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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)
Sources of antioxidants in the diet
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)
Antioxidants and more…Phylloquinones and tocopherols are part
phenolic, part isoprenoid
Coenzyme Q10:
Redox carrier for
electrons in human
mitochondrial ETS
Vitamin K1
Sources: plants, primarily green veggies
Role: blood clotting – needed for carboxylation of Glutamate
residues in prothrombin
Inhibited by warfarins (coumadin)
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
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
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
1) decarboxylated
2) oxidized to quinone
3) methylated
4) hydroxylated
5) O-methylated
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 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
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
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
are aofmajor
component
of:
In theacids
early days
antioxidant
research,
lipids /LDL oxidation was considered the
major
Lipoproteins,
especiallydue
LDL
lipoproteins)
health complication
to (low-density
oxidative stress
and first step in atherosclerosis.
Now
Cellwemembranes--oxidation
degrades
making
them
know that most diseases
of aging,membranes
including many
cancers
andless fluid
neurodegenerative
Oils and fats in food;
oxidation
causes
“rancidity”
diseases
are also
associated
with long-term oxidative stress
and associated inflammation
Basic free radical scavenging: phenols form
resonance-stabilized radicals
Phenoxyl radicals are
usually further oxidized
to quinones
Structures of some “polyphenolic” antioxidants found
in fruits, vegetables & legumes
OH
quercetin,
a "flavonol"
HO
caffeic acid, a phenolic acid
OH
O
OR
OH
Found in blueberries,
blackberries and cranberries
OH
catechins
OH
HO
O
Found in berries, onions,
and citrus fruit
resveratrol, a
"stilbene"
OH
genistein, an
"isoflavone"
HO
HO
O
Found in herbs, coffee
and fruits
O
OR
OH
Found in chocolate
and tea
OH
Found in red wine,
peanuts
OH
O
Found in soy products
and legumes
OH
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
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
Despite its mythical powers, flavonoids identified in
Acai are similar to those found in other fruits
OH
cyanidin, an
"anthocyanin"
HO
OH
O
OR
OH
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
R = glucose or rutin
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
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)
Antioxidants in cranberries (Vaccinium macrocarpon):
Flavonol and anthocyanin glycosides
myricetin
quercetin
methyl
quercetin
cyanidin
peonidin
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
OH
OH
OH
OH
B-type
linkage
O
O
HO
HO
4
OH
HOOH
HO
HO
HO
8
OH
OH
4
OH
OH
OH
OH
O
O
8
4
OH
OH
OH
OH
HO
HO
4
OH
OH
6
6
OH
OH
O
O
HO
HO
4
8
8
7
7
HO
HO
HO
HO
O
O
A-type
linkage
4
2
2
O
O
OH
OH
OH
OH
Tetramer of catechin & epicatechin units
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)
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
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.
Authors group antioxidant assays into two categories:
1. H atom transfer reactions (HAT) – monitor rxn kinetics
2. Electron transfer reactions (ET) – involve a redox rxn with the
oxidant that can be monitored
Review discusses
the pros and cons
of each type of
antioxidant assay
Total phenolics determination: FolinCiocalteu 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 electrontransfer 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:
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.
DPPH: 2,2-diphenyl-1picrylhydrazyl radical
lmax = 517nm
O2N
.
(Ph)2-N-N
H
NO 2
O2N
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
Cranberry flavonoids were more effective
free radical scavengers than Vitamin E
Compound
EC50 for DPPH assay
(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
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
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
How do these
popular
antioxidants
and beverages
stack up in
protecting
plasma lipids?
J. Agric. Food Chem (2004) 52:5843-48
TBARS assay was used to
determine 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 azocompounds such as 2,2’-azobis(2amidino-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.
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)
Fe induced formation of
hydroxyl radical
Fenton Reaction:
Fe2+
+ H2O2
H. J. H. Fenton, J. Chem. Soc., Trans. 1894: 899
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.
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 RSBE/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)
Reperfusion
Reactive oxygen
species
[O2-., H2O2]
Oxidative
damage, cell
death (Chan, 2001)
An in vitro model that can predict stroke damage
Cerebellar granule neurons from neonatal rat brain
are cultured at 37oC 7-10 days
Oxygen Glucose
Deprivation 6 hr
Simulated ischemia
Control
6 hr
Cranberry
phenolic
extract
Flavonols
and/or
Anthos
Cranberry
phenolic
extract
PACs
n = 6/group
Flavonols
and/or
Anthos
PACs
1 mM H2O2
6 hr
Reperfusion
(oxidative stress)
Cranberry
phenolic
extract
Flavonols
and/or
Anthos
PACs
After incubation, samples are collected and analyzed for
markers of necrosis (LDH) or apoptosis (caspase-3)
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
oxidative stress (reperfusion) 42.7%
oxygen/glucose deprivation
(simulated ischemia)
48.5%
Apoptosis
36.5%
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?
OH
cyanidin
HO
OH
O
OR
OH
R = gal, ara
quercetin
* P < 0.05
OH
OH
HO
O
OR
OH
O
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