Antioxidant and eicosanoid enzyme inhibition properties of
pomegranate seed oil and fermented juice flavonoids
Shay Yehoshua Schubert a, Ephraim Philip Lansky b, Ishak Neeman a,*
a Laboratories of Food Engineering and Biotechnology, Technion—Israel Institute of Technology, Haifa 32000, Israel
b Rimoni Corporation, Science Park, Nesher, Israel
Received 8 April 1998; received in revised form 9 November 1998; accepted 20 November 1998
Abstract
The antioxidant and eicosanoid enzyme inhibition properties of pomegranate (Punica granatum) fermented juice
and seed oil flavonoids were studied. The pomegranate fermented juice (pfj) and cold pressed seed oil (pcpso) showed
strong antioxidant activity close to that of butylated hydroxyanisole (BHA) and green tea (Thea sinensis), and
significantly greater than that of red wine (Vitis 6itifera). Flavonoids extracted from pcpso showed 31–44% inhibition
of sheep cyclooxygenase and 69–81% inhibition of soybean lipoxygenase. Flavonoids extracted from pfj showed
21–30% inhibition of soybean lipoxygenase though no significant inhibition of sheep cyclooxygenase. The pcpso was
analyzed for its polyphenol content and fatty acid composition. Total polyphenols in pcpso showed a concentration
by weight of approximately 0.015%.
Pcpso fatty acid composition showed punicic acid (65.3%) along with palmitic
acid (4.8%), stearic acid (2.3%), oleic acid (6.3%), linoleic acid (6.6%) and three unidentified peaks from which two
(14.2%) are probably isomers of punicic acid (El-Shaarawy, M.I., Nahpetian, A., 1983). Studies on pomegranate seed
oil. Fette Seifen Anstrichmittel 83(3), 123–126). © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords : Pomegranate; Cyclooxygenase; Lipoxygenase; Antioxidant; Eicosanoids; Punica granatum
1. Introduction
Pomegranate (Punica granatum), a small tree
originating in the Orient, belongs to the Punicaceae
family (Harde et al., 1970). Pomegranate is
grown mainly in Iran, India and the USA, but
also in most Near and Far East countries. The
main use of pomegranate is as table fruit, but
large amounts are used in the beverage and liquor
industries (Nagy et al., 1990). The pericarp, containing
up to 30% tannins, is used in tanning
leather (Duke and Ayensu, 1985).
In folk medicine, pomegranate preparations,
especially of the dried pericarp, but also of the
roots, barks of the tree and roots, and the juice of
the fruit, are employed as per orum medication in
the treatment of colic, colitis, diarrhea, dysentery,
leucorrhea, menorrhagia, oxyuriasis, paralysis and
rectocele, and as external applications to caked
breast (Duke and Ayensu, 1985) and to the nape
of the neck in mumps (Boulos, 1983) and
headache (Ayensu, 1981). Further, a number of
therapeutic actions of these materials have been
described including vermifugal, taenicidal, astringent,
antispasmodic, antihysteric, diuretic, carminative.
sudorific, galactogogue and emmenagogue
(Bianchini and Corbetta, 1979).
Flavonoids, a broad class of polyphenolic compounds
widely distributed among photosynthesizing
cells, possess an impressive array of
pharmacological activity (Hasten, 1983). These
include: free radical scavenging, inhibition of a
vast spectrum of enzymes, and estrogenic activity.
Consequently, a potential role for these compounds
in several therapeutic functions is apparent.
As anti-inflammatory agents, flavonoids may
be effective against parodentitis and local pain,
without the gastric irritating effects of aspirin and
other non-steroidal anti-inflammatory drugs
(which also act through inhibition of cyclooxygenase-
catalyzed prostaglandin formation).
Flavonoids have also been suggested as cancerprotective
agents, if not therapeutic ones (Hasten,
1983) and the consumption of dietary flavonoids
was inversely correlated with coronary heart disease
in a population of elderly men (Hertog et al.,
1993). In the present work we studied pcpso and
pfj for their antioxidant activity (Hammerschmidt
and Pratt, 1978) and inhibitory effects on lipoxygenase
and cyclooxygenase, key enzymes in the
eicosanoids pathway. Lipoxygenase inhibition was
determined using soybean 5-lipoxygenase (Grossman
and Zakut, 1979) and cyclooxygenase inhibition
using sheep cyclooxygenase from sheep
vesicular glands (Van der Ouderaa et al., 1977).
2. Materials and methods
2. 1. Plant material
Plant material was collected by one of the
authors (E. Lansky) through the courtesy of the
late Professor Dan Palevitch from the cultivar
collection from the Neve Yaar Research Station,
Volcani Agricultural Research Organization,
Ministry of Agriculture, State of Israel in the
southern Galilee. A sample of mixed cultivars was
employed.
2. 2. Preparation of fermented plant juice (pfj )
The seeds of the fruit containing the intact juice
sacs were manually separated from the pericarps,
and the sacs ruptured by very light agitation in an
electric blender for 2–3 s. The mixture of the juice
and the seeds was then added to a high quality
sterilized plastic jug (ordinarily used for storing
spring water). To 16 l of this mixture was added 5
g of wine yeast, Saccharomycs bayanus (Lalvin
EC-1118) obtained from Lallemand, Montreal,
Canada. A sterile surgical glove was affixed to the
neck of the bottle with a rubber band which
served as a pressure release valve, and fermentation
was allowed to proceed at room temperature
until complete (10 days). A portion of the wine
was then decanted and gradually evaporated to
one-tenth of its original volume to yield the pfj
extract used in the study.
2. 3. Preparation of cold pressed pomegranate seed
oil (pcpso )
After the completion of fermentation of the
juice, the seeds were removed by straining and
dried in the sun, or alternatively, over an electric
radiator. The dried seeds were then cold pressed
in a Tiby Press Type 55 machine with a 7-mm
nozzle manufactured by Skeppsta Maskin of Orebro,
Sweden. A 5.3% yield of oil per dry weight of
seeds was obtained.
2. 4. Fla6onoid extraction from pcpso
Flavonoid extraction from the pcpso was accomplished
with the method previously described
for olive oil (Vazues et al., 1973). A 10-g aliquot
of pcpso was moved with 50 ml hexane in a
separation funnel and polyphenols extracted with
three volumes of 60% methanol. The methanol
phase was then moved to a second separation
funnel and washed with 20 ml hexane. The methanol phase was then collected and dried
with anhydrous Na2SO4 and again dried in a
vacuum evaporator at 40°C. The resultant
polyphenols were resuspended in methanol and
extracted with three portions of chloroform,
each half the volume of the methanol phase.
The chloroform was removed and the methanol
dried again in the vacuum evaporator at 40°C.
The polyphenols were resuspended in water and
extracted with petrol ether (60–80) until a clear
organic phase was obtained. The water phase
was saturated with NaCl and extracted with
four portions of ethyl acetate (EA), each a third
of the water phase volume. The EA fractions
were collected and dried with anhydrous
Na2SO4. The EA was dried in a vacuum evaporator
and the polyphenols resuspended in
methanol and kept at 20°C.
2. 5. Fla6onoid extraction from pjf
The pomegranate fermented juice extract was
combined with two times its volume of EA,
shaken vigorously, and left for 8 h. The EA
phase was then dried in the vacuum evaporator
at 40°C, and polyphenols resuspended in
methanol.
2. 6. Determination of antioxidant acti6ity
Antioxidant activity was determined by measuring
the coupled oxidation of carotene and
linoleic acid (Fluka, Germany), a modification
of a method previously reported (Hammerschmidt
and Pratt, 1978). Approximately 10 mg
trans-b-carotene (type 1 synthetic, Sigma, St
Louis, MO) was dissolved in 10 ml of chloroform.
The carotene–chloroform solution, 0.2 ml,
was pipetted into a boiling flask containing 20
ml linoleic acid and 200 ml Tween-40 (Sigma).
After removal of the chloroform with N2, 50 ml
of double distilled water (DDW) was added to
the flask with vigorous swirling. To tubes containing
the putative antioxidants in 2 ml
ethanol, 5 ml of the aliquots of these emulsions
were each added to final concentrations by
weight of 0.01%. Spectrophotometric readings at
470 nm (Ultraspec II spectrophotometer) were
taken immediately after addition of the emulsion
to the antioxidant solution against a blank containing
absolute ethyl alcohol (Carlo Erba,
Italy). The tubes were stoppered and placed in a
water bath at 50°C, with readings taken at 15-
min intervals for 90 min. Controls consisted of
butylated hydroxyanisole (BHA, Sigma), green
tea (Bi Luo Chun, Hua Sheng Wen Ju Factory,
Su Zhou, China) and red wine (Cabernet Sauvignon,
Barkan Winery, Israel, 1995).
2. 7. Polyphenol determination
Polyphenols were determined using a spectrophotometric
method (AOAC, 1990). Folin
Danis reagent (Na2WO4·2 H2O 100 g) and phosphomolybdic
acid (20 mg, 50 ml) were distilled
for 2 h in reflux, chilled and diluted to 1 liter in
DDW. Subsequently, 35 g Na2CO3 was dissolved
in 100 ml DDW, left overnight for crystallization
and filtered.
To obtain a calibration curve, to different
concentrations of tannic acid were added 0.5 ml
Folin Danis, then 1 ml Na2CO3 solution, followed
by DDW until a total volume of 10 ml
was achieved. Readings were taken at 760 nm
after 30 min. Polyphenols were determined in a
similar manner, but instead of tannic acid the
flavonoid sample was used.
2. 8. Cyclooxygenase preparation
Cyclooxygenase was obtained from sheep
vesicula seminalis (Yamamoto, 1982). Ten vesicles
from freshly slaughtered sheep were homogenized
in three volumes of potassium phosphate
buffer, 50 mM, pH, 7.4, containing 1 mM
EDTA (Fluka Germany). The homogenate was
centrifuged at 12000cg for 15 min and the
surfactant centrifuged at 100000cg for 1 h.
The pellet containing the microsomal fraction
was dissolved in Tris–HCl buffer (Sigma) containing
1% Tween 20 (Sigma), 0.1 mM EDTA
and 20% glycerol, centrifuged at 27000cg for
30 min, and the surfactant containing the dissolved
enzyme was collected into small containers
and kept at 70°C. 2. 9. Determination of the acti6ity of
cyclooxygenase
The activity of cyclooxygenase was determined
using a polarographic assay employing an O2
electrode. Oxygen uptake was measured as the
change in dissolved oxygen concentration catalyzed
by cyclooxygenase and measured using a
Clark (O2) electrode. The substrate was arachidonic
acid 90% purity (Sigma), 0.1 mM in Tris–
HCl, pH 8.0 buffer and Hemin (chlorid) (Fluka
Germany) 1 M. The enzyme was preincubated for
2 min with the inhibitor, then added to the reaction
cell containing the substrate at 30°C. Hydroquinone
(Fluka Germany), 0.041 mg:ml, was
added immediately prior to the reaction. Indomethacin
(Sigma), a known cyclooxygenase inhibitor,
was used as a positive control.
2. 10. Determination of the acti6ity of
lipoxygenase
The activity of soybean lipoxygenase (Sigma)
was similarly determined using a polarographic,
oxygen-measuring assay. Oxygen uptake was assessed
as the change in dissolved oxygen concentration
catalyzed by lipoxygenase and measured
using the aforementioned Clark electrode. The
substrate in this case was linoleic acid, 7.5 mM,
dispersed in water with the help of Tween 20, and
diluted with 0.2 M sodium phosphate buffer to
pH 6.5. The enzyme was preincubated for 2 min
with each putative inhibitor and then added to the
reaction cell containing the substrate at 30°C.
Green tea, red wine and BHA were employed as
positive controls.
2. 11. Determination of pcpso fatty acids
composition
Following extraction of polyphenols, 0.5 ml
pcpso was refluxed for 2 h with 100 ml of 1%
H2SO4. After cooling, the mixture was placed in a
separation funnel and 300 ml H2O added. The oil
was extracted with three volumes of 50 ml petrol
ether 40–60 (Frutaroum Israel). The fatty acid
methyl esters were then analyzed in an HP 5890
series II gas chromatograph equipped with a
flame ionization detector and coupled to a Kunirun
computing integrator. Column used
6%c0.25E` c2 mm Chromosorb W-HP 100:120
coated with 10% FFAP. Column temperature was
programmed from 190 to 210°C. Nitrogen was
the carrier gas. Mixtures of authentic standard
fatty acids methyl esters were chromatographed
under the same conditions for comparison.
3. Results and discussion
In Fig. 1, the antioxidant activities of
pomegranate fermented juice (pjf) extract and
pomegranate cold pressed seed oil extract (pcpso)
are compared with the chemical antioxidant standard,
BHA, and the most popular botanical antioxidants,
green tea and red wine. As can be
noted, the antioxidant activity of both
pomegranate fractions was significantly superior
to that of red wine. Conversely, the antioxidant
activity of the pomegranate fractions approached,
but did not surpass, the antioxidant activity of
either a premium green tea or BHA.
The measurement of antioxidant activity depicted
in the figure is accomplished through a
coupled oxidation of linoleic acid to a variety of
future oxidation-provoking oxidation products,
and b-carotene, whose pigment is readily and
quantifiably detectable with spectrophotometry.
As the b-carotene loses its color, oxidation is
proceeding, not only of the b-carotene itself, but
Fig. 1. Comparison of antioxidant activity of pfj extract and
pcpso extract to BHA, green tea and red wine extracts. Antioxidant
concentration, 0.01%. Negative control, ethanol (n
3). also of linoleic acid. Thus the more bleached out
the solution, and the lower the values in the
figure, the greater is presumed to be the oxidant
activity. The values are an expression of the measurable
optical density (OD) of the solution over
time (T), i.e. OD:T.
Fig. 2 depicts the inhibition of the eicosanoids
pathway enzyme cyclooxygenase, responsible for
the ‘cyclic’ transformation of arachidonic acid to
prostaglandins and thrombaxane. The
prostaglandins, so named because they were originally
discovered in prostate glands, are key mediators
of inflammation, which is why the so-called
non-steroidal anti-inflammatory drugs (NSAIDs)
such as aspirin, acetyl-salicylic acid (ASA) and
indomethacin are effective—because they inhibit
cyclooxygenase. Prostaglandins, as well as thrombaxane,
are involved in clotting mechanisms,
again why aspirin is used prophylactically to prevent
thromboses. Here, the pomegranate fractions
from both the pfj and pcpso are employed at a
standard weight of 5 mg total polyphenols, obtained
as previously described. The height of the
bar graphs is proportional to the degree of activity
of cyclooxygenase, and inversely proportional
to the degree of enzyme inhibition. As can be
readily observed, the activity of this enzyme was
eliminated with the NSAID, indomethacin. The
pomegranate fermented juice fraction (pjf) failed
to show any inhibition, but pomegranate cold
pressed seed oil (pcpso) fraction effected 37%
inhibition of cyclooxygenase (i.e. 63% of total
cyclooxygenase activity).
In Fig. 3, the activity of the second major
eicosanoid pathway enzyme, lipoxygenase, is expressed
by the height of the bar graphs. The
industrial antioxidant BHA effected a 92% inhibition
of this enzyme, the pomegranate fermented
juice fraction (pjf) a 23.8% inhibition, and 75%
inhibition by the pcpso fraction.
Lipoxygenase also catalyzes transformations of
the starting substrate arachidonic acid, but in a
parallel ‘linear’ rather than ‘cyclic’ pathway, to
produce the leukotrienes (Johnson et al., 1983).
Like prostaglandin and thrombaxane,
leukotrienes also play important, though as yet
incompletely understood, roles in inflammation,
atheromatous plaque formation and platelet aggregation,
and also, apparently, asthma (Spector,
1995).
Table 1 reveals the result of quantitative analysis
of the cold pressed pomegranate seed oil
(pcpso) by gas chromatography (GC) and mass
spectrometry (MS). A full 65.3% of this oil, in
agreement with previous investigation (El-
Shaarawy and Nahpetian, 1983), is shown to be
punicic acid, a fatty acid which seems to be
unique to pomegranate seed oil. The full meaning
Table 1
Pcpso fatty acid compositiona
Fatty acid Percent of total oil
4.8 16:0 palmitic
2.3 18:0 stearic
6.3 18:1 oleic
6.6 18:2 linoleic
65.3 18:3 punicic
0.4 Unknown
8.3 Unknown
6.0 Unknown
and implications of this compound in human
physiology, nutrition and medicine remains to
be elucidated. Further, 14.3% of the mixture of
fatty acids remained unidentifiable, even though
both the GC and MS were run twice with the
sample on separate occasions.
4. Conclusion
This study clearly demonstrates a decided antioxidant
activity of a pomegranate fermented
juice and seed preparation and also of cold
pressed pomegranate seed oil. Consequently, a
role for these materials as potential natural food
preservatives and:or health protective or therapeutic
agents is suggested.
The enzyme inhibition properties of the fermented
juice preparation and cold pressed
pomegranate seed oil remain to be amplified in
future investigations. Cold pressed pomegranate
seed oil possesses uniqueness both in fatty acid
composition and also range of estrogenic compounds
including the isoflavonic phytoestrogens,
another important phytoestrogen, coumestrol,
and the steroidal estrogen estrone (Moneam et
al., 1988), and to exert a potent estrogenic effect
in vivo in two different animal models (Sharaf
and Nigm, 1964). In this study, a potential role
for pomegranate seed oil as a cardioprotective
and also as an anti-inflammatory medicament
for internal and:or external applications, is suggested.
The procedure for drying the seeds in the sun
was less than ideal, and may have hindered,
though most likely not potentiated, the antioxidant
and enzyme inhibition properties of the oil.
In the future, less potentially physiologically disruptive
methods of drying should be explored.
The power of the fermented juice is less clear.
In the present study, fermentation of the juice
was undertaken both to conform to the parallel
used in wine, the source of the so-called French
paradox, whereby the cardioprotective effect of
red wine in revelers of high fat foods has been
attributed to its antioxidant activities (Ramarathnam
et al., 1995), and also as a means of
effecting a gentle ethanolic:aqueous extraction of
the seeds. It should be recalled that the juice
was fermented here with the seeds inside, and
also aged for an additional few months, again
with the seeds still contained with the juice.
Hence, we are as yet unable to differentiate between
the partial extraction of the seed oil into
the fermented juice, and the actual antioxidant
and enzyme inhibition properties of the juice itself,
both in an unfermented and fermented
state. In future studies, we plan to study the
unfermented pomegranate juice separately, the
fermented pomegranate juice from which the
seeds were removed prior to fermentation, and
also the juice fermented and aged with the seeds
inside as was used in this study.
Finally, even though pfj may not in the end
be an inhibitor of cyclooxygenase catalyzed
prostaglandin formation, it may still have an indirect
role to play in inhibition of inflammation,
as well as in inhibiting the pathogenesis of more
complex disease patterns such as AIDS, carcinogenesis,
atherosclerosis and diabetic sequellae
through a more general antioxidant effect (Sen
and Packer, 1996). A rapidly growing body of
work strongly suggests that the overall reduction–
oxidation (redox) state in the cytoplasm
may itself act profoundly in activating and deactivating
certain genes. Specifically, reactive oxygen
species (ROS) such as H2O2 in high enough
concentrations may act as ‘signal transduction
messengers’ to promote the activity of at least
two factors, nuclear factor NF-kB and activator
protein AP-1, whose receptor sites are located
on the promotor regions of different genes involved
in HIV replication, atherosclerotic mechanisms,
carcinogenesis and diabetic changes. In
short, suppression of intracellular oxidation significantly
reduces the transcription of several
key proteins (Barnes and Karin, 1997), including
‘leukocyte-endothelial adhesion molecules’
(Collins et al., 1995), cyclooxygenase (Newton et
al., 1997), lipoxygenase and NO synthase.
Through this mechanism, as well as via the suppression
of lipoxygenase-catalyzed leukotriene
formation, pfj and other natural antioxidants
may in the end still act as anti-inflammatory
agents in addition to their traditional role in
preventing the oxidation of lipids. Acknowledgements
This research was made possible through the
generous support ofLisa Schwartz Reik of Beit
Yannay, Israel. Special appreciation also to the
family of the late Professor Dan Palevitch, who
supplied not only pomegranates but also clues to
their true pharmacognostic potential, to Eli
Merom of Kibbutz Sde Eliahu for carefully salvaging
organically grown pomegranate seeds from
commercial juice extractions, and to Bengt Jonsson
of Skeppsta Maskin Company, Orebro, Sweden,
for the cold pressing of the dried
pomegranate seeds to obtain the oil.
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http://www.a-o-t.com
pomegranate seed oil and fermented juice flavonoids
Shay Yehoshua Schubert a, Ephraim Philip Lansky b, Ishak Neeman a,*
a Laboratories of Food Engineering and Biotechnology, Technion—Israel Institute of Technology, Haifa 32000, Israel
b Rimoni Corporation, Science Park, Nesher, Israel
Received 8 April 1998; received in revised form 9 November 1998; accepted 20 November 1998
Abstract
The antioxidant and eicosanoid enzyme inhibition properties of pomegranate (Punica granatum) fermented juice
and seed oil flavonoids were studied. The pomegranate fermented juice (pfj) and cold pressed seed oil (pcpso) showed
strong antioxidant activity close to that of butylated hydroxyanisole (BHA) and green tea (Thea sinensis), and
significantly greater than that of red wine (Vitis 6itifera). Flavonoids extracted from pcpso showed 31–44% inhibition
of sheep cyclooxygenase and 69–81% inhibition of soybean lipoxygenase. Flavonoids extracted from pfj showed
21–30% inhibition of soybean lipoxygenase though no significant inhibition of sheep cyclooxygenase. The pcpso was
analyzed for its polyphenol content and fatty acid composition. Total polyphenols in pcpso showed a concentration
by weight of approximately 0.015%.
Pcpso fatty acid composition showed punicic acid (65.3%) along with palmitic
acid (4.8%), stearic acid (2.3%), oleic acid (6.3%), linoleic acid (6.6%) and three unidentified peaks from which two
(14.2%) are probably isomers of punicic acid (El-Shaarawy, M.I., Nahpetian, A., 1983). Studies on pomegranate seed
oil. Fette Seifen Anstrichmittel 83(3), 123–126). © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords : Pomegranate; Cyclooxygenase; Lipoxygenase; Antioxidant; Eicosanoids; Punica granatum
1. Introduction
Pomegranate (Punica granatum), a small tree
originating in the Orient, belongs to the Punicaceae
family (Harde et al., 1970). Pomegranate is
grown mainly in Iran, India and the USA, but
also in most Near and Far East countries. The
main use of pomegranate is as table fruit, but
large amounts are used in the beverage and liquor
industries (Nagy et al., 1990). The pericarp, containing
up to 30% tannins, is used in tanning
leather (Duke and Ayensu, 1985).
In folk medicine, pomegranate preparations,
especially of the dried pericarp, but also of the
roots, barks of the tree and roots, and the juice of
the fruit, are employed as per orum medication in
the treatment of colic, colitis, diarrhea, dysentery,
leucorrhea, menorrhagia, oxyuriasis, paralysis and
rectocele, and as external applications to caked
breast (Duke and Ayensu, 1985) and to the nape
of the neck in mumps (Boulos, 1983) and
headache (Ayensu, 1981). Further, a number of
therapeutic actions of these materials have been
described including vermifugal, taenicidal, astringent,
antispasmodic, antihysteric, diuretic, carminative.
sudorific, galactogogue and emmenagogue
(Bianchini and Corbetta, 1979).
Flavonoids, a broad class of polyphenolic compounds
widely distributed among photosynthesizing
cells, possess an impressive array of
pharmacological activity (Hasten, 1983). These
include: free radical scavenging, inhibition of a
vast spectrum of enzymes, and estrogenic activity.
Consequently, a potential role for these compounds
in several therapeutic functions is apparent.
As anti-inflammatory agents, flavonoids may
be effective against parodentitis and local pain,
without the gastric irritating effects of aspirin and
other non-steroidal anti-inflammatory drugs
(which also act through inhibition of cyclooxygenase-
catalyzed prostaglandin formation).
Flavonoids have also been suggested as cancerprotective
agents, if not therapeutic ones (Hasten,
1983) and the consumption of dietary flavonoids
was inversely correlated with coronary heart disease
in a population of elderly men (Hertog et al.,
1993). In the present work we studied pcpso and
pfj for their antioxidant activity (Hammerschmidt
and Pratt, 1978) and inhibitory effects on lipoxygenase
and cyclooxygenase, key enzymes in the
eicosanoids pathway. Lipoxygenase inhibition was
determined using soybean 5-lipoxygenase (Grossman
and Zakut, 1979) and cyclooxygenase inhibition
using sheep cyclooxygenase from sheep
vesicular glands (Van der Ouderaa et al., 1977).
2. Materials and methods
2. 1. Plant material
Plant material was collected by one of the
authors (E. Lansky) through the courtesy of the
late Professor Dan Palevitch from the cultivar
collection from the Neve Yaar Research Station,
Volcani Agricultural Research Organization,
Ministry of Agriculture, State of Israel in the
southern Galilee. A sample of mixed cultivars was
employed.
2. 2. Preparation of fermented plant juice (pfj )
The seeds of the fruit containing the intact juice
sacs were manually separated from the pericarps,
and the sacs ruptured by very light agitation in an
electric blender for 2–3 s. The mixture of the juice
and the seeds was then added to a high quality
sterilized plastic jug (ordinarily used for storing
spring water). To 16 l of this mixture was added 5
g of wine yeast, Saccharomycs bayanus (Lalvin
EC-1118) obtained from Lallemand, Montreal,
Canada. A sterile surgical glove was affixed to the
neck of the bottle with a rubber band which
served as a pressure release valve, and fermentation
was allowed to proceed at room temperature
until complete (10 days). A portion of the wine
was then decanted and gradually evaporated to
one-tenth of its original volume to yield the pfj
extract used in the study.
2. 3. Preparation of cold pressed pomegranate seed
oil (pcpso )
After the completion of fermentation of the
juice, the seeds were removed by straining and
dried in the sun, or alternatively, over an electric
radiator. The dried seeds were then cold pressed
in a Tiby Press Type 55 machine with a 7-mm
nozzle manufactured by Skeppsta Maskin of Orebro,
Sweden. A 5.3% yield of oil per dry weight of
seeds was obtained.
2. 4. Fla6onoid extraction from pcpso
Flavonoid extraction from the pcpso was accomplished
with the method previously described
for olive oil (Vazues et al., 1973). A 10-g aliquot
of pcpso was moved with 50 ml hexane in a
separation funnel and polyphenols extracted with
three volumes of 60% methanol. The methanol
phase was then moved to a second separation
funnel and washed with 20 ml hexane. The methanol phase was then collected and dried
with anhydrous Na2SO4 and again dried in a
vacuum evaporator at 40°C. The resultant
polyphenols were resuspended in methanol and
extracted with three portions of chloroform,
each half the volume of the methanol phase.
The chloroform was removed and the methanol
dried again in the vacuum evaporator at 40°C.
The polyphenols were resuspended in water and
extracted with petrol ether (60–80) until a clear
organic phase was obtained. The water phase
was saturated with NaCl and extracted with
four portions of ethyl acetate (EA), each a third
of the water phase volume. The EA fractions
were collected and dried with anhydrous
Na2SO4. The EA was dried in a vacuum evaporator
and the polyphenols resuspended in
methanol and kept at 20°C.
2. 5. Fla6onoid extraction from pjf
The pomegranate fermented juice extract was
combined with two times its volume of EA,
shaken vigorously, and left for 8 h. The EA
phase was then dried in the vacuum evaporator
at 40°C, and polyphenols resuspended in
methanol.
2. 6. Determination of antioxidant acti6ity
Antioxidant activity was determined by measuring
the coupled oxidation of carotene and
linoleic acid (Fluka, Germany), a modification
of a method previously reported (Hammerschmidt
and Pratt, 1978). Approximately 10 mg
trans-b-carotene (type 1 synthetic, Sigma, St
Louis, MO) was dissolved in 10 ml of chloroform.
The carotene–chloroform solution, 0.2 ml,
was pipetted into a boiling flask containing 20
ml linoleic acid and 200 ml Tween-40 (Sigma).
After removal of the chloroform with N2, 50 ml
of double distilled water (DDW) was added to
the flask with vigorous swirling. To tubes containing
the putative antioxidants in 2 ml
ethanol, 5 ml of the aliquots of these emulsions
were each added to final concentrations by
weight of 0.01%. Spectrophotometric readings at
470 nm (Ultraspec II spectrophotometer) were
taken immediately after addition of the emulsion
to the antioxidant solution against a blank containing
absolute ethyl alcohol (Carlo Erba,
Italy). The tubes were stoppered and placed in a
water bath at 50°C, with readings taken at 15-
min intervals for 90 min. Controls consisted of
butylated hydroxyanisole (BHA, Sigma), green
tea (Bi Luo Chun, Hua Sheng Wen Ju Factory,
Su Zhou, China) and red wine (Cabernet Sauvignon,
Barkan Winery, Israel, 1995).
2. 7. Polyphenol determination
Polyphenols were determined using a spectrophotometric
method (AOAC, 1990). Folin
Danis reagent (Na2WO4·2 H2O 100 g) and phosphomolybdic
acid (20 mg, 50 ml) were distilled
for 2 h in reflux, chilled and diluted to 1 liter in
DDW. Subsequently, 35 g Na2CO3 was dissolved
in 100 ml DDW, left overnight for crystallization
and filtered.
To obtain a calibration curve, to different
concentrations of tannic acid were added 0.5 ml
Folin Danis, then 1 ml Na2CO3 solution, followed
by DDW until a total volume of 10 ml
was achieved. Readings were taken at 760 nm
after 30 min. Polyphenols were determined in a
similar manner, but instead of tannic acid the
flavonoid sample was used.
2. 8. Cyclooxygenase preparation
Cyclooxygenase was obtained from sheep
vesicula seminalis (Yamamoto, 1982). Ten vesicles
from freshly slaughtered sheep were homogenized
in three volumes of potassium phosphate
buffer, 50 mM, pH, 7.4, containing 1 mM
EDTA (Fluka Germany). The homogenate was
centrifuged at 12000cg for 15 min and the
surfactant centrifuged at 100000cg for 1 h.
The pellet containing the microsomal fraction
was dissolved in Tris–HCl buffer (Sigma) containing
1% Tween 20 (Sigma), 0.1 mM EDTA
and 20% glycerol, centrifuged at 27000cg for
30 min, and the surfactant containing the dissolved
enzyme was collected into small containers
and kept at 70°C. 2. 9. Determination of the acti6ity of
cyclooxygenase
The activity of cyclooxygenase was determined
using a polarographic assay employing an O2
electrode. Oxygen uptake was measured as the
change in dissolved oxygen concentration catalyzed
by cyclooxygenase and measured using a
Clark (O2) electrode. The substrate was arachidonic
acid 90% purity (Sigma), 0.1 mM in Tris–
HCl, pH 8.0 buffer and Hemin (chlorid) (Fluka
Germany) 1 M. The enzyme was preincubated for
2 min with the inhibitor, then added to the reaction
cell containing the substrate at 30°C. Hydroquinone
(Fluka Germany), 0.041 mg:ml, was
added immediately prior to the reaction. Indomethacin
(Sigma), a known cyclooxygenase inhibitor,
was used as a positive control.
2. 10. Determination of the acti6ity of
lipoxygenase
The activity of soybean lipoxygenase (Sigma)
was similarly determined using a polarographic,
oxygen-measuring assay. Oxygen uptake was assessed
as the change in dissolved oxygen concentration
catalyzed by lipoxygenase and measured
using the aforementioned Clark electrode. The
substrate in this case was linoleic acid, 7.5 mM,
dispersed in water with the help of Tween 20, and
diluted with 0.2 M sodium phosphate buffer to
pH 6.5. The enzyme was preincubated for 2 min
with each putative inhibitor and then added to the
reaction cell containing the substrate at 30°C.
Green tea, red wine and BHA were employed as
positive controls.
2. 11. Determination of pcpso fatty acids
composition
Following extraction of polyphenols, 0.5 ml
pcpso was refluxed for 2 h with 100 ml of 1%
H2SO4. After cooling, the mixture was placed in a
separation funnel and 300 ml H2O added. The oil
was extracted with three volumes of 50 ml petrol
ether 40–60 (Frutaroum Israel). The fatty acid
methyl esters were then analyzed in an HP 5890
series II gas chromatograph equipped with a
flame ionization detector and coupled to a Kunirun
computing integrator. Column used
6%c0.25E` c2 mm Chromosorb W-HP 100:120
coated with 10% FFAP. Column temperature was
programmed from 190 to 210°C. Nitrogen was
the carrier gas. Mixtures of authentic standard
fatty acids methyl esters were chromatographed
under the same conditions for comparison.
3. Results and discussion
In Fig. 1, the antioxidant activities of
pomegranate fermented juice (pjf) extract and
pomegranate cold pressed seed oil extract (pcpso)
are compared with the chemical antioxidant standard,
BHA, and the most popular botanical antioxidants,
green tea and red wine. As can be
noted, the antioxidant activity of both
pomegranate fractions was significantly superior
to that of red wine. Conversely, the antioxidant
activity of the pomegranate fractions approached,
but did not surpass, the antioxidant activity of
either a premium green tea or BHA.
The measurement of antioxidant activity depicted
in the figure is accomplished through a
coupled oxidation of linoleic acid to a variety of
future oxidation-provoking oxidation products,
and b-carotene, whose pigment is readily and
quantifiably detectable with spectrophotometry.
As the b-carotene loses its color, oxidation is
proceeding, not only of the b-carotene itself, but
Fig. 1. Comparison of antioxidant activity of pfj extract and
pcpso extract to BHA, green tea and red wine extracts. Antioxidant
concentration, 0.01%. Negative control, ethanol (n
3). also of linoleic acid. Thus the more bleached out
the solution, and the lower the values in the
figure, the greater is presumed to be the oxidant
activity. The values are an expression of the measurable
optical density (OD) of the solution over
time (T), i.e. OD:T.
Fig. 2 depicts the inhibition of the eicosanoids
pathway enzyme cyclooxygenase, responsible for
the ‘cyclic’ transformation of arachidonic acid to
prostaglandins and thrombaxane. The
prostaglandins, so named because they were originally
discovered in prostate glands, are key mediators
of inflammation, which is why the so-called
non-steroidal anti-inflammatory drugs (NSAIDs)
such as aspirin, acetyl-salicylic acid (ASA) and
indomethacin are effective—because they inhibit
cyclooxygenase. Prostaglandins, as well as thrombaxane,
are involved in clotting mechanisms,
again why aspirin is used prophylactically to prevent
thromboses. Here, the pomegranate fractions
from both the pfj and pcpso are employed at a
standard weight of 5 mg total polyphenols, obtained
as previously described. The height of the
bar graphs is proportional to the degree of activity
of cyclooxygenase, and inversely proportional
to the degree of enzyme inhibition. As can be
readily observed, the activity of this enzyme was
eliminated with the NSAID, indomethacin. The
pomegranate fermented juice fraction (pjf) failed
to show any inhibition, but pomegranate cold
pressed seed oil (pcpso) fraction effected 37%
inhibition of cyclooxygenase (i.e. 63% of total
cyclooxygenase activity).
In Fig. 3, the activity of the second major
eicosanoid pathway enzyme, lipoxygenase, is expressed
by the height of the bar graphs. The
industrial antioxidant BHA effected a 92% inhibition
of this enzyme, the pomegranate fermented
juice fraction (pjf) a 23.8% inhibition, and 75%
inhibition by the pcpso fraction.
Lipoxygenase also catalyzes transformations of
the starting substrate arachidonic acid, but in a
parallel ‘linear’ rather than ‘cyclic’ pathway, to
produce the leukotrienes (Johnson et al., 1983).
Like prostaglandin and thrombaxane,
leukotrienes also play important, though as yet
incompletely understood, roles in inflammation,
atheromatous plaque formation and platelet aggregation,
and also, apparently, asthma (Spector,
1995).
Table 1 reveals the result of quantitative analysis
of the cold pressed pomegranate seed oil
(pcpso) by gas chromatography (GC) and mass
spectrometry (MS). A full 65.3% of this oil, in
agreement with previous investigation (El-
Shaarawy and Nahpetian, 1983), is shown to be
punicic acid, a fatty acid which seems to be
unique to pomegranate seed oil. The full meaning
Table 1
Pcpso fatty acid compositiona
Fatty acid Percent of total oil
4.8 16:0 palmitic
2.3 18:0 stearic
6.3 18:1 oleic
6.6 18:2 linoleic
65.3 18:3 punicic
0.4 Unknown
8.3 Unknown
6.0 Unknown
and implications of this compound in human
physiology, nutrition and medicine remains to
be elucidated. Further, 14.3% of the mixture of
fatty acids remained unidentifiable, even though
both the GC and MS were run twice with the
sample on separate occasions.
4. Conclusion
This study clearly demonstrates a decided antioxidant
activity of a pomegranate fermented
juice and seed preparation and also of cold
pressed pomegranate seed oil. Consequently, a
role for these materials as potential natural food
preservatives and:or health protective or therapeutic
agents is suggested.
The enzyme inhibition properties of the fermented
juice preparation and cold pressed
pomegranate seed oil remain to be amplified in
future investigations. Cold pressed pomegranate
seed oil possesses uniqueness both in fatty acid
composition and also range of estrogenic compounds
including the isoflavonic phytoestrogens,
another important phytoestrogen, coumestrol,
and the steroidal estrogen estrone (Moneam et
al., 1988), and to exert a potent estrogenic effect
in vivo in two different animal models (Sharaf
and Nigm, 1964). In this study, a potential role
for pomegranate seed oil as a cardioprotective
and also as an anti-inflammatory medicament
for internal and:or external applications, is suggested.
The procedure for drying the seeds in the sun
was less than ideal, and may have hindered,
though most likely not potentiated, the antioxidant
and enzyme inhibition properties of the oil.
In the future, less potentially physiologically disruptive
methods of drying should be explored.
The power of the fermented juice is less clear.
In the present study, fermentation of the juice
was undertaken both to conform to the parallel
used in wine, the source of the so-called French
paradox, whereby the cardioprotective effect of
red wine in revelers of high fat foods has been
attributed to its antioxidant activities (Ramarathnam
et al., 1995), and also as a means of
effecting a gentle ethanolic:aqueous extraction of
the seeds. It should be recalled that the juice
was fermented here with the seeds inside, and
also aged for an additional few months, again
with the seeds still contained with the juice.
Hence, we are as yet unable to differentiate between
the partial extraction of the seed oil into
the fermented juice, and the actual antioxidant
and enzyme inhibition properties of the juice itself,
both in an unfermented and fermented
state. In future studies, we plan to study the
unfermented pomegranate juice separately, the
fermented pomegranate juice from which the
seeds were removed prior to fermentation, and
also the juice fermented and aged with the seeds
inside as was used in this study.
Finally, even though pfj may not in the end
be an inhibitor of cyclooxygenase catalyzed
prostaglandin formation, it may still have an indirect
role to play in inhibition of inflammation,
as well as in inhibiting the pathogenesis of more
complex disease patterns such as AIDS, carcinogenesis,
atherosclerosis and diabetic sequellae
through a more general antioxidant effect (Sen
and Packer, 1996). A rapidly growing body of
work strongly suggests that the overall reduction–
oxidation (redox) state in the cytoplasm
may itself act profoundly in activating and deactivating
certain genes. Specifically, reactive oxygen
species (ROS) such as H2O2 in high enough
concentrations may act as ‘signal transduction
messengers’ to promote the activity of at least
two factors, nuclear factor NF-kB and activator
protein AP-1, whose receptor sites are located
on the promotor regions of different genes involved
in HIV replication, atherosclerotic mechanisms,
carcinogenesis and diabetic changes. In
short, suppression of intracellular oxidation significantly
reduces the transcription of several
key proteins (Barnes and Karin, 1997), including
‘leukocyte-endothelial adhesion molecules’
(Collins et al., 1995), cyclooxygenase (Newton et
al., 1997), lipoxygenase and NO synthase.
Through this mechanism, as well as via the suppression
of lipoxygenase-catalyzed leukotriene
formation, pfj and other natural antioxidants
may in the end still act as anti-inflammatory
agents in addition to their traditional role in
preventing the oxidation of lipids. Acknowledgements
This research was made possible through the
generous support ofLisa Schwartz Reik of Beit
Yannay, Israel. Special appreciation also to the
family of the late Professor Dan Palevitch, who
supplied not only pomegranates but also clues to
their true pharmacognostic potential, to Eli
Merom of Kibbutz Sde Eliahu for carefully salvaging
organically grown pomegranate seeds from
commercial juice extractions, and to Bengt Jonsson
of Skeppsta Maskin Company, Orebro, Sweden,
for the cold pressing of the dried
pomegranate seeds to obtain the oil.
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