Antioxidants – an overview
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Transcript of 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)• 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 dietSources of antioxidants in the diet
Sources of antioxidants in the diet:Sources of antioxidants in the diet:
Polyphenols,Polyphenols, carotenoidscarotenoids & vitamins& 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)
OH
HO
Lutein, a carotenoid
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: 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 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
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
Radicals and ROSThe 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 DNAAging = cumulative damage over the years
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
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
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
Basic free radical scavenging: phenols form resonance-stabilized radicals
OH O. O
R.
O
O
OH O
O
OH OR
O2
O
O
-ROH
-HR
Phenoxyl radicals areusually further oxidizedto quinones
caffeic acid, a phenolic acid
O
OH
OH
OR
HO
OH
cyanidin, an "anthocyanin"
Structures of some “polyphenolic” antioxidants found in fruits, vegetables & legumes
Found in blueberries, blackberries and cranberries
O
OH
OH
OR
HO
OH
quercetin,a "flavonol"
O
Found in berries, onions,and citrus fruit
O
OH
OH
OR
HO
OH
catechins
Found in chocolate and tea
OH
HO
OH
resveratrol, a"stilbene"
Found in red wine,peanuts
O
OH
HO
OH
genistein, an"isoflavone"
O
Found in soy productsand legumes
Found 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 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.)
• -unsaturated ketone and 3-OH on C-ring
O
OH
OH
OR
HO
OH O
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 sugaris attached to a C instead of O, making it more difficult to hydrolyze the glycosidic linkage
O
OH
OH
OR
HO
OH
cyanidin, an"anthocyanin"
R = glucose or rutin
PACs
The “French Paradox”The “French Paradox”
In certain regions of France, the incidence of cardiovascular disease is relatively low, despite a diet high in saturated fats
ResveratrolResveratrol&&FlavonoidsFlavonoids
Flavonoids protect against effects Flavonoids protect against effects of cardiovascular diseaseof 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)
O
OH
H
H
OH
H
HHO H
OH
H
H
H OH
HO HO
HO
O
OH
OH
OR
HO
OH O
OH
gal=galactopyranose ara = arabinofuranose
O
OH
OMe
OR
HO
OH O
O
OH
OH
OR
HO
OH O
R = gal, ara R = gal, ara, rhamno, xyl
O
OH
H
H
OH
H
HHO H
Me
rham = rhamnopyranose
O
OH
H
H
OH
H
HHO
xyl = xylose
R = gal
O
OH
OH
OR
HO
OH
R = gal, ara
O
OH
OMe
OR
HO
OH
R = gal, ara
cyanidin
quercetinmyricetin
methyl quercetin
peonidin
Antioxidants in cranberries (Vaccinium macrocarpon):Flavonol and anthocyanin glycosides
Cranberry PACsCranberry 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 activity
HO
OH
OH
OH
OH
O
HO
OH
OH
OH
OH
O
OHHO
OH
OH
HO O
OHO
OHHO
HOO
4
8
4
6
7
84
2
Tetramer of catechin & epicatechin units
Cranberry proanthocyanidins
B-type linkage
A-type linkage
Resveratrol…Resveratrol…the fountain of youth?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)
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 PPAR
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. • Resveratrol’s effects were associated with an induction of genes for oxidative phosphorylation and mitochondrial biogenesis• A resveratrol-mediated decrease in acetylation of PGC-1 (controls mitochondrial biogenesis and function) and an increase in PGC-1 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:1.H atom transfer reactions (HAT) – monitor rxn kinetics2.Electron transfer reactions (ET) – involve a redox rxn with the oxidant that can be monitored
Review discussesthe pros and cons of each type of antioxidant assay
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 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
1) Free – radical scavenging (DPPH or TEAC assay)
2) Lipid oxidation / peroxidation assay (TBARS)
3) LDL oxidation assays
4) ORAC assay
5) Assays measuring redox reactions of iron (FRAP)
6) 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.
General free radical-scavenging ability:General free radical-scavenging ability:the DPPH Assaythe DPPH Assay
Antioxidant 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 radical
NO2
O2N
O2N
(Ph)2-N-N
DPPH: 2,2-diphenyl-1-picrylhydrazyl radical
max = 517nm.H
Cranberry flavonoids were more effective Cranberry flavonoids were more effective free radical scavengers than Vitamin Efree radical scavengers than Vitamin E
ECEC5050 for DPPH assay for DPPH assay
Compound Compound ( (g/mL)g/mL) ((M)M)
myricetin-3-arabinosidemyricetin-3-arabinoside 7.8 7.8 17.0 17.0
quercetin-3-galactosidequercetin-3-galactoside 9.6 9.6 20.7 20.7
cyanidin-3-galactosidecyanidin-3-galactoside 3.5 3.5 7.7 7.7
Trolox/Vit E (standard)Trolox/Vit E (standard) 7.5 7.5 30.0 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
CompoundCompound ICIC5050 ( (M)M)
myricetin-3-arabinosidemyricetin-3-arabinoside 3.5 3.5
quercetin-3-galactosidequercetin-3-galactoside 4.3 4.3
cyanidin-3-galactosidecyanidin-3-galactoside 1.5 1.5
Vitamin E (standard)Vitamin E (standard) 2.4 2.4
Inhibition of low-density lipoprotein oxidation Inhibition of low-density lipoprotein oxidation in vitroin vitro by by cranberry flavonoids is comparable to Vitamin Ecranberry 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) 52:5843-48
TBARS assay was used todetermine antioxidant activity
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)
Protecting against iron-induced lipid oxidation
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.
ORAC values are expressed as mmoles of Trolox equivalents per unit mass or volumeTrolox = water-soluble Vitamin E analog
Use of ORAC to compare antioxidant power of foods or change in plasma antioxidant capacity over time in response to a treatment
Source: Brunswick labs (http://brunswicklabs.com/app_orac.shtml)
Fe induced formation of hydroxyl radical
Fe2+ + 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 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.
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
Cellular Antioxidant Activity (CAA) assay Can 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
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
Ischemic Stroke
Hypoglycemia & Hypoxia
Loss of cell homeostasis, cell death by two paths:necrosis and apoptosis (Martin, 1998)
Reactive oxygen species[O2
-., H2O2]
Oxidative damage, cell death (Chan, 2001)
Reperfusion
An in vitro model that can predict stroke damage
After 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 days
Control6 hr
Oxygen Glucose Deprivation 6 hr
Simulated ischemia
1 mM H2O2
6 hrReperfusion(oxidative stress)
Cranberryphenolic extract
Flavonolsand/or Anthos
PACs
Cranberryphenolicextract
PACs
Flavonolsand/orAnthos
Cranberryphenolicextract
Flavonolsand/or Anthos
PACsn = 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
Necrosis Apoptosisoxidative 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?Which compounds were most effective?
O
OH
OH
OR
HO
OH R = gal, ara
cyanidin
* P < 0.05
O
OH
OH
OR
HO
OH O
quercetin
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???
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
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