Granatapfel Punica Granatum Granatapfelkernöl Pomegranate

Die Wiederentdeckung des Granatapfels

Wusste bereits die griechische Göttin Aphrodite, welcher Reichtum sich in den Früchten jenes Baumes verbarg, den sie einst auf Zypern pflanzte? Punica Granatum, der Granatapfelbaum, birgt einen seltenen Schatz in der Schale seiner dunkelroten Granatäpfel: Eine nur in dieser Frucht vorkommende Fettsäure, die sich äußerst positiv auf unsere Gesundheit auswirkt. Weitere höchst wirksame Inhaltsstoffe, z.B. Flavonoide,die entzündungshemmend wirken, sind im Granatapfel enthalten. Die stark rote Frucht besteht aus einer harten Schale und zahlreichen Samenkernen. Aus diesen Samen pressen wir in einem besonders schonenden Verfahren unter Ausschluss von Luft, Licht und bei niedrigen Temperaturen ein extrem hochwertiges Kernöl. Diese Rarität ist besonders reich an einer in der Natur ausschließlich im Granatapfel vorkommenden Fettsäure (Gamma-Linolensäure), die für den menschlichen Körper besonders förderlich sein soll. Hervorzuheben ist hierbei die sowohl präventive als auch heilende Wirkung bei Krebserkrankungen. Der Granatapfelrohsaft ist höchst wirksam als Antioxidans.



Der Genuss des Saftes beugt Alterserscheinungen und Arteriosklerose vor und ergänzt die Wirkung des Öls bei Krebserkrankungen. Die Schale, reich an Flavonoiden, eignet sich sehr gut für Teeaufgüsse. Das Granatapfelmehl ist ein Nahrungsergänzungsmittel für die Hormontherapie während der Wechseljahre. Wir von All Organic Trading sind stolz darauf, Ihnen dieses wunderbare Öl sowie weitere Produkte exklusiv im deutschen Markt anbieten zu können.

Die besondere Wirkung auf die Gesundheit Der Granatapfel hält eine Fülle positiver Effekte für unsere Gesundheit parat: starke Wirkung als Antioxidans Das einmalige Fettsäurenspektrum der Samen hat die Fähigkeit, schädliche Radikale im Körper abzufangen. starke antikanzerogene Wirkung gegen Krebs Studien haben gezeigt, dass die besondere Fettsäure des Kernöls in der Lage ist, sowohl Hautkrebs als auch Brustkrebs vorzubeugen oder sogar deren Krebszellen unschädlich zu machen. Vorbeugung von Arteriosklerose Untersuchungen zufolge soll der Granatapfel Arteriosklerose vermeiden helfen oder auch eine bereits begonnene Erkrankung rückgängig machen. Entzündungshemmung Die Fettsäure des Granatapfels ist mit für die Bildung von Prostaglandinen im menschlichen Körper verantwortlich. Prostaglandine sind für den Stoffwechsel und die Immunabwehr äußerst wichtige Hormone, die der Körper selbst bildet.


Gegen Hautalterung Auch wurde eine stärkende Wirkung auf die Epidermis und eine faltenreduzierende Wirkung nachgewiesen. Hormontherapie Unangenehme Hitzewallungen während der Wechseljahre sollen durch die Inhaltsstoffe des Granatapfels verhindert werden. Dadurch eignet der Granatapfel sich sehr gut als Ersatz für die umstrittene Östrogen-Therapie.
Wir bieten Ihnen exklusiv im deutschen Markt folgende Produkte des Granatapfels an: Granatapfelkernöl, kbA Granatapfelkernöl Kapseln, kbA Granatapfelrohsaft, kbA Granatapfelkern Mehl entölt, microfein, kbA Granatapfelschalen Mehl , kbA Granatapfelschalen, fein und grob, kbA

So verwenden Sie die Produkte:

Unsere hochwertigen Granatapfel Produkte sind Nahrungsergänzungsmittel.

Das bedeutet, dass sie weder vorher erwärmt noch gekocht, sondern in purer Form über fertig zubereitete Speisen gegeben werden. Das Granatapfelkernöl kann pur (etwa 1 Teelöffel täglich) oder in Kapselform eingenommen werden. Zur Hautpflege wird das reine Öl aufgetragen oder einem Trägeröl, z.B. Jojoba- oder Mandelöl beigemischt. Der Granatapfelrohsaft kann mit Honig verfeinert, anderen Säften oder Aloe Vera Gel beigemischt werden. Die Schalen eignen sich zur Zubereitung hochwertiger Tees und Teemischungen. Das Granatapfelmehl wird als Nahrungsergänzungsmittel Speisen beigegeben. Wichtige Hinweise zur Aufbewahrung: Das hochwertige Granatapfelkernöl sollte an einem kühlen, dunklen Ort gelagert werden, damit seine Wirkung erhalten bleibt.

Die hochwertigen Inhaltsstoffe des Granatapfels

Bei den wirksamen Inhaltsstoffen des Granatapfels unterscheidet man vor allem zwei Gruppen: Den hohen Gehalt an Flavonoiden und die hohe Konzentration von der exklusiv im Granatapfel vorkommenden Fettsäure, der Gamma-Linolensäure, einer Form der für den menschlichen Körper essentiellen Linolensäure. Fettsäuren Das Granatapfelkernöl ist besonders reichhaltig an der im Pflanzenreich hier einmalig vorkommenden Gamma-Linolensäure, einem Isomer der Linolensäure. Davon enthält das Öl über 60%. Weitere ungesättigte Fettsäuren dieses Öls sind Linolsäure und Ölsäure. Was ist nun das besondere an dieser seltenen Fettsäure? Gamma-Linolensäure gehört mit sogar 3 Doppelbindungen zu den seltenen mehrfach ungesättigten Fettsäuren, die für die Stoffwechselprozesse im Körper so äußerst wichtig sind. Die eigentliche Rarität besteht in der besonderen cis-trans-cis- Anordnung der Doppelbindungen, die sie zur konjugierten Fettsäure mit spezieller Wirkung auf den menschlichen Organismus macht. Weitere bioaktive Substanzen: Flavonoide Flavonoide kommen in vielen Pflanzen, vornehmlich in der Schale, vor. Sie wirken krankheitsvorbeugend und heilend bei bestimmten Erkrankungen. So wurde eine antimikrobielle Wirkung bei Zahnfleisch- oder Harnwegsentzündungen festgestellt.

Cytotoxic effect of conjugated trienoic fatty acids on mouse tumor and human monocytic leukemia cells.

Suzuki R, Noguchi R, Ota T, Abe M, Miyashita K, Kawada T.

Division of Marine Bioscience, Graduate School of Fisheries Science, Hokkaido University, Hakodate, Japan.




The cytotoxicity of fatty acids from seed oils containing conjugated linolenic acids (CLN) was studied. Fatty acids from pomegranate, tung, and catalpa were cytotoxic to human monocytic leukemia cells at concentrations exceeding 5 microM for pomegranate and tung and 10 microM for catalpa, but fatty acids from pot marigold oil had no effect at concentrations ranging up to 163 microM. The main conjugated fatty acids of pomegranate, tung, catalpa, and pot marigold were cis(c)9,trans(t)11,c13-CLN (71.7%), c9,t11,t13-CLN (70.1%), t9,t11,c13-CLN (31.3%), and t8,t10,c12-CLN (33.4%), respectively. Therefore, the cytotoxicities of fatty acids from pomegranate, tung, and catalpa were supposed to be due to 9,11,13-CLN isomers. To elucidate the cytotoxicity of these CLN, we separated each CLN isomer from the fatty acid mixtures by high-performance liquid chromatography and analyzed its cytotoxicity. The cytotoxicities of c9,t11,c13-CLN, c9,t11,t13-CLN, and t9,t11,c13-CLN were much stronger than that of t8,t10,c12-CLN. Therefore, the higher cytotoxicity of fatty acids from pomegranate, tung, and catalpa than those from pot marigold would be derived from the different activities of 9,11,13-CLN and 8,10,12-CLN. Since there was little difference in the cytotoxicities of c9,t11,c13-CLN,c9,t11,t13-CLN, and t9,t11,c13-CLN, it is suggested that the cis/trans configuration of 9,11,13-CLN isomers had little effect on their cytotoxic effects. The mechanism of the cytotoxicity of the four fatty acids above may involve lipid peroxidation, because the order of toxicity of the fatty acids was consistent with their susceptibility to peroxidation in aqueous phase. This was supported by the decrease in the cytotoxicity of the fatty acids by addition of butylated hydroxytoluene.

PMID: 11432460 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov

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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.
References
AOAC, 1990. Official Methods of Analysis, 952.03, 15th edn.
Association of Official Analytical Chemists, Washington,
DC.
Ayensu, S.E., 1981. Medicinal Plants of the West Indies.
Reference Publications, Algonac, MI.
Barnes, P.J., Karin, M., 1997. Nuclear factor-KB: a pivotal
transcription factor in chronic inflammatory diseases. New
England Journal of Medicine 336, 1066–1071.
Bianchini, F., Corbetta, F., 1979. Health Plants of the World.
Newsweek, New York.
Boulos, L., 1983. Medicinal Plants of North Africa. Reference
Publications, Algonac, MI.
Collins, T., Read, M.A., Niesh, A.S., Whitley, M.Z., Thanos,
D., Maniatis, T., 1995. Transcriptional regulation of endothelial
cell adhesion molecules: NF-KB and cytokine-inducible
enhancers. FASEB Journal 9, 899–909.
Duke, A.J., Ayensu, S.E., 1985. Medicinal Plants of China.
Reference Publications, Algonac, MI.
El-Shaarawy, M.I., Nahpetian, A., 1983. Studies on
pomegranate seed oil. Fette Seifen Anstrichmittel 83 (3),
123–126.
Grossman, S., Zakut, R., 1979. Determination of the activity
of lipoxygenase. Methods of Biochemical Analysis 25,
303–309.
Hammerschmidt, P.A., Pratt, D.E., 1978. Phenolic antioxidants
of dried soybean. Journal of Food Science 43, 556–
559.
Harde, H., Schumacher, W., Firbas, F., Denffer, D., 1970.
Strasburg’s Textbook of Botany. Chaucer, London, p. 2.
Hasten, B., 1983. Flavonoids: a class of natural products of
high pharmacological potency. Biochemical Pharmacology
32 (7), 1141–1148.
Hertog, M.G.L., Feskens, E.M., Hollman, P.C.H., Katen,
M.B., Kromhout, D., 1993. Dietary antioxidant flavonoids
and risk of coronary heart disease: the Zutphen Elderly
Study. Lancet 342, 1007–1011.
Johnson, M., Carey, F., McMillan, R.M., 1983. Alternative
pathways of arachidonate metabolism: prostaglandins,
thromboxane and leukotrienes. Essays in Biochemistry 19,
41–139.
Moneam, N.M.A., El Sharaky, A.S., Badreldin, M.M., 1988.
Oestrogen content of pomegranate seeds. Journal of Chromatography
438, 438–442.
Nagy, P., Shaw, P.E., Wardowski, W.F., 1990. Fruits of
Tropical and Subtropical Origin. Florida Science Source,
Florida, USA, pp. 328–347.
Newton, R., Kuitert, L.M., Bergmann, M., Adcock, I.M.,
Barnes, P.J., 1997. Evidence for involvement of NF-KB in
the transcriptional control of COX-2 gene expression by
IL-1B. Biochemical and Biophysical Research Communications
237 (1), 28–32.
Ramarathnam, N., Osawa, T., Ochi, H., Kawakishi, S., 1995.
The contribution of plant food antioxidants to human
health. Trends in Food Science and Technology 6, 75–82.
Sen, C.K., Packer, L., 1996. Antioxidant and redox regulation
of gene transcription. FASEB Journal 10, 709–720.
Sharaf, A., Nigm, S.A.R., 1964. The oestrogenic activity of
pomegranate seed oil. Journal of Endocrinology 29, 91–92.
Spector, S.L., 1995. Leukotriene inhibitors and antagonists of
asthma. Annals of Allergy, Asthma and Immunology 75,
463–470.
Van der Ouderaa, F.J., Buytenhek, M., Nugteren, X., Van
Dorp, D.A., 1977. Purification and characterization of
prostaglandin endoperoxide synthetase from sheep vesicular
glands. Biochimica et Biophysica Acta 487, 315–331.
Vazues, R.A., Janar del Valle, C., Janer del Valle, L.M., 1973.
Determination of total phenols in olive oils. Grasasy
Aceites 24, 350.
Yamamoto, S., 1982. Purification and assay of PGH synthase
from bovine seminar vesicles. Methods in Enzymology 86,
55–60.



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FAQ: Frequently Asked Questions
Pomegranate Seed Oil, Conjugated Fatty Acids and CLA

1. What is CLA? CLA is an abbreviation for Conjugated Linoleic Acid. CLA was discovered
about 25 years ago in burned beef by researchers looking for the compounds that make
burned beef carcinogenic (cancer-causing). CLA has been shown to one of the most
powerful cancer protective compounds ever discovered in Nature.





2. Is CLA a pure chemical? No. Actually, CLA is a combination of different fatty acids,
which all have in common the fact that they possess conjugated double bonds. This means
that these fatty acids contain double bonds (more than one) that alternate, with single bonds
in between.
3. Is CLA sold as a nutritional supplement? Yes, CLA, derived from the milk and meat of
ruminant animals (animals which chew their cud) such as sheep and cows, is one of the most
popular nutritional supplements sold in health food stores throughout the world.
4. What are the uses for CLA? CLA has been extensively studied throughout the world in
over 100 investigative trials in both people and animals. It has been shown to powerfully
eliminate or prevent atherosclerosis, to prevent cancers and to help the action of insulin in the
body, leading to redistribution of body fat.
5. Can CLA lower blood sugar in diabetics? CLA has been shown in animal studies to be
capable of lowering blood sugar in diabetic animals.
6. Is CLA also used externally? Yes, CLA is a popular cosmeceutical ingredient used in
anti-aging preparations for the skin. It has an anti-inflammatory effect which is desirable in
making the skin appear more youthful.
7. What is pomegranate seed oil and how is it similar to CLA? Pomegranate seed oil
(PSO) is the oil which can be cold-pressed from dried, clean pomegranate seeds. PSO is
comprised of 80% conjugated fatty acids which are technically a kind of CLA. It is one of the
only plant sources of conjugated fatty acids. However, the conjugated fatty acids in PSO
contain three double bonds, while the CLA's of animal origin contain only two double bonds.
8. How does PSO compare with animal-derived CLA in potency? A Japanese study in
2000 compared conjugated fatty acids possessing three double bonds (such as occur in
pomegranate seed oil) with conjugated fatty acids with two double bonds (as from animal
sources and which are currently sold in health food stores as CLA). The fatty acids with three
conjugated double bonds were considerably more effective in killing leukemia cells than were
the ordinary two double bond CLA's.
9. What other plants besides pomegranate contain conjugated fatty acids? There are
only about ten species of plants worldwide which contain conjugated fatty acids. One of the
best known is the Chinese tung tree, whose nuts are rich in a three double bond conjugated
fatty acid. The main use of tung oil throughout history has been as a preservative for wood
and an ingredient in paints and varnishes. Another plant which contains conjugated fatty
acids is the Chinese bitter cucumber (Trichosanthis sp), which has been shown to help in the
treatment of AIDS.
10. Has pomegranate seed oil been studied by medical scientists? Yes, although the
study of pomegranate seed oil by medicine is still in its infancy. One group found
pomegranate seed oil to cause breast cancer cells to self-destruct (undergo "apoptosis").
This group compared pomegranate seed oil in this regard with pure chemicals such as a rare
form of Vitamin E, delta tocopherol, which is known to cause apoptosis in breast cancer cells.
Pomegranate seed oil outperformed the pure compounds. A more recent study showed that
applying 5% pomegranate seed oil to the skin of mice prevent these mice from developing
cancerous skin tumors.
11. Is PSO safe and are there any known contraindications? Pomegranate seed oil is a
natural substance with no known side effects or toxicity. People have eaten pomegranates
for thousands of years, often chewing and swallowing the seeds. If fresh pomegranates are
consumed in this way, the eater will also get a good dose of pomegranate seed oil. However,
because pomegranate seed oil also contains small amounts of natural estrogens, it is
probably best to avoid consuming it on a daily basis during the first trimester of pregnancy.
This is common sense for any medicinal substance.
12. How much PSO is contained in a normal pomegranate? In general, it takes about
500 kg of fresh pomegranates to produce 1 kg of pomegranate seed oil. A small 100 g
pomegranate contains about 200 mg of oil.
13. How much PSO should a person take on a daily basis? Follow the example of eating
one pomegranate a day. For an average 250 g pomegranate, the daily dose would be about
500 mg of oil per day. This comes out to about 7-8 drops of oil on a daily basis, more or less
depending on body weight.
14. What is the relationship between PSO and pomegranate juice? Research with
prostate cancer cells showed that the cancer-suppressive effects of PSO were considerably
enhanced when used in combination with extracts from pomegranate fermented juice and
pomegranate peels. It is likely that the flavonoids and other polyphenols in pomegranate juice
synergistically augment the effects of PSO.
15. How could PSO and pomegranate juice help diabetes?
In a 2003 study in diabetic "Zucker" rats, a Japanese research team showed CLA to help
alleviate "insulin resistance," an important cause of adult diabetes. Further, the study
suggested that this improvement could also help prevent hypertension and obesity in these
animals.
Pomegranate juice contains antioxidants which prevent glycosylation, the adherence of sugar
to hemoglobin, and the start of many secondary effects of diabetes, such as eye problems.
However, ordinary pomegranate juice or pomegranate juice concentrate itself contains much
sugar, and is not recommended for diabetic patients. Only the de-alcoholized, fermented
juice is completely sugar-free.
16. To review, what are the potential uses of PSO? PSO has only begun to be studied, so
there is much we do not know. However, taking the experience with CLA as a guide, it is
likely that PSO may be useful for prevention of atherosclerosis and cancer, for normalizing
blood sugar and for improving the function of the immune system.
Rimonest Ltd.
TECHNICAL DATA SHEET
PO Box 9 45, Haifa, ISRAEL

http://www.a-o-t.com

Matuškovič Ján
Slovak University of Agriculture in Nitra, Slovakia




Abstract
We solve a problem of obtaining of Punica granatum L. mutants from 1991. Used chemical
mutagen in experiment was NaN3 (sodium azidimide) in 0,3; 0,5 and 0,7 mM concentrations.
Control variant was without use of mutagen. Seeds were macerated in solution of sodium
azidimide, rinsed, dried, sowed and plants were cultivated four years in greenhouse.
Qualitative traits – height, thickness of trunks and others were evaluated. We evaluate
suitability and intensity of vegetative propagation from mutants as mother plants. Cultivated
mother plants were hardened from spring to autumn in 1998 out of greenhouse and
transplanted in repository between greenhouses. Adaptability of plants was good – without
loss of plants. Formation of new shoots and in 2001 formation of generative buds and flowers
in 2001 confirms of plants vitality.
Keywords
pomegranate, Punica granatum, mutations, sodium azidimide
The culture of the decorative sorts in residential spaces belongs to the important part of the
ecology aesthetics. In this area a very large space and possibilities have been being created for
miniatures and later bonsai shapes. The interest in these ones has increased very much in our
country. ŠTOLC, 1990 and ŘIHA, 1991, meanwhile there are not enough plants of this type
available and the prices are high.
However, there are possibilities how to get over the period of the deficiency of suitable
material PFISTERER, 1991 and MATUŠKOVIČ, BRINDZA, 1994, for the flat miniaturesbonsai.
In general, however, the two problems have been being persisted, namely, the system
of reproduction and the new decorative types creation. The second problem can be solved
more over with the help of cultivation, in which the mutation phenomenon has been
successfully exploited MATUŠKOVIČ, BRINDZA, 1992, 1997 and 1999. The mentioned
question has been being attended by us since 1991.
We have chose the gender Punica, as a model plant, which is the only gender in the family of
Puniciacae, and has only two species. The first is Punica granatum L., so called ”granate
punic” and Punica protopnica Balf., so called ”granate sokotra, that is the endemic of the
island Sokotra POSPÍŠIL, HRACHOVÁ, 1989.
Historically, the granate represents the early cultural wood which had been grown by old
Egyptians long ago, the Greek used it as a symbol of fortility. The Roman imported the fruits
from Punicia – North Africa from here was the name Punica. The most characteristic is like
the thorny shrub with shiny subsidable counter standing foliage, brichred flowers and
bloodred apples RYŠAN, 1991.
The time and concentrate of chemical mutagenes application, which is our case, is wideranging
and in general depends on the evolutionary state of organism, its biological
peculiarities and chemical quality of mutagen. The works with the inducing of mutation by
various mutagenes are based in most cases with the aim of obtaining new signs at plant
organisms.
In practical cultivation it is possible rather wide exploitation of the experimental mutagen,
which is based on the knowledge, that mutagen is effective in increasing of diverseness of
starting material for selection, the genetic variability of biological material signs is getting
wider. For this activity various organ are used, or parts of the plants – seeds, pollen, buds,
trimmings and others. In the forms of amount various varieties of granates punic, taken by
chemical or physical mutagenes attending, according to MAMEDOVA, 1984, the function of
chromosome abnormalities was higher than with non-controlled attending.
The mutagenesis and concretely radiation with subtropical plants, among which also granates,
studied AKHUNDZADZE, 1981. It was about the influence of gamma-ray on the trimmings,
seeds and pollen. Alltogether there were recorded 12 types of mutate changes and the most
frequent change was stuntedity and with irradiate of trimmings the acceleration of fertility
was recorded. The mutantes of all examined models were used on with hybridization.
Also Kergadze, 1987 studied the radiation mutagenesis with the help of gamma-ray on the
granates. With the help of this method he cultivated new variety of granate punic mutant ”
KARABAKH ” .
MATERIALS AND METHODS
We obtained the seeds of the granate punic from the fruits carried from Jalta in 1991.
Chosen chemo-mutagen NaN3 – sodium azidimide
It belongs to the group folded of very miscellaneous chemical compounds. It is one of the
most effective mutagens of cultural plants, which is exploited for cultivate aims. From the
chemical point of view NaN3 is white, very well water – soluble crystalline substance. It is
prepared by the reaction of molten sodium amide with nitrooxygen. It can be melted without
decomposition but following heating causes outbreak.
NaNH2 + N2O –––> NaN3+H2O
Chosen variations:
I. Check – without sodium azidimide
II. Concentration 0.3 mM of solution of sodium azidimide
III. Concentration 0.5 mM of solution of sodium azidimide
IV. Concentration 0.7 mM of solution of sodium azidimide
• Seeds were divided into 4 groups by 100 pieces
• For 20 hours they were mazering in chosen concentrations
• Twice washed in destil water each time during 1 hour
• We dried them on the filter paper
• Planting of seeds 16.4.1991
Signs rated:
• the high of plants
• number of branching
• thickness of stems
• increases of summer growth
• increase of one-year-old wood
In winter 1992 we tried concentriations NaN3 0.1, 0.9 and 1.1 mM on the very small pattern
of seeds carried from Libya ( 50 pieces ).
In the next years we decided to verify suitability and intensity of vegetative multiplied
biological material from the maternal plants of already mentioned varieties.
• take of biological material was done from 3 groups of applicated NaN3, concentrations
0.3, 0.5 and 0.7 mM in summer and autumn period • trimming used – top
- middle
• number of varieties – 6
• repetitions 4
• number of trimmings – 10 pieces in each repetition
• alltogether was tested – 240 trimming pieces
• without growth stimulators
Watching and checked factors:
• ability to create callose and roots
• regenerative ability – top and middle trimmings
• the most suitable concentration NaN3 for taking roots trimming
• determination of basic factors for securing intensive growth of multiplied plants
• to make an attempt to specify the factors, conditioning dwarf forms of growth
• results rating – Chi – quadrate by test
- with signification to 0.01 highty proved
to 0.05 proved
and over 0.05 not proved
In 1999, 28 th May we planted maternal plants into repositorium which was organised
between two greenhouses.
From schoolyears 2000 to 2001 there was invited tenders on a doctorand work under the title
”Study of mutants of granate punic (Punica granatum L.)” which is still going on.
RESULTS ACHIEVED
I have been fumbling with the idea of creating and forming some species of decorative plants
for possible culture in the form of miniature-bonsai since 1980.
But later, in 1990, together with Mr.lecturer Brindza, we started to experiment the possibility
of verifying the influence of chemo-mutagen NaN3 – sodium azidimide on the seeds of Punica
granatum L. with the aim of obtaining mutate changes in the habit of plants and in other signs,
especially of decorative character.
In 1991, when the seeds of chosen varieties were sown, we got at control 15 plants with
concentration 0.3 mM – 19 plants, 0.5 mM – 49 plants and 0.7 mM – 21 plants. We recorded
the loss at 0.5 mM – 5 plants and at 0.7 mM – 1 plant.
In the next year 1992, I did the selection from each variety that was examined by phenological
watching and measuring.
At measuring of the plant height we found out quicker increase at control and varieties with
lower concentration NaN3.
The creation of branching number does not have positively linear character, although, we
found out rather big differences between the applications NaN3. Higher concentrations
limitated number of growing branches expressively.
The thickness of stems is developing almost straight-linear with seoson period while higher
concentration suppresses thickness expressively in comparison with control.
At measuring of increases of summer-growths we can positively prove inhibitive influence
of NaN3 in comparison with control.
A very interesting knowledge was acheived while rating the growth of length in one-year-old
wood. Although this sign is of little influence in the range of time period, higher
concentrations were more successful against control. Beside phenological watching and measuring, we have found out following visual changes on
the set of model plants:
• at the control variety the plants distinguish natral, quicker increase of biomass. The wood
colour is ashen – pale, the leaves have normal size without amy change.
• at varieties with the application of NaN3, the slower increase of biomass is found out. It is
evident at thinner stems, smaller leaves – which turn only little, they are fatter and their
colour and wood colour is darker.
The induction of mutants Punica granatum L. – granate punic is, according the effect of
chosen concentrations NaN3, successful by vegetative reproduction.
At rating results we started from individual varieties, their best and less successful ability to
take roots, presented in pieces, per centages and mathematic-statistic method Chi – quadrate
by test.
We have come to the following conclusion:
• Callose creation
From the terms of taking trimming the most successful was the seemd ( autumn) period with
the effect of callose creation 36.2 per cent against the first ( summer ) with the effect only
15.4 per cent.
From the exploited biological material of mutant the callose was best made with the top
trimming, from 32.5 per cent to 57.5 per cent. The middle trimmings were lower successful,
from 22.5 per cest to 40.0 per cent.
The sequence of influence of concentration NaN3 on the creation of callose is following:
- variety 0.7 next 0.3 and 0.5 mM concentration
• The roots creation
From the terms of taking the trimmings more successful was the first ( summer ) period with
the effect of creation roots of 35.0 per cent against the second ( autumn ) with the effect of
only 27.5 per cent.
From the exploited biological material of mutants the best roots were created from the middle
trimmings (middle) from 85.0 per cent to 60.0 per cent. At the top trimmings the effect was
substendially lower, from 20.0 per cent to 7.5 per cent.
The sequence of concentration influence of NaN3 on the creation of roots is following:
- variety 0.7 next 0.5 and 0.3 mM of concentration.
• The trimmings loss ( died away)
From the terms of taking trimmings there was the second ( autumn ) period more successful
with the loss of 36.2 per cent against the first term ( summer ), when the loss was 49.6 per
cent of trimmings.
From the exploited biological mutant material the least loss was from the middle trimmings
from 12.1 per cent to 6.7 per cent. From the top trimmings died away more, from 37.5 per
cent to 29.6 per cent.
The sequence of concentration influence of NaN3 on the loss of trimmings is following:
- variety 0.3 nex 0.5 and 0.7 mM of concentration
• At rating of regenerative ability of the top and middle trimmings from the mutants Punica
granatum L. by the method chi – quadrate by test, was proved the economic
effectiveness of obtaining regenerats of these mutants by middle trimmings in both
periods of harvest.
After two years of plants hardiness ( spring – autumn ) out of greenhouse, we finally planted
these maternal plants into the prepared reprositorium. It is the space between two
greenhouses.There were planted 33 eight – year – old maternal plants. According to varieties we have
chosen individually following numbers:
• control ( checked ) variety 6 plants
• mutants 0.1 mM 6 plants
• mutants 0.3 mM 5 plants
• mutants 0.5 mM 8 plants
• mutants 0.7 mM 8 plants
ABSTRACT
We started to work with the possibility of obtaining mutants Punica granatum L. in 1991.
We use NaN3 as the chemo-mutagen – sodium azidimide in 0.3 – 0.5 and 0.7 mM
concentration and the control variety without NaN3. With chemomutagen NaN3 we mazered
the seed material, that was, after the procedure of dripping, washing and drying obtained
mutants, planted aned later cultivated till the fourth year of life. At the same time we rated the
qualitative signs ( height of plants, thickness of stems and others ). Next we verified
suitability and intensity of vegetative reproduction from already maternal plants of mutants.
The cultivated maternal plants of mutants we planted in 1999 only in conditions of
greenhouse, after the hardiness out of greenhouse in the previous year ( spring-autumn), into
the repositorium between the two greenhouses. Their adaptability to outside is going on
excellently without loss. On the contrary, their increases in wood, this year 2001 also the
amount of developed flower buds and flowers prove their vitality.
References:
AKUZNDZADZE, J.M.: Radiačná mutagenéza subtropických plodín. 1.Vses.konf.po
prikl.radiobiol. Teor. Prikl. Aspekty radiats. Biol. technol., 10-12 no, 1981. Tez.
Dokl.1981, 50-51.Kischonev, Moldavian.
KERKADZE, J.G.: Radiačná mutagenéza pri subtropických ovocných drevinách. Radiat
sionnyi mutagenez i ego rol v evoluyutsii i selektissii, 1987, s.231-354, Moscow,
USSR.
MAMEDOV, G.M.: Meotické abnormality v indukovaných formách granátovníka púnskeho
v dôsledku ošetrenia semien rôznymi dávkami chemických a fyzikálnych
mutagenov.2.Vses.konf.po s. – kh.radiol., Tez. Doz. T.2 1984, s.63-64, Obninsk,
USSR.
MATUŠKOVIČ, J., BRINDZA, J.: Vplyv azidu sodného na formovanie habitusu rastlín pri
granátovom jablku ( Punica granatum L.). Zborník AF, VŠP Nitra, 1992, s.51-57.
MATUŠKOVIČ, J., BRINDZA, J.: Tvorba a formovanie vybraných druhov rastlín pre
kultiváciu v tvare bonsaj. Záverečná správa, marec 1994, 18s.
MATUŠKOVIČ,J., BRINDZA,J.: Indukcia mutácií ovocného klonu granátovníka púnskeho.
Záverečná správa, december 1999, 31 s.
PFISTERER,J.: Zimmer bonsai, Gu-Pflanzen, Ratgeber, München 1991.
POSPÍŠIL, F., HRACHOVÁ, B.: Úžitkové rostliny jižních zemí. ČSAV,Praha 1989.
RYŠAN, M.: Bonsaj, SZZ, Bratislava 1991, 176 s.
ŘÍHA, P.: Rok s bonsají, Prospektrum-Praha 1991.
ŠTOLC, K.J.: Bonsaj, Bonsaj klub Bratislava 1990 Danubiaprint.
Author's contact:
Doc. Ing. Ján Matuškovič, PhD.
Katedra ovocinárstva, vinohradníctva a vinárstvaTr.A.Hlinku 2
SPU 949 76 Nitra
e-mail: Jan.matuskovic@-uniag.sk.

ABSTRACT Pomegranate seed oil was investigated for possible skin cancer chemopreventive efficacy in mice. In the main
experiment, two groups consisting each of 30, 4–5-week-old, female CD1 mice were used. Both groups had skin cancer initiated
with an initial topical exposure of 7,12-dimethylbenzanthracene and with biweekly promotion using 12-O-tetradecanoylphorbol
13-acetate (TPA). The experimental group was pretreated with 5% pomegranate seed oil prior to each TPA application.





Tumor incidence, the number of mice containing at least one tumor, was 100% and 93%, and multiplicity, the
average number of tumors per mouse, was 20.8 and 16.3 per mouse after 20 weeks of promotion in the control and pomegranate
seed oil-treated groups, respectively (P , .05). In a second experiment, two groups each consisting of three CD1 mice
were used to assess the effect of pomegranate seed oil on TPA-stimulated ornithine decarboxylase (ODC) activity, an important
event in skin cancer promotion. Each group received a single topical application of TPA, with the experimental group
receiving a topical treatment 1 h prior with 5% pomegranate seed oil. The mice were killed 5 h later, and ODC activity was
assessed by radiometric method. The experimental group showed a 17% reduction in ODC activity. Pomegrante seed oil (5%)
significantly decreased (P , .05) tumor incidence, multiplicity, and TPA-induced ODC activity.
INTRODUCTION
SKIN CANCER is the most common type of cancer in the
United States,1 with more than a million reported cases2
and 9,000 deaths per year.3 Increasing incidence of these
cancers due to constant exposure of skin to environmental
carcinogens, including both chemical agents and ultraviolet
radiation, provides a strong basis for chemoprevention with
both synthetic and natural, and internal and topical, remedies.
4 Further, skin cancer chemoprevention is a useful
model for cancer chemoprevention in general.5
Chemical and UVB radiation-induced skin carcinogenesis
in murine skin and possibly human skin is a stepwise
process of at least three distinct stages: initiation, promotion,
and progression. Experimental initiation in vivo is accomplished
by the topical application of a single dose of
a skin carcinogen such as 7,12-dimethylbenzanthracene
(DMBA), and is essentially irreversible. However, an initiation
dose of carcinogen may not produce visible tumors,
resulting only following prolonged and repeated application
of a tumor promoter such as 12-O-tetradecanoylphorbol 13-
acetate (TPA) to initiated skin.6,7 Promoters like TPA induce
ornithine decarboxylase (ODC), the rate-limiting enzyme
in the synthesis of polyamines8 and an important
molecular target for skin cancer chemoprevention.9 Other
targets may also involve promotion, or initiation or progression
events in the multistage process of neoplastic development.
Our previous work has highlighted the efficacy of topically
applied natural products derived from onion and garlic
oils,10 and more recently sandalwood oil11,12 and its constituent,
13 in preventing skin tumors in CD1 and SENCAR
mice. In the present work, we bring this experience to bear
on the study of pomegranate seed oil as a potential skin cancer
chemopreventive product.
Pomegranate fruit (Punica granatum) has been used
worldwide as an item of diet and medicine for millenia, and
has also been regarded as an important symbol in world religions
and mythologies and of medicine itself.14 We previously
demonstrated potent antioxidant and prostaglandininhibitory
activities for polyphenols extracted from pomegranate
seed oil and pomegranate fermented juice,15 as well
as a wide range of human breast cancer suppressive properties
in vitro, including promotion of apoptosis and inhibi-
tion of proliferation and invasion by the seed oil, and inhibition
of DMBA-initiated carcinogenesis in a mouse mammary
organ culture (MMOC) by the fermented juice
polyphenols.16 We recently showed chemopreventive activity
of the whole seed oil in the MMOC to be even stronger,
weight per weight, than that of the purified fermented juice
polyphenols.17
Pomegranate seed oil consists of .80% conjugated fatty
acids, the most important of which is the octadecatrienoic
acid, punicic acid. Punicic acid, like the ,1% polyphenols
in pomegranate seed oil, is an inhibitor of prostaglandin
biosynthesis.18 Punicic acid is also cytotoxic to mouse
leukemia cells, possibility related to inhibition of lipid peroxidation.
19 Pomegranate is one of only about a half dozen
plants known to contain conjugated fatty acids. A possible
relationship between the relative botanical isolation of
pomegranate and its singular chemistry and anticancer properties
has been noted.20
The purpose of the present investigation was to study
the chemopreventive effects of pomegranate seed oil on
DMBA-initiated and TPA-promoted skin tumor development
during the initiation and promotion phases in CD1
mice. Further, the effects of pomegranate seed oil on weight
gain and ODC activity in the experimental animals were also
evaluated.
MATERIALS AND METHODS
Pomegranate seed oil
Pomegranate seed oil was provided by Rimonest Ltd. (Rimonest
Ltd., Haifa, Israel; www.rimonest.com) from pomegranates
of the “Wonderful” cultivar, organically grown at
Kibbutz Sde Eliahu, Israel, in the year 2000. Seeds were
separated from their juice sacs, washed in water, and dried
in a convection current solar dryer. Oil extrusion was by
“cold press” at 80°C, using a Type 40A electric screw press
(Skeppsta Maskin, Orebro, Sweden). The oil was assayed
by an independent laboratory (Mylnfield Research Services,
Invergowrie, Dundee, Scotland) and shown to contain not
less than 80% conjugated fatty acids as triglycerols, diglycerols,
and monoglycerols.
Tumorogenesis protocol
The skin cancer protocol of Dwivedi et al.13 was used. In
brief, 4–6-week-old CD1 mice were divided into two groups,
each group containing 30 mice, as indicated in Table 1. The
mice were kept in an environmentally controlled room with
temperature, humidity, and light regulated. The backs of the
mice were shaved carefully with an electric clipper to avoid
cuts. The mice were allowed to rest for 2 days before carcinogenesis
was initiated.
Carcinogenesis was initiated with DMBA (200 nmol in
100 mL of acetone) applied topically. One week later, carcinogenesis
was promoted with TPA (5 nmol in 100 mL of
acetone), applied topically twice weekly. TPA treatment
continued throughout the duration of the experiment (20
weeks). Mice in group 1 served as the control and were pretreated
topically with 100 mL of acetone 1 h prior to each
TPA application. Mice in group 2 were pretreated topically
with 100 mL of 5% pomegranate seed oil in acetone 1 h
prior to each TPA application. Tumor counts and group
weights were taken on a weekly basis. Tumor incidence and
multiplicity were calculated and analyzed statistically.
ODC assay
Mice were divided into two groups, each containing three
mice. The backs of the mice were shaved carefully with an
electric clipper to avoid cuts. Mice in group 1 received 100
mL of acetone before TPA (5 nmol in 100 mL of acetone)
treatment topically. Mice in group 2 received 100 mL of 5%
pomegranate seed oil in acetone, before topical TPA (5 nmol
in 100 mL of acetone) treatment.
Mice were killed 5 h after the topical applications of TPA.
The dorsal epidermis was removed and homogenized in
phosphate buffer (pH 7.2) containing 0.1 mM pyridoxal
phosphate and 0.1 mM EDTA. The homogenate was centrifuged
at 105,000 g for 90 min and the supernatant collected
and used for the ODC assay. The assay mixture in
the main part of a Warburg flask was composed of 40 mL
of phosphate buffer (pH 7.2), 25 mL of pyridoxal phosphate,
25 mL of dithiothreitol, 25 mL of EDTA, 10 mL of L-ornithine
containing 0.5 mCi of DL[1-14C]ornithine, and 200
mL of epidermal supernatant.
The center well of the Warburg flask contained 400 mL
of ethanolamine and methoxyethanol used to absorb the
14CO2 produced in the main compartment. After incubation
at 37°C for 1 h, the reaction was stopped by the addition of
500 mL of citric acid. The mixture was stored in a dark place
overnight to ensure complete absorption of 14CO2 in the center
well. The contents of the center well were transferred to
a scintillation vial. The center well was washed with 0.5 mL
of ethanol four times, and the wash also added to the scintillation
vial, along with 10 mL of scintillation fluor. Radioactivity
was counted with a Beckman LS6000SE liquid
scintillation counter. The disintegrations per minute were
quantified. Assessment of ODC activity was accomplished
by measuring the production of 14CO2 from DL-[1-14C]ornithine.
Protein assay
Protein was assayed in the supernatant with a Bio-Rad
Protein Assay Kit. A standard curve was obtained using
bovine serum albumin. Absorbance values at 595 nm were
determined using the spectrophotometer. Protein concentra-
tions of the supernatant were extrapolated from the standard
curve data.
Statistical analysis
The INSTAT software (GraphPad, San Diego, CA,
U.S.A.) was used for the data analysis. x2 was used for the
comparison of papilloma incidence and Student’s t test for
tumor multiplicity and ODC activity. Significance was considered
at P , .05.
RESULTS
The effects of pomegranate seed oil treatment on the incidence
of skin tumors in CD1 mice are shown in Fig. 1.
Skin tumors appeared in the sixth week of promotion after
the initial DMBA application in the control and treated
groups. Pomegranate seed oil treatment did not delay the appearance
of tumors, but significantly decreased (P , .05)
the rate at which the tumors developed. Skin tumor incidence
after 20 weeks of promotion was 100% and 93% for
the control and 5% pomegranate seed oil-treated groups, respectively.
The effects of pomegranate seed oil treatment on tumor
multiplicity in CD1 mice are shown in Fig. 2. Pomegranate
seed oil treatment significantly decreased (P , .05) the tumor
multiplicity throughout the 20 weeks of promotion. The
mean number of tumors per mouse was 20.8 and 16.3 for
the control and 5% pomegranate seed oil-treated groups, respectively.
Topical application of 5% pomegranate seed oil also significantly
inhibited (P , .05) TPA-induced epidermal ODC
activity. Fig. 3 illustrates the effects of pomegranate seed
oil treatment on TPA-induced epidermal ODC activity. The
ODC activity was 18.49 and 14.84 nmol of 14CO2/mg/h in
the control and 5% pomegranate seed oil-treated groups, respectively.
The pomegranate seed oil group has significantly
(P , .05) decreased ODC activity. Topical application of
5% pomegranate seed oil alone did not induce any epidermal
ODC activity. Topical application of 5% pomegranate
seed oil also did not have any effect on weight gain, as indicated
in Fig. 4.
CONCLUSIONS
Pomegranate seed oil (5%) topical applications significantly
decreased the incidence of skin tumor development,
skin tumor multiplicity, and ornithine decarboxylase activity
during 20 weeks of promotion. It is thus likely that the
inhibition of ornithine decarboxylase by the pomegranate
seed oil was at least partially responsible for the chemopreventive
effect.
As noted, pomegranate seed oil is very rich in punicic
acid, a known inhibitor of prostaglandin biosynthesis,
specifically by inhibiting cyclooxygenase (Cox 1 and Cox
2) and lipoxygenase.21 Pomegranate seed oil also inhibits the
upstream eicosanoid enzyme, phospholipase A2, expressed
by human prostate cancer cells.22 That prostaglandins at very
low concentrations promote ornithine decarboxylase23 suggests
that the inhibition of prostaglandin biosynthesis by
pomegranate seed oil might also contribute to its inhibition
of ornithine decarboxylase and, ultimately, to inhibition of
skin cancer promotion.
Overall, pomegranate seed oil appears to be a benign natural
product with potential as a topical chemopreventive
agent against skin cancer. More in-depth investigations, including
clinical studies, are warranted to evaluate this hypothesis
further.
ACKNOWLEDGMENTS
The authors wish to thank Mr. Eli Merom of Kibbutz Sde
Eliahu, Israel, for supplying the organically grown pomegranates
used in this study. Thanks also to Alexander
Botvinnik for technical assistance in the preparation of the
manuscript.
REFERENCES
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3. Cancer Facts & Figures—1996. American Cancer Society Publication
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4. Gupta S, Mukhtar H: Chemoprevention of skin cancer: current
status and future prospects. Cancer Metastasis Rev 2002;21:
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5. Richmond E, Viner JL: Chemoprevention of skin cancer. Semin
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6. Agarwal R, Mukhtar H: Cutaneous chemical carcinogens. In:
Pharmacology of the Skin (Mukhtar H, ed.). CRC Press, Boca Raton,
FL, 1991, pp. 371–387.
7. Boutwell RK: Some biological aspects of skin carcinogenesis.
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8. O’Brien TG, Simsiman RC, Boutwell RK. Induction of the
polyamine-biosynthetic enzymes in mouse epidermis by tumorpromoting
agents. Cancer Res 1975;35:1662–1670.
9. Stratton SP, Dorr RT, Alberts DS. The state-of-the-art in chemoprevention
of skin cancer. Eur J Cancer 2000;36:1292–1297.
10. Dwivedi C, Rohlfs S, Jarvis D, Engineer FN. Chemoprevention
of chemically induced skin tumor development by diallyl sulfide
and diallyl disulfide. Pharm Res 1992;9:1668–1670.
11. Dwivedi C, Abu-Ghazaleh A. Chemopreventive effects of sandalwood
oil on skin papillomas in mice. Eur J Cancer Prev 1997;
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12. Dwivedi C, Zhang Y: Sandalwood oil prevents skin tumour development
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13. Dwivedi C, Guan X, Harmsen WL, Voss AL, Goetz-Parten D, Koopman
EM, Johnson KM, Valluri HB, Matthees DD: Chemopreventive
effects of a-santalol on skin tumor development in CD1 and
Sencar mice. Cancer Epidemiol Biomarkers Prev 2003;12:151–156.
14. Langley P. Why a pomegranate? BMJ 2000;321:1153–1154.
15. Schubert SY, Lansky EP, Neeman I. Antioxidant and eicosanoid
enzyme inhibition properties of pomegranate seed oil and fermented
juice flavonoids. J Ethnopharmacol 1999;66:11–17.
16. Kim ND, Mehta R, Yu W, Neeman I, Livney T, Amichay A,
Poirier D, Nicholls P, Kirby A, Jiang W, Mansel R, Ramachandran
C, Rabi T, Kaplan B, Lansky E. Chemopreventive and adjuvant
therapeutic potential of pomegranate (Punica granatum)
for human breast cancer. Breast Cancer Res Treat 2002;71:
203–217.
17. Unpublished data presented at the 13th Annual Meeting of the
North American Menopause Society, Chicago 2002. Rajendra
Mehta, Department of Surgical Oncology, University of Illinois
at Chicago.
18. Nugteren DH, Christ-Hazelhof E. Naturally occurring conjugated
octadecatrienoic acids are strong inhibitors of prostaglandin
biosynthesis. Prostaglandins 1987;33:403–417.
19. Suzuki R, Noguchi R, Ota T, Abe M, Miyashita K, Kawada T: Cytotoxic
effect of conjugated trienoic fatty acids on mouse tumor and
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20. Longtin R: The pomegranate: nature’s power fruit? J Natl Cancer
Inst 2003;95:346–348.
21. Unpublished data, Robert Newman, Department of Pharmacology,
MD Anderson Cancer Center, Houston, TX.
22. Unpublished data, Wenguo Jiang, Department of Surgery, Cardiff
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POMEGRANATE SEED OIL AND SKIN CANCER IN MICE 161

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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