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Antioxidants – an overview

Antioxidants – an overview. Antioxidants are molecules capable of reducing the causes or effects of oxidative stress Oxidative stress can be caused by environmental factors, disease, infection, inflammation, aging (ROS production)

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Antioxidants – an overview

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  1. Antioxidants – an overview • Antioxidants are molecules capable of reducing the causes • or effects of oxidative stress • Oxidative stress can be caused by environmental factors, • disease, infection, inflammation, aging (ROS production) • ROS or “reactive oxygen species” include free radicals and • other oxygenated molecules resulting from these factors • The body produces some endogenous antioxidants, but • dietary antioxidants may provide additional line of defense • Flavonoids & other polyphenolics, Vitamins C & E, and • carotenoids are the most common dietary antioxidants • Many herbs and botanicals also contain antioxidants • Resources: • Gordon, M. H., “Dietary Antioxidants in Disease Prevention,” Natural Product Reports (1996) 13: 265-273; Pietta, P.-G., “Flavonoids as Antioxidants”, Journal of Natural Products (2000) 63: 1035-1042; Scalbert, A., Johnson, I.T., Saltmarsh, M. “Polyphenols: antioxidants and beyond,” American Journal of Clinical Nutrition (2005) 81: 215S-217S; Huang, D., Ou, B., Prior, R. “The Chemistry Behind Antioxidant Capacity Assays”, J. Agric. Food Chem. (2005) 53:1841-56.

  2. Sources of antioxidants in the diet

  3. Sources of antioxidants in the diet:Polyphenols,carotenoids& vitamins • Red wine (tannins, resveratrol, flavonoids) • Cranberries & blueberries (flavonoids & tannins) • Strawberries (ellagic acid, ellagitannins) • Tea (EGCG & other catechins, tannins) • Chocolate (catechins) • Onions (quercetin) • Spinach & leafy greens (lutein & zeaxanthin) • Eggs (lutein) • Citrus fruits (Vitamin C) • Plant oils (Vitamin E & omega-3)

  4. Antioxidants and more…Phylloquinones and tocopherols are part phenolic, part isoprenoid Coenzyme Q10: Redox carrier for electrons in human mitochondrial ETS Vitamin E Sources: cereals, seed oils eggs, soybean, corn oil, barley --Free radical scavenger --Protects lipids in LDL and cell membranes from oxidation --decreases coronary artery lesions, but effect on CVD mortality still not proven Vitamin K1 Sources: plants, primarily green veggies Role: blood clotting – needed for carboxylation of Glutamate residues in prothrombin Inhibited by warfarins (coumadin)

  5. Biosynthesis of Vitamin E: Shikimate pathway-derived 4-hydroxyphenyl pyruvic acid is alkylated with isoprenoid chain from mevalonate pathway Rings are methylated by SAM Cyclization of phenol with chain forms chroman ring Tocopherols differ in pattern of methylation on the ring

  6. Oxidation of phenolic ring to quinone forms plastoquinones, ubiquinones. • Coenzyme Q10 comes from • 4-hydroxybenzoic acid precursor. • After attachment of the isoprene chain, the ring is • decarboxylated • oxidized to quinone • methylated • hydroxylated • O-methylated

  7. Not all natural plant antioxidants are phenolics... • Derived from 40 carbon isoprenoid chain precursor (phytoene) through the mevalonate pathway • Conjugation gives the molecule high antioxidant capacity and ability to absorb harmful UV light • Role in plant: carotenoidsact as light-harvesting pigments, protect against photo-damage by scavenging peroxyl and singlet oxygen • In humans, carotenoids are carried in the LDL along with tocopherol • Lutein and zeaxanthin are present in the human eye (macula) and are thought to protect the retina from oxidative stress • Other observed beneficial bioactivities may or may not be linked to the antioxidant properties

  8. Defining “antioxidant” • The term “antioxidant” has many definitions • Chemical definition: “a substance that opposes oxidation or inhibits reactions promoted by oxygen or peroxides” • Biological definition: “synthetic or natural substances that prevent or delay deterioration of a product, or are capable of counteracting the damaging effects of oxidation in animal tissues” • Institute of Medicine definition: “a substance that significantly decreases the adverse effects of reactive species such as ROS or RNS on normal physiological function in humans Huang, et al, J. Agric. Food Chem. 2005, 53: 1841-1856

  9. Radicals and ROS The enemy: “Reactive Oxygen Species” (ROS) are highly reactive free radicals • Superoxide (O2-.) – protonation forms .OOH • Hydroxyl radical (.OH) most reactive • Peroxyl radicals (.OOH,.OOR) more selective • Alkoxy radicals (.OR) • Peroxynitrite (ONOO-) They form as the result of stress, inflammation, and the human body’s natural defenses in vivo, many are formed in the mitochondria, by phagocytes and peroxisomes, and by CYP450 activities. They target tissue, proteins, lipids and DNA Aging = cumulative damage over the years

  10. What do antioxidants do? • Prevent formation of ROS • Inhibit xanthine oxidase, COX, LOX, GST monooxygenases, chelate metals • Scavenge/remove ROS before they can damage important biomolecules • Aid the human body’s natural defenses • Upregulate superoxide dismutase (O2-.), catalase (H2O2), glutathione peroxidase (endogenous AO) • Repair oxidative damage • Eliminate damaged molecules • Prevent mutations

  11. Lipid oxidation: a radical mechanism • PUFAs (R-H) are major target due to reactivity of C=C • Initiation: X· + RH  X-H + R· ROOH  RO· + HO· or 2 ROOH  RO· + ROO· + H2O • Propagation R· + O2 ROO· ROO· + R’H  ROOH + R’· • Termination ROO· + R’OO·  ROOR’ + O2 RO· + R’·  ROR’ • Hydroperoxides (ROOH) also oxidize to aldehydes and ketone by-products

  12. In the early days of antioxidant research, lipids /LDL oxidation was considered the major health complication due to oxidative stress and first step in atherosclerosis. Now we know that most diseases of aging, including many cancers and neurodegenerative diseases are also associated with long-term oxidative stress and associated inflammation

  13. Basic free radical scavenging: phenols form resonance-stabilized radicals Phenoxyl radicals are usually further oxidized to quinones

  14. Structures of some “polyphenolic” antioxidants found in fruits, vegetables & legumes caffeic acid, a phenolic acid Found in blueberries, blackberries and cranberries Found in berries, onions, and citrus fruit Found in herbs, coffee and fruits Found in chocolate and tea Found in red wine, peanuts Found in soy products and legumes On a molecular level, all of these compounds “absorb” harmful free radicals and chelate pro-oxidant metal ions Most of them also modulate cellular biochemical reactions and the expression of genes and proteins associated with oxidative stress

  15. Flavonoids as antioxidants Flavonoids are especially effective because of structural features including: • Conjugation to further stabilize radicals • ortho-dihydroxysubstituted B ring allows for chelation of pro-oxidant metal ions (Fe2+, Fe3+, Cu2+, etc.) • a,b-unsaturated ketone and 3-OH on C-ring

  16. Despite its mythical powers, flavonoids identified in Acai are similar to those found in other fruits R = glucose or rutin Orientin = luteolin-8C-glucoside (above) Homoorientin = luteolin-6C-glucoside (below) Luteolin is a flavone. These compounds are unusual because the sugar is attached to a C instead of O, making it more difficult to hydrolyze the glycosidic linkage PACs

  17. The “French Paradox” In certain regions of France, the incidence of cardiovascular disease is relatively low, despite a diet high in saturated fats Resveratrol & Flavonoids

  18. Flavonoids protect against effects of cardiovascular disease The Zutphen Elderly Study • A large cohort of Dutch men aged 50 to 69 years were examined in 1970 and followed up for 15 years for dietary factors and incidence of disease • Dietary intake of flavonoids correlated with reduced incidence of stroke and reduced coronary heart disease mortality (Hertog, et al, Lancet 1993)

  19. Antioxidants in cranberries (Vaccinium macrocarpon): Flavonol and anthocyanin glycosides myricetin quercetin methyl quercetin cyanidin peonidin

  20. Cranberry proanthocyanidins Cranberry PACs are oligomers of epicatechin units that contain both A and B-type linkages between units Extracts contain oligomers up to 12 DP These compounds have antibacterial, antitumor and antioxidant activity B-type linkage A-type linkage Tetramer of catechin & epicatechin units

  21. Resveratrol…the fountain of youth? • Produced by plants in response to stress • Found in red wine, grape and cranberry juice, legumes • Thought to contribute to the “French Paradox” • Decreases lipoprotein oxidation leading to cardiovascular disease (early 90’s) • Anticancer & antiinflammatory activity (1997) • Extends lifespan through sirtuin activation, enhancing mitochondrial function (2006)

  22. Extending lifespan: the sirtuins • Sir2 family of proteins (silent information regulator) regulate aging & longevity in lower organisms • NAD+-dependent protein acetylases that regulate gene silencing, DNA repair & recombination • Sirtuins mediate life-extending effect of caloric restriction • Analogous SIRT1 gene found in mammals • SIRT1 is a key regulator of energy and metabolic homeostasis. • May regulate apoptosis (modulation of p53 tumor suppressor) and differentiation • Modulates adipogenesis by deactivating PPARg, triggering loss of fat, similar to caloric restriction de la Lastra & Villegas (2005) Mol. Nutr. Food Res. 49: 405-430

  23. Reduction of oxidative stress in mitochondria: • Abstract: • Diminished mitochondrial oxidative phosphorylation and aerobic capacity are associated with reduced longevity. We tested whether resveratrol, which is known to extend lifespan, impacts mitochondrial function and metabolic homeostasis. • Treatment of mice with resveratrol significantly increased their aerobic capacity, as evidenced by their increased running time and consumption of oxygen in muscle fibers. • Resveratrol’s effects were associated with an induction of genes for oxidative phosphorylation and mitochondrial biogenesis • A resveratrol-mediated decrease in acetylation of PGC-1a (controls mitochondrial biogenesis and function) and an increase in PGC-1a activity was observed. • This mechanism is consistent with resveratrol being a known activator of the protein deacetylase, SIRT1. • Importantly, resveratrol treatment protected mice against diet-induced-obesity and insulin resistance.

  24. Authors group antioxidant assays into two categories: • H atom transfer reactions (HAT) – monitor rxn kinetics • Electron transfer reactions (ET) – involve a redoxrxn with the oxidant that can be monitored Review discusses the pros and cons of each type of antioxidant assay

  25. Total phenolics determination: Folin-Ciocalteu reaction • Developed by Singleton and Rossi (1965) • Measures total phenolics content of a preparation, expressed as gallic acid equivalents (or other standard) • Total phenolic content often correlates well with antioxidant capacity (not always however) • Yellow reagent prepared from solution of Na2WO4 and Na2MoO4dihydrates, and is thought to contain heteropolyphospho-tungstates-molybdates (PMoW11O40)4- • Phenolate anions reduce Mo(VI) to Mo (V) by electron-transfer reaction, producing a blue color that is quantified at 750 nm – species thought to be (PMoW11O40)4-?

  26. Evaluation of antioxidant efficacy: Selected antioxidant assays • Free – radical scavenging (DPPH or TEAC assay) • Lipid oxidation / peroxidation assay (TBARS) • LDL oxidation assays • ORAC assay • Assays measuring redox reactions of iron (FRAP) • Cellular antioxidant assay (CAA) Resources: excerpts from: Yan, X., Murphy, B.T., Hammond, G.B., Vinson, J. A., Neto, C.C. “Antioxidant activities and antitumor screening of extracts from cranberry fruit” J. Agric. Food Chem. (2002) 50: 5844-5849. Seeram, N. and Nair, M. “Inhibition of lipid peroxidation and structure-activity related studies of the dietary constituents anthocyanins, anthocyanidins and catechins” J. Agric. Food Chem (2002) 50: 5308-5312. Vinson, J. et al, “Vitamins and especially flavonoids in common beverages are powerful in vitro antioxidants which enrich LDL and increase their oxidative resistance after ex vivo spiking in human plasma” (1999) J. Agric. Food Chem. 47: 2502-2504. Wolfe, K. and Liu, R.H. “Cellular Antioxidant Activity (CAA) Assay for Assessing Antioxidants, Foods, and Dietary Supplements” J. Agric. Food Chem. (2007) 55, 8896–8907.

  27. General free radical-scavenging ability: the DPPH Assay • Antioxidant activity of extracts and compounds can be • evaluated by a general radical-scavenging assay that • predicts ability to quench OH., ROO. and other ROS. • antioxidant • Violet ------------------> Yellow • Radical-scavenging activity is determined by measuring degree • of absorbance quenching for varying sample concentrations • Activity expressed as EC50 = concentration required to quench 50% of DPPH radical DPPH: 2,2-diphenyl-1-picrylhydrazyl radical lmax = 517nm . H

  28. Cranberry flavonoids were more effective free radical scavengers than Vitamin E EC50 for DPPH assay Compound (mg/mL) (mM) myricetin-3-arabinoside 7.8 17.0 quercetin-3-galactoside 9.6 20.7 cyanidin-3-galactoside 3.5 7.7 Trolox/Vit E (standard) 7.5 30.0 Yan, X., Murphy, B. T., Hammond, G. B., Vinson, J. A. and Neto, C. C. J. Agric. Food Chem (2002) 50: 5844-5849

  29. Inhibition of low-density lipoprotein oxidation in vitro by cranberry flavonoids is comparable to Vitamin E Compound IC50 (mM) myricetin-3-arabinoside 3.5 quercetin-3-galactoside 4.3 cyanidin-3-galactoside 1.5 Vitamin E (standard) 2.4 Yan, X., Murphy, B. T., Hammond, G. B., Vinson, J. A. and Neto, C. C. J. Agric. Food Chem (2002) 50: 5844-5849

  30. Lipid peroxidation as the target • TBARS assay for LDL oxidation: Joe Vinson, Univ. of Scranton • Used to test flavonoids and other dietary antioxidants • for ability to prevent lipoprotein oxidation • LDL / VLDL are reacted with varying concentrations of antioxidant in the presence of cupric ion (Cu2+) to induce formation of oxidation products from unsaturated FA • After 6 hrs @ 37oC, thiobarbituric acid (TBA) added • Formation of conjugated diene oxidation products measured by fluorescence • % inhibition = control - native LDL – sample fluorescence x 100/control fluorescence Oxidation products of lipids or LDL react with TBA to form colored adducts that can be detected by absorbance or fluorescence

  31. How do these popular antioxidants and beverages stack up in protecting plasma lipids?

  32. J. Agric. Food Chem (2004) 52:5843-48 TBARS assay was used to determine antioxidant activity

  33. Pro-oxidant or anti-oxidant? • Cu(II)-initiated oxidation of LDL produces decomposition products like hexanal • Their production is measured using GC and correlated with level of oxidation • Complication: Cu(II)-catalyzed oxidation can be promoted by the presence of excess antioxidants (e.g. tocopherols, which donate an e- to produce Cu(I), and the resulting radical reacts with the lipids)

  34. Protecting against iron-induced lipid oxidation

  35. ORAC assay • ORAC (oxygen radical absorbance capacity) assay is used extensively to compare antioxidant activities of foods, beverages, and antioxidant capacity of human blood samples in a clinical setting. • ORAC is based on the inhibition of peroxyl-radical-induced oxidation initiated by thermal decomposition of azo-compounds such as 2,2’-azobis(2-amidino-propane) dihydrochloride (AAPH) • Free radical damage to a fluorescent probe is quantified by measuring the change in its fluorescence intensity. • The inhibition of free radical damage by an antioxidant is assessed by comparing probe fluorescence in presence or absence of the antioxidant. • Grandfathers of ORAC: method was developed by Dr. Guohua Cao in 1992. In 1995, Dr. Cao joined Dr. Ronald L. Prior's group at Jean Mayer USDA Human Nutrition Research Center on Aging to develop a semi-automated ORAC assay.

  36. Use of ORAC to compare antioxidant power of foods or change in plasma antioxidant capacity over time in response to a treatment ORAC values are expressed as mmoles of Trolox equivalents per unit mass or volume Trolox = water-soluble Vitamin E analog Source: Brunswick labs (http://brunswicklabs.com/app_orac.shtml)

  37. Fe induced formation of hydroxyl radical Fenton Reaction: H. J. H. Fenton, J. Chem. Soc., Trans. 1894: 899 Fe2+ + H2O2 Fe3+ + OH_ + OH Ascorbate(AscH-) + Fe3+→•Asc- + Fe2+ Hydroxyl radical OH, very reactive, t1/2 ca 10-9 s Consequences: Oxidative DNA damage, protein modification, lipid peroxidation, etc. Even small amounts of ferrous iron in the body can lead to the production of a large number of hydroxyl radicals.

  38. Fluorescent sensing of iron-induced oxidation in cells (Guo, 2010) Fig. 3. The RS-BE sensor can detect iron/H2O2-induced oxidative stress in live cells. Confocal fluorescence images of live human SH-SY5Y cells with the treatment of RS-BE/Fe/ H2O2 (scale bar 10 µm). (a) DIC; (b) the cells incubated with 10 µM RS-BE for 30 min; (c) the cells were then incubated with 10 µM Fe(8-HQ) for 30 min; (d) and (e) the cells were further treated with 100 µM H2O2 for 10 and 25 min, respectively, (f) Integrated emission (547-703 nm)  intensity of (a), (b), (c), (d) and (e) images.

  39. FRAP and similar assays measure ability to reduce Fe3+  Fe2+ • Benzie & Strain (1999) Methods in Enzymology • FRAP reagent contains ferric (Fe3+ ) tripyridyl triazine complex • Reduction to ferrous (Fe2+) tripyridyl triazine forms a blue complex • Reducing capacity of compounds or mixtures are measured based on change in absorbance at 593 nm

  40. Cellular Antioxidant Activity (CAA) assayCan antioxidant activity be measured directly inside cells? • Dye precursor DCFH diffuses into the cell • Cells treated with ABAP, azo compound that forms peroxyl radicals • Peroxyl radicals oxidize dye to fluorescent form • Cells are treated with antioxidants • If AO makes it into cell and scavenges the radicals, fluorescence decreases

  41. A study of oxidative stress • Canadian researchers found that wild blueberries decreased damage to brain cells caused by stroke-like conditions • Sweeney, M. et al: Nutritional Neuroscience, 2002 • Collaborative study (Neto & Sweeney) investigated whether cranberry could also prevent this type of damage

  42. Reperfusion Ischemic Stroke Reactive oxygen species [O2-., H2O2] Hypoglycemia & Hypoxia Loss of cell homeostasis, cell death by two paths: necrosis and apoptosis (Martin, 1998) Oxidative damage, cell death(Chan, 2001)

  43. An in vitro model that can predict stroke damage Cerebellar granule neurons from neonatal rat brain are cultured at 37oC 7-10 days 1 mM H2O2 6 hr Reperfusion (oxidative stress) Control 6 hr Oxygen Glucose Deprivation 6 hr Simulated ischemia Cranberry phenolic extract Cranberry phenolic extract Cranberry phenolic extract Flavonols and/or Anthos Flavonols and/or Anthos Flavonols and/or Anthos PACs PACs PACs n = 6/group After incubation, samples are collected and analyzed for markers of necrosis (LDH) or apoptosis (caspase-3)

  44. Whole Cranberry Extract Protected Neurons from Stress-Induced Death Percent decrease in both types of stroke-induced damage at the highest dosage level of crude extract (0.3 mg/mL) Necrosis Apoptosis oxidative stress (reperfusion) 42.7% 36.5% oxygen/glucose deprivation (simulated ischemia) 48.5% 50.0% Neto, C., Lamoureaux, T., Kondo, M., Sweeney-Nixon, M., Solomon, F., MacKinnon, S. Phenolics in Foods and Natural Health Products Symposium, ACS Books (2005).

  45. Which compounds were most effective? * P < 0.05

  46. Indications from tissue culture model: • Whole cranberry extract can inhibit brain cell death in vitro by up to 50% • The anthocyanins contributed most strongly to protection, particularly in the oxidative stress model • The whole cranberry extract is more protective against apoptosis than the fractions, suggesting that the phenolics work synergistically and that the mechanism is more than just free-radical scavenging But what happens in vivo???

  47. 6-week feeding study (Sweeney):Rats on a diet of feed supplemented with commercial cranberry powder (equivalent to 2.8 cups/day human dosage) n = 5 - 7 Possible treatment effect, but not statistically significant

  48. What happens in vivo? • Anthocyanins do get into the brain (ACS, JAFC, 2005) but the bioavailability of other flavonoids to brain is unknown • Most anthocyanins get broken down to smaller phenolics • Plasma levels of intact anthocyanins (nmol/L) are too low for radical-scavenging but may be sufficient to modulate cell signalling and gene expressiona • Other mechanisms: bilberry anthocyanins (Vaccinium myrtillus) decrease capillary fragility and permeability • Antiinflammatory properties: inhibition of COX-2 and prostacyclin activity may relax blood vessels • Combination of antioxidant and other effects aMilbury, et al, 2010, Journal of Nutrition

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