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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 • 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

  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. Radicals The enemy: “Reactive Oxygen Species” (ROS) are highly reactive free radicals • Superoxide (O2-.) • Hydroxyl radical (.OH) • Peroxyl radicals (.OOH) They form as the result of stress, inflammation, and the human body’s natural defenses 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

  5. 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 SOD, catalase, glutathione peroxidase • Repair oxidative damage • Eliminate damaged molecules • Prevent mutations

  6. Structures of some “polyphenolic” antioxidants found in fruits, vegetables & legumes caffeic acid, a phenolic acid Found in herbs, coffee and fruits Found in blueberries, blackberries and cranberries Found in berries, onions, and citrus fruit Found in chocolate and tea Found in red wine, peanuts Found in soy products and legumes On a molecular level, all of these compounds react with harmful free radicals and can chelate metal ions that act as pro-oxidants Flavonoids are especially effective due to because of structural features including: Conjugation, o-dihydroxysubstituted B ring, a,b-unsaturated ketone, 3-OH on C-ring They also modulate cellular biochemical reactions and the expression of genes and proteins associated with oxidative stress

  7. 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)

  8. 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 • May regulate apoptosis (downregulation 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

  9. Despite its mythical powers, flavonoids identified in Acai are similar to those found in other fruits 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 R = glucose or rutin

  10. Evaluation of antioxidant efficacy: Antioxidant assays • Free – radical scavenging (DPPH or TEAC assay) • Lipid oxidation / peroxidation assay (TBARS) • LDL oxidation assay • ORAC assay • Cellular antioxidant assay (CAA) • Assays measuring redox reactions of iron 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.

  11. 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

  12. 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

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

  14. Protecting against iron-induced lipid oxidation

  15. 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) • ORAC measures free radical damage to a fluorescent probe through the change in its fluorescence intensity, an index of the degree of free radical damage. • The inhibition of free radical damage by an antioxidant is reflected in its protection against the change of probe fluorescence in the ORAC assay. • 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.

  16. 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)

  17. 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

  18. 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.

  19. 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.

  20. 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 foods or plasma are measured based on change in absorbance at 593 nm

  21. Antioxidants and more…quinones and isoprenoids 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 (carboxylation of glu in prothrombin Inhibited by warfarins (coumadin) Derived from 40 carbon isoprenoid chain (phytoene) through the mevalonate pathway; conjugation makes good antioxidant.

  22. Biosynthesis of Vitamin E: Shikimate pathway-derived 4-hydroxyphenyl pyruvic acid is alkylated with isoprenoid chain made through mevalonate pathway Rings are then methylated by SAM Cyclization of phenol with chain gives tocopherol chroman ring Tocopherols differ in pattern of methylation on the ring Oxidation to quinone occurs in plastoquinones, ubiquinone Coenzyme Q10 comes from 4-hydroxybenzoic acid which is decarboxylated, oxidized to quinone.

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