milk, and vegetables
(beetroot, celery, spinach).
●​ Tobacco: Contains 400 compounds (e.g., carbon monoxide, cyanide, nitrosamines,
superoxide, nitric oxide) inducing respiratory burst in alveoli.

4. Disease Processes by Free Radicals and ROS

Mechanisms of Damage

●​ Lipid Peroxidation: Free radicals abstract hydrogen from membrane phospholipids (rich
in polyunsaturated fatty acids), forming reactive free radicals. Oxygen adds to form
peroxides, initiating a chain reaction that fragments membranes, affecting structural
integrity and permeability.
 ●​ Protein Damage:
○​ Free radicals modify, oxidize, hydrolyze, fragment, or inactivate proteins in
membranes and cytoplasm.
○​ Oxidized LDL protein mediates cholesterol deposition, leading to heart attack and
stroke.
●​ DNA Damage:
○​ Causes strand breaks, mutations (leading to cancer), and interferes with growth,
differentiation, cell division, and cell death regulation.

Sites and Pathological Changes

●​ DNA:
○​ Damage to nucleotides, thiol-dependent enzymes, protein cross-linking.
○​ Leads to mutations, cancer, metabolic disturbances (inflammation, diabetes,
heart disease, asthma).
●​ Extracellular Components:
○​ Damage to hyaluronic acid, lipid peroxidation of membranes.
○​ Causes joint diseases, allergies, inflammation, edema, degenerative changes,
reduced immunity.

5. Markers of Oxidative Stress

●​ Immunoassay of Oxidized LDL: Specific to cardiovascular disease (CVD) risk, linked

to dietary/supplemental antioxidant consumption.
●​ Isoprostane F₂α:
○​ Measures free radical oxidation of polyunsaturated fatty acids.
○​ Structurally similar to prostaglandins, used to assess oxidative stress in infants
receiving therapeutic oxygen.

6. Antioxidant Defense Systems

●​ Comprises endogenous (synthesized by cells) and exogenous (dietary) antioxidants

protecting cell membranes, lipoproteins, DNA, and RNA.
●​ Exogenous Antioxidants: Include nutrients (ascorbic acid, tocopherols, β-carotene)
and non-nutrients (polyphenols, flavonols).

Primary and Secondary Antioxidants

●​ Primary Antioxidants (first line of defense):

○​ Vitamin E, vitamin C, carotenoids, flavonoids, polyamines, melatonin, estrogen,
ubiquinone, lipoic acid, uric acid, glutathione.
○​ Enzymes: Superoxide dismutase (Cu, Zn, Mn), glutathione peroxidase (Se),
catalase (Fe).
 ●​ Secondary Antioxidants:
○​ Copper, glutathione reductase, ascorbate reductase, glucose-6-phosphate
dehydrogenase, ceruloplasmin, transferrin, metallothionein, albumin, bilirubin,
N-acetylcysteine.

Non-Enzymatic Antioxidants

●​ End Ascorbic Acid (Vitamin C):

○​ Water-soluble, found in cytosol, plasma, and body fluids.
○​ Scavenges ROS (superoxide, hydroxyl, peroxyl radicals, singlet oxygen).
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○​ Inhibits nitroso compound formation from dietary nitrates.
○​ Regenerates vitamin E.
○​ May prevent chronic diseases (cancer, CVD, cataracts).
●​ Vitamin E:
○​ Phenolic antioxidant, donates hydrogen to free radicals, preventing lipid
peroxidation.
○​ Protects β-carotene from autooxidation.
○​ Reduces LDL oxidation, inflammatory cell adhesion, platelet aggregation, and
endothelial dysfunction.
○​ Located in cell/organelle membranes, low concentration (1 molecule per 2000
phospholipids), regenerated by vitamin C.
○​ Ameliorates selenium deficiency and vice versa.
●​ Carotenoids (e.g., β-carotene, lycopene, lutein, zeaxanthin, astaxanthin):
○​ Scavenge singlet oxygen, effective at low oxygen partial pressures.
○​ Tissue-specific accumulation (lycopene in testes, zeaxanthin/lutein in retina
macula).
○​ Complement vitamin E in preventing lipid peroxidation.
●​ Ubiquinone:
○​ Lipophilic quinone, electron carrier in mitochondrial electron transport.
○​ Antioxidant properties in vitro, synthesis decreases with age.
●​ Glutathione (GSH):
○​ Endogenous, contains sulfur amino acids with reducing thiol groups.
○​ Regenerated by glutathione reductase (requires niacin, riboflavin).
●​ Sulfur-Containing Amino Acids: Provide reducing thiol groups for proteins and GSH.

Antioxidant Enzymes

●​ Superoxide Dismutase (SOD): Converts superoxide to hydrogen peroxide (requires

Cu, Zn, Mn).
●​ Catalase: Splits hydrogen peroxide to water and oxygen (requires Fe).
●​ Glutathione Peroxidase (GPx): Detoxifies hydrogen peroxide and lipid hydroperoxides
(requires Se).
 ●​ Phase I and II Metabolism:
○​ Phase I: Mixed Function Oxidases (MFO) detoxify chemicals, producing ROS.
○​ Phase II: Non-enzymatic molecules (glucuronic acid, glutathione, glycine) bind
toxins for excretion, supported by enzymes like glutathione-S-transferase,
NADPH, quinone reductase (require Se, Zn, Cu, Fe).

7. Mechanisms of Combating Free Radicals and ROS

First Line of Defense: Prevention

●​ Mitochondrial Electron Transport: Cytochromes and metalloenzymes reduce oxygen

to water, preventing superoxide release.
●​ Metal Ion Sequestration: Proteins like transferrin and ceruloplasmin bind Fe and Cu to
prevent ROS formation.

Second Line of Defense: Antioxidant Enzymes

●​ Superoxide Dismutase: Converts superoxide to hydrogen peroxide.

○​ Reaction: 2O₂ + 2H⁺ → H₂O₂ + O₂.
●​ Catalase: Converts hydrogen peroxide to water and oxygen.
○​ Reaction: 2H₂O₂ → 2H₂O + O₂.
●​ Glutathione Peroxidase (GPx):
○​ Detoxifies hydrogen peroxide using glutathione (GSH).
○​ GSH exists in reduced (2GSH) and oxidized (GSSG) forms, regenerated by
glutathione reductase (requires niacin, riboflavin).

Third Line of Defense: Free Radical Scavengers

●​ Vitamin E: Inactivates free radicals on cell membranes, regenerated by vitamin C.

●​ Vitamin C and Glutathione: Regenerate vitamin E, reduce oxidized glutathione via
glucose metabolism (requires niacin, riboflavin).

Final Defense: Damage Repair

●​ Phospholipases: Remove lipid peroxidation (LPO) products from membranes, replaced

with fresh fatty acids.
●​ Proteases: Digest damaged proteins.
●​ DNA Repair: Cleaves and repairs damaged DNA, though repair is limited, leading to
diseases.

Antioxidant Mechanisms

●​ Decrease localized oxygen concentrations to reduce oxidation.

●​ Scavenge free radicals to prevent hydrogen abstraction.
●​ Bind transition metals (Cu, Fe) to prevent radical formation.
 ●​ Decompose peroxides to prevent conversion to radicals.
●​ React with peroxyl/alkoxyl radicals to stop chain propagation.

8. Antioxidants and Disease

Inflammation

●​ Chronic Inflammation: Exacerbated by excess serum glucose, lipids, and central fat,
leading to conditions like CVD, arthritis, obesity, Alzheimer’s, immune dysfunction,
diabetes (Figure 24b).
●​ Antioxidant Role: Dietary antioxidants (vitamins C, E, β-carotene, phenolics)
delay/prevent inflammatory conditions (Figure 24c).
●​ Mechanisms:
○​ Phenolics act as free radical scavengers and inhibit
lipoxygenase/cyclooxygenase (Cox-2).
○​ Influence gene expression to control inflammation.

Immunity and Aging

●​ Defenses: Skin, mucous membranes, acidic secretions, T-cell-mediated immunity,

antibodies.
●​ Supporting Nutrients: Vitamin A (β-carotene, carotenoids), vitamins E, C, B₆, folate, Zn,
Se, Cu.
●​ Aging Effects:
○​ NIN studies: 25% decrease in glutathione reductase activity in old age,
increasing degenerative disease risk.
○​ Free radicals cause immune system degeneration, cataracts, atherosclerosis,
arthritis, Parkinson’s.
○​ 200 mg/day vitamin E enhances immune responsiveness in the elderly.
○​ 800 IU vitamin E for 30 days increases interleukin II production.
○​ Vitamin B₆ depletion reduces interleukin II, reversible with repletion.
●​ Resveratrol: Found in groundnut skin, activates proteins to slow aging.

Cataract

●​ Cause: Oxidative stress from UV light and free radicals reduces lens transparency.
●​ Lens Composition: High-density crystalline proteins; high reduced glutathione (GSH)
protects against damage.
●​ Biochemical Change: Insoluble proteins accumulate, causing opacity.
●​ NIN Findings:
○​ Cataract patients have lower riboflavin, Cu, Zn levels; 80% show riboflavin
deficiency.
○​ Low antioxidant enzyme levels (catalase, SOD, GPx).
○​ Low intake of β-carotene, riboflavin, vitamin C, Cu, Zn, Mn.
 ●​ Prevention: High vitamin C, E, carotenoid, and omega-3 fatty acid intake delays
cataract progression.
●​ Eugenol (Cloves): Protects lens biochemically in vitro.
●​ Inositol (Vitamin B Complex): Mitigates cataracts via antioxidative/antiglycating
properties.

Cancer

●​ Mechanism: Free radicals cause DNA damage, leading to mutations and malignancy.
●​ Antioxidants
○​ β-carotene: Reduces risk of lung, stomach, cervix, esophageal, throat cancers.
○​ Vitamin C: Reduces risk of upper GI tract, cervix cancer, CVD.
○​ Vitamin E: Decreases oral/pharyngeal cancer, CVD risk.
○​ Selenium: Reduces esophageal/stomach cancer risk.
○​ Resveratrol: Inhibits prostate tumor formation.
○​ Curry Leaf Extract: Polyphenol-rich, prevents cancer (NIN 2017-18).
○​ Soya Isoflavones: Reduce breast cancer mortality risk.
●​ Therapeutic Inhibitors: Vitamin E, vitamin C, dimethyl thiourea, dimethyl sulfoxide,
mannitol, allopurinol, SOD.

Protein Energy Malnutrition (PEM)

●​ Oedema in Kwashiorkor:
○​ Linked to low plasma Cu, ceruloplasmin, and RBC SOD in marasmic-kwashiorkor
children.
○​ Causes: Recurrent infections, diarrhea, inadequate dietary intake.
○​ Contributes to oedema formation.

Neurological Conditions

●​ Blood-Brain Barrier: Tight microglial junctions protect CNS; permeable in Alzheimer’s,

stress.
●​ Antioxidants:
○​ α-tocopherol: Slows Alzheimer’s progression.
○​ Folate, vitamin C, β-carotene support cognitive ability.
○​ Vitamins B₆, B₁₂, folate maintain memory.
●​ Parkinson’s Disease:
○​ Oxidative stress damages dopaminergic neurons.
○​ Multiple antioxidants at appropriate doses improve L-dopa therapy efficacy.

Cardiovascular Disease (CVD)

●​ Factors: High linoleic acid in LDL and low antioxidant availability increase LDL oxidation.
●​ Antioxidant Enzymes: SOD, catalase, GPx lowered in ischemic heart disease; Se
deficiency implicated.
 ●​ Vitamin E:
○​ Most concentrated antioxidant in LDL (20-300 times higher than others).
○​ Prevents PUFA oxidation in cell membranes.
○​ More effective than β-carotene or vitamin C for CVD.
●​ Phenolic Compounds (e.g., in olive oil):
○​ Increase HDL, decrease oxidative stress markers.
○​ Higher flavonoid intake reduces CVD risk.
●​ French Paradox: Low CHD rates despite high saturated fat intake, possibly due to
antioxidant consumption.

9. Effect of Exercise

●​ Endurance Exercise: Increases oxygen utilization (10-20 times resting state),

generating more free radicals.
●​ Regular Exercise: Enhances antioxidant defenses, protecting against damage.
●​ Intense Exercise in Untrained Individuals: Overwhelms defenses, increasing damage.
●​ Vitamin E: Protects against exercise-induced oxidative damage.
●​ Recommendation: Regular exercise with 5 servings/day of fruits/vegetables;
anthocyanidins help athletes.

10. Requirements

●​ No Fixed RDA: Recommendations focus on antioxidant-rich foods.

●​ Expert Committee 2020: 500 g/day of fruits/vegetables for β-carotene, vitamin C,
polyphenols, flavonoids, complemented by vegetable oil for vitamin E.

11. Sources of Antioxidants

Nutrient Antioxidants

●​ β-carotene: Green leafy vegetables, ripe yellow fruits/vegetables (papaya, muskmelon,

mango, pumpkin, carrots).
●​ Vitamin C: Orange, lemon, sweet lime, guava, gooseberry, sprouted pulses.
●​ Vitamin E: Cereals, cereal products, oilseeds, nuts.
●​ Selenium and Zinc: Meats, seafood, cereals.
●​ Copper: Oysters, liver, mushrooms, nuts, chocolate.
●​ Iron: Meat, liver, green leafy vegetables, cereals, millets, pulses.

Whole Grains and Pulses

●​ Rich in vitamin E, selenium (selenomethionine, 85-100% bioavailability).

●​ Vitamin E inhibits nitrosamine formation at low pH.
●​ Soybean: Contains isoflavones, phytoestrogens, anthocyanin (black soybean seed
coat).
 ●​ Rajamah: High antioxidant compounds.

Nuts and Oilseeds

●​ Contain phytosterols, ellagic acid, flavonoids, phenolic compounds, luteolin, tocopherols.

●​ Groundnut Skin: Contains resveratrol, reduces hypertension, diabetes, aging-related
metabolic steps, has anti-inflammatory, antitumor, antiplatelet properties.
●​ Walnuts: Highest antioxidant activity (2-15 times more potent than vitamin E).
●​ Antioxidant Activity (Figure 24f, DPPH Trolox equiv. mg/100g):
○​ Walnuts: 1541, groundnut: 747, cashew nuts: 670, almond: 634, etc.

Whey Proteins

●​ Stimulate cell-mediated/humoral immunity, increase tissue glutathione, inhibit tumor

growth.
●​ Found in low molecular weight ultrafiltration permeate from milk/whey.

Fruits and Vegetables

●​ Contain vitamins C, β-carotene, tocopherols, flavonoids, polyphenols (nutraceuticals).

●​ Curry Leaves: Polyphenol-rich, anti-inflammatory, prevent breast cancer.
●​ Lycopene (Tomatoes): Affects gene expression, protects against prostate cancer,
reduces CVD risk in type 2 diabetes.
●​ Quercetin: Bioflavonoid in citrus fruits, leafy vegetables, reduces oxidative
stress/inflammation.
●​ Mushrooms: Polyphenols, ergothioneine provide antioxidant effects.
●​ Phytochemicals:
○​ Carotenoids (α/β-carotene, lutein, lycopene, cryptoxanthin, zeaxanthin): Mango,
papaya, spinach, tomatoes, curry leaves.
○​ Lycopene: Tomatoes, watermelon, red peppers, pink grapes; cooking enhances
bioavailability.
○​ Lutein: Green leafy vegetables.
○​ Resveratrol: Red grapes, pomegranate, red wine.
○​ Polyphenols: Berries, strawberries.
○​ Flavonoids: Apples, pears, citrus fruits, onions, broccoli.
○​ Phytoestrogens: Soybean, yam, cabbage, chickpeas.
○​ Terpenes: Citrus fruits, carrots, broccoli.
●​ Antioxidant Activity (Figure 24i, DPPH Trolox equiv. mg/100g):
○​ Guava: 891, pomegranate: 500, mango: 330, orange: 176, banana: 32.

Oils: Rice Bran Oil: High tocotrienols, oryzanol.

●​ Palm Oil: Rich in tocopherols, tocotrienols.

●​ Sesame Oil: Stable due to lignans (sesamol, sesamin, sesamolin).
●​ Olive Oil: Contains vitamin E.
Jaggery: Higher antioxidant value than sugar/honey (jaggery: 208.1 Trolox equiv.; honey:
19-62; sugar: 15-16, NIN 2009-10).

11.8 Spices

●​ Saffron, Annatto: Contain antioxidant carotenoids.

●​ Ginger: Stimulates xenobiotic metabolism enzymes.
●​ Curcumin (Turmeric):
○​ Inhibits lipid peroxidation, scavenges superoxide/hydroxyl radicals.
○​ Anticancer, anti-inflammatory, reduces mutagenicity in smokers, enhances
chemotherapy/radiotherapy efficacy.
●​ Eugenol (Cloves): High antioxidant activity.
●​ Coriander Seeds: 136 ng/g selenium.
●​ Mustard Seeds: 128 ng/g selenium.
●​ Garlic: Allyl compounds, antitumor properties.
●​ Antioxidant Activity : Cloves: 16532, cinnamon: 8030, star anise: 6174, mace: 1438,
turmeric: 1088.

Tea

●​ Green/Black Tea: Flavonoids have 400% greater antioxidant potency than orange juice.
○​ 200 mg flavonoids/cup.
○​ Green tea: Rich in catechins, gallic acid, increases glutathione peroxidase
activity.
○​ Black tea: Contains thearubigins, theaflavins, reduces LDL oxidation (via EC,
EGCG).
○​ 1-6 cups/day increases plasma antioxidant capacity.

Coffee

●​ Chlorogenic Acid: Polyphenol with antioxidant value.

●​ Caffeine: Scavenges hydroxyl radicals, reduces ROS-mediated disease risk (CVD,
diabetes, cancers).
●​ Robusta Beans: Twice the caffeine of Arabica.

Red Wine

●​ Resveratrol: Delays aging, reduces cholesterol/LDL in high-cholesterol individuals.

●​ Proanthocyanidins: Flavonoids in grape seeds/skins.

Herbs
 ●​ Contain monoterpenes, sesquiterpenes, flavonoids, diterpenoids, ursolic acid,
coumarins, phthalides, polyacetylenes (basil, dill, fennel, marjoram, rosemary, sage,
anise, caraway, celery seeds, cilantro).

Aquatic Organisms

●​ Astaxanthin: Carotenoid in salmon, shrimp, lobsters, with high antioxidant activity due
to oxygenated ring structures.

Ayurvedic Medicines

●​ Mundi (Sphaerantus hirtus): Anti-arthritis, good in vitro antioxidant activity.

●​ Haritaki (Terminalia chebula): Hepatoprotective, good antioxidant activity (NIN
2000-01).

12. Effect of Processing

●​ Sprouting/Malting: Increases phenolic content in wheat.

●​ Boiling/Pressure Cooking: Increases phenolic content in whole grains (green gram,
black gram, Bengal gram), brinjal, French beans.
●​ Dry Roasting: Increases phenolic content in dehusked legumes.
●​ No Effect: Tomato, onion, groundnut, sesame phenolic content unchanged by
processing.
●​ Deep-Frying: Decreases antioxidant activity in soaked black gram dal.
●​ Roasting Nuts: Reduces antioxidant quality; raw walnuts retain full effectiveness.
●​ Baking Bread: Produces pronyl-lysine (antioxidant, 8 times more in crust than crumb)
via Maillard reaction.

13. Recommendations

●​ Food-Based Approach: Prioritize antioxidant-rich foods over supplements to prevent

degenerative diseases.
●​ Diet Composition: Minimum 5 servings/day of fruits/vegetables, whole grains, nuts,
oils, spices, tea, coffee to delay aging, reduce cancer, CVD, and other disorders.
●​ Avoid: High sugar, fat, salt foods to maximize micronutrient and antioxidant intake.