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.

Sources of antioxidants in the diet

Sources of antioxidants in the diet:

Polyphenols, carotenoids & vitaminsRed 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)

Antioxidants and morePhylloquinones and tocopherols are part phenolic, part isoprenoidCoenzyme Q10:

Redox carrier for electrons in humanmitochondrial ETSVitamin ESources: cereals, seed oilseggs, soybean, corn oil, barley--Free radical scavenger--Protects lipids in LDL and cellmembranes from oxidation--decreases coronary arterylesions, but effect on CVDmortality still not provenVitamin K1Sources: plants, primarily green veggiesRole: blood clotting needed for carboxylation of Glutamate residues in prothrombinInhibited by warfarins (coumadin)

Biosynthesis of Vitamin E:

Shikimate pathway-derived 4-hydroxyphenyl pyruvic acid is alkylated with isoprenoid chainfrom mevalonate pathwayRings are methylated by SAM

Cyclization of phenol with chainforms chroman ringTocopherols differ in pattern of methylation on the ring

Oxidation of phenolic ring toquinone forms plastoquinones, ubiquinones.

Coenzyme Q10 comes from4-hydroxybenzoic acid precursor.After attachment of the isoprene chain, the ring is decarboxylatedoxidized to quinonemethylatedhydroxylatedO-methylated

Not all natural plant antioxidants are phenolics...

Derived from 40 carbon isoprenoid chain precursor (phytoene) through the mevalonate pathwayConjugation gives the molecule high antioxidant capacity and ability to absorb harmful UV lightRole in plant: carotenoids act as light-harvesting pigments, protect against photo-damage by scavenging peroxyl and singlet oxygen In humans, carotenoids are carried in the LDL along with tocopherolLutein and zeaxanthin are present in the human eye (macula) and are thought to protect the retina from oxidative stressOther observed beneficial bioactivities may or may not be linked to the antioxidant properties

Defining antioxidantThe term antioxidant has many definitionsChemical definition: a substance that opposes oxidation or inhibits reactions promoted by oxygen or peroxidesBiological 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 tissuesInstitute 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

Radicals and ROSThe enemy: Reactive Oxygen Species (ROS) are highly reactive free radicals Superoxide (O2-.) protonation forms .OOHHydroxyl radical (.OH) most reactivePeroxyl radicals (.OOH,.OOR) more selectiveAlkoxy radicals (.OR)Peroxynitrite (ONOO-) They form as the result of stress, inflammation, and the human bodys natural defenses in vivo, many are formed in the mitochondria, by phagocytes and peroxisomes, and by CYP450 activities.They target tissue, proteins, lipids and DNAAging = cumulative damage over the years

What do antioxidants do?Prevent formation of ROSInhibit xanthine oxidase, COX, LOX, GST monooxygenases, chelate metalsScavenge/remove ROS before they can damage important biomolecules Aid the human bodys natural defensesUpregulate superoxide dismutase (O2-.), catalase (H2O2), glutathione peroxidase (endogenous AO) Repair oxidative damage Eliminate damaged molecules Prevent mutations

Lipid oxidation: a radical mechanismPUFAs (R-H) are major target due to reactivity of C=CInitiation:X + RH X-H + RROOH RO + HO or 2 ROOH RO + ROO + H2OPropagationR + O2 ROOROO + RH ROOH + RTerminationROO + ROO ROOR + O2RO + R RORHydroperoxides (ROOH) also oxidize to aldehydes and ketone by-products

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

Chemistry of TAG:

a) Saponification:

Base-catalyzed hydrolysis (NaOH) produces fatty acid salts + glycerol

b) Hydrogenation:

Reaction with H2/Pt converts unsaturated carbons to saturated

c) Catabolism:TAG undergo acid-catalyzed hydrolysis in stomach (digestion) Fatty acids are broken down 2 C at a time to acetyl-CoA which enters citric acid cycle

Lipid peroxidation

and antioxidants

Polyunsaturated fatty acids are easily

oxidized by O2 or oxygen free radicals:

a peroxy radical

an alkyl hydroperoxide

Health ramifications:Oxidation of LDL initiates formation of plaque (solid buildup) in blood vessels and onset of atherosclerosis/heart disease.

Fatty acids are a major component of:

Lipoproteins, especially LDL (low-density lipoproteins)

Cell membranes--oxidation degrades membranes making them less fluid

Oils and fats in food; oxidation causes rancidity

Antioxidant compounds react with free radicals (often by forming a more stable free radical) and remove them from the site before damage occurs.

Common antioxidants include substituted phenols, vitamins E, A & C

Basic free radical scavenging: phenols form resonance-stabilized radicalsPhenoxyl radicals areusually further oxidizedto quinones

caffeic acid, a phenolic acidStructures of some polyphenolic antioxidants found in fruits, vegetables & legumesFound in blueberries, blackberries and cranberriesFound in berries, onions,and citrus fruitFound in chocolate and teaFound in red wine,peanutsFound in soy productsand legumesFound in herbs, coffeeand fruits On a molecular level, all of these compounds absorb harmful free radicals and chelate pro-oxidant metal ionsMost of them also modulate cellular biochemical reactions and the expression of genes and proteins associated with oxidative stress

Flavonoids as antioxidantsFlavonoids are especially effective because of structural features including: Conjugation to further stabilize radicalsortho-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

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

The French ParadoxIn certain regions of France, the incidence of cardiovascular disease is relatively low, despite a diet high in saturated fats

Resveratrol&Flavonoids

Flavonoids protect against effects of cardiovascular diseaseThe Zutphen Elderly StudyA 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 diseaseDietary intake of flavonoids correlated with reduced incidence of stroke and reduced coronary heart disease mortality (Hertog, et al, Lancet 1993)

cyanidinquercetinmyricetinmethyl quercetinpeonidinAntioxidants in cranberries (Vaccinium macrocarpon):Flavonol and anthocyanin glycosides

Cranberry PACsare oligomers of epicatechin units thatcontain both A and B-typelinkages between units

Extracts contain oligomers up to 12 DP

These compounds have antibacterial, antitumor and antioxidant activityTetramer of catechin & epicatechin unitsCranberry proanthocyanidinsB-type linkageA-type linkage

Resveratrolthe fountain of youth?Produced by plants in response to stressFound in red wine, grape and cranberry juice, legumesThought to contribute to the French ParadoxDecreases lipoprotein oxidation leading to cardiovascular disease (early 90s) Anticancer & antiinflammatory activity (1997)Extends lifespan through sirtuin activation, enhancing mitochondrial function (2006)

Extending lifespan: the sirtuinsSir2 family of proteins (silent information regulator) regulate aging & longevity in lower organisms

NAD+-dependent protein acetylases that regulate gene silencing, DNA repair & recombinationSirtuins mediate life-extending effect of caloric restrictionAnalogous SIRT1 gene found in mammalsSIRT1 is a key regulator of energy and metabolic homeostasis.May regulate apoptosis (modulation of p53 tumor suppressor) and differentiationModulates adipogenesis by deactivating PPARg, triggering loss of fat, similar to caloric restriction de la Lastra & Villegas (2005) Mol. Nutr. Food Res. 49: 405-430

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. Resveratrols 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. Reduction of oxidative stress in mitochondria:

Authors group antioxidant assays into two categories:H atom transfer reactions (HAT) monitor rxn kineticsElectron transfer reactions (ET) involve a redox rxn with the oxidant that can be monitoredReview discussesthe pros and cons of each type of antioxidant assay

Total phenolics determination: Folin-Ciocalteu reactionDeveloped 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 Na2MoO4 dihydrates, 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-?

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, 88968907.

General free radical-scavenging ability:the DPPH AssayAntioxidant activity of extracts and compounds can beevaluated 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 radicalDPPH: 2,2-diphenyl-1-picrylhydrazyl radicallmax = 517nm.H

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

CompoundIC50 (mM)myricetin-3-arabinoside 3.5quercetin-3-galactoside 4.3cyanidin-3-galactoside 1.5Vitamin E (standard) 2.4Inhibition of low-density lipoprotein oxidation in vitro by cranberry flavonoids is comparable to Vitamin E Yan, X., Murphy, B. T., Hammond, G. B., Vinson, J. A. and Neto, C. C.J. Agric. Food Chem (2002) 50: 5844-5849

Lipid peroxidation as the targetTBARS 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 oflipids or LDL react with TBA to form colored adducts that can be detected by absorbance or fluorescence

How do these popular antioxidantsand beverages stack up inprotecting plasma lipids?

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

Pro-oxidant or anti-oxidant?Cu(II)-initiated oxidation of LDL produces decomposition products like hexanalTheir 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)

Protecting against iron-induced lipid oxidation

ORAC assayORAC (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.

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

Fe induced formation of hydroxyl radicalFe2+ + H2O2 Fe3+ + OH_ + OH Fenton Reaction: 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. Ascorbate(AscH-) + Fe3+ Asc- + Fe2+H. J. H. Fenton, J. Chem. Soc., Trans. 1894: 899

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 m). (a) DIC; (b) the cells incubated with 10 M RS-BE for 30 min; (c) the cells were then incubated with 10 M Fe(8-HQ) for 30 min; (d) and (e) the cells were further treated with 100 M H2O2 for 10 and 25 min, respectively, (f) Integrated emission (547-703 nm)intensity of (a), (b), (c), (d) and (e) images.

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

Cellular Antioxidant Activity (CAA) assay Can antioxidant activity be measured directly inside cells?Dye precursor DCFH diffuses into the cellCells treated with ABAP, azo compound that forms peroxyl radicalsPeroxyl radicals oxidize dye to fluorescent formCells are treated with antioxidantsIf AO makes it into cell and scavenges the radicals, fluorescence decreases

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

An in vitro model that can predict stroke damageAfter incubation, samples are collected and analyzed for markers of necrosis (LDH) or apoptosis (caspase-3) Cerebellar granule neurons from neonatal rat brain are cultured at 37oC 7-10 daysControl6 hrOxygen Glucose Deprivation 6 hrSimulated ischemia1 mM H2O26 hrReperfusion(oxidative stress)Cranberryphenolic extractFlavonolsand/or AnthosPACsCranberryphenolicextractPACsFlavonolsand/orAnthosCranberryphenolicextractFlavonolsand/or AnthosPACsn = 6/group

Percent decrease in both types of stroke-induced damageat the highest dosage level of crude extract (0.3 mg/mL)Whole Cranberry Extract Protected Neurons from Stress-Induced Death NecrosisApoptosisoxidative 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).

Which compounds were most effective?* P < 0.05

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 scavengingBut what happens in vivo???

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 - 7Possible treatment effect, but not statistically significant

What happens in vivo?Anthocyanins do get into the brain (ACS, JAFC, 2005) but the bioavailability of other flavonoids to brain is unknownMost anthocyanins get broken down to smaller phenolicsPlasma 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 permeabilityAntiinflammatory 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

Hydroxyl radical is the most reactive and may act as initiator for lipid peroxidation. Peroxyl radicals act more slower, more selectively over a longer distance. Superoxide is less likely to target lipids, but if protonated it forms OOH, which does target lipids. Alkoxy and peroxy radicals can form from iron-catalyzed decomposition of hydroperoxides (ROOH) **Resveratrol is one of the most potent plant-derived activators of SIR2, the yeast sirtuin gene that mediates the life-extending effects of calorie restriction by nutrient withdrawal. In yeast, resveratrol mimics calorie restriction, increases DNA stability and extends lifespan by 70%. It has been shown to activate SIRT1, a mammalian homolog of Sir2, improving mitochondrial function and energy homeostasis.***Early studies showed the flavonoids effective at inhibiting LDL oxidation in vitro, similar studies by Reed, Porter showed PACs have similar activityJoe has some more results to confirm this with other flavonoids

****Antho/flavonol fraction is more effective than crudeAnthos alone protect by up to 30%PACs not very effective (not significant)Ursolic and esters no effect*