Evaluation of antioxidant properties of pomegranate peel extract
in comparison with pomegranate pulp extract
Yunfeng Li, Changjiang Guo
, Jijun Yang, Jingyu Wei, Jing Xu, Shuang Cheng
Department of Nutrition, Institute of Hygiene and Environmental Medicine, Tianjin 300050, PR China
Received 19 July 2004; received in revised form 9 February 2005; accepted 9 February 2005
Pomegranate is an important source of bioactive compounds and has been used for folk medicine for many centuries. Pomegran-
ate juice has been demonstrated to be high in antioxidant activity and is eﬀective in the prevention of atherosclerosis. In a previous
study, we found that pomegranate peel had the highest antioxidant activity among the peel, pulp and seed fractions of 28 kinds of
fruits commonly consumed in China as determined by FRAP (ferric reducing antioxidant power) assay. In this study, we extracted
antioxidants from pomegranate peel, using a mixture of ethanol, methanol and acetone, and the antioxidant properties of the extract
were further investigated as compared with the pulp extract. The contents of total phenolics, ﬂavonoids, proathocyanidins and
ascorbic acid were also measured. The results showed that pomegranate peel extract had markedly higher antioxidant capacity than
the pulp extract in scavenging or preventive capacity against superoxide anion, hydroxyl and peroxyl radicals as well as inhibiting
-induced LDL oxidation. The contents of total phenolics, ﬂavonoids and proathocyanidins were also higher in peel extract
than in pulp extract. The large amount of phenolics contained in peel extract may cause its strong antioxidant ability. We concluded
that pomegranate peel extract appeared to have more potential as a health supplement rich in natural antioxidants than the pulp
extract and merits further intensive study.
Ó2005 Elsevier Ltd. All rights reserved.
Keywords: Pomegranate peel; antioxidant activity; LDL oxidation
Antioxidant is deﬁned as any substance that, when
present at low concentrations compared with those of
an oxidizable substrate, signiﬁcantly delays or prevents
oxidation of that substrate (Halliwell, 1995). Epidemio-
logical studies have shown that consumption of fruits
and vegetables is negatively associated with the morbid-
ity and mortality of cardio- and cerebro-vascular dis-
eases and certain types of cancers (Johnsen et al.,
2003; Rissanen et al., 2003; Temple & Gladwin, 2003),
and the antioxidants contained in fruits and vegetables,
including ascorbic acid, carotenoids, ﬂavonoids, hydro-
lysable tannins, are supposed to play an important role
in the prevention of these diseases (Huxley & Neil, 2003;
Knekt et al., 2002; Lampe, 1999). Evidence from animal
and human experiments also reveals that some natural
antioxidants other than ascorbic acid, carotenoids and
vitamin E could be absorbed signiﬁcantly and act as po-
tent antioxidants in vivo (Cao, Muccitelli, Sanchez-
Moreno, & Prior, 2001; Pataki et al., 2002; Scalbert &
Williamson, 2000; Su et al., 2003). These results have
been the driving force for many recent studies on the
antioxidant activity of diﬀerent foods of plant origin
and the derived products, as well as the eﬀective constit-
uents, responsible for this activity.
Pomegranates (Punica granatum) have been used
extensively in the folk medicine of many cultures
0308-8146/$ - see front matter Ó2005 Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +86 22 84655429; fax: +86 22
E-mail address: email@example.com (C. Guo).
Food Chemistry 96 (2006) 254–260
(Longtin, 2003). Recently, pomegranate juice, and even
fermented pomegranate juice, were demonstrated to be
high in antioxidant activity (Gil, Tomas-Barberan,
Hess-Pierce, Holcroft, & Kader, 2000; Schubert, Lan-
sky, & Neeman, 1999). Pomegranate juice also displays
potent antiatherogenic action in atherosclerotic mice
and humans (Aviram et al., 2000; Kaplan et al., 2001).
All these activities may be related to diverse phenolic
compounds present in pomegranate juice, including
punicalagin isomers, ellagic acid derivatives and antho-
cyanins (delphinidin, cyanidin and pelargonidin 3-gluco-
sides and 3,5-diglucosides). These compounds are
known for their properties in scavenging free radicals
and inhibiting lipid oxidation in vitro (Gil et al., 2000;
Noda, Kaneyuki, Mori, & Packer, 2002).
Previously, we found that pomegranate peel had the
highest antioxidant activity among the peel, pulp and
seed fractions of 28 kinds of fruits commonly consumed
in China, as determined by FRAP (ferric reducing anti-
oxidant power) assay (Guo et al., 2003). It seems, there-
fore, that pomegranate peel may be a rich source of
natural antioxidants and worthy of further study. It is
interesting that pomegranate peels have been used since
antiquity in the Middle East as colorant for textiles be-
cause of their high tannin and phenolic contents. Singh,
Murthy, and Jayaprakasha (2002) recently reported that
methanol extract of pomegranate peel had much higher
antioxidant capacity than that of seeds, as demonstrated
by using the b-carotene–linoleate and DPPH model sys-
tems. This pomegranate peel extract could eﬀectively
protect (after oral administration) against CCl
hepatotoxicity, in which ROS damage was intensively
involved (Murthy, Jayaprakasha, & Singh, 2002). In
the current study, we optimized the antioxidant extract-
ing procedure, based on FRAP value of the extracts.
The peel extract, with high FRAP value, that we ob-
tained was further measured for the contents of total
phenolic, ﬂavonoids, proanthocyanidins and ascorbic
acid and evaluated by using diﬀerent in vitro models
for its antioxidant capacity, including scavenging or pre-
ventive capacity against superoxide anion, hydroxyl and
peroxyl radicals, as well as inhibiting CuSO
LDL oxidation in comparison with the pulp extract.
The objectives of this study were to establish an eﬃcient
antioxidant extracting procedure and to explore the pos-
sibility of developing a nutraceutical agent rich in natu-
ral antioxidants from the pomegranate peel.
2. Materials and methods
Catechin, rutin, quercetin, 2,4,6-tripyridyl-s-triazine
(TPTZ) were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). Trolox, ascorbic acid, and disodium
ﬂuorescein (FL) were obtained from Aldrich (Milwau-
kee, WI, USA). The 2,20-azobis (2-amidinopropane)
dihydrochloride (AAPH) was from Wako Chemicals
USA (Richmond, VA). All other chemicals used were
of analytical grade.
2.2. Antioxidants extraction
Ripened pomegranates were obtained from Lintong,
Shanxi Province of China. The peel and pulp were sep-
arated manually. The fresh peels collected were cut into
pieces and extracted with diﬀerent solvents, including
methanol, ethanol, acetone and their combinations.
The extracts were ﬁltered through Whatman No. 41 ﬁl-
ter paper. The residues were re-extracted by the same
solvent. All extracts were pooled together and concen-
trated under vacuum at 60 °C, and the concentrates
were powdered and stored in a desiccator. The pulps
were similarly extracted.
2.3. Determination of total phenolics, ﬂavonoids,
proanthocyanidins and ascorbic acid
The content of phenolic compounds in the extracts
was determined according to the method of Jayapraka-
sha, Singh, and Sakariah (2001). The extracts were dis-
solved in water. Aliquots of 0.5 ml samples were mixed
with 2.5 ml of 10-fold-diluted Folin–Ciocalteu reagent
and 2 ml of 7.5% sodium carbonate. The mixture was al-
lowed to stand for 30 min at room temperature before
the absorbance was measured at 760 nm spectrophoto-
metrically. The ﬁnal results were expressed as tannic
The ﬂavonoid content of the extracts was measured
using a modiﬁed colorimetric method (Jia, Tang, &
Wu, 1999). A quantity of 0.5 g of extracts was dissolved
in 10 ml water and extracted by 10 ml n-butanol, three
times. The extracts were pooled and concentrated under
vacuum at 60 °C. The residue was re-dissolved in 5 ml of
60% ethanol and washed twice with 5 ml of 30% etha-
nol. All three parts were pooled together and ﬁltered.
The ﬁltrate was diluted, up to 25 ml, with 30% ethanol.
A volume of 0.5 ml of the solution was transferred to a
test tube containing 4.5 ml of 30% ethanol and mixed
with 0.3 ml of 5% sodium nitrite for 5 min. Then,
0.3 ml of 10% aluminium nitrate were added. After
6 min, the reaction was stopped by adding 2 ml of 1 M
sodium hydroxide. The mixture was further diluted with
30% ethanol up to 10 ml. The absorbance of the mixture
was immediately measured at 510 nm. The ﬂavonoid
content was calculated and expressed as rutin
Determination of proanthocyanidins was based on
the procedure reported by Sun, Ricardo-Da-Silva, and
Spranger (1998). A volume of 0.5 ml of 50 mg/l of
extract solution was mixed with 3 ml of 4% vanillin–
Y. Li et al. / Food Chemistry 96 (2006) 254–260 255
methanol solution and 1.5 ml hydrochloric acid and the
mixture was allowed to stand for 15 min. The absor-
bance at 500 nm was measured and the ﬁnal result was
expressed as catechin equivalents.
A colorimetric procedure for the determination of to-
tal ascorbic acid, including dehydroascorbic acid, in
fruits, vegetables and derived products was followed
(GB12392-90, 1990). This procedure had been standard-
ized and was approved by the Ministry of Public Health,
PR China for national implementation in 1990.
2.4. FRAP assay
The procedure described by Benzie and Strain (1996)
was followed. Brieﬂy, the FRAP reagent contained
2.5 ml of 10 mM TPTZ solution in 40 mM HCl plus
2.5 ml of 20 mM FeCl
and 25 ml of 0.3 M acetate buf-
fer, pH 3.6, and was freshly prepared and warmed at
37 °C prior to use. Aliquots of 40 ll diluted sample solu-
tion were mixed with 0.2 ml distilled water and 1.8 ml
FRAP reagent. The absorbance of reaction mixture at
593 nm was measured spectrophotometrically after
incubation at 37 °C for 10 min. The 1 mM FeSO
used as the standard solution. The ﬁnal result was ex-
pressed as the concentration of antioxidants having a
ferric reducing ability equivalent to that of 1 mM
2.5. Superoxide radical ðO
Superoxide radical-scavenging activity was deter-
mined, using a commercial kit (Jiancheng Bioengineer-
ing Institute, Nanjing, China). The superoxide radicals
were generated by the xanthine/xanthine oxidase system
and reacted with 2,4-iodiphenyl-3,4-nitrophenyl-5-phe-
nyltetrazolium chloride to form formazan, a coloured
compound which can be spectrophotometrically quanti-
ﬁed at 550 nm . The production of formazan is inversely
related to the superoxide radical-scavenging activity of
the samples tested. The ﬁnal results were expressed as
the inhibition degree of formazan production.
2.6. Hydroxyl radical (
OH) prevention activity
Hydroxyl radical prevention activity was measured
based on the method reported by Ou et al. (2002). The
sample (200 ll), and FL (3.6 ml, 86.1 nM) were pipetted
into a test tube and incubated at 37 °C for 15 min.
Thereafter, 100 llof4%H
were added and vortexed,
and the initial ﬂuorescence intensity was recorded on a
Hitachi spectroﬂuorophotometer. The excitation and
emission wavelengths were 493 and 515 nm, respec-
tively. 100 ll of 9.2 mM CoSO
were added to initiate
the reaction. Fluorescence intensity readings were taken
every three minutes until zero ﬂuorescence intensity was
reached. The ﬁnal results were calculated by using a
regression equation between the catechin concentration
and the net area under the FL decay curve and ex-
pressed as catechin equivalents.
2.7. Peroxyl radical (ROO
Peroxyl radical-scavenging activity was determined
by an improved oxygen radical absorbance capacity
(ORAC) assay (Ou, Hampsch-Woodill, & Prior, 2001).
The sample (200 ll), phosphate buﬀer (3.5 ml, 75 mM,
pH 7.4), and FL (100 ll, 35 nM) were mixed in a test
tube and incubated at 37 °C for 5 min before the initial
ﬂuorescence intensity was recorded. AAPH (200 ll,
75 g/l) was added to initiate the reaction. Fluorescence
readings were taken every three minutes until zero ﬂuo-
rescence intensity was reached. The excitation and emis-
sion wavelengths were 493 and 515 nm, respectively.
Trolox was also used as a standard to calibrate the ﬁnal
2.8. Inhibition of low density lipoprotein (LDL) oxidation
Inhibition of LDL oxidation was determined accord-
ing to the method of Princen, Van Poppel, Vogelezang,
Buytenhek, and Kok (1992). Rat serum was collected
and diluted by phosphate buﬀer (50 mM, pH 7.4) to
the concentration of 0.6%. Aliquots of 5.0 ml diluted
serum were mixed with 10 ll DMSO or 10 ll DMSO
containing various concentrations of extracts. The
solution (20 ll of 2.5 mM) was added to initiate
the reaction. The absorbance at 234 nm was recorded
immediately and was taken every 20 min thereafter for
200 min at room temperature. The net area under the
curve was calculated and treated as the ﬁnal result.
3. Results and discussion
Both pomegranate pulp and peel contain many diﬀer-
ent kinds of antioxidants, including those possibly not
so far well characterized . Gil et al. (2000) identiﬁed sev-
eral phenolic compounds from pomegranate juice, such
as anthocyanins, punicalagins, ellagic acids, and hydro-
lysable tannins, Noda et al. (2002) reported that three
major anthocyanidins found in pomegranate juice were
delphinidin, cyanidin and pelargonidin. Pomegranate
peel, also, had been shown to be rich in polyphenols
(Ben, Ayed, & Metche, 1996). It is time-consuming to
purify all antioxidants, one by one, from pomegranate
peel. From the practical point of view, a suitable extract-
ing procedure should be developed to recover as many
antioxidants as possible before an extract rich in natural
antioxidants could be further explored for possible
application in health-promoting supplements for the
food industry. Singh et al. (2002) extracted antioxi-
dants from pomegranate peel and seed with the use of
256 Y. Li et al. / Food Chemistry 96 (2006) 254–260
methanol, acetone or water and found that methanol
gave maximum antioxidant yield. We consider that a
combination of diﬀerent solvents may be more eﬃcient
for extracting antioxidants because antioxidants may
diﬀer in their solubility in diﬀerent solvents. In the pres-
ent study, the peel extract obtained by use of a mixture,
composed of methanol, ethanol, acetone and water, was
signiﬁcantly higher in FRAP value than those obtained
using individual solvents, namely using methanol, etha-
nol or acetone (Fig. 1). This result indicates that the
mixture of diﬀerent solvents is more powerful in recov-
ering antioxidants than are individual solvents. Based
on fresh weight, the yields of dried extracts from peel
and pulp were 31.5 ± 3.1% and 14.5 ± 1.7%, respec-
tively, by the extracting procedure developed in this
study. A document based on this extracting procedure
is being prepared to be submitted for Chinese Patent
We measured some antioxidant fractions present in
the peel and pulp extracts we obtained. As shown in
Table 1, the total phenolics content of peel extract was
nearly 10-fold as high as that of pulp extract. The con-
tents of ﬂavonoids and proanthocyanidins were also
higher in peel extract than in pulp extract. This result
clearly indicates that peel extract contains more antiox-
idants than does the pulp extract. This is consistent with
the data reported by Tomas-Barberan et al. (2001), who
found that peel tissues usually contained larger amount
of phenolics, anthocyanins and ﬂavonols than did ﬂesh
tissues in nectarines, peaches and plums. However,
ﬂavonoids or proanthocyanidins account for only a
small part of total phenolics present in the peel extract.
In addition, both peel and pulp extracts contained a
small amount of ascorbic acid. Therefore, ascorbic acid
could not be an important antioxidant, either in the peel
or pulp extract that we obtained.
The FRAP assay treats the antioxidants contained in
the samples as reductants in a redox-linked colorimetric
reaction and the value reﬂects the reducing power of the
antioxidants. The procedure is relatively simple and easy
to standardize. Thus, it has been used frequently in the
assessment of antioxidant activity of various fruits and
vegetables and some biological samples, though we
understand that it has some limitations (Guo et al.,
2003; Halvorsen et al., 2002; Pulido, Bravo, & Saura-
Calixo, 2000). Based on FRAP value, the peel extract
was much stronger than the pulp extract in reducing
power in a dose-dependent manner (Fig. 2), indicating
that peel extract has more potential antioxidant activity.
We further compared the scavenging or preventive
capacity of peel and pulp extracts against several com-
mon free radicals in vitro. The superoxide anion is a
well-recognized free radical species and is generated con-
tinuously by several cellular processes, including the
Fig. 1. Comparison of antioxidant extracting eﬃciency from pome-
granate peel by diﬀerent solvents based on FRAP value, n= 4. Solvent
1, methanol; solvent 2, ethanol; solvent 3, acetone; solvent 4, mixture
of methanol, ethanol, acetone and water. Data were analyzed by one
way analysis of variance. *P< 0.05, solvent 4 vs. solvents 1, 2 or 3.
Analysis of main antioxidant fractions contained in pomegranate peel and pulp extracts ð
Extract Yield (%) Phenolics (mg/g) Flavonoids (mg/g) Proanthocyanidins(mg/g) Ascorbic acid (mg/g) Water (%)
Pulp 14.5 ± 1.7 24.4 ± 2.7 17.2 ± 3.3 5.3 ± 0.7 0.85 ± 0.02 10.9 ± 1.1
Peel 31.5 ± 3.0 249.4 ± 17.2 59.1 ± 4.8 10.9 ± 0.5 0.99 ± 0.02 8.0 ± 0.8
Phenolics, tannic acid equivalents; ﬂavonoids, rutin equivalents; proanthocyanin, catechin equivalents.
y = 4.4593x + 0.1003
y= 0.3084x + 0.053
FRAP value (mmol/L)
Fig. 2. Antioxidant activity of pomegranate peel and pulp extracts as
measured by FRAP assay. The result is expressed as the concentration
of antioxidants having a ferric reducing ability equivalent to that of
1.0 mM FeSO
Y. Li et al. / Food Chemistry 96 (2006) 254–260 257
microsomal and mitochondrial electron transport sys-
tems. Although the superoxide anion is limited in activ-
ity, it may combine with other reactive species, such as
nitric oxide, produced by macrophages, to yield a more
reactive species (Fridovich, 1995). The results of our
study showed that the peel extract presented rather more
superoxide radical-scavenging ability than the pulp ex-
tract (Fig. 3), based on the inhibition of superoxide rad-
ical-related formazan production. At a concentration of
50 g/l, the inhibition activities were 43.0% and 37.7% for
the peel and pulp extracts, respectively.
The hydroxyl radical is a highly reactive free radical
species and capable of damaging almost every molecule
found in living cells. It can be generated in vivo in the pres-
ence of both superoxide radicals and transition cations,
such as iron or copper via the Haber–Weiss reaction
(Castro & Freeman, 2001). We used the hydroxyl radical
prevention capacity assay, as developed by Ou et al.
(2002), to compare the preventive capacity of peel and
pulp extracts against hydroxyl radicals. The results
revealed that the peel extract possessed about 25 times
higher activity than the pulp extract (Fig. 4). Since the
procedure is based on the metal-chelating property of
the antioxidants, the so-called preventive capacity against
hydroxyl radicals is actually related to the metal-chelating
capability of the samples tested (Ou et al., 2002).
The peroxyl radicals occur during oxidation of lipids
in oxidative stress. They may diﬀuse a considerable dis-
tance and can react avidly with sulfhydryl groups (Tho-
mas, 1999). The improved ORAC assay used in this
study is basically similar to the hydroxyl radical preven-
tion capacity assay in principle, in which the ﬂuorescein
is employed as the sensitive probe for free radical attack.
However, AAPH is used, instead of the H
system, to generate peroxyl radicals in this procedure.
Again, the peel extract appeared to be more eﬀective than
the pulp extract in scavenging peroxyl radicals (Fig. 5).
The ‘‘oxidative modiﬁcation of lipoproteins’’ hypoth-
esis proposes that LDL oxidation plays a key role in
early atherosclerosis. The oxidized LDL (Ox-LDL) is
atherogenic because it is cytotoxic toward arterial cells
and stimulates the monocytes to be adhesive to the
endothelium. The uptake of Ox-LDL, via scavenger
receptors, by the monocytes promotes cholesterol accu-
mulation and foam cell formation, which leads to the
development of atheromatous plaques. Thereby, inhibi-
tion of LDL oxidation is supposed to be one of the cru-
cial steps in retarding the foam cell formation and
development of aortic lesions (Chisolm & Steinberg,
2000). Aviram et al. (2000) reported that pomegranate
juice could eﬀectively protect LDL against oxidation
in vitro, which was attributed to the polyphenols and
ascorbic acid contained in the juice. Further studies
from the same laboratory demonstrated that pomegran-
ate juice consumption reduced the LDL susceptibility to
macrophage-mediated oxidation in atherosclerotic E
mice and healthy human subjects (Aviram et al., 2000;
Kaplan et al., 2001). In the current study, we also con-
ﬁrmed the inhibitive action of pomegranate pulp extract
-induced LDL oxidation, as evidenced by
decreased conjugated dienes production in a dose-
dependent fashion. As compared to the pulp extract,
y = 13.46Ln (x)- 13.67
y = 11.88Ln (x)- 11.87
Inhibition degree (%)
Fig. 3. Dose-dependent superoxide radical scavenging ability of
pomegranate peel or pulp extract as determined by xanthine/xanthine
oxidase method. The result is expressed as the inhibition degree of
formazan production. The curves were simulated logarithmically.
0 6 12 18 24 30 36 42 48 54 60
FL intensity (AU)
catechin 100 µ mol/L
catechin 200 µ mol/L
catechin 400 µ mol/L
y=260.82x + 27.355
y= 202.3x -191.39
0 0.5 1 1.5 2 2.5 3
Fig. 4. Hydroxyl radical prevention activity of pomegranate peel and
pulp extracts as determined by hydroxyl radical prevention capacity
assay. The upper is the decay curve of ﬂuorescein (FL) in the presence
or absence of catechin. The below is the dose-dependent hydroxyl
radical prevention activity of peel or pulp extract calculated as catechin
258 Y. Li et al. / Food Chemistry 96 (2006) 254–260
the peel extract acted more dramatically in protecting
LDL against oxidation (Fig. 6), indicating a possibility
that pomegranate peel extract may be more promising
in the prevention of atherosclerosis by inhibiting LDL
Phenolic compounds, or polyphenols, constitute one
of the most numerous and widely distributed groups
of substances in the plant kingdom. They can range
from simple molecules, such as phenolic acids, to highly
polymerized compounds, such as tannins. Flavonoids
are reported to be the most abundant polyphenols in hu-
man diets. The reasons for recent renewed interest in
phenolics are that most phenolics possess strong antiox-
idant capacity in vitro and some of them have been dem-
onstrated to be signiﬁcantly bioavailable in vivo (Bravo,
1998; Jialal & Devaraj, 1996; Rice-Evans, Miller, &
Paganga, 1996; Scalbert & Williamson, 2000). Prior
et al. (1998) reported that the content of total phenolics
was well correlated with the antioxidant capacity of
fruits. It has also been shown that phenolics from red
wine, green tea and chocolate could inhibit LDL oxida-
tion signiﬁcantly in vitro (Teissedre, Frankel, Water-
house, Peleg, & German, 1996; Waterhouse, Shirley, &
Donovan, 1996; Yoshida et al., 1999). Given the higher
amount of phenolics contained in peel extract, it is not
surprising that peel extract displays higher activity, in
scavenging or preventive capacity against free radicals
and inhibiting LDL oxidation, than the pulp extract.
Although both peel and pulp extracts contain certain
amount of ﬂavonoids and proanthocyanidins, we are
not sure how much they may contribute to the antioxi-
dant activity presented by the peel or pulp extract.
Moreover, the possible synergistic action among diﬀer-
ent antioxidants contained in the extracts cannot cur-
rently be ruled out . Further study should be carried
out to identify the predominant phenolics responsible
for the antioxidant activity of peel extract.
In conclusion, we used a mixture of methanol, ethanol,
acetone and water to extract antioxidants from the pome-
granate peel and the extract we obtained possessed stron-
ger antioxidant properties than the pulp extract, including
scavenging or preventive capability against several reac-
tive oxygen species and inhibiting LDL oxidation. The
high antioxidant activity of the peel extract appeared to
be attributed to its high phenolics content. We consider
that this peel extract deserves more intensive study,
including its antioxidant composition, bioavailability
and possible protection against cardiovascular diseases.
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Fig. 6. Dose-dependent inhibition of CuSO
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