To characterize the antioxidants in food, it is necessary to know: -How much antioxidant is contained in the food. -Are the antioxidants absorbed from the food to the blood? -If the antioxidants in the blood make their way to the intended body cells. -Do the antioxidants help the cells to defend themselves against oxygen radicals? May it be given by increasing the production of the cell's own defense enzymes or by direct chemical neutralization of the radicals? For all these above-mentioned steps different measurement techniques exist.

2. LABORATORY ANALYSIS OF
ANTIOXIDANTS IN FOOD
• To assess the efficacy of an antioxidant for food preservation à it is
enough to check if the food is in fact preserved.
• To assess the efficacy of antioxidant with respect to its effects in the
human body à more complex tests are required.

3. To characterize the antioxidants in food, it is necessary to
know:
1. How much antioxidant is contained in the food.
2. Are the antioxidants absorbed from the food to the blood?
3. If the antioxidants in the blood make their way to the intended body
cells.
4. Do the antioxidants help the cells to defend themselves against oxygen
radicals? May it be given by increasing the production of the cell's own
defense enzymes or by direct chemical neutralization of the radicals?
ØFor all these above-mentioned steps different measurement techniques
exist.

4. HOW MUCH ANTIOXIDANT IS
CONTAINED IN THE FOOD?
There are a multitude of
antioxidants in such foods as:
• Fruits
• Vegetables
• Cereals
• Mushrooms
• Beverages
• Spices
• Medicinal herbs
These natural antioxidants are
mainly:
• Polyphenols (phenolic acids,
flavonoids, anthocyanins,
lignans and stilbenes)
• Carotenoids (xanthophylls and
carotenes)
• Vitamins (vitamin E and C)
(Douglas et al., 2017; Dong-Ping et al., 2017)
à Higher intake of antioxidant-rich foods is associated with better health
and functional longevity.

5. Antioxidants have a wide range of biological effects:
• anti-inflammatory
• Antibacterial
• Antiviral
• anti-aging
• Anticancer
• Oppose ROS action and limit oxidative stress
Due to their various nutritional function and health benefits, antioxidants
have been increasingly investigated.
HOW MUCH ANTIOXIDANT IS
CONTAINED IN THE FOOD?
(Dong-Ping et al., 2017)

6. To know which antioxidants and how much are present in foods, it is
necessary to carry out specific tests.
This is usually done by:
üenzymatic reactions (commercially available enzymatic kits
üchromatographic methods such as HPLC.
There are different commercially available test methods to determinate the
antioxidant capacity.
HOW MUCH ANTIOXIDANT IS
CONTAINED IN THE FOOD?
(Zainol et al., 2019)

7. OXYGEN RADICAL ABSORBANCE
CAPACITY (ORAC)
• Prior to the ORAC measurement, solid samples are extracted with
acetone and water. If necessary, liquid samples must be filtered. The
extract is mixed with fluorescein.
• Apph (2,2’-azobis(2-amidinopropane) dihydrochloride) is added resulting
in the formation of peroxyl radicals. These radicals oxidize the fluorescein
to a non-fluorescent molecule. It is observed by measuring the decreasing
fluorescence in time intervals (1 or 2 minutes).
(Aurelia et al., 2016; Zhong et al., 2015)

8. • The fluorescence measurement continues until all fluorescein is oxidized
(10 to 60min). The antioxidants of the sample delay or slow down the
decay of fluorescence.
• Standard solutions of trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid) are measured in the same way. The linear relation
between the area under the curve of a given sample and the area under
the curve of the trolox allows for the calculation as a “relative”
antioxidant capacity.
• The antioxidant capacity is expressed as µmol trolox equivalents (TE) per
g or mL of sample (or equivalent units)15.
OXYGEN RADICAL ABSORBANCE
CAPACITY (ORAC)
(Zhong et al., 2015; Barba et al., 2013)

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

10. ORAC values of some foods.
Following (David B. Haytowitz and Seema, 2010)

11. DPPH is a stable compound with an unpaired electron (a radical).
Its colour 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.
DPPH
(2,2 DIPHENYL-1-PICRYLHYDRAZYL)
(Sagar et al., 2011)

12. The absorbance is often read after 6 min. It must be considered that 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.
Therefore, hydrophilic antioxidants are underestimated.
DPPH
(2,2 DIPHENYL-1-PICRYLHYDRAZYL)
(Sagar et al., 2011; Sharma et al., 2009)

13. • 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 chemical principle is the same as in the DPPH assay:
• A coloured radical (formed by the reaction of DPPH and persulfate) is
bleached by the reaction with the antioxidants of a sample. The
decreasing colour (the decreasing absorbance) is measured.
ABTS AND TEAC
(Moniruzzaman et al., 2012)

14. The result can be calculated as IC50 or by comparing with trolox
concentrations. In the latter case the acronym TEAC is used for the assay.
ØIC50 (Half-maximal inhibitory concentration) is the most used and
informative measure of a drug's efficacy. It indicates how much of a
substance is needed to inhibit a biological process or biological
component by 50%. The values are expressed as molar concentration.
The biological component could be an enzyme, cell, cell receptor or
microorganism.
ABTS AND TEAC
(Moniruzzaman et al., 2012; Aykul et al., 2016)

15. This assay was intended and is often described as a polyphenol
determination but, it reacts with any antioxidant.
A mixture of sodium tungstate and sodium molybdate is reduced by the
antioxidants of a sample at basic ph. The reduced form is measured
photometrically at 760 nm after completion of the reaction (usually 2 h).
The absorbance is then compared to that of a series of gallic acid or
(epi)catechin standards.
The result is expressed as mg gallic acid or (epi)catechin equivalents (equiv.)
per g or mL of sample. Other polyphenols (quercetin or chlorogenic acid)
can be also used as a reference.
FOLIN - CIOCALTEU
(Kadriye et al., 2013)

16. 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
(Ayse et al., 2009; Huang et al., 2005)

17. Bioassays are quantitative biological assays used to determine
concentration or potency of a substance by its effect on living animals (in
vivo) or tissue/cell culture systems (in vitro).
The bioassays for determination of the antioxidant capacity use the whole
cell or its derived biological material treated with the antioxidant alongside
with a generator of free radicals. That is done to provoke an oxidation
reaction.
It is measured through the detection of a molecular marker of cellular
stress.
BIOASSAYS FOR THE DETERMINATION
OF ANTIOXIDANT CAPACITY
(Ursula., 2018)

18. There are many quantitation procedures with both direct and indirect
measurements:
• the activity of SOD
• catalase
• glutathione
• the activity of oxidases,
• the oxidation inhibition using external stressors
Different biological models combined with different bioassays produce
different results à It is imprudent to compare data obtained with different
conditions.
(Ursula, 2018; Meghri et al., 2019)
BIOASSAYS FOR THE DETERMINATION
OF ANTIOXIDANT CAPACITY

19. The in vitro antioxidant potential of a compound provides information on
how the cells react to the antioxidant based on the condition that the
antioxidants reaches the cells.
The in vitro antioxidant potential does not describe, however, whether this
compound will be absorbed or metabolized in vivo.
(Aruoma, 1996)
BIOASSAYS FOR THE DETERMINATION
OF ANTIOXIDANT CAPACITY

20. Enzymatic systems (superoxide dismutase, catalase and glutathione
peroxidase) act as endogenous antioxidants in cells.
In normal healthy conditions, the endogenous antioxidant systems can take
under control 99% of the produced ROS.
The biological activity of the endogenous enzymatic systems can be
modulated by exogenous compounds, thus increasing or decreasing their
activities.
The increase of the antioxidant enzymes activity and the reduction of
oxidases activity contribute to the protection of molecules.
ANTIOXIDANT ENZYMES
(Ursula, 2018; Ighodaro & Akinloye, 2018)

21. The most important endogenous antioxidant enzymes are:
• SOD - catalyses the dismutation of superoxide into hydrogen peroxide or
molecular oxygen.
• Catalase - catalyses the decomposition of hydrogen peroxide to water
and molecular oxygen.
ANTIOXIDANT ENZYMES
(Ighodaro & Akinloye, 2018; Weydert & Cullen, 2010)

22. SOD activity can be measured by activity assays or activity gels.
Biochemical method: xanthine-xanthine oxidase is used to generate
superoxide (O2•−). Nitroblue tetrazolium (NBT) reduction is used as an
indicator of O2•− production.
SOD will compete with NBT for O2•−. The percent inhibition of NBT
reduction is a measure of the amount of SOD present. Catalase is included
to remove H2O2 produced by SOD.
The original concentration of sample protein should be around 20 μg/μl.
Various amounts of protein are added into tubes until maximum inhibition
of NBT reduction is obtained.
ANTIOXIDANT ENZYMES
(Weydert & Cullen, 2010)

23. The SOD activity gel assay is based on the inhibition of the reduction of
NBT by SOD.
The principle of this assay is based on the ability of O2•− to interact with
NBT reducing the yellow tetrazolium within the gel to a blue precipitate.
Areas where SOD is active develop a clear area (achromatic bands)
competing with NBT for the O2•−. Once run, the gels are stained for SOD
activity.
Stained native activity gels will have a light to dark purple appearance with
clear bands representing the area where SOD enzymes are present.
ANTIOXIDANT ENZYMES
(Weydert & Cullen, 2010)

24. Catalase activity can be measured by a spectrophotometric
procedure measuring peroxide removal.
It is a direct assay with pseudo-first order kinetics. The rate of peroxide
removal by catalase is exponential. Catalase begins to be inactivated by
H2O2 (at levels greater than 0.1 M).
By the end of the assay, H2O2 is consumed and catalase is inactivated.
ANTIOXIDANT ENZYMES
(Ighodaro & Akinloye, 2018)

25. Catalase activity gels can also be used.
These will be in a green-blue colour with white broad bands where the
enzyme is present. After separation of native protein, the catalase enzyme
removes the peroxides from the area of the gel it occupies.
Removal of peroxide prevent the reduction of potassium ferricyanide into
potassium ferrocyanide, which reacts with ferric chloride to form a Prussian
blue precipitate.
Catalase gels will have one band that rarely saturates getting larger with
increasing catalase activity.
ANTIOXIDANT ENZYMES
(Ighodaro & Akinloye, 2018)

26. The enzymatic assays measure the consumption of the substrate or the
production of the reaction product over time.
Colorimetric methods are widely used to indirectly measure the
enzymatic activity or its inhibition concentrations. This process involves a
dye substrate that is oxidized upon reaction with one of the molecules
implicated in the enzymatic catalysis.
ANTIOXIDANT ENZYMES
(Ursula, 2018)

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