ORAC - CSIC

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ORAC - CSIC

COST-927 Action
THERMALLY PROCESSED FOODS: Possible health implications
Building Skills on the Determination of the Overall Antioxidant Capacity of
Thermally Processed Foods
Olsztyn, 26 - 30 January 2009
Institute of Animal Reproduction and Food Research, Department of Food
Technology in Olsztyn, Poland
Dr. Dolores del Castillo
Antioxidant activity by the
Oxygen Radical Absorbance Capacity assay
ORAC assay
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STARTING POINT
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DID EXIST ANY PREVIOUS JOB?
Title: PHYCOERYTHRIN FLUORESCENCE-BASED ASSAY FOR
PEROXY-RADICALS - A SCREEN FOR BIOLOGICALLY RELEVANT
PROTECTIVE AGENTS
Author(s): DELANGE RJ, GLAZER AN
Source: ANALYTICAL BIOCHEMISTRY Volume: 177 Issue:
2 Pages: 300-306 Published: MAR 1989
Times Cited: 99
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WHAT ARE THE MAIN
DISADVANTAGES OF THAT
METHOD ?
1. IT IS NOT CAPABLE OF QUANTITATING RESULTS
2. IT IS LIMITATED ONLY TO SCREENING THE
RADICAL SCAVENING CAPACITY OF THE SAMPLE
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Basic definitions for easy understanding of the
PRINCIPLES of different analytical methods for
assessing total antioxidant capacity
REDUCTION= A GAIN OF ELECTRONS
OXIDATION= A LOSS OF ELECTRONS
REDUCTANT OR REDUCING AGENT= A SUBSTANCE THAT DONATES
ELECTRONS AND CAUSES TO ANOTHER REACTANT TO BE REDUCED
OXIDANT OR OXIDATING AGENT= A SUBSTANCE THAT ACCEPTS
ELECTRONS AND CAUSES ANOTHER REACTANT TO BE OXIDIZED
ANTIOXIDANT= SUBSTANCE THAT WHEN PRESENT AT LOW
CONCENTRATIONS COMPARED WITH THOSE OF AN OXIDIZABLE
SUBSTRATE, SIGNIFICANTLY PREVENTS OR DELAYS A PRO-OXIDANT
INITIATED OXIDATION OF THE SUBSTRATE
PRO-OXIDANT= SUBSTANCE THAT CAN CAUSE OXIDATIVE DAMAGE TO
LIPIDS, PROTEINS AND NUECLEIC ACIDS. IT SYNONYM FOR REACTIVE
SPECIES. CHEMICALLY, IT IS AN OXIDANT. AN ANTIOXIDANT CAN
EFFICIENTLY REDUCE A PRO-OXIDANT
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REACTIVE SPECIES IN BIOLOGICAL SYSTMES:
HYDROXYL RADICALS (•OH)
SUPEROXIDE ANIONS (O2-)
SINGLET OXYGEN (1O2)
HYDROGEN PEROXIDES (H2O2)
ORGANIC PEROXIDES (R-OOH)
NITRIC OXIDE
PEROXYNITRITE
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DEFINITION OF ANTIOXIDANT ACTIVITY
ABILITY OF A COMPOUND TO REDUCE PROOXIDANTS OR REACTIVE SPECIES OF
PATHOLOGIC SIGNIFICANCE
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AN ANTIOXIDANT IS A REDUCTANT, BUT A
REDUCTANT IS NOT NECESSARILY AN ANTIOXIDANT
Na, GLUCOSE, ETHANOL AND MANY
OTHER REDUCTANT ARE NOT
ANTIOXIDANTS
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DIETARY ANTIOXIDANTS
ESSENTIAL
VITAMINE E (TOCOPHEROL)
VITAMINE C (ASCORBIC ACID)
VITAMIN A (RETINOL AND CAROTENOIDS)
NUMEROUS MINERALS (Cu, Mn, Zn, Se, Fe)
NON-ESSENTIAL
GLUTATHIONE, SMALL PEPTIDES
PHYTOCHEMICALS (THOUNDS IN FOOD SUPPLY)
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Basis of the ORAC assay
Reactive Oxygen Species (ROS) generators
1. Thermal control chemical generator
a. Aqueous soluble
b. Lipid soluble
2. Hydrogen peroxide, superoxide generator
3. Radiation related hydroxyl radical generator
ROS (R•, ROO•, O2•-, H2O2, •OH)
ANTIOXIDANTS
λExc=540 nm
OXIDATION
βPE INDICATOR
PROTEIN Oxidation
Decreased 565 nm emission fluorescence light
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Properties of Phycobiliproteins
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Absorbance
maximum1
(nm)
Fluorescence
emission2
(nm)
Molecular
weight3
(kda)
Absorbtivity4
(L/g-cm)
Molar
absorptivity4
(M-cm)-1(10-6)
Fluorescence:
Absorbance5
(relative to RPE)
RPhycoerythrin
565 (495)
575
240
8.2
1.97
1.00
BPhycoerythrin
545
575
240
10.0
2.40
1.40
Y-Phycoerythrin
~495 (545)
~563
C-Phycocyanin
615
647
220
7.0
1.54
0.15
R-Phycocyanin
617 (555)
637
100
7.0
0.70
0.14
Allophycocyanin
652
660
100
7.3
0.73
0.30
Phycoerythrin
566
566
617
55
8.0
0.44
0.25
Phycoerythrocy
anin
575
625
100
8.5
0.85
0.50
Pigment
1
0.50
Values in parentheses indicate secondary absorbance maxima.
Phycobiliproteins are aggregates of subunits, and various aggregates may occur in aqueous solution. Values given are
for most common reported aggregates; aggregates of both larger and smaller size may occur.
3
Value for Phycoerythrin 566 is an estimate.
4
An approximate relative indicator of the quantum efficiency of the pigment (measured at absorbance and emission
maxima).
2
Phycobiliproteins are water soluble
fluorescent proteins derived from
cyanobacteria and eukaryotic algae.
Nomenclature: Phycobiliproteins are classified on the
basis of their color into two large groups, the
phycoerythrins (red) and the phycocyanins (blue).
Absorption maxima for phycoerythrins lie between 490
and 570 nm while absorption maxima for phycocyanins
are found between 610 and 665 nm. These large groups
have been subdivided to reflect variation among the
proteins in the exact location of the absorbance
maximum and the specific shape of the absorbance
spectrum.
The B-phycoerythrin (B-PE) protein is the major light-harvesting pigment
of red microalgae Porphyridium cruentum. B-PE finds application mainly
in the pharmaceutical sector as fluorescent probe in biomedical analysis,
and also in the food and cosmetics industries. In spite of its numerous
uses, however, its large-scale purification remains problematic and
expensive.
B-PE is used in the pharmaceutical industry as a fluorescent probe and
reagent. It is also utilised as a natural dye in the food and cosmetics
industries due to its high fluorescence efficiency. The protein's
purification from algal extracts involves a number of often-crude, highly
complex steps, many of which are non-scaleable and result in
unsatisfactory yields.
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WHY DID THE AUTHORS SELECT B-PE AS
FLUORESCENCE PORBE?
1. B-PE´S DISTINCT EXCITATION AND EMISSION
WAVELENGTHS
2. HIGH FLUORESCENCE YIELD
3. SENSITIVITY TO ROS
4. WATER SOLUBILITY
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How to classify ORAC assay ?
INHIBITION METHOD
Involve a pro-oxidant and
oxidizable substrate
Antioxidant capacity of the
antioxidant
The pro-oxidant induces oxidative
damage to the substrate which is
inhibited in the presence of an
antioxidant
ORAC ASSAY MEASURES THE CAPICITY OF THE
ANTIOXIDANT TO DIRECTLY QUENCH FREE RADICALS
Very high molar ratio of AAPH to
antioxidants (mora than 2000)
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ADVANTAGES COMPARED TO PREVIOUS METHODS
1. The use of peroxyl and hydroxyl radicals as pro-oxidants
in the ORAC assay makes its different and unique from
the assays that involve oxidants that are necessarily prooxidants.
2. An improve in quantitation is achieved in ORAC assay by:
a) Taking the reaction between substrate and free radicals to
completation
b) Using an area-under-curve technique for quantitation
compared to the assay that measure the lag phase.
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ANTIOXIDANT ASSAYS
Involving oxidants that are not
necessarily pro-oxidants
Involving oxidants that are prooxidants
TRAP assay
FRAP assay
Luminol-based assays
TEAC assay
Dichlorofluorescin-diacetate
(DCFH-DA) based assay
Cyclic voltammetry assay
Crocin based assay
Phycoerythrin (PE) based assay
ORAC assay
Prior and Cao. Free Radical Biology & Medicine 27 (11/12), 1173-1181, 1999
Title: OXYGEN-RADICAL ABSORBENCY CAPACITY
ASSAY FOR ANTIOXIDANTS
Author(s): CAO GH, ALESSIO HM, CUTLER RG
Source: FREE RADICAL BIOLOGY AND
MEDICINE Volume: 14 Issue: 3 Pages: 303311 Published: MAR 1993
Times Cited: 441
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THE AMOUNT OF SAMPLE CAN BE
SUSTANTIALLY
REDUCED
AND
THE
SIMULTANEOUS KINETIC ANALYSIS OF
MANY SAMPLES IS MADE POSSIBLE BY
USSING
FLUORIMETRY
MICROPLATE
READER USING A 96-WELL PLATE
Cao, G, Verdon, CP, Wu AH, Wang H and Prior RL. Automated
assay of oxigen radical absorbance capacity with COBAS FARA II.
Clinical Chemistry 41, 1738-1744, 1995.
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Title: AUTOMATED-ASSAY OF OXYGEN RADICAL ABSORBENCY
CAPACITY WITH THE COBAS FARA-II
Author(s): CAO G, VERDON CP, WU AHB, et al.
Source: CLINICAL CHEMISTRY Volume: 41 Issue: 12 Pages: 17381744 Part: Part 1 Published: DEC 1995
Times Cited: 218
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Title: Development and validation of an improved oxygen radical
absorbance capacity assay using fluorescein as the fluorescent probe
Author(s): Ou BX, Hampsch-Woodill M, Prior RL
Source: JOURNAL OF AGRICULTURAL AND FOOD
CHEMISTRY Volume: 49 Issue: 10 Pages: 4619-4626 Published:
OCT 2001
Times Cited: 216
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WHAT DID THEY PROPOSE THIS TIME ?
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QUANTITATION
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SCHEMATIC ILUSTRATION OF THE PRINCIPLE
OF THE ORACFL ASSAY
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HOW DOES THE CALIBRATION CURVE LOOK?
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Molecular formula
Molar mass
C20H12O5
332.306 g/mol
Chemical and physical properties
1. Very high fluorescence
2. Excitation occurs at 494 nm and emission
at 521.
3. pKa of 6.4
4. Ionization equilibrium leads to pHdependent absorption and emission over
the range of 5 to 9
5. Fluorescence lifetimes of the protonated
and deprotonated forms of fluorescein
are approximately 3 and 4 ns
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07367 Fluka B-Phycoerythrin
concentration~10.0 mg/mL
(UV)fluorescenceλex 542 nm; λem 576 nm in 0.1 M
phosphate pH7.2
storage temp.2-8°C
Prices: 1mg 82.80 euros; 5mg 318.00 euros
P128 Sigma
B-Phycoerythrin from Porphyridium cruentum
(lyophilized powder)
1mg 356 euros
46960 Fluka Fluorescein sodium salt
Fluorescence λex 490 nm; λem 514 nm in 0.1 M Tris pH 8.0
Prices: 25 g 12.90 euros, 100 g 37.10 euros, 500 g 142.50
euros
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ARE THERE OTHER REASONS FOR CHANGING THE
FLUORESCENCE PROBE?
1. DIFFERENT PEs, SUCH AS B-PE AND R-PE,
POSSESS DIFERENT FLUORESCENCE INTENSITY
AND REACTIVITY TO PEROXY RADICAL
2. DIFFERENCES EXIST IN THE SAME PE WITH
DIFFERENT LOTS
3. A SINGLE PE LOT NUMBER SHOULD BE USED FOR
A PLANNED PROJECT
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HOW TO EXPLAIN THE INCONSISTENCY OF
PE FROM LOT TO LOT?
PURIFICATION
PROCESS
B-PE 30% PURE
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MORE?
B-PE INTERACT WITH
POLYPHENOLS
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COMPARISON OF FL WITH B-PE
ANALYSIS IN ABSENCE OF AAPH
B-PE
FL
STRONG PROTEIN BINDING
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B-PE DECLINED MORE RAPIDLY THAN DID FL FLUORESCENCE
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THE FL YIELDS A
CONSISTENTLY HIGHER
ORAC VALUE AS COMPARED
TO B-PE (~1.6-3.5 FOLD)
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HOW DO THEY INTERACT?
HYDROPHOBIC INTERACTION
HYDROGEN BONDING
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ORACFL MECHANISM
HAT= HYDROGEN ATOM TRANSFER
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ORACFL MECHANISM
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ORACFL MECHANISM
THE FOLLOWINGS EQUATIONS ILUSTRATES
THE STEPWISE PROCESS OF HAT
RADICAL
INICIATOR
ABSTRACTS A HYDROGEN
ATOM FROM THE ANTIOXIDANT
REACTION
BEWTEEN ROO•
AND THE
TARGET
MOLECULE IS
INHIBITED
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SUMMARY OF IMPROVEMENTS
1. ROGGEDNESS
2. FL DOES NOT INTERACT WITH ANTIOXIDANT
SAMPLES
3. FL SHOWS EXCELLENTE PHOTOSTABILITY SO THAT
THE ORACFL ASSAY CAN BE TRANSFERRED TO A 96WELL PLATE READER.
4. THE USE OF FL REDUCES THE COST OF THE
EXPERIMENT
5. ORACFL ASSAY DIRECTLY ESTIMATES THE CHAINBREAKING ANTIOXIDANT ACTIVITY
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WHAT DOES ORACFL ASSAY MEASURE?
ORACFL ASSAY MEASURES HYDROPHYLIC
ANTIOXIDANT ACTIVITY AGAINST PEROXYL
RADICAL
ORACFL ASSAY CANNOT BE CONSIDERED A “TOTAL ANTIOXIDANT
ACTIVITY ASSAY”
IT IS IMPOSSIBLE TO MEASURE TOTAL
ANTIOXIDANT ACTIVITY USING A SINGLE
ASSAY
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FOOD APPLICATION
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FOOD APPLICATION
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WHAT DID THE AUTHORS CONCLUDE?
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FURTHER IMPROVEMENTS
Times Cited: 103
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ADVANTAGES
1. THE EFFICIENCY IS IMPROVED WITH AT
LEAST A 10 FOLD INCREASE IN SAMPLE
PREPARATION AND IN INSTRUMENT
UTILIZATION (130 SAMPLES/DAY)
2. THE USE OF THE ROBOTIC LIQUID
HANDLING AND PLATE READER
TECHNOLOGIES HAS NOT
COMPROMISED THE QUALITY OF DATA
OBTAINED
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NEXT STEP
Volume: 50 Issue: 7 Pages: 1815-1821 Published: MAR 27 2002
Times Cited: 90
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WHAT DID THEY DO?
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ORACFL-LIPO ASSAY
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MECHANISMS FOR OXIDATION
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MECHANISMS FOR OXIDATION
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FOOD APPLICATION
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MAIN IMPROVEMENT
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FURTHER DATA ON FOOD
DATA ON LIPOPHILIC AND HYDROPHILIC
ORACFLVALUES FOR OVER 100 COMMON FOODS IN
U.S. MARKETS
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FURTHER DATA ON FOOD
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WHAT ABOUT CONCLUSIONS?
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Oxygen Radical Absorbance Capacity (ORAC) of
Selected Foods - 2007
http://www.ars.usda.gov/Services/docs.htm?docid=15866
This database contains ORAC data for 277 food items and represents a
collaboration between the Nutrient Data Laboratory, Beltsville Human
Nutrition Research Center and the Arkansas Children's Nutrition Center
in Little Rock, Arkansas.
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The database will be used by scientists to help guide ongoing research into how antioxidants
may correlate to health benefits. For example, many fruits and vegetables are known to be
good sources of antioxidant vitamins, such as E, C, and beta carotene, a form of vitamin A.
But these natural foods also contain other compounds, collectively known as phytonutrients,
that may contribute to health.
http://www.ars.usda.gov/is/pr/2007/071106.htm
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How to access to the databse?.
Go to:
http://www.ars.usda.gov/nutrientdata/ORAC
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CAN FOOD PROCESSING GIVE RISE
NEOANTIOXIDANTS?
SEVERAL CHEMICAL EVENTS
CONTRIBUTE TO THE OVERALL
ANTIOXIDANT PROPERTIES OF
PROCESSED FOODS
CAN BE MEASURED THE
NEOANTIOXIDANTS BY ORAC ASSAY?
KEY CHEMICAL EVENTS INDUCING
FORMATION OF NEW ANTIOXIDANTS
DURING THERMAL FOOD PROCESSING
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PAY ATENTION TO SAMPLES PREPARATION
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DO YOU HAVE ANY FURTHER
QUESTION?
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HMW MRPs contain reductone and amino reductone structures, which provide a potential for reducing and
metal chelation activities, both of which are relevant to the antioxidant mechanisms noted forMRPs. In the
present study, all three sugar-lysine HMW MRPs displayed a similar, significant capacity to scavenge
DDPH radicals, which was also found for earlier products of the MR generated from heated histidine-xylose
reactants. Measuring total antioxidant activity of MRPs by the ORAC method provided an excellent
basis for assessing MRP scavenging capacity for oxygen radicals between different sugar reactant
sources. It is of interest that the ORAC activity of all three HMW MRPs was not only similar when
expressed in Trolox equivalents, but also agree well with the DPPH measurements. Moreover, the Trolox
equivalent for all three MRP HMW mixtures far exceeds the total antioxidant activity determined previously
for ORAC measures of different soft fruits, such as strawberries (approx. 150 μmol Trolox/g dry matter
[dm]), blueberries (approx. 200 μmol Trolox/g dm), and raspberries (approx.100 μmol Trolox/g dm).
The potential for a large contribution from HMW MRPs to total antioxidant intake, in terms of ORAC
values, suggests that the estimated 1.2–1.7 mmol Trolox/day value proposed for US consumers may
be underestimated if MRPs derived from processed foods are considered in the ORAC range for
total antioxidant intake.
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WHAT OTHER REACTIONS MAY CONTRIBUTE TO
THE OVERALL ANTIOXIDANT ACTIVITY OF
PROCESSED FOODS?
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WHAT DID WE DO?
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DOES THE METHOD NEED FURTHER
IMPROVEMENTS?
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WHAT DO THEY THINK ABOUT THAT?
Oxygen radical absorbance capacity (ORAC) values have been obtained for a series of
teas and herbal infusions employing 2,20-azobis (2-amidinopropane) as free radical
source, and fluorescein and pyrogallol red as target molecules. The amounts of phenols
in the extracts were evaluated by Folin’s methodology. ORAC values are extremely
dependent upon the employed target molecule. Even more, relative ORAC values
measured for different infusions depend upon the employed methodology. For example,
ORAC-fluorescein value of Aloysia citriodora is larger than that of green tea, while if
pyrogallol red is employed as target molecule green tea appears as nearly nine times
more efficient. Similarly, for extracts with comparable amounts of phenols, herbal
infusions are more efficient than teas by ORAC fluorescein, while opposite conclusions
are obtained if ORAC-pyrogallol red values are considered. Extreme care must then be
taken for conclusions obtained from ORAC values estimated by employing a single
target molecule.
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IS THERE MORE WORK LEFT TO BE
DONE?
WHAT ELSE CAN WE DO?
FEEL FREE YOURSELF TO MAKE
PROPOSALS. THE DISCUSSION
SECTION IS OFICIALLY OPENED
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ORAC - CSIC

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