Granatapfel Punica Granatum Granatapfelkernöl Pomegranate

The antioxidant activity of pomegranate juices was evaluated by four different methods (ABTS,
DPPH, DMPD, and FRAP) and compared to those of red wine and a green tea infusion. Commercial
pomegranate juices showed an antioxidant activity (18-20 TEAC) three times higher than those of
red wine and green tea (6-8 TEAC). The activity was higher in commercial juices extracted from
whole pomegranates than in experimental juices obtained from the arils only (12-14 TEAC). HPLCDAD
and HPLC-MS analyses of the juices revealed that commercial juices contained the
pomegranate tannin punicalagin (1500-1900 mg/L) while only traces of this compound were detected
in the experimental juice obtained from arils in the laboratory. This shows that pomegranate
industrial processing extracts some of the hydrolyzable tannins present in the fruit rind. This could
account for the higher antioxidant activity of commercial juices compared to the experimental ones.
In addition, anthocyanins, ellagic acid derivatives, and hydrolyzable tannins were detected and
quantified in the pomegranate juices.




INTRODUCTION
Epidemiological studies show that consumption of
fruits and vegetables with high phenolic content correlate
with reduced cardio- and cerebrovascular diseases
and cancer mortality (Hertog et al., 1997a,b). Phenolic
compounds may produce their beneficial effects by
scavenging free radicals. In the past few years there has
been an increasing interest in determining relevant
dietary sources of antioxidant phenolics. Thus, red fruit
juices such as grape and different berry juices have
received attention due to their antioxidant activity.
Pomegranate juice has become more popular because
of the attribution of important biological actions (Lansky
et al., 1998). Thus, the antioxidant and antitumoral
activity of pomegranate bark tannins (punicacortein)
(Kashiwada et al., 1992; Su et al., 1988) and the
antioxidant activity of the fermented pomegranate juice
(Schubert et al., 1999) have been reported. However,
detailed investigations of the phenolic compounds and
the antioxidant activity of the juice have not yet been
carried out.
Pomegranate juice is an important source of anthocyanins,
and the 3-glucosides and 3,5-diglucosides of
delphinidin, cyanidin, and pelargonidin have been
reported (Du et al., 1975). It also contains 1 g/L citric
acid and only 7 mg/L ascorbic acid (El-Nemr et al.,
1990). In addition, pomegranate bark (Tanaka et al.,
1986b), leaf (Tanaka et al., 1985; Nawwar et al., 1994b),
and the fruit husk (Mayer et al., 1977) are very rich in
ellagitannins and gallotannins. Several apigenin and
luteolin glycosides from pomegranate leaves (Nawwar
et al., 1994a) and the hydrolyzable tannins punicalagin
and punicalin from pomegranate husk have previously
been identified (Mayer et al., 1977; Tanaka et al.,
1986a).
We report here on the evaluation of the antioxidant
activity of pomegranate juice extracted by different
procedures and the identification of the compounds
responsible for this activity.
MATERIALS AND METHODS
Materials. Four types of pomegranate juices were produced
from “Wonderful” pomegranates harvested in California during
October 1998. Juice 1 was obtained in the laboratory from
pomegranate arils by a hand press reaching a soluble solids
(SS) value of 15.5%. Juice 2 was obtained as for juice 1, but in
this case, the arils were frozen and stored for 9 months at -20
°C prior to juice preparation with a SS content of 16.6%. Juice
3 was a single-strength commercial juice produced by Green-
Valley Packers (Arvin, CA) with a 16.6% SS, and juice 4 was
a commercial concentrate juice produced by the same company.
The juice 4 was reconstituted in the laboratory by adding water
to decrease SS from 65.0 to 16.3% as in the original juice. Both
commercial and experimental juices were stored frozen (-20
°C) until analyzed.
HPLC-DAD Analyses. Three replicates from each juice
were centrifuged in an eppendorf tube (2 min at 1400 rpm)
and filtered through a 0.45 ím filter. Samples of 20 íL of juice
were analyzed using an HPLC system (Hewlett-Packard 1050
pump) coupled with a photodiode array detector (DAD) (series
1040M, series II) and an autosampler (series 1050), operated
by HP ChemStation software. A reversed-phase C18 Nucleosil
column (150 4.6 mm; particle size 5 ím) with a guard column
containing the same stationary phase (Safeguard holder 5001-
CS) was used. A quatranary pump was used for mixing the
mobile phase to avoid pressure fluctuations due to the mixing
of methanol (MeOH) in water. Acetic acid (2.5%) was added
to water and methanol to increase peak resolution before
preparing the following mobile phases: water (A); 88% water
+ 12% MeOH (B); 20% water + 80% MeOH (C); MeOH (D).
All solvents were HPLC grade. Elution started with 100% A,
which remained isocratic until 5 min. A gradient was then
installed to reach 100% B at 10 min, holding it isocratic for 3
more minutes. From 13 to 35 min, a linear gradient was
installed to reach 50% B and 50% C, and then 100% C at 40
min. The column was then washed with 100% D at 42 min.
The flow rate was 1 mL min-1 and chromatograms were
recorded at 510, 350, and 280 nm. The UV spectra of the
different compounds were recorded with a diode array detector.
HPLC-MS Analyses. Electrospray mass spectrometric
analyses were performed using a Hewlett-Packard 5989A
quadrupole instrument equipped with a electrospray interface
(HP 59987A). Nitrogen was used as a nebulizing gas at a
pressure of 50 psi and a temperature of 300 °C. The same
column and chromatographic conditions as for the HPLC-DAD
analyses were used.
Phenolic Compounds Identification and Quantification.
The phenolic compounds in pomegranate juice were
identified by their UV spectra, recorded with a diode-arraydetector,
and HPLC-MS (electrospray), and, wherever possible,
by chromatographic comparisons with authentic markers.
Individual anthocyanins were quantified by comparisons with
an external standard of cyanidin 3-glucoside (Apin Chemicals
Ltd., U.K.). Ellagic acid derivatives as an external standard
of ellagic acid, hydrolyzable tannins as gallic acid, and gallagicderived
tannins as punicalagin (isolated in the present work).
Concentrations were expressed as micrograms per milliliter
of juice. Reproducibility of the analyses was (5%.
Antioxidant Activity Evaluation. Four methods were
used to test the antioxidant activity of pomegranate juices
including three based on the evaluation of the free-radical
scavenging capacity of the juices, and one based on measuring
their iron-reducing capacity. The antioxidant activity of the
different pomegranate juices was compared to those of red
wine and green tea, two well-known food antioxidants (Ghiselli
et al., 1998; Yokozawa et al., 1998; Cao et al., 1996). A
commercial 1997 Cabernet Sauvignon wine from California
was used as red wine. Infusions of 1 g of green tea brewed for
5 min with 100 mL of boiling water were prepared. In addition,
the antioxidant activity of a water extract of pomegranate husk
(1 g of fruit rind homogenized with 10 mL of water) was also
tested. The first method generated the ABTS¥+ by addition of
H2O2 and horseradish peroxidase (Cano et al., 1998), which is
a colored free radical, whose neutralization was easily followed
by reading the decrease in absorbance at 414 nm after the
addition of the antioxidant. This assay is similar to that
described by Rice-Evans and Miller (1994) and to the commercial
RANDOX method. The second method assayed used
a commercially available free radical (DPPH¥+, 2,2 diphenyl-
1-picrylhydrazyl) which is soluble in methanol (Brand-Williams
et al., 1995), and the antioxidant activity measured by
decrease in absorbance at 515 nm. The third radical-scavenging
method generates a colored free radical (DMPD¥+) by
addition of Fe3+ to p-phenylene diamine (Fogliano et al., 1999),
and the absorbance at 505 nm was measured. The FRAP
method was developed to measure the ferric reducing ability
of plasma at low pH (Benzie and Strain, 1996). An intense
blue color is formed when the ferric-tripyridyltriazine (Fe3+-
TPTZ) complex is reduced to the ferrous (Fe2+) form and the
absorption at 593 nm was recorded. Standard solutions of 5.7
mM L-ascorbic acid (Aldrich, Germany) in deionized water and
10 mM TROLOX (6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid, Aldrich, Germany) in methanol were prepared.
For all the antioxidant activity methods, diluted samples in
water of 1:20 (v:v) for red wine and green tea, 1:5 and 1:10 for
husk and 1:50 for pomegranate juices were used, except when
using the DMPD method in which dilutions of 1:100 to 1:200
of pomegranate juices were needed. Diluted standards or
diluted juice samples were used on the day of preparation
except the ascorbic acid solutions that were used within 1 h
of preparation. Fifty microliters of diluted standards (or juice
samples) was mixed in an Eppendorf tube with 950 íL of the
free-radical (or Fe3+) solutions. These solutions were left to
react for a period of time (15 min for the DPPH method, 4
min for the FRAP assay, and 10 min for the DMPD and the
ABTS methods) under continuous stirring. The changes in
absorbance were then measured at 25 °C. The results were
expressed as Trolox equivalent antioxidant capacity (TEAC)
and ascorbic acid equivalent antioxidant capacity (AEAC)
(Cano et al., 1998; Cao et al., 1998; Cao and Prior, 1998; Wang
et al., 1996).
Cation-Exchange Resin. A Bio-Rad cation-exchange resin
AG50W-X8 was used to remove the anthocyanins from the
pomegranate juices. Activation of the resin was carried out
for 15 min with 1N HCl followed by another 15 min with 1 N
NaOH and repeated three times. Then, the resin was washed
with water to end the activation with acetate buffer (pH 4.0).
Pomegranate juice was loaded onto the resin, and the anthocyanins
were retained. The supernatant was passed through
an activated C-18 solid-phase extraction cartridge. The eluted
water and methanol fractions were analyzed with DPPH,
FRAP, and DMPD methods for antioxidant activity.
Total Phenolics. For total phenolic determinations, dilutions
of 1:10 and 1:20 for red wine, green tea, and 1:5 and 1:10
for husk and pomegranate juices were used. Total phenolics
were determined by the Folin-Ciocalteu reagent (Singleton
and Rossi, 1965). Dilutions were carried out per duplicate and
calculated by a calibration curve obtained with p-coumaric
acid. The absorbance was measured at 660 nm.
RESULTS AND DISCUSSION
Antioxidant Activity of Pomegranate Juices. The
antioxidant activity shown in Figures 1 and 2 are
equivalent to those of Trolox and ascorbic acid solutions
with the indicated concentrations in millimolar. Red
wine and green tea provided similar results using all
four antioxidant activity methods and TEAC and AEAC
values were within the ranges previously published:
6-12 TEAC for red wine (Fogliano et al., 1999; Ghiselli
et al., 1998) and 8.3 TEAC for green tea infusions (Prior
and Cao, 1999). The analyzed Cabernet Sauvignon red
wine showed a free-radical scavenging activity equivalent
to that of a Trolox solution 6 mM with the ABTS
method, 7.5 mM with the DPPH method, 8.7 mM with
the DMPD method, and 9.4 mM with the FRAP method.
The values calculated as ascorbic acid equivalents were
very similar to those found for Trolox equivalents. Green
tea showed antioxidant activity values similar to those
of red wine, with the exception of the values obtained
with the DMPD method in which the green tea values
were significantly lower (4.4 TEAC) than those found
for red wine (8.7 TEAC). The differences observed could
probably be explained by the interference of organic
acids present in wine (tartaric) with the DMPD method
(see below).
Using the ABTS and the DPPH methods, the antioxidant
activity of the experimental pomegranate juice
obtained from fresh arils (Juice 1) was twice those of
red wine and green tea (Figure 1). The activity was
lower in the experimental juice prepared from frozen
arils (Juice 2), showing that during the freezing process
some antioxidant compounds are degraded or transformed,
but this juice still showed a higher antioxidant
activity than red wine and green tea. The antioxidant
activity of both commercial pomegranate juices (Juices
3 and 4) was even higher (nearly three times that of
wine and tea) (Figure 1) and suggested that the industrial
process to obtain the juices either increased the
content of pomegranate antioxidants or enhanced their
activity. The FRAP method also showed a higher
antioxidant capacity for the experimental juice produced
from fresh arils with respect to red wine or green tea,
and a smaller activity for the juice produced from frozen
arils (Juice 2). Again, the activity of the commercial
juices (3 and 4) was 2-fold that of the experimental ones
(1 and 2), supporting that the method of juice extraction
has an important role in this activity.
When measuring the antioxidant activity of pomegranate
juices by the DMPD method (Figure 1), an
extraordinary high activity was observed compared to
the other free radical scavenging activity methods.
However, the antioxidant activities of red wine, green
tea, and pomegranate husk extract measured by the
DMPD method were in the same ranges as those
measured with the ABTS, DPPH, and FRAP methods.
This clearly shows that there is something in pomegranate
juice that neutralizes the DMPD free radical,
and that this juice constituent is not a main constituent
in wine, tea, or the water extract of pomegranate husk.
The antioxidant activity for the four analyzed juices
evaluated by the DMPD method was in the same range.
This antioxidant test was repeated at least four times
to confirm the observed high activity. To determine the
reason for the high activity observed in pomegranate
juices with the DMPD method, the commercial single
strength juice (3) was fractionated. A cation-exchange
resin was used to remove anthocyanins from the juice
and the remaining supernatant was fractionated by
filtration trough a C-18 solid-phase extraction cartridge.
The water-soluble compounds of the supernatant were
eluted from the cartridge with water and the phenolic
compounds retained were then eluted with methanol.
The percentage of the antioxidant inhibition of the
fractions evaluated with DPPH, FRAP, and DMPD
methods are shown in Table 1. After removing the
anthocyanins, the compounds remaining in the supernatant
fraction conferred a 28% of the total antioxidant
activity of the commercial pomegranate juice for the
DPPH and FRAP methods. However, when the DMPD
method was assayed, the supernatant fraction was the
responsible of the 63% of the total antioxidant activity
of the juice. When the supernatant was fractionated
through the sep-pack, only the methanol fraction showed
activity when the DPPH and FRAP methods were used.
The water fraction did not contain any compound with
antioxidant activity for DPPH and FRAP methods.
However, a high activity of 74.6% was shown when the
water fraction was evaluated with the DMPD method,
and only a 15.2% of the activity was due to the methanol
soluble compounds. This fact suggested that some water
soluble constituents of pomegranate juice reacted with
the DMPD free radical and showed an enhanced antioxidant
activity. These water soluble compounds did not
show free-radical scavenging activity with the other two
methods. This prompted us to study the free radical
scavenging activity of the main organic acid in pomegranate
(citric acid). In addition, the activity of malic
acid, a common organic acid of many other fruits, and
tartaric acid, the main acid in grapes and red wine, were
also tested. None of the organic acids showed antioxidant
activity when were evaluated with the DPPH and
FRAP methods. However, citric, malic and tartaric acids
showed antioxidant activity when the DMPD method
was tested. Citric acid had an important activity neutralizing
the DMPD radical, while the other assayed
organic acids showed considerably less activity (data not
shown). These results show that the DMPD method
should be used with caution for evaluation of total
antioxidant capacity, especially in those food products
which are rich in organic acids (especially citric acid).
We conclude that both experimental and commercial
pomegranate juices showed an antioxidant activity that
was always higher than those of red wine and green
tea. In addition, the commercial single-strength juice
(juice 3) and the juice from concentrate (juice 4) had a
higher antioxidant activity than those experimental
juices (1 and 2) obtained by pressing the arils without
including the rind or husk.
HPLC Qualitative Analysis of Pomegranate
Juices and Phenolic Compounds Identification.
Due to the differences observed in the antioxidant
activity of experimental and commercial juices, their
phenolic compounds were studied by HPLC on reversedphase
reversedphase
column coupled with diode array detector (HPLCDAD)
and mass spectrometry detector (HPLC-MS). In
addition, the phenolic compounds present in water
extracts of pomegranate rinds were also analyzed. Both
commercial and experimental pomegranate juices were
characterized for their typical red color produced by a
combination of delphinidin, cyanidin and pelargonidin
3-glucosides and 3,5-diglucosides, which were easily
detected in the HPLC chromatograms recorded at 510
nm (Gil et al., 1995). Pelargonidin 3,5-diglucoside was
only present as traces in the different juices and this
prevented its quantification. In addition, another anthocyanin
with larger retention time and UV-vis
spectra as a delphinidin derivative was detected in
minor amounts, but its small concentration prevented
its identification.
Both colored and noncolored phenolics are clearly
shown in the chromatograms of the different pomegranate
juices (Figure 2). The experimental juices obtained
directly from arils are characterized by two main
compounds A and C, in addition of the anthocyanin
peaks (E, delphinidin 3-glucoside; F, cyanidin 3-glucoside),
and many other minor peaks that were observed
in the chromatograms (Figure 2A). The 3,5-diglucosides
of delphinidin and cyanidin which were clearly resolved
in the chromatogram at 510 nm, appeared as broad
peaks that overlapped with other phenolic peaks in the
chromatograms at 280 nm (tR 20-25 min). The chromatograms
of commercial juices showed in addition two
other main peaks (B and D) and a minor, but quite
distinctive, peak at higher retention time (G) (Figure
2B). The water extract of pomegranate rind (Figure 2C)
was characterized by the presence of B, D, and G and
the absence of A and C and the typical pomegranate
juice anthocyanins. This clearly shows that the phenolic
pattern of the commercial pomegranate juice includes
additional phenolics to those present in the arils juice
and that the industrial process to produce pomegranate
juices also extracts some phenolic compounds from the
fruit rind.
The main phenolic compounds in pomegranate juice
were identified by their MS fragments and UV spectra
(Table 2). Compound A had a UV spectrum with a
maximum at 278 nm, with a shape similar to that of
gallic acid but with a slight shift in its maximum (Figure
3A). This compound was quite water soluble and was
not adsorbed on the solid-phase extraction cartridges
(RP-18), where it eluted together with all the water
soluble compounds (sugars, organic acids, etc.). Its
HPLC-MS (electrospray) analysis showed a quasi-molecular
ion at 333 m/z (M-H)-, in accordance with
galloyl-glucose, a common constituent of plants containing
hydrolyzable tannins.
Compound C was the main UV absorbing compound
in the aril juices. This compound showed a UV spectrum
with a maximum at 266 nm (Figure 3C). This compound
was partially purified from the aril juice by removing
the anthocyanins using ion-exchange chromatography,
solid-phase extraction on a reversed-phase cartridge,
and LH-20 chromatography with methanol. This produced
an enriched fraction that was HPLC-MS analyzed.
This compound gave a quasi-molecular ion at
1397 and characteristic fragments at 935, 785, 765, 613,
451, and 301 m/z. A fragment at 173, corresponding to
gallic acid, was also observed. These analyses showed
that this was a hydrolyzable tannin containing at least
a molecule of ellagic acid (301 m/z) a molecule of gallic
acid (173 m/z) and a molecule of tertgallic acid (451m/z),
and the absence of gallagyl residues (lack of 601 m/z
fragment). The MS spectrum of this compound was
consistent in all the HPLC-analyses carried out with
the different juices and fractions, in which the same
molecular ion and fragments were observed. Its UV
spectrum supported that this was an ellagitannin
containing at least one galloyl residue and lacking
gallagyl residues. The presence of a gallagyl residue in
C would render the compound yellow, and with a UV
spectrum showing maxima around 375 and 265 nm (as
compounds B and D) (Figure 4D). The ion at 935 was
consistent with a molecule containing two hexahydroxydiphenyl
molecules (precursors of ellagic acid) and one
gallic acid residue on glucose. The peak at 783 was
consistent with a galloyl loss, and the peak at 765
entails an additional loss of a water molecule. An
additional loss of a galloyl residue leads to fragment 613,
and the loss of glucose yields the peak at 451 that can
further fragment to render ellagic acid (301 m/z). So this
compound was tentatively identified as a digalloyl,
tertgalloyl ester of glucose. Its full identification will
only be possible after NMR studies of the isolated
compound.
Compounds B and D had the same UV spectra with
maxima at 378 and 258 nm (characteristic of gallagic
acid derivatives) (Figure 3D). These compounds were
the main constituents of a water extract of pomegranate
husk, and had a characteristic yellow color. They were
purified from the water husk extracts by LH-20 chromatography
with methanol. Both compounds interconverted
rapidly when in solution to render approximately
30% B and 70% D, this behavior is similar to that
already described for R and â isomers of punicalagin
(position isomers at the anomeric carbon of the glucose)
by Doig et al. (1990). The HPLC-MS analyses of
compounds B and D showed identical spectra for both
compounds with a quasimolecular ion at 1083 m/z (MH)
in accordance to punicalagin (glucose + gallagyl +
hexahydroxydiphenoyl) (Figure 4). This is a complex
ellagitannin characteristic of pomegranate peel, which
contains glucose, ellagic acid, and gallagic acid. Several
isomers have been previously described in pomegranate
fruit peel and also in leaves and bark. Fragments for
the loss of ellagic acid (781 m/z) and for the gallagic (601
m/z) and ellagic acid (301 m/z) residues were the main
fragments observed in the HPLC-MS spectrum, supporting
the nature of these compounds.
In addition, two other ellagic acid derivatives (with
characteristic UV spectra showing maxima at 362, 346,
300, and 256 nm) (Figure 3G) were detected. One of
them was identified as ellagic acid (MS 301 m/z) (G),
and another as ellagic acid hexoside (MS 463 m/z and a
fragment at 301 for the loss of a hexose), and was
tentatively identified as ellagic acid glucoside. In addition,
an ellagic acid pentoside (M-H, 433 m/z), and an
ellagic acid rhamnoside (M-H, 447 m/z) were also
detected in some samples.
Quantitation of Phenolic Compounds in the
Juices and Antioxidant Activity of the Isolated
Phenolics. The main phenolic compounds present in
pomegranate juice can be arranged into four groups. A
first group includes the anthocyanin pigments, which
are easily quantified by HPLC with detection in the
visible region at 510 nm, using cyanidin 3-glucoside as
an external standard. The second group includes the
hydrolyzable tannins of the gallagyl type, which are
characterized by a typical UV spectrum with two
maxima at 378 and 258 nm. This group includes the
punicalagin isomers (B and D), punicalin (gallagylglucose),
and other related compounds, and they were
quantified as punicalagin by HPLC with UV detection
at 350 nm. All these compounds showed HPLC-MS
spectra with the characteristic fragment al 601 m/z
corresponding to gallagic acid. The third group of
pomegranate juice phenolics includes ellagic acid (G)
and its glycosides. These compounds are characterized
by the typical UV spectrum of ellagic acid (UV max, 362,
346, 300, 256) and by HPLC-MS spectra with the
fragment at 301 m/z corresponding to ellagic acid. These
compounds were quantified at 350 nm as ellagic acid.
The fourth group of pomegranate phenolics includes a
very wide group of hydrolyzable tannins with undefined
UV spectra showing only maxima below 280 nm. These
compounds are different combinations of glucose, gallic
acid, hexahydroxydiphenic acid (which gives rise to
ellagic acid after hydrolysis), and tertgallic acid. Their
HPLC-MS spectra are characterized by the presence of
fragments at 173 (gallic), 301 (ellagic), and 451 (tertgallic)
mass units. All these compounds, which include
A and C, were quantified at 280 nm as gallic acid.
Both experimental and commercial juices had the
same anthocyanin pigments, but significant quantitative
differences were found (Table 3). It seems that when
arils are frozen and stored prior to juice extraction (juice
2), the anthocyanins are partly degraded and/or transformed
into other products. Something similar was
observed in the anthocyanins of the commercial juice
obtained from concentrate (juice 4). However, the experimental
juice obtained from arils (juice 1) and the
commercial single-strength juice (juice 3) contained
similar amounts of anthocyanins (Table 3).
The main difference observed between the commercial
and the experimental juices was the high content of
punicalagins and ellagic acid derivatives in the commercial
juices. The other hydrolyzable tannins remained
quite constant in the different juices. One possible
explanation for the high content of the rind constituents
(punicalagin isomers and ellagic acid derivatives) in the
commercial juices is that the hydrostatic pressure to
crush the whole fruit to release the juice from the arils,
also extracts the water-soluble ellagitannins from the
rind that pass to the juice in proportion to the force used.
Other factors include the juice processing conditions
such as added enzymes, thermal treatments and concentration
process.
The phenolic content calculated by the Folin-Ciocalteu
method of pomegranate juice, both experimental and
commercial, was in the same range as red wine (generally
above 2000 mg/L) and in this case it was twice that
found in green tea (Table 4). The total phenolics
calculated as an addition of the individual phenolics in
the HPLC chromatogram of single-strength pomegranate
juice, reached 2487 mg/L, which was in good
agreement with the value found with the Folin-Ciocalteu
method (2566 mg/L). This analysis also confirmed
that the commercial juices had a higher phenolic content
(above 2500 mg/L) than the juices produced in the
laboratory from arils (1800-2100 mg/L) (Table 4).
To calculate the contribution of the different groups
of phenolics to the total antioxidant activity of pomegranate
juice, the antioxidant capacity values of 1 mM
solutions of gallic acid, cyanidin 3-glucoside, ellagic acid,
and punicalagin were calculated and quoted as TEAC
and AEAC. The concentration of Trolox or ascorbic acid
required giving the same radical scavenging capacity
as 1 mM test substance is shown in Table 5. The higher
antioxidant activity was observed for punicalagin, as
could be expected for a large molecule with 16 phenolic
hydroxyls per molecule. Cyanidin 3-glucoside was the
less active showing antioxidant activity in the same
range as ellagic acid (both having four free phenolic
hydroxyls). Gallic acid showed a relatively high antioxidant
activity (2.5 TEAC) although it had only three
free phenolic hydroxyls per molecule. This value is in
agreement with previously reported data that determined
a TEAC of 2.6 for methyl gallate (Hagerman et
al., 1998).
The antioxidant activity of the individual compounds
was then used to calculate the contribution of the
different phenolic compounds to the total antioxidant
capacity of the single-strength commercial pomegranate
juice (Table 6). The total antioxidant activity of this
pomegranate juice was equivalent to that of a solution
20.5 mM of Trolox calculated experimentally by the
DPPH method. When the contribution of the different
phenolics groups to the juice antioxidant activity was
calculated, the anthocyanins accounted for only 1.4 mM
of Trolox, and the ellagic acids only 0.5 mM of Trolox.
The punicalagins, however, accounted for 9.9mMTrolox
and the other hydrolyzable tannins (calculated as gallic
acid) reached 6.2 mM of Trolox. When all the calculated
activities were added this reached 17.9 mM of Trolox,
which explains 87% of the antioxidant activity experimentally
determined for this juice. This clearly shows
that the antioxidant capacity of pomegranate juices is
mainly due to the hydrolyzable tannins including punicalagins.
The increase observed in punicalagin derivatives
in commercial juices is responsible for their higher
antioxidant activity when compared with the juices
obtained experimentally from arils, which have only the
antioxidant activity due to the other hydrolyzable
tannins. These results support previously reported work
in which the antioxidant capacity of high molecular
weight polyphenolics (tannins) was reported to be 15-
30 times more effective at quenching peroxyl radicals
than simple phenolics or Trolox (Hagerman et al., 1998).
These results are especially interesting as indirect
evidence shows that pomegranate tannins can be absorbed
in the intestine (Filippich et al., 1991). In fact it
has been reported that the ellagitannins of pomegranate
are hydrolyzed extensively in mice, leading to the
excretion of ellagic acid in the feces and urine (Castonguay
et al., 1994).
Commercial pomegranate juices show an antioxidant
activity three times higher than red wine and a green
tea infusion. The activity was higher in commercial
juices than in the experimental ones obtained in the
laboratory by hand pressing the arils. This difference
in activity seems to be due to the presence of pomegranate
rind tannins in commercial juice. The main antioxidant
compounds in pomegranate juice are hydrolyzable
tannins, but anthocyanins and ellagic acid derivatives
also contribute to the total antioxidant capacity
of the juice. From the methodological point of view the
DPPH and FRAP methods are recommended as easy
and accurate methods for measuring the antioxidant
activity of fruit and vegetable juices or extracts. The
DPPH method is less sensitive than the other methods
for hydrophilic antioxidants, while FRAP is a simple test
with a wide dilution juice range. The results are highly
reproducible and comparable to other free radical
scavenging methods such as ABTS. The DMPD method
should be used with caution in those extracts rich in
organic acids.
ABBREVIATIONS USED
ABTS, 2,2¢-azinobis(3-ethylbenzothiazoline)-6-sulfonic
acid; DPPH, R,R-diphenyl-â-pycrylhydrazyl; DMPD,
N,N-dimethyl-p-phenylenediamine; FRAP, ferric reducing
ability of plasma; TROLOX; TEAC, Trolox equivalent
antioxidant capacity; AEAC, ascorbic acid equivalent
antioxidant capacity.
ACKNOWLEDGMENT
This research project was supported, in part, by a
grant from Paramount Farming Co., Bakersfield, CA.
We thank Andrew L. Waterhouse and Paedar Cremin,
Department of Viticulture and Enology, University of
California at Davis, for discussion of relevant analytical
methods and access to the HPLC-MS system in their
laboratory.
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Received for review March 29, 2000. Revised manuscript
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JF000404A

Antioxidant Activity of Pomegranate Juice and Its Relationship
with Phenolic Composition and Processing
Marı´a I. Gil,† Francisco A. Toma´s-Barbera´n,† Betty Hess-Pierce,‡ Deirdre M. Holcroft,§ and
Adel A. Kader*,‡
Department of Pomology, University of California, Davis, California 95616, Department of Food Science
and Technology, CEBAS (CSIC), P.O. Box 4195, Murcia 30080, Spain, and Department of Horticultural