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ANALYTICAL PROGRESS
Summer 2001 Takes you into the Heart of a Giant Resource Volume 19 Number 2

ANTIOXIDANT ACTIVITY
by Aruna Prakash, PhD

WHAT ARE ANTIOXIDANTS? There are a number of clinical studies suggesting

that the antioxidants in fruits, vegetables, tea and
Antioxidant compounds in food play an important red wine are the main factors for the observed
role as a health-protecting factor. Scientific efficacy of these foods in reducing the incidence of
evidence suggests that antioxidants reduce risk for chronic diseases including heart disease and some
chronic diseases including cancer and heart cancers. The free radical scavenging activity of
disease. Primary sources of naturally occurring antioxidants in foods have been substantially
antioxidants are whole grains, fruits and investigated and reported in the literature
vegetables. Plant sourced food antioxidants like by Miller and Rigelhof et.al (1,2).
vitamin C, vitamin E, carotenes, phenolic acids, .
phytate and phytoestrogens have been recognized
as having the potential to reduce disease risk. Most
of the antioxidant compounds in a typical diet are METHOD CONSIDERATIONS
derived from plant sources and belong to various
classes of compounds with a wide variety of Various antioxidant activity methods have been
physical and chemical properties. Some used to monitor and compare the antioxidant
compounds, such as gallates, have strong activity of foods. In recent years, oxygen radical
antioxidant activity, while others, such as the absorbance capacity assays and enhanced
mono-phenols are weak antioxidants chemiluminescence assays have been used to
evaluate antioxidant activity of foods, serum and
The main characteristic of an antioxidant is its other biological fluids. These methods need
ability to trap free radicals. Highly reactive free special equipment and technical skills for the
radicals and oxygen species are present in analysis. These types of methods published in the
biological systems from a wide variety of sources. literature for the determinations of antioxidant
These free radicals may oxidize nucleic acids, activity of foods involve electron spin resonance
proteins, lipids or DNA and can initiate (ESR) and chemiluminescence methods. These
degenerative disease. Antioxidant compounds like analytical methods measure the radical-scavenging
phenolic acids, polyphenols and flavonoids activity of antioxidants against free radicals like
scavenge free radicals such as peroxide, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical,
hydroperoxide or lipid peroxyl and thus inhibit the the superoxide anion radical (O2), the hydroxyl
oxidative mechanisms that lead to degenerative radical (OH), or the peroxyl radical (ROO). The
diseases. various methods used to measure antioxidant

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activity of food products can give varying results low density lipoprotein oxidation mediated by
depending on the specificity of the free radical cupric ions.
being used as a reactant. There are other methods
which determine the resistance of lipid or lipid A rapid, simple and inexpensive method to
emulsions to oxidation in the presence of the measure antioxidant capacity of food involves the
antioxidant being tested. The malondialdehyde use of the free radical, 2,2-Diphenyl-1-
(MDA) or thiobarbituric acid-reactive-substances picrylhydrazyl (DPPH). DPPH is widely used to
(TBARS) (1) assays have been used extensively test the ability of compounds to act as free radical
since the 1950’s to estimate the peroxidation of scavengers or hydrogen donors, and to evaluate
lipids in membrane and biological systems. These antioxidant activity of foods. It has also been used
methods can be time consuming because they to quantify antioxidants in complex biological
depend on the oxidation of a substrate which is systems in recent years. The DPPH method can be
influenced by temperature, pressure, matrix etc. used for solid or liquid samples and is not specific
and may not be practical when large numbers of to any particular antioxidant component, but
samples are involved. Antioxidant activity applies to the overall antioxidant capacity of the
methods using free radicals are fast, easy and sample. A measure of total antioxidant capacity
simple. The ABTS [2,2’-azinobis(3- will helps us understand the functional properties
ethylbenzothiazoline-6-sulfonic acid)] radical of food.
cation (2) has been used to screen the relative
radical-scavenging abilities of flavonoids and Antioxidant activity has been expressed in various
phenolics through their properties as electron- or ways including the percentage of the reagent used,
H-donating agents. the oxidation inhibition rate and so on.

Prior et al. (3) have used the Oxygen Radical An easier way to present antioxidant activity of
Absorbance Capacity (ORAC) procedure to foods would be to reference a common reference
determine antioxidant capacities of fruits and standard. One common reference standard,
vegetables. In the ORAC method, a sample is (S)-(-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-
added to the peroxyl radical generator, 2,2’- carboxylic acid, also known as Trolox, serves as
azobis(2-amidinopropane)dihydrochloride such a common reference standard.
(AAPH) and inhibition of the free radical action is
measured (4) using the fluorescent compound, B-
phycoerythrin or R-phycoerythrin. THE DPPH METHOD

Phenolic and polyphenolic compounds constitute A simple method that has been developed to
the main class of natural antioxidants present in determine the antioxidant activity of foods utilizes
plants, foods, and beverages and are usually the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH)
quantified employing Folin’s reagent. Vinson et radical. The structure of DPPH and its reduction by
al. (5) have measured phenolics in fruits and an antioxidant are shown above. The odd electron
vegetables colorimetrically using the Folin- in the DPPH free radical gives a strong absorption
Ciocalteu reagent and determined the fruit and maximum at 517 nm and is purple in color. The
vegetable’s antioxidant capacity by inhibition of color turns from purple to yellow as the molar
3
absorptivity of the DPPH radical at 517 nm reduces (TE) per 100 gm of sample or Trolox units per 100
from 9660 to 1640 when the odd electron of DPPH gm.
radical becomes paired with a hydrogen from a free
radical scavenging antioxidant to form the reduced A difference between this method and other
DPPH-H. The resulting decolorization is published methods is carrying out the reaction of
stoichiometric with respect to number of electrons the sample itself with DPPH in methanol/water.
captured. Reacting an aqueous-methanolic DPPH solution
with the sample for 4 hours at 35 °C facilitates the
Antioxidant compounds may be water-soluble extraction of antioxidant compounds from the
lipid-soluble, insoluble, or bound to cell walls. sample thereby increasing the measured
Hence, extraction efficiency is an important factor antioxidant activity of the sample. Determination
in quantification of antioxidant activity of foods. of antioxidant activity of various types of foods
Trolox (as the reference standard) and the sample using DPPH is comparable to other methods. It is
are reacted with DPPH solution in methanol/water probable each of these methods measure a
for four hours at 35°C in a vessel mounted on a somewhat different profile of antioxidant
rotary shaker and the absorbance changes are compounds. Antioxidant analysis by other
measured at 517 nm. The quantity of sample published methods is limited to those compounds
necessary to react with one half of the DPPH is soluble in the selected solvent. Antioxidant
expressed in terms of the relative amount of activity of insoluble compounds was not
Trolox reacted. Antioxidant activity of a sample is accounted in a single extraction method.
expressed in terms of micromole equivalents of Extraction techniques using different solvents and
Trolox (TE) per 100 grams of sample, or simply concentrating the solvent is time consuming. In
this method, DPPH is allowed to react with the
whole sample. Sufficient time allows DPPH to
react slowly with weak antioxidants.

The kinetics of flavanones, flavanols and various

phenolic compounds scavenging DPPH radicals
Trolox units per 100 gm or TE/100g. were studied by electron spin resonance
spectrometry and other techniques. Compounds
with similar structures follow similar trends using
various methods.
RESULTS AND DISCUSSION
Antioxidant activity of grains, dry beans, fresh
The reaction of DPPH with numerous antioxidants vegetables and fruits were analyzed using the
has been published and the stoichiometry DPPH method. Some of these results, along with
characterized (6,7). As mentioned above, results for some standard antioxidant compounds
antioxidants in food may be water soluble, fat are presented in Tables 1 and 2. DPPH results for
soluble, insoluble, or bound to cell walls and thus standard phenolic compounds follow similar
not necessarily freely available to react with DPPH, trends as observed by other methods. Antioxidant
hence they react at different rates i.e. differing content of vegetables and fruits was previously
kinetics, and the reaction will often not go to reported using ORAC and other methods (8,9).
completion in a reasonable assay time. Therefore, Similar trends in antioxidant activity were
the sample size that can lower the initial observed for grains, vegetables and fruits
absorbance of DPPH solution by 50% has been comparing those results with those obtained using
chosen as the endpoint for measuring the DPPH.
antioxidant activity. This change is compared to
the change induced by Trolox, the reference Antioxidant activity of dry beans increases with
standard, and the antioxidant activity of the sample the red color of the beans, red kidney beans being
is expressed in micromoles of Trolox equivalents the highest. Vinson et al. also observed that red
 4
kidney beans had higher activity than other beans various food products are tested in biological
(4). Similarly, red grapes have higher antioxidant models for chronic disease. It is reasonable to
activity than green grapes, and red cabbage is expect that high antioxidant foods have greater
higher than green cabbage. potential to reduce free radicals in the body than do
Antioxidant capacity of blueberries and low antioxidant foods. Thus it is important to know
strawberries containing phenolic anthocyanins are the antioxidant content of foods, in addition to
high in the range of 3100-5100 TE/100gm. Dried knowing the basic nutritional information such as
fruits like raisins and dates have high antioxidant the protein, fiber, mineral and vitamin contents.
activities, 5900-6600 TE/100gm, and these results
are consistent with published ORAC data. (1) Miller, H.E., Rigelhof, F., Marquart, L.,
Prakash, A., and Kanter, M. (2000) Cereal
Antioxidant activities of various fractions of grain Foods World 45(2), 59-63.
during milling process were studied and the results (2) Miller, H.E., Rigelhof, F., Marquart, L.,
indicate that bran has the highest antioxidant Prakash, A., and Kanter, M. (2000) J. Am.
activity and refined flour had the lowest activity. Coll. Nutr. 19(3), 312S-319S.
Antioxidant activities were also studied for ready (3) Hodges, D.M., DeLong, J.M., Forney, C.F. &
to eat oat, wheat, corn, and rice cereals. In whole Prange, R.K. (1999) Planta 207, 604-611.
grain cereals, bran and germ are intact, hence (4) Pellegrini, N., Re, R., Yang, M., & Rice-
antioxidant activity of whole grain cereals is Evans, C., (1999) Methods in Enzymology,
higher than it is in refined grain cereals. Volume 299, 379-389.
Antioxidant activity of whole grain wheat and (5) Prior, R.L., Cao, G., Martin, A., Sofic, E.,
whole grain oat cereals is in the range 2200-3600 McEwen, J., O’Brien, C., Lischner, N.,
TE/100gm. Refined grain corn and rice cereals are Ehlenfeldt, M., Kalt, W., Krewer, G., &
in the range 1400 - 2000 TE/100gm, as a result of Mainland, C.M., (1998) J. Agric. Food Chem.
bran removal. Hence, the refined grain cereals 46, 2686-2693.
have lower antioxidant activity. Similar trends (6) Cao, G., Verdon, C.P., Wu, A.H.B., Wang, H.,
have been observed with whole wheat bread, 2000 & Prior, R.L. (1995) Clin, Chem., 41, 1738-
TE/100gm versus white bread, 1200 TE/100gm. 1744.
(7) Vinson, J.A., Hao, Y., Su, X., & Zubik, L.
(1998) J. Agri. Food Chem. 46, 3630-3634.
CONCLUSION (8) Cuvelier, M. E., Richard, H., & Berset, C.
(1992) Biosci. Biotech. Biochem. 56, 324-325.
The antioxidant activity of various foods can be (9) Hogg, J. S., Lohmann, D. H., & Russell, K. E.
determined accurately, conveniently, and rapidly (1961) Can. J. Chem. 39, 1588-1594.
using DPPH. The trend in antioxidant activity (10) Cao, G., Sofic, E., & Prior, R. L. (1966) J.
obtained by using the DPPH method is Agric. Food Chem. 44, 3426-3431.
comparable to trends found using other methods (11) Wang, H., Cao, G., & Prior, R.L. (1996) J.
reported in the literature. This method can be used
successfully for solid samples without prior
extraction and concentration, which saves time.
The reaction time of four hours and a temperature
of 35°C facilitates the extraction and reaction of
antioxidant compounds with DPPH. Antioxidant
activity measured using DPPH accounts partially
for the bound and insoluble antioxidants. Relative
antioxidant content provides an indication of
importance of each of the foods. Antioxidant
activity and nutritional labeling data including
vitamins, fibers, minerals will aid in the
interpretation of clinical results obtained as Agric. Food Chem. 44, 701-705.
5

ANTIOXIDANT
ACTIVITY
FOOD (TE/100 Grams)

Red Grapes 1350

Red Cabbage 1000

Broccoli Flowers 500

Spinach 500

Green Grapes 400

ANTIOXIDANT
STANDARD ACTIVITY Tomato 300
ANTIOXIDANTS (TE/100 Grams)
Green Beans 175
Ascorbate 442,000
Green Cabbage 150
Trolox 400,000
Lima Beans 1055
Vitamin E 201, 000
Red Beans 11459
BHT 395, 000
Blueberries 3300

Raisins 5900

Wheat Bran 4620

Wheat Flour 600

(refined)
 6
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