This document discusses various methods for analyzing antioxidant capacity, including ORAC, HORAC, CUPRAC, DPPH, PFRAP, fluorometry, FRAP, and ABTS assays. The ORAC assay measures antioxidant scavenging against peroxyl radicals using fluorescein as a fluorescent probe. The HORAC assay similarly uses a fluorescent probe to measure protection against hydroxyl radicals. CUPRAC determines antioxidant capacity based on copper ion reduction. DPPH measures hydrogen or electron donation through color change. PFRAP analyzes reducing ability through complex formation. Fluorometry quantifies antioxidants in biodiesel. FRAP detects redox potentials below 0.7 V through color change.

1. ANTIOXIDANT CAPACITY
ANALYSIS
By
Shune Lei Thu
Cahyuning Isnaini
MSc Food Technology Program School of Agro Industry
Mae Fah Luang University

2. Antioxidants are compounds capable to
either delay or inhibit the oxidation
processes which occur under the
influence of atmospheric oxygen or
reactive oxygen species.
ANTIOXIDANT

4. ORAC (oxygen radical absorption
capacity) assay
The ORAC assay provides a controllable source of
peroxyl radicals that model reactions of antioxidants with
lipids in both food and physiological systems, and it can
be adapted to detect both hydrophilic and hydrophobic
antioxidants by altering the radical source and solvent
Method measures the antioxidant scavenging activity against the peroxyl radical
Induced by 2,2’- azobis-
(2-amidino-propane)
dihydrochloride
(AAPH), at 37oC
Fluorescein --------> fluorescent probe ---------> reaction
with the peroxyl radical ------> loss of fluorescence
(indicator)
Determine the quantity of antioxidant decomposition amount
Use to study the AOC of many compounds and
food samples. Industry has accepted the method
to the point that some nutraceutical
manufacturers are beginning to include ORAC
values on product labels. 4
ROS (relative oxygen species)  Free Radical (if over necessary intermediate)
 cancer, againg [degenerative diseasecardiovascular, Alzheimer]

5. HORAC (hydroxyl radical antioxidant
capacity) assay
The initial fluorescence was measured, after which the readings were taken every minute after
shaking. Gallic acid solutions were used for building the standard curve.
Measurement of the antioxidant capacity of biological fluids, bio cells, and extracts under the conditions of
Fenton-like reactions
Use a Co(II) complex to evaluate the protecting ability against the formation of hydroxyl radical
Fluorescein is incubated with the sample to be analyzed
added
Fenton mixture (generating hydroxyl radicals)
HORAC antioxidant capacity  cell lysate, plasma, serum, tissue homogenates, and food extracts (fast and reliable)

6. CUPRAC (cupric reducing antioxidant
power) assay
• CUPRAC  method for polyphenol and flavonoid number and plasma serum antioxidant
capacity
• Standard antioxidants or extracts are mixed with CuSO4 and neocuproine.
• After 30min, the absorbance was measured at 450nm.
• In the assay, Cu(II) is reduced to Cu(I) through the action of electrondonating
antioxidants.
• Results are expressed in milligrams of Trolox per liter of extract.

7. DPPH method (standard 2,2-Diphenyl-1-
picrylhydrazyl)
Formation of DPPH upon absorption of hydrogen
from an antioxidant. Therefore, the antioxidant
concentration effect can be easily evaluated by
following the decrease of UV absorption at 517
nm. Trolox is used as standard antioxidant
Measure the antioxidant activity of tissue extracts Act as free radical scavengers or hydrogen donors
DPPH• reacts with a
hydrogen donor of
antioxidant/reducing
compound
Reduced (molecular) form (DPPH) is generated
color changing from
purple (strong absorption,
517nm) to yellow
Stable free radical
Antioxidant capacity determination in fruit juices and fruit (guava) extracts

8. The PFRAP (Potassium ferricyanide reducing
power) method
• An absorbance increase can be correlated to the reducing ability of
antioxidants/antioxidant extracts. The compounds with antioxidant
capacity react with potassium ferricyanide, to form potassium
ferrocyanide. The latter reacts with ferric trichloride, yielding ferric
ferrocyanide, a blue coloured complex, with a maximum absorbance at
700 nm.
Potassium ferricyanide reduction by
antioxidants and subsequent reaction of
potassium ferrocyanide with Fe3+

9. Fluorometry
• The fluorescence spectroscopy has been applied for the determination of phenolic
compounds in oils. A method based on fluorescence is proposed to quantify the
butylhydroxyanisole (BHA) and tert-butylhydroquinone (TBHQ) antioxidant
concentration in biodiesel produced from sunfower and soybean oils.
• It was verified that the fluorescence intensity around 330nm increases linearly as
function of antioxidant concentration with correlation coefficient of about 1,
regardless of the oil source and antioxidants
Fluorescence and excitation spectra of the
solutions were recorded at room
temperature using a spectrofluorimeter.
The emission spectra were obtained under
excitation at about 310 nm and
fluorescence in the 320-800nm range was
evaluated.
Biodiesel samples without BHA and
TBHQ showed fluorescence band at
about 420nm, which can be attributed to
tocopherols, inherent to the vegetable
oils used in the biodiesel production.
The addition of BHA and/or TBHQ is
responsible for the occurence of a
fluorescence band around 330nm.

10. FRAP (Ferric Reducing Antioxidant Power) assay
The reaction detects compounds with redox potentials of
<0.7 V (the redox potential of Fe3+-TPTZ), so FRAP is a
reasonable screen for the ability to maintain redox status
in cells or tissues.
The standard curve was linear between 25 and 800 mM Trolox.
Results are expressed in mM TE/g fresh mass. Additional dilution was
needed if the FRAP value measured was over the linear range of the
standard curve.
The FRAP assay was originally developed by Benzie and Strain to
measure reducing power in plasma, but the assay subsequently has
also been adapted and used for the assay of antioxidants in botanicals.
The reaction measures reduction of ferric 2,4,6-
tripyridyl-s-triazine (TPTZ) to a colored product
taken at 593 nm.
Reducing power appears to be related to the degree
of hydroxylation and extent of conjugation in
polyphenols.
However, FRAP cannot detect compounds that act
by radical quenching (H transfer), particularly
thiols and proteins. This causes a serious
underestimation in serum.
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11. ABTS assay (2, 2’-Azinobis (3-etilbenzatiazolin)-
6-sulfonat acid)
A simpler and more frequently applied approach, is the
decolorization of preformed ABTS. An obvious drawback
of ABTS-based assays is the promiscuity of reactions of
ABTS• which is a nonphysiological free radical
The peroxidase substrate 2,2'-azino-bis(3-ethylbenzthiazoline- 6-
sulphonic acid) (ABTS), forming a relatively stable radical (ABTS)
upon one-electron oxidation, has become a popular substrate for
estimation of total antioxidant capacity. Kinetic assays, including the
commercialized TAS assay (Randox), are based on the inhibition of
the formation of ABTS by one-electron oxidants.
.
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12. TRAP (Total Peroxyl Radical Trapping
Antioxidant Parameter) assay
According to Ciz (2010), It was impossible to detect the
antioxidant capacity using TRAP method in samples
where the quantity of polyphenols was equal to or less
than 35 mg per 100 g of fresh weight.
The luminol-enhanced chemiluminescence (CL) was used to follow up the peroxyl radical reaction. The CL
signal is driven by the production of luminol derived radicals from thermal decomposition of AAPH. The TRAP
value is determined from the duration of the time period (Tsample) during which the sample quenched the CL
signal due to the present antioxidants. A known quantity (8.0 nM) of trolox, a water-soluble analogue of
tocopherol was used as a reference inhibitor (Ttrolox) instead of the sample.
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13. Methods of Total Antioxidant Capacity Assessment

14. Reference
• Pisoschi A.M. 2011, Methods for Total Antioxidant Activity
Determination: A Review, Pisoschi and Negulescu, Biochem & Anal
Biochem, 1:1.
• Reşat Apak, 2007, Mechanism of antioxidant capacity assays and the
CUPRAC (cupric ion reducing antioxidant capacity) assay,
Microchimica Acta, April 2008, Volume 160, Issue 4, pp 413-419
(http://link.springer.com/article/10.1007%2Fs00604-007-0777-0)
• www.cellbiolabs.com/sites/default/files/STA-346-horac-assay-kit.pdf
• www.bmglabtech.com/en/technology/assay-technologies/orac/

15. THANK YOU VERY MUCH