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Badarinath, 2010 PDF
Badarinath, 2010 PDF
Badarinath, 2010 PDF
Abstract: This stimuli article provides an overview general aspect, their comparisons, correlations and considerations
of various In-Vitro methods to measure the antioxidant defense system and to discuss a number of updated In-Vitro
methods used for detection of antioxidant properties. This review article gives the information regarding the different
methods that are used to perform the In-Vitro antioxidant activity. It emphasizes the method simplicity, time required,
instrumentation which makes us to decide which method to be fallowed to perform antioxidant property based on the
feasibilities afforded to determine it. It makes a glance regarding the advantage of different methods and which is most
common method used in present days for effective analysis. On the other hand as there are advantage there may be some
disadvantages which are also mentioned in this article. The research studies should also be carried mostly on the
accurate methods for exact results which also act as a good reference for the further researches.
Key words: In-Vitro antioxidant methods, Cellular antioxidant activity, Cellular antioxidant activity, Semi quantitative
analysis, Folin-Ciocalteu method.
Introduction
A antioxidant is a chemical that prevents the oxidation ONOO [peroxy nitrate], NO2 [nitrogen dioxide] and
of other chemicals. They protect the key cell N2O3[dinitrogen trioxide].4,5 In a normal cell, there are
components by neutralizing the damaging effects of appropriate oxidant: antioxidant balance. However,
free radicals, which are natural by- products of cell this balance can be shifted, when production species is
metabolism. 1,2 Free radicals form when oxygen is increased or when levels of antioxidants are
metabolized or formed in the body and are chemical diminished. This stage is called oxidative stress.
species that posses an unpaired electron in the outer Oxidative stress results in the damage of biopolymers
(valance) shell of the molecule. This is the reason, why including nucleic acids, proteins, polyunsaturated fatty
the free radicals are highly reactive and can react with acids and carbohydrates. Lipid peroxidation is
proteins, lipids, carbohydrates and DNA. These free oxidative deterioration of polyunsaturated lipids and it
radicals attack the nearest stable molecules, stealing its involves ROS and transition metal ions. It is a
electron. When the attacked molecule loses its molecular mechanism of cell injury leading yield a
electron, it becomes a free radical itself, beginning a wide range of cytotoxic products, most of which are
chain reaction, finally resulting in the description of a aldehydes, like malondialdehyde (MDA), 4-
living cell3. Free radicals may be either oxygen derived hydroxynonrnal(HNE), Oxidative stress causes serious
(ROS, reactive oxygen species) or nitrogen derived cell damage leading to a variety of human diseases6
(RNS, reactive nitrogen species). The oxygen derived like Alzheimers disease, Parkinsons disease,
molecules are O-2 [superoxide], HO[hydroxyl] ,HO2 atheroscleorosis, cancer, arthritis, immunological
[hydroperoxyl], ROO[peroxyl], RO[alkoxyl] as free incompetence and neurodegenerative disorders, etc.
radical and H2O2 oxygen as non-radical. Nitrogen Nutritional antioxidant deficiency also leads to
derived oxidant species are mainly NO [nitric oxide], oxidative stress, which signifies the identification of
A.V.Badarinath et al /Int.J. PharmTech Res.2010,2(2) 1277
natural anti-oxidative agents present in die consumed immerse in 0.2% DPPH methanol solution and sample
by human population. 7, 8 spots were evaluated for radical scavenging activity.
The same method can be implemented to detect total
1) Comparisons phenolic and total flavonoid content just by changing
In-Vitro determination of antioxidant capacity the mobile phase solvent system and visualizing agent.
This approach has benefits over simply quantifying Vanillin /H2SO4 reagent is sprayed on the plate and
antioxidant components as it provides a measure of heating it at 1100C, for 5 minutes and observed to
their effectiveness. The various conventional and latest detect different groups of compounds. Orange-yellow
methods comes under invitro are listed in table no.1. It spots indicate poly phenolic compounds. The silicagel
is very difficult to select a suitable antioxidant assay plate was sprayed with natural products-PEG reagent
method. Antioxidants act by several mechanisms and and observed at UV-365 nm, to detect the flavonoids
no one assay can capture the different modes of action as they appear as yellow-orange fluorescent spots.
of antioxidant. Conventional cuvette assay of radical
scavenging activity is replaced by 96-well plate titer Semi quantitative analysis
assay from past couple of years. Cuvette assay method The diameter and intensity of the yellow spot depends
uses UV-Visible spectrophotometer to see the on the amount of known amount and concentrations
absorbance, where as 96-well plate method uses of the solutions. We can judge the potency of the test
ELISA plate reader for absorbance. The first method is sample. Further more, Rf values for each hand can
very tedious, time consuming method, allows only 1 be calculated and photographs can be taken.
sample to read a time and requires high quantity of 40 g shows equal antioxidant property with that of
reagent where as the second method is time saving and 50 g of rutin by this method10. A method was
it reads about 96 samples at a time, with small amount developed to measure the radical scavenging activity
of reagent. of compounds separated by reversed-phase TLC (RP-
TLC) using phenolic acids as model analytes. TLC
a) TLC Autography technique separation was followed by dipping the plate in a
The antiradical screening by thin layer 0.04% (wt/vol) solution of 1, 1 diphenyl-2-
chromatography (TLC) autography technique provides picrylhydrazyl (DPPH) in methanol. Reversed phase
an easy, effective and rapid way to study plant extract technique was applied for the measurement of free
profiles. No sample purification is needed as this radical-scavenging activity of rapeseed meal
technique provided a simultaneous separation and fractions.11 Bleaching of -carotene on TLC is
radical scavenging activity measurement of anti- another method. After developing and drying, plates
oxidative compounds in plant extract.9 qualitative as were sprayed with a 0.02% solution of -carotene in
well as semi quantitative analysis of antioxidants can CH2Cl2. plates were placed under natural light until
be done by this technique. discoloration of background. The yellow spots
remaining indicated the presence of antioxidant
Qualitative analysis substances. Absorbance at 517 nm was determined
In order to detect the antioxidant activity, a method after 30 min and the percentage of activity was
based on the reduction of 2,2-diphenyl-1- calculated.12 Using TLC autography, a total of 58
picrylhydrazyl(DPPH) can be carried out. DPPH is a extracts from various organs (aerial parts, leaves,
free radical stable at room temperature, which flowers, fruits, roots) of 16 Turkish plants were tested
produces a violet solution in methanol. When the free for their antibacterial, antifungal, acetylcholinesterase
radical reacts to an antioxidant, its free radical property inhibitory, antioxidant and radical scavenging
is lost due to chain breakage and its color changes to activities. Antioxidant and radical scavenging
light yellow. In the DPPH free radical scavenging activities were found to be predominant in highly
capacity assay by TLC, the extracts that produced polar extracts.13 TLC DPPH combined with Video
yellow are white spots in the purple background were scanning Documentation system including a HV-C20
considered as antioxidants. 3 x CCD video camera (Hitachi, Japan) can be
Procedure in brief is-extracts resolved in the solvent is connected with Reprostar 3 transilluminator cabinet,
spotted on the silica-gel 60F 254 plates and develop and for TLC plate imaging software supplied by
the chromatogram in adequate solvent systems. Now Camag, Muttenz, Switzerland can be used.
all the plates can be sprayed with a methanolic solution The method was successfully applied to rapeseed meal
of DPPH (2mg/ml). Thus, antioxidants appear as extract. A RPTLC method combined with video
yellow bands on a light purple background. After scanning detection for quantitative evaluation of free
spotting the extracts on the TLC plates, even uneluted radical scavenging activity of antioxidative fractions
plates also can be used to determine the qualitative from rapeseed meal by using DPPH is reported. The
antioxidant analysis. The uneluted plates also can activity was evaluated by measuring the area of bright
A.V.Badarinath et al /Int.J. PharmTech Res.2010,2(2) 1278
yellow bands against the purple background by a CCD method provides for concurrent multisample analysis
video camera after dipping the plate in 0.04% (w/v) with automated data storage, regression analyses, and
DPPH solution. DPPH scavenging activity of L- calculation of oxidation inhibition rates.
ascorbic acid and 17 well-known phenolic compounds Dihydrorhodamine was selected as the preferred
including -tocopherol, phenolic acids and flavonoids substrate for screening crude extracts, and typical
was determined by this TLC-DPPH method. assay results are presented. Novel lead antioxidants
Advantages are selected from active extracts by chromatographic
This technique is easy, effective, and rapid way to analysis with electrochemical detection. 15
study plant extract profiles. No sample separation is Advantages
needed. Potency of sample can be known. It provides fro concurrent multisample analysis with
automated data storage, regression analyses, and
b) Cellular antioxidant activity (CAA) assay calculation of oxidation inhibition rates. For screening
Recently, scientists at cornell University proposed a crude extracts and typical assay results are presented.
new measure of antioxidant activity called the cellular
antioxidant activity (CAA) assay, which they dubbed d) Cupric Ion Reducing antioxidant capacity
the next step in quantifying antioxidant activity. The (CUPRAC)
new CAA method tests antioxidant compounds CUPRAC method is Novel hydroxyl radical
activity inside cells: An approach that probably scavenging antioxidant activity assay for water-soluble
provides a more accurate gauge of the antioxidant antioxidants. Reactive oxygen species (ROS) may
power of whole foods and individual antioxidant attack biological macromolecules giving rise to
nutrients and compounds. Kellys Wolfe and Rui Hai oxidative stress-originated diseases. Since OH is very
Liu of cornell universitys food science lab developed short-lived, secondary products resulting from OH
the new method in which antioxidant reaction will attack to various probes are measured. Although the
takes place inside the cell. This new approach is more measurement of aromatic hydroxylation with HPLC /
biologically relevant as it accounts for uptake, electrochemical detection is more specific than the
metabolism, distribution and activity of antioxidant low-yield TBARS test, it requires sophisticated
compounds in cells versus solely looking at instrumentation. As a more convenient and less costly
antioxidant value. They applied the new technique to alternative, we can use p-aminobenzoate, 2,4- and 3,5-
equal amount s (100 grams ) of wild blueberries, dimethoxybenzoate probes for detecting hydroxyl
cranberries, apples, red and green grapes. The results radicals generated from an equivalent mixture of Fe(II)
placed wild blueberries on top, followed by + EDTA with hydrogen peroxide. The produced
cranberries, apples, red grapes and green grapes.14 hydroxyl radicals attacked both the probe and the
Advantages water soluble antioxidants in 370C incubated solutions
More accurate guage of antioxidant power of whole for 2 h. The CUPRAC absorbance of the ethylacetate
foods and individual antioxidant nutrients and extract due to the reduction of Cu (II)-neocuproine
compounds. This approach is more biologically reagent by the hydroxylated probe decreased in the
relevant as it accounts for uptake, metabolism, presence of OH scavengers, the difference being
distribution.14 proportional to the scavenging ability of the tested
Disadvantages compound. A rate constant for the reaction of the
Time consuming, costly. scavenger with hydroxyl radical can be deduced from
the inhibition of color formation. The second-order
c) Dye-substrate oxidation method rate constants of the scavengers were determined with
A novel microtiter plate assay was developed to competition kinetics by means of a linear plot a A0/A
determine the total peroxyl radical trapping activity of as a function of C scavenger / C probe where A0 and A are
antioxidant extracted from marine organisms by the CUPRAC absorbences of the system in the absence
measuring the inhibition rate of dye-substrate and presence of scavenger, respectively and C is the
oxidation. They compared use of dihydrorhodamine- molar concentration of relevant species. The 2,4 and
123, dihydrofluorescein and 3, 5-dimethoxybenzoates were the best probes in terms
dichlorodihydrofluorescein as reduced substrates for of linearity and sensitivity. Iodide, metabisulfite,
oxidation by peroxyl radicals generated from 2,2- hexacyanoferrate (II), thiourea, formate, and dimethyl
azobis(2-amidinopropane) dihydrochloride. The sulfoxide were shown by the modified CUPRAC assay
oxidation products of these highly reactive substrates to be more effective scavengers than mannitol,
are intensely colored dyes that absorb maximally in the glucose, lysine, and simple alcohols as in the TBARS
wavelength region, 489 to 512 nm, and their assay. The developed method is less lengthy, more
concentrations were determined photometrically using specific, and of a higher yield than the classical
a 96-well, microtiter plate reader. The microtiter plate TBARS assay. The hydroxyl radical scavenging rate
A.V.Badarinath et al /Int.J. PharmTech Res.2010,2(2) 1279
constants of ascorbic acid, formate and chelating capability of the compounds.19 The hydroxyl
hexacyanoferrate(II) that caused interference in other radical scavenging capacity and efficacy of a novel
assays could be easily found with the proposed organosiliceous anionic hydride compound, silica
procedure. 16 Apricots as five varieties of Malatya hydride, were quantified by a recently developed
region are screened for antioxidant capacity by using method. The method measures a direct relationship
CUPRAC. The novel reagent for the CUPRAC total between the hydroxyl radical scavenging capability of
antioxidant capacity assay, bis(neocuproine) copper(II) the antioxidant compound and the linear decrease in
chloride, was easily accessible, stable, selective and signal from a fluorescent 2-hydroxyterephthalate
responding to all antioxidants. Sulphite (normally product created by reacting a Fe2+-EDTA complex in
contributing to the colour formed in the CUPRAC the presence of a potential radical scavenger. A
assay) was removed prior to assay on a strongly basic fluorescence signal half-inhibition, IC 50, value of 1.4
anion exchanger at pH 3 in the form of HSO. The 0.1 m was obtained for silica hydride compounds.
CUPRAC findings correlated well with the results of The validity of the analysis was verified by electron
ABTS /TEAC and Folin assays. This work reports for spin resonance spectroscopy, spectrophotometric
the first time the use of a novel spectrophotometric analysis of NAD+ / NADH ratios, mitochondrial
method (CUPRAC) for the assay of both total membrane potential measurements and assays of
antioxidant capacity and sulphite levels of diverse reductions of both cytochrome C (Fe3+) to cytochrome
apricot samples.17 c (Fe2+) and epinephrine to adenochrome reductions. 20
Advantages Advantages
It requires sophisticated instrumentation. As a more Antioxidant capacity of number of non-refined seed
convenient and less costly alternative. The developed oils is compared with that of refined oils by using this
method is less lengthy, more specific and of a higher simple technique.
yield than the classical TBARS assay.
Disadvantages f) Enhanced chemiluminescence (ECL)
Sophisticated instruments are required which are more ECL has been used to measure antioxidant capacity in
expensive. biological fluids. The assay involves the
chemiluminescent substrate luminal. Light emission
e) Cellular antioxidant activity occurs when the luminal is oxidized by hydrogen
This method contains different principles to detect peroxide that is generated in a reaction catalyzed by
antioxidant property. It is based on solubilisation of the horseradish peroxidase ( fluid because the reaction
oils in aqueous buffer, labeling of the resulting HRP). This method can quantify the antioxidant
emulsions with a suitable reporter fluorophore, which capacity of a is sensitive to radical scavenging
reflects lipid oxidation, and continuous monitoring of antioxidants that reduce the light output. A method of
the decomposition process. Antioxidant capacity of assay of the antioxidant activity of biological sample
number of non-refined seed oils is compared with that suspected of having such activity, is under patent and
of refined oils by using this simple technique. And it this method comprises the steps of (a) initiating a
was found that, most of the antioxidative components chemiluminescent reaction and allowing said reaction
are removed from edible oils during refining process. to progress, thereby to generate a level of
18
A novel fluorometric method has been developed to luminescence, said level being selected from the group
evaluate hydroxyl radical is generated by a Co(II) consisting of (i) A rising level between 90 to 100 %
mediated Fenton-like reaction, and the hydroxyl of maximum, (ii) The maximum (iii) A post-
radical formation under the experimental condition is maximum substantially constant plateau level: (b)
indirectly confirmed by the hydroxylation of p- Adding said sample to said progressing
hydroxybenzoic acid. The fluorescence decay curve of chemiluminescent reaction, said sample causing said
FL is monitored in the absence or presence of level of luminescence generated by said reaction to
antioxidant, the area under the fluorescence decay change when said sample has antioxidant activity: (c)
curve (AUC) is integrated, and the net AUC, which is Monitoring said change in the level of luminescence:
an index of the hydroxyl radical prevention capacity, and (d) Determining the antioxidant activity of said
is calculated by subtracting the AUC of the blank from sample assayed by reference to that of samples of
that of the antioxidant. Gallic acid is chosen as a known antioxidant activity subjected to steps (a) to (c)
reference standard, and the activity of sample is above. The principle behind the enhanced
expressed as gallic acid equivalents. The method is chemiluminescent assay for TAC measurement is best
rigorously validated through linearity, precision, described in the work by (Whitechead, et.al, 1992). To
accuracy and ruggedness. A wide range of phenolic perform the enhanced chemiluminescence assay, a
antioxidants is analyzed and the hydroxyl radical signal reagent (luminal plus para-iodophenol), which
prevention capacity is mainly due to the metal- is a source of chemiluminescence, is mixed with
A.V.Badarinath et al /Int.J. PharmTech Res.2010,2(2) 1280
horseradish peroxidase (HRP)-linked immunoglobulin protein R-phycoerythrin (R-PE). In the TRAP assay
to produce ROS, which in turn is mixed with a the lag-phase induced by plasma is compared with that
substrate, hydrogen peroxide (H2O2). The power of the induced by Trolox in the same plasma sample.24
antioxidants in the seminal plasma to reduce the Advantages
chemiluminescence of the signal reagent is compared Used for measurements of in-vivo antioxidant capacity
with that of Trolox (6-hydroxy-2,5,7,8-tetramethyl in serum or plasma because it measures
chroman-2-carboxylic acid), a water-soluble nonenzymatic antioxidants such as glutathione,
tocopherol analogue, and is measured as molar Trolox ascorbic acid.24
equivalents. Although accurate, this method is Disadvantages
cumbersome and time consuming, because fresh Many different end points have been used, so
signaling reagent solution must be prepared each time comparisons between laboratories are difficult. It is
the assay is performed standardized with Trolox. relatively complex and time consuming. It also
Moreover, the signal reagent may reduce in intensity, requires a high degree of expertise and experience.
adding another technical problem. Finally, expensive
instrumentation (eg, luminometer) is needed to i) Oxygen radical absorbing capacity (ORAC) assay
measure the chemiuluminescence, which means that Basically the same principle is applied as in the TRAP
this assay is often not readily available in a assay. The ORAC assay is another commonly applied
physicians office. 21 antioxidant assay based on the ability of a test
Advantages substance to inhibit the oxidation of B-phycoerythrin
ECL has been used to measure antioxidant capacity of by reactive oxygen species, relative to Trolox.
biological fluids. This method can quantify the Proteins interfere with the analysis, partially protecting
antioxidant capacity of a is sensitive to radical R-PE when all plasma antioxidants are exhausted.
scavenging antioxidants that reduce the light output. Determination of the lag-phase TRAP and ORAC
Disadvantages assays can be performed with different radicals and
This method is cumbersome and time-consuming thus different results will be obtained depending on
because fresh signaling reagent solution must be the radical. For these reasons, results obtained with
prepared. Finally, expensive instrumentation (eg. the TRAP or the ORAC assay in plasma have to be
Luminometer) is needed to measure the interpreted with care.25
chemiluminescence. Advantages
The advantage of the AUC approach is that it implies
g) Ferric-raducing antioxidant power (FRAP) assay equally well for both antioxidants that exhibit distinct
In order to assess the modifying effect of tea lag phase and those that have no lag phases. ORAC
flavonoids on plasma antioxidant status, a variety of assay has been broadly applied in academy and in the
methods has been employed. Commonly used is the food and dietary supplement industries as a method of
FRAP assay. This is a colorimetric assay that choice to quantify AOC.25
measures the ability of plasma to reduce the intense Disadvantages
blue ferric tripyridyltriazine complex to its ferrous ORAC is limited to measurement of hydrophilic chain
form, thereby changing its absorbance.22 but ignores lipophilic antioxidants. It requires
Advantages fluorometers, which may not be routinely available in
It is simple, speedy, inexpensive, and robust does not analytical laboratories. Temperature control decreases
required specialized equipment. It can be performed reproducibility.
using automated, semiautomated, or manual
methods.22 j) Trolox equivalent antioxidant capacity (TEAC)
Disadvantages This assay is based on the ability of molecules to
FRAP cannot detect species that act by radical scavenge the stable free radical of 2,2- azinobis (3-
quenching (H transfer), particularly SH group ethylbenzothiozoline-6-sulfonic acid) in comparison
containing antioxidants like thiols, such as glutathione with Trolox, a water soluble analogue of vitamin E.
and proteins. 23, 24 The activity of a compound is therefore expressed as
TEAC. Of these assay, the ECL seems the least
h) Total radical trapping antioxidant parameter suitable to determine plasma antioxidant capacity
(TRAP) because it relies on enzymatic activity. This technique
another assay which has been applied in human plasma has not been widely applied, which limits the
is the total radical trapping antioxidant parameter possibility to compare results from different studies.
(TRAP). In this assay, the rate of peroxidation induced All the other assays have been applied in plasma
byAAPH(2-azobis(2-amidinopropane) hydrochloride) reproducibility.
is monitored through the loss of fluorescence of the
A.V.Badarinath et al /Int.J. PharmTech Res.2010,2(2) 1281
directly relates to the hydrogen atom donating ability foods in digestion may differ from liberation of same
of a compound and is not correlated to the redox in extraction for antioxidant tests. Further, dietary
potentials alone. 32 antioxidants have to be absorbed and localized in
3) Considerations of in-vitro antioxidant assay active forms in the oxidation site to enable antioxidant
methods effect. Antioxidants act by several mechanisms e.g. by
Understanding of roles of various antioxidants and donating hydrogen to radicals, reducing power, free
their activities is challenging. The use of one radical scavenging activity, metal chelating ability,
dimensional method to evaluate multifaceted inhibition of -carotene bleaching and quenching
antioxidants is not a complete analytical system. Due singlet oxygen. But unfortunately, most of the assay
to the complexity of the composition of plant methods measures any one of the following.
products, separating each antioxidant compound and a) Measuring its ability to donate an electron or
studying it individually is costly and inefficient, not hydrogen atom to a specific reactive oxygen species
withstanding the possible synergistic interactions or to any electron acceptor. b) Testing its ability to
among the antioxidant compounds in plant products. remove any source of oxidative initiation. In addition
The biggest problem is the lack of a validated assay food antioxidants may inhibit oxidation by several
that can reliably measure the antioxidant capacity of mechanisms. The dominant mechanism depends on
foods and biological samples. Several reviews have conditions. For example in case of liberation of
been published, and the opinions vary considerably. antioxidant from food into stomach, physical
There seems to be no consensus of opinions, most structure of food and temperature of the meal influence
probably due to the fact that the area of antioxidants is the dominant mechanism. But these factors do not
such a complex topic. There is considerable debate take into the account in in-vitro antiradical activity
about which method is best and it is critical to methods. Further more, we are using oxidizing
understand that these tests are done in test tubes, not in substrates, initiators, and other components in in-vitro
people, since, there are different ways to measure assay, which may not be present in the digestive
antioxidant power, leaving research people seriously system. Hence, partitioning of oxidizing substrates,
confused. The method used to measure and calculate antioxidants and provident in the studied food is
the antioxidant activity has a major impact on the critical. Antioxidant activity may be distinctly
results because, both in foods and in vitro, oxidation different in bulk oils and multiphase foods, such as
reactions are complex. Liberation of antioxidants from aemulsions.33, 34, 35, 36, 37
Conclusion
Factors affecting oxidation reactions and antioxidant heterogeneous and the reaction cannot be easily
activities in foods and in vitro differ. The current monitored in real time. Conventional chemical
approaches have still leaved many open questions. In analysis on these matrixes requires tedious sample
vitro assays can only rank antioxidant activity for treatments. Thus the efficiency is fairly low and the
their particular reaction system and their relevance to results are only qualitative. Our aim is to develop a
in vivo health protective activities is uncertain. high through out assay that can be used to monitor
Therefore, it is prodent to use more than one type of oxidation progress of an emulsion system in real time
antioxidant assay to measure antioxidant activities, and taking advantage of oxygen sensor coated micro-plate.
to include at least one assay that has biological The capacity of antioxidants in emulsion can thus be
relevance. Currently there is no convenient assay to quantified and ranked. The assay will be a valuable
evaluate antioxidant capacity in a food system, tool for identifying better antioxidants for food
particularly in emulsions and foams, which are preservations and cosmetic products.
A.V.Badarinath et al /Int.J. PharmTech Res.2010,2(2) 1284
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