This document provides information on several common methods used to measure antioxidant capacity in biological samples and foods. It describes the principles, procedures, and key measurements of assays such as ORAC, DPPH, ABTS, Folin-Ciocalteu, FRAP, CAA, HPLC, UPLC, and ELISA. These assays utilize different chemical reactions and detection methods to quantify a sample's ability to inhibit oxidation initiated by free radicals.

1. INSTITUT KURZ
GENERAL LABORATORY
ASSAYS
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2. OXYGEN RADICAL ABSORBANCE
CAPACITY (ORAC)
Oxygen Radical Absorbance Capacity (ORAC) is a a method that
measures the antioxidant capacity in biological samples and foods.
It is based on the inhibition of the peroxyl-radical-induced oxidation
initiated by thermal decomposition of azo-compounds.
(Ronald L. Prior et al., 2003)

3. 1. The extract is mixed with fluorescein
2. Apph (2,2’-azobis(2-amidinopropane)dihydrochloride) is added
resulting in the formation of peroxyl radicals.
3. These radicals oxidise the fluorescein to a non-fluorescent
molecule.
4. The fluorescence measurement continues until all fluorescein is
oxidised.
→ The antioxidants of the sample delay or slow down the decay of
fluorescence.
OXYGEN RADICAL ABSORBANCE
CAPACITY (ORAC)
(Zhong et al., 2015; Barba et al., 2013)

4. ORAC assay measures the antioxidant capacity of:
• Types of antioxidants that delay the reaction between radical and
fluorescein (visible as a delay time) → the reaction starts later.
• Types of antioxidants that slow this reaction (visible as a slower decrease
in fluorescence) → the reaction starts last but evolves slowly.
The course of the reaction is tracked until the complete oxidation of
fluorescein.
OXYGEN RADICAL ABSORBANCE
CAPACITY (ORAC)
(Ou et al., 2001; Ayse et al., 2010)

5. DPPH (2,2-diphenyl-1-picrylhydrazyl) is a stable compound with an
unpaired electron (a radical).
This method consists of neutralizing or reducing the free radical DPPH by
transferring electrons and or a hydrogen atom.
DPPH
(2,2 DIPHENYL-1-PICRYLHYDRAZYL)
(Sagar et al., 2011; Huang & Prior, 2005)

6. DPPH color disappears after reaction with an antioxidant (or with another
radical). The reaction between the antioxidants of a sample with DPPH is
observed by absorbance of the solution at 515 nm and is read after 6 min.
→ The reaction time is different for different antioxidants and can be
longer than 30 min.
Only organic solvents can be used due to the solubility of DPPH
→ Hydrophilic antioxidants are underestimated.
DPPH
(2,2 DIPHENYL-1-PICRYLHYDRAZYL)
(Sagar et al., 2011; Sharma et al., 2009)

7. • ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid))
• TEAC (Trolox Equivalent Antioxidant Capacity)
ABTS and TEAC are the same assay, but the way that the results are
expressed is different.
The method is based on the capacity of an antioxidant to stabilize the
ABTS colored cation radical. It can be previously formed by the oxidation
of ABTS by methemoglobin and hydrogen peroxide.
ABTS AND TEAC
(Moniruzzaman et al., 2012; Londoño, 2012)

8. The addition of antioxidants to this previously obtained radical follows an
electron transfer mechanism, which is visualized as a discoloration
corresponding to when the radical ABTS it is reduced by antioxidant.
The degree of discoloration makes it possible to evaluate the percentage of
inhibition of the ABTS cation radical, which is determined as a function of
the antioxidant concentration and the reaction time.
The result can be calculated as IC50 or by comparing with trolox
concentrations (TEAC).
ABTS AND TEAC
(Moniruzzaman et al., 2012; Floegel et al., 2011)

9. Spectrophotometric method based on the interaction of reducing
substances with the Folin-Ciocalteu reagent.
• An alkaline pH medium reduces substances, leading to the formation of
the phenolate anion.
• This anion reduces the Folin-Ciocalteu reagent, forming tungsten oxide
and molybdenum oxide.
• These oxides have a blue coloring detectable in the 760 nm spectrum
band, enabling their quantification through spectrophotometry.
• The absorbance is compared to that of a series of gallic acid or
(epi)catechin standards.
FOLIN - CIOCALTEU
(Kadriye et al., 2013; Genovese et al., 2003)

10. This assay is based on the reduction of Fe3+ to Fe2+.
2,4,6-tripyridyl-s-triazine (TPTZ) forms a complex with both Fe3+ and
Fe2+, of which the latter absorbs at 595 nm. Absorption is measured after 4
min assuming that the reaction is completed.
The reaction mechanism is a pure electron transfer. Antioxidants reacting
by hydrogen atom transfer (glutathione) do not reduce Fe(3).
Due to the endpoint at 4 min reaction time, slowly reacting antioxidants are
underestimated.
FERRIC REDUCING ANTIXIDANT POWER
(Benzie & Strain, 1999; Cerretani & Bendini, 2010)

11. CELLANTIOXIDANT ACTIVITY (CAA)
(Reşat et al., 2016)
Cell Antioxidant activity (CAA) is an assay for quantifying the
antioxidant activity of phytochemicals, food extracts, and dietary
supplements.
It is an in-vitro-measurement method that uses human or animal cell
colonies and does not require in-vivo evolvement of probands.

12. CELLANTIOXIDANT ACTIVITY (CAA)
(Wolfe & Liu, 2007)
The procedure include the following steps:
1. Sample is incubated with the cells and with a precursor probe.
2. Precursor 2′,7′-dichlorofluorescin diacetate (DCFH-DA) traverses the
cell membrane and is deacetylated by cellular esterases.
3. Intracellular ROS oxidize the deacetylated precursor (DCFH) to
fluorescent dichlorofluorescein (DCF).
4. ROS generator is added to the culture media, passes through the cell
membrane and the oxidation reaction is accelerated.
5. Fluorescence is measured at 3–5min intervals for 90min or until the
curves reaches a plateau.

13. CELLANTIOXIDANT ACTIVITY (CAA)

14. HIGH PRESSURE LIQUID
CHROMATOGRAPHY (HPLC)
High pressure liquid chromatography (HPLC) separates, identifies and
quantifies components dissolved in a liquid solvent with a high analytical
resolution103.
It requires a:
• Stationary phase – spherical solid porous particles (3-10µm), usually
made of silica, that is packed into stainless-steel columns (with an internal
diameter between 2-5mm).
• Mobile phase – liquid solvent.
(Skoog et al., 1997)

15. HIGH PRESSURE LIQUID
CHROMATOGRAPHY (HPLC)
(Skoog et al., 1997)
HPLC measurement goes through the following steps:
1. Mobile phase is introduced into the system and is pumped through the
pumps, passing continuously by the column under high pressure and
with a controlled flow.
2. Injector extract a sample volume and introduces it into the mobile
phase.
3. Sample passes through the pores (between the microspheres of the
stationary phase) with a certain pressure. → It separates the sample’s
components based on their ability to partition between the mobile and
stationary phase.
4. After leaving the column, the compounds are detected, identified and
quantified by the detector that transfer the information to a
chromatogram.

16. HIGH PRESSURE LIQUID
CHROMATOGRAPHY (HPLC)

17. ULTRA PERFORMANCE LIQUID
CHROMATOGRAPHY (UPLC)
The principle of UPLC for the separation of components in a matrix is the
same as HPLC. → The main difference is in the particle size of sorbent of
the column.
Ultra performance liquid chromatography - tandem mass
spectrometry (UPLC-MS/MS) uses separation media with a particle size
<2μm.
It operate under very high pressure to accommodate the use of small
particles and combines a <2μm reverse-phase packing material with a
chromatographic system that operates at pressures between 6000–15000psi.
UPLC offers better resolution, speed and sensitivity than HPLC.
(Trenerry & Rochfort, 2010)

18. ENZYME-LINKED IMMUNOSORBENT
ASSAY (ELISA)
Enzyme-linked immunosorbent assay (ELISA) is a plate-based assay
that detects and quantifies peptides, proteins, antibodies and hormones.
It depends on specific antibodies to bind the target antigen and a detection
system to indicate the presence and quantity of antigen binding.
→ In this assay, an antigen is immobilized to a solid surface and then
complexed with an antibody that is linked to an enzyme. The detection is
accomplished by assessing the conjugated enzyme activity via incubation
with a substrate to produce a measurable product.
(British Society of Immunology; Crowther, 2009)

19. ENZYME-LINKED
IMMUNOSORBENT
ASSAY (ELISA)

20. ENZYME-LINKED IMMUNOSORBENT
ASSAY (ELISA)
ELISA can be performed with some modifications to the basic procedure:
• Direct - uses a labeled primary
antibody that reacts directly with
the antigen. The immobilization
of the antigen of interest is done
by direct adsorption to the assay
plate.
(Crowther, 1995)

21. ENZYME-LINKED IMMUNOSORBENT
ASSAY (ELISA)
• Indirect - uses a labeled
secondary antibody for detection.
The immobilization of the
antigen of interest is done
indirectly via a capture antibody
that has been attached to the
plate
(Crowther, 1995)

22. ENZYME-LINKED IMMUNOSORBENT
ASSAY (ELISA)
• Sandwich - the analyte to be
measured is bound between two
primary antibodies (the capture
and the detection antibody).
(Crowther, 1995)

23. ENZYME-LINKED IMMUNOSORBENT
ASSAY (ELISA)
• Competitive - is used when the
antigen is small and has only one
epitope, or antibody binding site.
(Crowther, 1995)

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Huang, Boxin Ou And Robert Jacob. Assays for Hydrophilic and Lipophilic Antioxidant Capacity (oxygen radical
absorbance capacity (ORACFL)) of Plasma and Other Biological and Food Samples. J. Agric. Food Chem. 2003, 51,
3273−3279.
• Barba, F.J., Esteve, M.J., Tedeschi, P. et al. A Comparative Study of the Analysis of Antioxidant Activities of Liquid Foods
Employing Spectrophotometric, Fluorometric, and Chemiluminescent Methods. Food Anal. Methods 6, 317–327 (2013).
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Methods (2009) 2:41–60.
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v. 53, n. 6, p. 1841-1856, 2005.
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REFERENCES

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