Antioxidant
Assays
Prepared by:
NENITO S. TOLEDO, RCh, PFT
Learning Outcomes
• Explain the principles of HAT- and SET based
methods of antioxidant assays
• Analyze the different applications of HAT- and
SET-based methods of antioxidant assays
Overview
• Antioxidants play an important role in food preservation by
inhibiting oxidation processes and contributing to health
promotion rendered by many dietary supplements,
nutraceuticals and functional food ingredients.
• Understanding the principle mechanisms, advantages and
limitations of the measurement assays is important for proper
selection of method(s) for valid evaluation of antioxidant
potential in desired applications
Overview
• Antioxidant activity can be monitored by a variety
of assays with different mechanisms, including
hydrogen atom transfer (HAT), single electron
transfer (ET), reducing power, and metal
chelation, among others.
Lipid oxidation and antioxidants
• Lipid oxidation is a major cause of food quality deterioration, and
has been a challenge for manufacturers and food scientists alike.
• Lipids are susceptible to oxidative processes in the presence of
catalysts such as heat, light, enzymes, metals, metalloproteins and
microorganisms, giving rise to the development of off-flavors in
foods and loss of essential amino acids, fat-soluble vitamins and
other bioactive molecules.
• In general, lipid oxidation produces lipid
hydroperoxides and conjugated dienes (or trienes) as
primary oxidation products, which are unstable and further
break down to a wide range of secondary oxidation
products, including alcohols, aldehydes, ketones,
hydrocarbons, volatile organic acids and epoxy
compounds, among others, some of which give rise to a
bad odor at very low threshold values and are responsible
for the “off-flavor” notes of oxidatively deteriorated foods.
What are antioxidants?
• Antioxidants may occur naturally in plants, animals and
microorganisms or may be synthesized by chemical
means.
• Higher plants and their constituents provide a rich source
of natural antioxidants, such as tocopherols and
polyphenols which are found abundantly in spices, herbs,
fruits, vegetables, cereals, grains, seeds, teas and oils.
What are antioxidants?
• Antioxidant means "against oxidation”.
• Antioxidants are compounds that inhibit oxidation, a
chemical reaction that can produce free radicals.
• Antioxidants play vital role in preserving the quality of food
and maintaining health of human being.
Examples of antioxidants include vitamins C
and E, selenium, and carotenoids, such as
beta-carotene, lycopene, lutein, and
zeaxanthin.
Why do we have to measure antioxidants?
• Measuring the antioxidant activity/capacity of foods and
biological samples is therefore essential not only
in ensuring the quality of functional foods, but more
importantly in studying the efficiency of food
antioxidants in preventing and treating the diseases
related to oxidative stress.
Hydrogen atom transfer (HAT) and Single
electron transfer (SET)
• Antioxidants can deactivate radicals by 2 major mechanisms.
The end results is the same, regardless of mechanism but
kinetics and potential for side reactions differ.
• Proton-coupled electron transfer and HAT reactions may occur
in parallel and the mechanism dominating in a given system will
be determined by antioxidant structure and properties, solubility,
and partition coefficient and system solvent.
• Bond dissociation energy (BDE) and ionization
potential (IP) are two major factors that determine
the mechanism and efficacy of antioxidants.
HAT-based methods
• Measures the classical ability of an antioxidant to
quench free radicals by hydrogen donation.
• Most relevant to reactions where antioxidants typically
act.
SET-based methods
• Detect the ability of a potential antioxidant to
transfer one electron to reduce any compound,
including metals, carbonyls and radicals.
Mechanisms of antioxidant reacting
with free radical: single electron transfer
(SET) and hydrogen atom abstraction
(HAT).
• In SET mechanism, the IP (ionization
potential) of the antioxidant is the
most important energetic factor in
evaluating the antioxidant action.
• In the HAT mechanism, the BDE
(bond dissociation enthalpy) of the
antioxidant is the important
parameter in evaluating the
antioxidant action.
ASSAY METHODS FOR ANTIOXIDANTS
1,1-diphenyl-2-picrylhydrazyl (DPPH) assay
Ferric ion reducing antioxidant power
(FRAP) assay
In this assay, reductants (antioxidants) in the sample reduce Fe(III) tripyridyltriazine
complex .
Oxygen radical absorbance capacity
(ORAC) assay
• The ORAC assay measures the radical chain breaking ability of
antioxidants by monitoring the inhibition of peroxyl radical-induced
oxidation.
• Peroxyl radicals are the predominant free radicals found in lipid
oxidation in foods and biological systems under physiological
conditions.
In this assay, the peroxyl radical produced by a generator reacts with a fluorescent
probe resulting in the loss of fluorescence, which is recorded with a fluorometer.
THANK YOU!
