Note: Descriptions are shown in the official language in which they were submitted.
CA 02389537 2002-05-21
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"MIXTURE OF POMEGRANATE SEED OIL AND JUICE PRODUCTS"
FIELD AND BACKGROUND OF THE INVENTION
s The present invention relates to a mixture of a pomegranate seed oil product
and a
pomegranate juice product and to a pharmaceutical composition containing same.
More
particularly, to a the mixture of the present invention has cancer preventing
properties and a
pharmaceutical composition containing the mixture may be advantageously
employed to treat
or prevent a variety of conditions, including but not limited to, cancer,
alzheimer's disease,
to climacteria, benign prostatic hyperplasia and estrogen deficiency.
Pomegranate (Punica granatum) has long been recognized as a fruit with many
benefits for health. t The plant is botanically unique, having actually only
one true botanical
relative, the pomegranate precursor, Punica protopunica, restricted to the
isolated island
Socotra off the coast of Yemen. Corresponding to this botanical uniqueness is
a parallel
15 distinctiveness in terms of its biochemistry. For example, pomegranate has
long been
recognized as the richest plant source of the female steroid hormone estrone,2
and recently,
the male hormone testosterone and another female steroid, estriol, have also
been discovered
in pomegranate seed oil.3 A wide range of polyphenolic compounds including
flavonoids,
anthocyanins and tannins have been characterized both in pomegranate juice4
and pericarp.5
2o Further, concentrations of these polyphenols extracted both from the
fermented juice and the
oil have been shown to be potently antioxidant in vitro and to additionally
inhibit the
eicosanoid enzyme lipoxygenase, and in the case of the polyphenols extracted
from
pomegranate seed oil, to also be significantly inhibitory of another
eicosanoid pathway
enzyme, cyclooxygenase,b
25 However, pomegranate products have not previously been demonatrated to
affect activity
of aromatase, an enzyme which catalyzes the transformation of andostenedione
to estrone, and of
Frawley, D and Lad, V. The Yoga of Herbs: An Ayurvedic Guide to Herbal
Medicine, Lotus Press, Twin Lakes,
WI 1986.
2 Moneam, N.M.A., EI Sharaky, A.S., and Badreldin, M.M. Oestrogen content of
pomegranate seeds. Journal of
Chromotography 438: 438-442, 1988.
3 Abd El Wahab, S.M., El Fiki, S.F., Mostafa, S.F. and Hassan, A.E.B.
Characterization of certain steroid hormones
in Punica eranatum L. seeds. Bulletin of the Faculty of Pharmacy of Cairo
University 36(1): 11-15, 1998.
4 Artik, N., Cemeroglu, B., Burakami, H., and Mori, T. Determination of
phenolic compounds in pomegranate juice
by HPLC. Fruit Process 8 ( 12): 492-499, 1998.
Ben Nasr, C., Ayed, N., and Metche, M. Quantitative determination of the
polyphenolic content of pomegranate
peel. ZLebensm Unters Forsch 203 (4): 374-378, 1996.
6 Schubert, S.Y., Lansky, E.P., and Neeman, I. Antioxidant and eicosanoid
enzyme inhibition properties of
pomegranate seed oil and fermented juice flavonoids. Journal
ofEthnopharmacology 66 (1): 11-17, 1999.
1
CA 02389537 2002-05-21
WO 01/37848 PCT/IL00/00800
testosterone to estradiol. In addition, there has been no experimental
analysis of the ability of
pomegranate fractions to interfere with the estrogenic activity of a compound
known to exert
estrogenic activity, namely 17-beta estradiol.
Demonstration of these capabilities woulde suggest potential utility in the
prevention and
treatment of cancer, including, but not limited to, estrogen dependent cancers
such as those of
breast and prostate, as well as for colon cancer. Such utility could easily
extend beyond cancer to
include a wide range of immune deficient and auto-immune based pathology, and
even to
Alzheimer's disease changes,'' g .
There is thus a widely recognized need for, and it would be highly
advantageous to
have, a mixture of a pomegranate seed oil product and a pomegranate juice
product and to a
pharmaceutical composition containing same possessing thes capabilities
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a cancer
chemo-preventive mixture. The mixture comprises a pomegranate seed oil product
and a
pomegranate juice product.
According to another aspect of the present invention there is provided a
pharmaceutical composition. The composition comprises physiologically active
amounts of
a pomegranate seed oil product and a pomegranate juice product and a
pharmaceutically
2o acceptable carrier.
According to further features in preferred embodiments of the invention
described
below, a pomegranate peel product is further included.
According to still further features in the described preferred embodiments the
pomegranate seed oil product is the result of a process selected from the
group consisting of
expeller pressing, supercritical fluid extraction with carbon dioxide, and
lyophilization.
According to still further features in the described preferred embodiments the
pomegranate seed oil product is produced from a material selected from the
group consisting
of pomegranate seeds and pomegranate seed cake.
' Bonnefont, A.B., Munoz, F.J_, and Inestrosa, N.C. Estrogen protects neuronal
cells from the cytotoxicity induced
by acetylcholinesterase-amyloid complexes. FEBSLetters 441: 220-224, 1998.
g Xu, H., Gouras, G.K., Greenfield, J.P., Vincent, B., Naslund, J.,
Mazzarelli, L., Fried, G., Jovanovic, J.N., Seeger,
M., Relkin, N.R., Liao, F., Checler, F., Buxbaum, JK.D., Chait, B.T.,
Thinakaran, G., Sisodia, S.S., Wang, R.,
Greengard, P. and Gandy, S. Estrogen reduces neuronal generation of Alzheimer
beta-amyloid peptides. Nature
Medicine 4: 447-451, 1998.
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According to still further features in the described preferred embodiments the
pomegranate seed oil product is selected from the group consisting of
pomegranate seed oil
and a non saponifiable fraction thereof.
According to still further features in the described preferred embodiments the
pomegranate juice product comprises at least one item selected from the group
consisting of
pomegranate juice, fermented pomegranate juice, dried pomegranate juice, dried
fermented
pomegranate juice, partially fermented pomegranate juice, partially dried
pomegranate juice,
partially fermented partially dried pomegranate juice, reduced pomegranate
juice, partially
reduced pomegranate juice and lyophylysates thereof .
to According to still further features in the described preferred the mixture
is provided in
a form selected from the group consistiong of a liquid, a powder, granules, a
tablet, a capsule,
a gel-tab, an ointment, a cream, a chewing gum, a food, a candy, an emulsion
and a
suppository.
According to still further features in the described preferred embodiments the
cancer
is a hormone dependent cancer.
According to still further features. in the described preferred embodiments
the
hormone dependent cancer is selected from the group consisting of breast
cancer and prostate
cancer.
According to still further features in the described preferred embodiments the
pomegranate peel product is selected from the group consisting of pomegranate
peel residue
present in pomegranate juice as a result of a juicing process, an aqueous
extract of
pomegranate peel, an alcohol extract of pomegranate peel, an extract performed
with an
organic solvent which is not alcohol, and a supercritical COZ extract of
pomegranate peel.
According to still further features in the described preferred the
pharmaceutical
composition is efficaciouly employed for treatment of a medical condition.
According to still further features in the described preferred embodiments the
medical
condition is selected from the group consisting of cancer, alzheimer's
disease, climacteria,
benign prostatic hyperplasia and estrogen deficiency.
According to still further features in the described preferred embodiments the
3o treatment is selected from the group consisting of a prophylactic
treatment, a palliative
treatment and a therapeutic treatment.
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According to still further features in the described preferred embodiments the
physiologic activity results from inhibition of an enzyme selected from the
group consisting
of aromatase and 17-beta-hydroxysteroid dehdrogenase (HSD) type 1.
According to still further features in the described preferred embodiments the
active
ingredients of the mixture or pharmaceutical composition comprise
dealcoholized
concentrated pomegranate wine, aqueous extract of pomegranate pericarp, and
seed cake
extract.
According to still further features in the described preferred embodiments the
physiologocally active ingredients comprise approximately 70% dealcoholized
concentrated
l0 pomegranate wine, approximately 10% aqueous extract of pomegranate
pericarp, and
approximately 20% seed cake extract.
According to still further features in the described preferred embodiments the
physiologocally active ingredients comprise approximately 30% dealcoholized
concentrated
pomegranate wine, approximately 10% aqueous extract of pomegranate pericarp,
and
l5 approximately 60% seed cake extract.
The present invention successfully addresses the shortcomings of the presently
known
configurations by providing a mixture of a pomegranate seed oil product and a
pomegranate
juice product, and pharmaceutical compositions containing same, which is has
potential
2o efficacy in prevention or treatment of cancer and other medical conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to
the
accompanying drawings. With specific reference now to the drawings in detail,
it is
25 stressed that the particulars shown are by way of example and for purposes
of illustrative
discussion of the preferred embodiments of the present invention only, and are
presented
in the cause of providing what is believed to be the most useful and readily
understood
description of the principles and conceptual aspects of the invention. In this
regard, no
attempt is made to show structural details of the invention in more detail
than is necessary
3o for a fundamental understanding of the invention, the description taken
with the drawings
making apparent to those skilled in the art how the several forms of the
invention may be
embodied in practice.
In the drawings:
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FIG. 1 is a histogram illustrating estrogenic activity of pomegranate juice
(P~
m a yeast estrogen screen;
FIG. 2 is a is a histogram illustrating aromatase inhibition by polyphenols
originating
in seed oil, peel extract and wine of pomegranates;
FIG. 3 is a table providing a numerical summary of the data of figue 2;
FIG. 4 is a histogram illustrating estrogenic activity of pomegranate non-
saponifiable
fraction (NSF) in a yeast estrogen screen;
FIG. 5 is a graph illustrating the effect of polyphenol fractions from
pomegranate seed oil, pericarp and fermented and fresh juice on proliferation
to of estrogen-dependent human breast cancer cells (MCF-7) in vitro;
FIG. 6 is is a graph illustrating the effect of effect of polyphenol
fractions from pomegranate seed oil, pericarp, and fermented and fresh
juice on proliferation of estrogen-independent human breast cancer cells
(MDA-MB-231 ) in vitro ;
FIG. 7 is a graph illustrating the effect of pomegranate pericarp
polyphenols on the proliferation of selected cancerous and normal cell
lines. Normal cell lines: Human Umbilical Vein Endothelium [HUVE] and Human
Foreskin Keratinocytes [HFK]). Cancerous lines marine melanocytic melanocytes
[B 16], human lung carcinoma [A549], human T cell leukemia [CCRF-HSB-2],
2o human gastric lymph node metastasis [TGBC11TKB]..;
FIG. 8 is a is a graph illustrating the effect of pomegranate fermented
juice polyphenols on the proliferation of selected cancerous and normal
cell lines (cell lines as detailed for figure 8);
FIG. 9 is a graph of the effect of pomegranate fresh juice polyphenols on
the proliferation of selected cancerous and normal cell lines (cell lines as
detailed for
figure 8). The effect here is clearly milder than for the fermented juice.
Again, the
most sensitive lines are the metastatic gastric and the leukemia. The normal
cell lines
are not affected.;
FIG. 10 is a graph of the effect of pomegranate pericarp polyphenols on
differentiation of
3o HL-60 human leukemia cells. The Y axis is proliferation, the absolute
amount of cell
growth. Cellular differentiation is observed most strongly in two of the
assays for cell
differentiation, namely nitro blue tetrazolium reducing activity and non-
specific esterase
activity. Less strong results were observed using the phagocytic acitivity and
specific
5
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esterase indices {NBT Nitro blue tetrazolium reducing activity; NSE Non-
specific
esterase activity; SE Specific esterase activity; PG Phagocytic activity; PR
Cellular
proliferation);
FIG. 11 is a graph of the effect of pomegranate fermented juice polyphenols on
differentiation of HL-60 human leukemia cells (assays as detailed for figure
11);
FIG. 12 is a graph of the effect of pomegranate fresh juice polyphenols on
differentiation of HL-60 human leukemia cells (assays as detailed for figure
11);
FIG.13 is a histogram of the effect of pomegranate seed oil on invasion
(metastasis)
of MCF-7 human estrogen-dependent breast cancer cells in vitro;
l0 FIG.14-18 are graphs of the effect of pure pomegranate seed oil on
proliferation of
human estrogen-dependent human breast cancer cells (MCF-7) in vitro (at 24,
72,
120, 168 and 26 hours of growth respectively);
FIG. 19 is a graph of the effect of pure pomegranate seed oil on proliferation
of
human LNCaP prostate cancer cells in vitro (at 120 hours);
FIG. 20 is a histogram illustrating aromatase inhibition by polyphenol
fractions
derived from seed oil, pericarp and fermented juice of pomegranate;
FIG. 21 is a histogram illustrating the effect of Pomegranate Fermented Juice
Polyphenols on proliferation of hPCPs (stromal benign prostatic hypertrophy)
Cells
FIG. 22 is a is a histogram illustrating the effect of pomegranate seed oil
polyphenols
on proliferation of hPCPs (stromal benign prostatic hypertrophy) cells;
FIG. 23 is a histogram illustrating the effect of combination of pomegranate
fermented
juice and seed oil polyphenols on proliferation of hPCPs (stromal benign
prostatic
hypertrophy) cells;
FIG. 24 is a histogram illustrating the effect of pomegranate fermented juice
polyphenols (W) on proliferation of human LNCaP human prostate cancer cells;
FIG. 25 is a graph of inhibition of proliferation of PC-3 human prostate
cancer cells at
progressively higher concentrations. Ethox = ethanol control, W = pomegranate
fermented juice polyphenols, P = pomegranate pericarp polyphenols, SCFO = pure
pomegranate seed oil;
3o FIG. 26 is a histogram of an alternative portrayal of inhibition of
proliferation of PC-3
human prostate cancer cells by pomegranate fractions. Ethox = ethanol control,
W =
pomegranate fermented juice polyphenols, P = pomegranate pericarp polyphenols,
SCFO = pure pomegranate seed oil.;
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FIG. 27 is a graph illustrating the effect of pomegranate pericarp extract
polyphenol
fraction of proliferation on poorly differentiation, androgen-independent PC-3
human
prostate cancer cells in vitro. The inhibition is concentration-dependent;
FIG. 28 is a graph illustrating the effect of pomegranate fermented juice
polyphenol
fraction (W) on the proliferation of PC-3 poorly differentiated, androgen-
independent
human prostate cancer cells in vitro. A concentration-dependent inhibition is
observed;
FIG. 29 is a graph illustrating the effect of effect of pure pomegranate seed
oil on
proliferation of human PC-3 poorly differentiated androgen-independent
prostate
l0 cancer cells in vitro;
FIG. 30 is a graph illustratring the concentration-dependent inhibition of
poorly
differentiation LNCaP human androgen-independent prostate cancer cells in
vitro by
pomegranate pericarp polyphenol fraction;
FIG. 31 is a graph illustrating the effect on proliferation of very poorly
differentiated
androgen-independent DU-145 human prostate cancer cells in vitro of
pomegranate
pericarp polyphenol fraction. The inhibition is concentration-dependent;
FIG. 32 is a graph illustrating the effect of a polyphenol fraction of
pomegranate
fermented juice on the proliferation of very poorly differentiated human
androgen-independent DU-145 prostate cancer cells in vitro. A
2o concentation-dependent inhibition is observed;
FIG. 33 is a graph illustrating the effect of pure pomegranate seed oil on the
proliferation of human very poorly differentiated androgen-independent DU-145
human prostate cancer cells in vitro. Inhibition is noted at the highest
concentration
tested;
FIG. 34 is a histogram illustrating the effect of pomegranate fermented juice
and
pericarp polyphenols on the Gl stage of the cell cycle in B16 melanin pigment
producing mouse melanoma cells. The y axis denotes % of cells at G1 phase at
time
of measurement. Increasing concentrations of the pomegranate fractions result
in an
increased number of cells at the Gl phase of arrest ;
FIG. 35 is a histogram illustrating the effect of pomegranate fermented juice
and
pericarp polyphenols on the G2 stage of B 16 melanin pigment producing mouse
melanoma cells. The y axis denotes % of cells at G2 at time of measurement.
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Increasing concentrations of the pomegranate fractions result in an increased
number
of cells at the G2 stage of arrest;
FIG. 36 is a histogram illustrating the effect of pomegranate fermented juice
and
pericarp polyphenols on the S stage of the cell cycle in B 16 melanin pigment
producing mouse melanoma cells. The y axis denotes % of cells at stage S
(synthesis
of DNA) of cell cycle at time of measurement. Increasing concentrations of the
pomegranate fractions result in decreased DNA synthesis;
FIG. 37 is a histogram summarizing overall effect on cell growth
(proliferation) of
B-16 marine melanin pigment producing melanoma cells by pomegranate fermented
to juice and pericarp polyphenol fractions. Increasing concentrations of the
active
materials result in an overall decrease in the growth of the cells ;
FIG. 38 is a histogram illustrating the effect on growth of HL-60 human
leukemia
cells in vitro by selected fractions of pomegranate fruit. [FJP = fermented
pomegranate juice polyphenol fraction; OP = pomegranate seed polyphenol
fraction.
EtOH (ethanol) control used here is at a much higher concentration that used
for
dissolving seed oil. OP and FJP are dissolved in DMSO (dimethyl sulfoxide).
Powerful inhibition is caused by FJP by fermented juice and by pomegranate
seed oil
at increasing concentrations;
FIG. 39 is a histogram illustrating the effect of selected pomegranate fruit
fractions on
2o the G1 phase of the cell cycle in HL-60 human leukemia cells. [ fractions
as in figure
38] Increasing the dose of the pomegranate fractions increases the percentage
of cells
at the G1 stage of arrest. Seed oil dissolved in 20 microliters per ml
ethanol; all
other fractions in DMSO 12.5 microliters per ml.;
FIG. 40 is a histogram illustrating the effect of pomegranate fruit fractions
on the G2
stage of cell division in human HL-60 promyelocytic leukemia cells. Only the
fermented juice polyphenols appear to have a significant effect in prolonging
this
stage;
FIG. 41 is a histogram illustrating the effect of selected pomegranate fruit
fractions on
the S phase of the cell cycle in HL-60 human promyelocytic leukemia cells. [
3o fractions as in figure 38] The fermented juice polyphenol fraction
completely
eliminates this phase. A similar, though attenuated, effect is observed for
the simple
concentrated fermented juice, as expected. The seed oil does not have this
effect;
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FIG. 42 is a histogram illustrating apoptosis in HL-60 human leukemia cells in
vitro
induced by selected pomegranate fruit fractions. [ fractions as in figure 38]
The
highest degree of apoptosis is observed for the whole pomegranate seed oil.
Solvent
for the OP, FJP and fermented juice is DMSO 12.5 micgroliters per ml. The seed
oil
is dissolved in ethanol. At the lower concentration of seed oil, the ethanol
concentration is 10 micrograms per ml. At the higher concentration, the
ethanol
concentration is 20 micrograms per ml;
FIG. 43 is a histogram illustrating inhibition of 17-beta-hydroxysteroid
dehydrogenase Type 1 by selected pomegranate fractions. [P = pericarp extract,
W =
fermented juice extract, SCFO = pomegranate seed oil extracted with
supercritical
COz, EM-251 = positive control (16-alpha-bromopropyl-estradiol)];
FIG. 44 is a graph of the effect of pomegranate fermented juice extract (W) on
proliferation of human multiple myeloma cell line HS-Sultan (HSS);
FIG. 45 is a graph of the effect of pomegranate fermented juice extract (W)
and
pomegranate pericarp extract (P) on proliferation of human multiple myeloma
cell
line MM.1 S;
FIG. 46 is a a graph of the effect of pomegranate fractions on proliferation
of human
multiple myeloma cell line U266. [W = fermented juice extract, P = pericarp
extract,
SESCO = supercritical COZ extracted seed oil, SEEE = ethanolic extract of seed
cake
(following oil extrusion)];
FIG. 47 is a histogram comparing pomegranate fermented juice extract (W) to a
known Vitamin D differentiation inducing agent (cont-D) with respect to the
prevention of carcinogenesis in a marine mammary gland organ culture;
FIG. 48 is a histogram illustrating dose-dependent inhibition of HT-29 human
colon
adenocarcinoma cells by pomegranate fermented juice (W) and pericarp (P)
extracts;
FIG. 49 is a is a histogram illustrating dose-dependent inhibition of
proliferation of
rapidly dividing WI38 human diploid normal embryonic lung tissue by
pomegranate
fermented juice (W) and pericarp (P);
FIG. 50 is a histogram illustrating dose-dependent inhibition on HPB-ALL human
3o thymoma cells of pomegranate fermented juice (W) and pericarp (P) extracts
relative
to quercetin (Q).;
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FIG. S 1 is a histogram illustrating a comparison of the anti-proliferative
effect of
pomegranate pericarp (P) and fermented juice (W) extracts on human thymoma
cells
(HPB-ALL) and their normal conterpats (PBL);
FIG. 52 is a flow diagram showing production steps in manufacture of 1000
doses
ofan elixir for women according to the present invention;
FIG. 53 is a is a flow diagram showing production steps in manufacture of 1000
doses
of an elixir for men according to the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
1 o The present invention is of a mixture of a pomegranate seed oil product
and a
pomegranate juice product, and pharmaceutical compositions containing same
which can be
used to prevent or treat a variety of medical conditions.
Specifically, the present invention can be used to prevent or treat cancer,
especially hormone
dependentcancer.
The principles and operation of a mixture of a pomegranate seed oil product
and a
pomegranate juice product, and pharmaceutical compositions containing same
according to
the present invention may be better understood with reference to the drawings
and
accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to
be
2o understood that the invention is not limited in its application to the
details of construction
and the arrangement of the components set forth in the following description
or illustrated in
the drawings. The invention is capable of other embodiments or of being
practiced or carried
out in various ways. Also, it is to be understood that the phraseology and
terminology
employed herein is for the purpose of description and should not be regarded
as limitingThe
present invention is of a cancer chemo-preventive mixture. 'The ability of
mixtures according
to the present invention to inhibit cancer stems from their ability to inhibit
cellular
proliferation and stimulate cellular differentiation as detailed hereinbelow
in the examples
section. These general cellular phenomena are explained by apparent influences
on estrogen
and aromatase activities as well as 17-beta-hydroxysteroid dehydrogenase Type
1 activity as
3o detailed in examoples hereinbelow. The mixture includes a pomegranate seed
oil product ,
and a pomegranate juice product. The pomegranate seed oil product may be, for
example,
the result of a process such as expeller pressing, supercritical fluid
extraction with carbon
dioxide, or lyophilization. The pomegranate seed oil product may be produced
from a
CA 02389537 2002-05-21
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material including, but not limited to, pomegranate seeds and pomegranate seed
cake. As
such, the pomegranate seed oil product may be, for example, pomegranate seed
oil or a non
saponifiable fraction thereof.
The pomegranate juice product may include, for example pomegranate juice,
fermented pomegranate juice, dried pomegranate juice, dried fermented
pomegranate juice,
partially fermented pomegranate juice, partially dried pomegranate juice,
partially fermented
partially dried pomegranate juice, reduced pomegranate juice, partially
reduced pomegranate
juice and lyophylysates thereof or any combination of these ingredients.
The present invention is further embodied by a pharmaceutical composition
including
physiologically active amounts of a pomegranate seed oil product and a
pomegranate juice
product as defined hereinabove. The pharmaceutical composition furthe includes
a
pharmaceutically acceptable carrier. In some cases, the mixture or
pharmaceutical
composition may further include a pomegranate peel product in order to
increase efficacy
thereof. The pomegranate peel product may be, for example the pomegranate peel
residue
present in pomegranate juice as a result of a juicing process, an aqueous
extract of
pomegranate peel, an alcohol extract of pomegranate peel, an extract performed
with an
organic solvent which is not alcohol, a supercritical COZ extract of
pomegranate peel or any
combination thereof.
The mixture or pharmaceutical composition may be provided in myriad forms,
includiang but not limited to, a liquid, a powder, granules, a tablet, a
capsule, a gel-tab, an _
ointment, a cream, a chewing gum, a food, a candy, an emulsion and a
suppository.
Because of the demonstrable effect of the components of the mixture of the
present
invention on estrogen and on enzymes involved in estrogen synthesis, the
present invention
will most likely have special efficacy in treatment of cancer which is hormone
dependent.
Such hormone dependent cancers include, but are not limited to breast cancer
and prostate
cancer.
Alternately or additionally, the pharmaceutical composition of the present
invention
may be efficaciouly employed for treatment of a medical condition including,
but not limited
to cancer, alzheimer's disease, climacteria, benign prostatic hyperplasia and
estrogen
3o deficiency. In vivo cell culture data on cell lines derived from cancers
including breast
cancer, prostate cancer, melanoma, human lung carcinoma, human T cell leukemia
, human
gastric lymph node metastasis and benign prostatic hypertrophy are presented
in examples
hereinbelow. The term "treatment", as used in this specification and the
accompanying
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claims is to be construed in its broadest possible sense. As such treatment
includes, but is not
limited to, prophylactic treatment, palliative treatment and therapeutic
treatment.
Based upon results presented in examples hereinbelow, the physiologic activity
of mixtures
and pharmaceutical compositions of the present invention is believed to result
from
inhibition of enzymes including, but not limited to, aromatase and 17-beta-
hydroxysteroid
dehdrogenase (HSD) type 1. However, the nature of the experimental work is
empirical, and
the possibility that additional metabolic pathways are involved is high.
Figures 1-51 are
explained in the context of examples 1-12 hereinbelow.
As disclosed in detail in example 13 hereinbelow (see also figures 52 and 53)
the
to invention 'is specifically embodied by a mixture or pharmaceutical
composition which
includes as active ingredients dealcoholized concentrated pomegranate wine,
aqueous extract
of pomegranate pericarp, and seed cake extract. According to specific
preferred
embodiments of the invention, the ingredients are present in a ratio of
approximately 70%
dealcoholized concentrated pomegranate wine, approximately 10% aqueous extract
of
pomegranate pericarp, and approximately 20% seed cake extract. According to
specific
preferred embodiments of the invention, the ingredients are present in a ratio
of
approximately 30% dealcoholized concentrated pomegranate wine, approximately
10%
aqueous extract of pomegranate pericarp, and approximately 60% seed cake
extract.
2o Additional objects, advantages, and novel features of the present invention
will
become apparent to one ordinarily skilled in the art upon examination of the
following
examples, which are not intended to be limiting. Additionally, each of the
various
embodiments and aspects of the present invention as delineated hereinabove and
as claimed
in the claims section below finds experimental support in the following
examples.
EXAMPLES
Reference is now made to the following examples, which together with the above
descriptions, illustrate the invention in a non limiting fashion.
General references to standard laboratory techniques are provided throughout
this
3o document. The procedures therein are believed to be well known in the art
and are provided
for the convenience of the reader. All the information contained therein is
incorporated
herein by reference.
Materials and methods:
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Reference is now made to the following materials and methods which are
employed
in the examples detailed hereinbelow.
The Yeast Estrogen Screen (YES) was performed according to the method
described by
Arnold et a19 . The yeast (strain DY150) contains the yeast expression plasmid
containing the
human estrogen receptor (hER) and the estrogen-sensitive Lac-Z reporter
plasmid. The special
yeast used was supplied courtesy of Dr. John McLachlan, Tulane-Xavier Center
for
Bioenvironmental Research, New Orleans, LA 70112, USA. The yeast was grown
overnight in
the presence or absence of 17-beta estradiol in a concentration of 0.4 gM. One
set of estradiol
samples was also incubated with freeze dried pomegranate juice (lmg / 1001,11
MeOH). Another
1o set of samples were incubated with the pomegranate juice only. All samples
were tested in
triplicates.
The aromatase assay was carried out by contract on coded samples of the
putative
inhibitors. The method depends of the release of tritiated water after
aromatization of
androstenedione, consistent with the method described by Rabe et a1.1°
The coded samples
l5 consisted of polyphenol fractions of the pomegranate seed oil, fermented
juice and pericarp
aqueous extract respectively. Aminoglutethamide, the known aromatase
inhibitor, was used as a
positive control at a concentration of 100 microM. The experimental
pomegranate fractions
were used at full strength, and at 50%, 10% and 5% dilutions.
Preparation of pome~ ate polyphenol fractions was according to the method
described
2o by Schubert et alb. For the fermented juice and aqueous extract of the
rinds, the original liquids
were combined with two times their volume of ethyl acetate, shaken vigorously,
and left for 8
hours. The ethyl acetate phase was then dried in the vacuum evaporator at 40
degrees centigrade,
and polyphenols resuspended in methanol.
Polyphenol extraction from cold pressed pomegranate seed oil was accomplished
by
25 moving a 10 gram aliquot 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 Na2S04 and again dried in a vacuum evaporator at 40
degrees. The
resultant polyphenols were resuspended in methanol and extracted with three
portions of
3o chloroform, each half the volume of the methanol phase. The chloroform was
removed and the
9 Arnold, S.F., Robinson, M.K., Notides, A.C., Guillette, L.J. and McLachlan,
J.A. A yeast estrogen screen for
examining the relative exposure of cells to natural and xenoestrogens.
Environmental Health Perspectives 104 (5):
544-548, 1996.
to Rabe, T., Rabe, D. and Runnebaum, B. New aromatase assay and its
application for inhibitory studies of
aminoglutethimide on microsomes of human term placenta. Journal of Steroid
Biochemistry 17: 305-309, 1982.
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methanol dried again in the vacuum evaporator at 40 degrees. 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 NaCI and extracted with four
portions of ethyl
acetate (EA), each a third of the water phase volume. The EA fraction was
collected and dried
with anhydrous Na2S04 . The EA was dried in a vacuum evaporator and the
polyphenols
resuspended in methanol.
A non-saponifiable fraction (NSF) of pome~yranate seed oil was prepared by
combining a quantity of ethanol extracted pomegranate seed oil with KOH to
produce a
saponified mixture. This mixture was washed repeatedly with petroleum ether to
obtain the
to NSF, which was subsequently dried with anhydrous sodium sulfate. According
to published
references, this NSF, when injected into mice and rabbits, exerted significant
estrogenic
activities as measured by ovarian weight and cornification of vaginal
epithelium. t t
The MTT assay was performed as secribed in Ruben, R.L. and Neubauer, R.H.
(1987) "Semiautomated colorimetric assay for in vitro screening of anticancer
compounds",
Cancer Treat. Rep. 71 (12):1141-9.
Nitro blue tetrazolium reducing activity was measured bythe method described
in
Kawaii S., Tomono Y., Katase E., Ogawa K. andYano M. (1999) "Effect of citrus
flavonoids
on HL-60 cell differentiation" Anticancer Res. 19(2A):1261-9.
2o Non-specific esterase activity was measured by the method of Rovera G.,
Santoli D.
and Damsky C. (1979) "Human promyelocytic leukemia cells in culture
differentiate into
macrophage-like cells when treated with a phorbol diester" Proc. Natl. Acad.
Sci. U S A
76(6):2779-83.
Specific esterase activity was measured by the method of Kawaii S., Tomono Y.,
Katase E., Ogawa K, and Yano M. (2000)"Effect of coumarins on HL-60 cell
differentiation" Anticancer Res. 20(4):2505-12.
Pha~ocytic activity was measured by the method of Kawaii S., Tomono Y., Katase
E.,
Ogawa K. and Yano M. (1999)"Isolation of furocoumarins from bergamot fruits as
HL-60
differentiation-inducing compounds" J. Agric. Food Chem. 47(10):4073-8.
3o Cellular proliferation in human promyelocytic leukemia cells was measured
by the
method of Kawaii S., Tomono Y., Katase E., Ogawa K., Yano M., Takemura Y., Ju-
ichi M.,
" Sharaf, A. and Nigm, S.A.R. The oestrogenic activity of pomegranate seed
oil. Journal of Endocrinology 29:
91-92, 1964.
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Ito C. and Furukawa H. (1999)"Acridones as inducers of HL-60 cell
differentiation" Leuk
Res. 23(3):263-9.
EXAMPLE 1:
s YEAST ESTROGEN SCREN OF POMEGRANATE DERIVATIVES
In order to test the anti-estrogenic activity of pomegranate juice (PJ) and
estrogenic
activity of NSF of pomegrate seed oil, a YES was performed. Results of the YES
assays are
presented in figures 1 and 4. Figure 1 shows inhibition of the estrogenic
activity of the estrogenic
standard 17-beta estradiol as a result of the addition of the pomegranate
juice. 17-beta estradiol
to activity of 100 Miller Units was reduced to 50 Miller Units by the added
pomegranate juice.
Using a similar screen. ( Figure 4) NSF was shown have an estrogenic activity
about 20% that of
a comparable concentration.
These data corroborate animal findings which suggest that pomegranate seed oil
may
have utility as an estrogen homologue with potential applications in treating
climacteric women
15 or male prostate cancer patients. Also corroborated is the earlier finding
that pomegranate juice
also may exert a weak estrogenic action parallel to its antiestrogenic
potential. t2
EXAMPLE 2:
ASSAY OF AROMATASE ACTIVITY
20 Results of the aromatase assay are summarized in Figures 2 and 3. The
positive
control aminoglutethamide (AGM) exhibited 66% inhibition of aromatase. The
pomegranate
seed oil extract (Sample One) showed a mean inhibition of 61 % which was
concentration
dependent and rapidly dropped off at lower concentrations. The aqueous
pericarp extract
(Sample Two) showed a mean inhibition of 80% at full concentration, and at SO%
actually
25 increased to 89%. This level of inhibition was consistent all the way down
to 5% dilution.
The fermented juice (wine) extract (Sample Three) showed 60% inhibition at
full strength,
70% inhibition at SO% dilution and 10%, and 88% inhibition at 5%. These
results confirm
an corroborate those of example 1 in suggesting potential medical utility for
the assayed
preparations of pomegranate. Aromatase inhibition and suppression of
endogenous
3o estrogenic activity are important factors in the control of breast cancer
growth.l3
'Z Miksicek, R.J. Commonly occurring plant flavonoids have estrogenic
activity. Molecular Pharmacology 44:
37-43, 1993.
~3 Dowsett, M., Macaulay, V., Gledhill, J., Ryde, C., Nicholls, J., Ashworth,
A., McKinna,
A. and Smith, LE. Control of aromatase in breast cancer cells and its
importance for tumor growth. Journal of
Steroid Biochemistry and Molecular Biology 44 (4-6): 605-609, 1993.
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Example 3:
C. Antiproliferative effects of pomegranate fermented juice and pericarp
extracts in
estrogen receptor positive (MCF-7) and estrogen receptor negative (MDA-MB-231)
human
~ breast cancer cells in culture
In order to test the effect of pomegranate fermented juice and pericarp
extracts in
estrogen receptor positive (MCF-7) and estrogen receptor negative (MDA-MB-231)
human
breast cancer cells in culture, polyphenol-rich fractions, consisting of
flavonoids and tannins,
were extracted from the seed oil, pericarp, unfermented and fermented juice of
the
l0 pomegranate, Punica granatum. The different fractions were incubated in
individual well
plates with both estrogen receptor positive (MCF-7) and estrogen receptor
negative
(MDA-MB-231) human breast cancer cells for 48 hours. Cell viability was
assessed with the
MTT assay. Results are summarized in figures 5 and 6. The fermented juice
exerted the
strongest overall anti-proliferative effect in both the MCF-7 and MDA-MB-231
lines. The
second strongest in both lines was the aqueous pericarp extract. The
unfermented juice also
exerted significant anti-proliferative activity of the MCF-7 cells, but only
mild
anti-proliferative activity in the MDA-MB-231 cells. Overall, the effect in
the MCF-7 lines
for all pomegranate materials was more pronounced than that for the MDA-MB-
231.
Polyphenol fraction isolated from the pomegranate seed oil failed to have anti-
proliferative
2o effect in either of the assays at the concentrations employed. The IC50 for
the fermented
pomegrante juice polyphenol / flavonoid fraction was about 40 micrograms / ml
for the
MCF-7 cells and 120 micrograms / ml for the MDA-MB-231. These findings
indicate that
pomegranate fermented juice and pericarp decoction inhibit proliferation of
both
estrogen-positive and estrogen-negative breast cancer cells, apparently by two
different
mechanisms. Synergy between these mechanisms remains to be investigated.
Example 4:
Differentiation promoting and anti-proliferative properties of pomegranate
fermented
juice and pericarp extracts in cancer and normal cell lines
3o In order to assess the ability of pomegranate fermented juice and pericarp
extracts to
promote differentiation and prevent proliferation, in cancer and normal cell
lines
polyphenol-rich fractions were extracted from fresh and fermented pomegranate
juice,
expeller-pressed pomegranate seed oil and an aqueous decoction of pomegranate
pericarps
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(rinds) utilizing chemical solvents. The four different fractions were then
individually tested
in proliferative assays in normal (human umbilical epithelium--HUVE, human
foreskin
keratinocyte--HFK) and cancer (human lung carcinoma--A-549, melanin pigment
producing
mouse melanoma--B16 melanoma--4A5, human T-cell leukemia--CCRF-HSB-2 and human
gastric cancer lymph-node metastasized--TGBC11TKB) cell lines. Differentiation-
inducing
activity was assessed by nitro blue tetrazolium reducing activity, non-
specific esterase
activity, specific esterase activity, phagocytic activity and cellular
proliferation in human
promyelocytic leukemia cells (HL-60). Results are summarized in figures 7-12.
Very strong
antiproliferative activity was observed for the fermented juice and aqueous
pericarp extract in
lymph node metastasized and human T-cell leukemia cell lines, and a milder
activity for the
non-fermented juice was observed in the same lines. Additional moderate
antiproliferative
activity was observed in mouse melanoma and human lung carcinoma cells with
the
pomegranate pericarp and fermented juice. The polyphenol fraction obtained
from the oil
was without effect in all cell lines, and the normal cells, i.e., the human
foreskin
~ 5 keratinocytes and the human umbilical vein epithelium, were essentially
unaffected by all -
pomegranate fractions though some mild antiproliferative activity was observed
for the
pericarp and fermented juice at very high concentrations. Differentiation-
inducing activity
was observed consistently for all parameters measured: fermented
juice>/=pericarp»unfermented juice»>oil. These results indicate that the
pomegrante
2o fermented juice and pericarp fractions can inhibit cancer cells by
promoting differentiation,
differentiation being inversely correlated with cancer virulence. Inhibition
of proliferation
was also demonstrated in a number of key human and one marine cell lines, and
an absence
of proliferation inhibition noted in two key normal human cell lines. Thus
from this data it
appears that the pomegranate pericarp fraction specifically signficantly
inhibit cancer cell
25 growth and that the the metastatic line was most strongly affected.
Similarly, the most
powerful antiproliferative effects are observed in the metastatic gastric
lymph node and the T
cell leukemia. The normal cell lines appear to be minimally affected with
respect to
proliferation. The effect of juice on proliferation is clearly milder than for
the fermented -
juice. Again, the most sensitive lines are the metastatic gastric and the
leukemiawhilethe
3o normal cell lines are not affected.
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Example 5:
Inhibition of invasion and proliferation of human MCF-7 breast cancer cells
and
inhibition of proliferation of LNCapFGC human prostate cancer cells by
pomegranate
seed oil in vitro
s Pure expeller-pressed pomegranate seed oil (PSO) dissolved in ethanol was
added to
human MCF-7 human breast cancer cells, pre-labelled with a non-toxic
flourescent dye
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine) to a tissue culture
of normal human
HUVEC vascular endothelial cells. The degree of endothelial cell / tumor cell
adhesions, a
pre-condition for invasion and metastasis, was quantified with a flourescent
plate reader.
The results (figure 13) demonstrated a significant inhibition of the
experimental invasion by
the PSO at dosages from Smicrograms / ml of PSO in the cell medium. The effect
increased
linearly until about lOmicrograms/ml; gradually increasing until 200
micrograms / ml and
then leveling off. An antiproliferative effect of the PSO employing a standard
MTT assay
(figures 14-18) was noted in the same MCF-7 cell line from about 50 micrograms
/ml,
increasing linearly with dose and leveling off at 10,000 micrograms /ml, and
in human
LNCapFGC from about 1 micrograms /ml leveling off by 10 micrograms /ml (figure
19).
These results demonstrate inhibition of breast cancer cell invasion by pure
pomegranate seed
oil at very low dosage. A tenfold greater concentration inhibits proliferation
of the same
cells. Inhibition of proliferation in prostate cancer cells was demonstrated
at a dose
2o approximately equivalent to that required for inhibition of invasion in
breast cancer cells. In
other words, prostate cancer cells were considerably more sensitive to the
effects of the oil
than the breast cancer cells.
Example 6:
Inhibition of estrogen synthetase (aromatase) by flavonoids derived from
selected
pomegranate fractions
In order to elecucidate the possible molecular mechanism of pomegranatae
fractions
on hormone dependent cancer cells, Polyphenol-rich fractions were extracted
from the seed
oil, fermented juice and pericarp of Punica granatum using chemical solvents.
Each fraction
was individually tested by administering 10 microliters into the testing well
in a human
placenta system for aromatase inhibition using 10 microliters of 100
microMolar
aminoglutethamide as a postive control. Samples were tested at 100%, 50%, 10%,
S%, 1%
and 0.1 % dilutions. Inhibition was recorded as the percentage of inhibition
achieved relative
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to that of the positive control, aminoglutethamide at 100 microMolar.
Inhibition was strong
in all fractions and was according to: pericarp > fermented juice » oil.
Inhibition was not
attentuated even at S% dilution with the pericarp and fermented juice
fraction, but the oil
showed a 50% attenuation by the 50% dilution, and no activity at 10% dilution
and lower.
Inhibition was still observed, though attenuated, at 1% and 0.1% dilution for
the pericarp
decoction and fermented juice polyphenol fractions. Results are summarized in
figure 20.
The P-450 enzyme, aromatase (estrogen synthetase), is responsible for
catalyzing the
biosynthesis of the steroidal estrogens estrone and 17-beta-estradiol from the
androgens
androestenedione and testosterone respectively in vivo. As this is a major
biosynthetic
1 o pathway for the production of these strong estrogens, inhibition of
aromatase is a popular and
proven pharmacological method of retarding the development of estrogen-
dependent breast
cancers. These results suggest utility of pomegranate fermented juice and
pericarp fractions
in the treatment or prevention of breast cancer.
Example 7:
Antiproliferative activity of pomegranate fermented juice and seed oil
flavonoids in human
prostate cancer (LNCaP, PC-3, DU-145) and human stromal benign prostatic
hypertrophy
(hPCPs) cells in vitro
Human epithelial prostate cancer cells (LNCaP) and human stromal benign
prostatic
2o hypertrophy (BPH) cells (hPCPs) were seeded into 96 well plates and grown
in the presence of
varying dilutions of polyphenol fractions extracted from pomegranate seed oil
and pomegranate
fermented juice, and also in the presence of pure pomegranate fermented juice
and pure
pomegranate seed oil. On days 2, 3 and 4 cells were fixed, stained and the
absorption measured.
For each extract dilution and control, 8 samples were measured and mean value
and standard
2s deviation calculated. Results showed a strong inhibition of proliferation
in both the prostate
cancer and BPH cells by the pomegranate fermented juice flavonoids, and a
milder inhibition by
the full fermented pomegrante juice. Results are summarized in figures 21-24.
In all instances,
the effect was more pronounced in the BPH cells (hPCPs) but was also strong in
the cancer cells
(LNCaP). Additional studies (Campbell, Geldof) focussed on the action of the
aforementioned
3o pomegranate compounds in more aggressive, more poorly differentiated,
androgen-independent
prostate cancer lines (i.e., PC-3 and DU-145). Results are summarized in
figures 25-33. In
these lines as well, a signficant inhibition of proliferation was noted with
the fermented juice and
pericarp extracts (W, P) but not with pomegranate seed oil. These results
demonstrate an
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antimitogenic (antiproliferative) activity of pomegranate fermented juice and
pomegranate
fermented juice polyphenol fraction both in human prostate cancer cells and in
human stromal
BPH cells. This suggests a potential utility for pomegranate fermented juice
fractions in the
prevention and possibly also the treatment both of human prostate cancer and
benign prostatic
hypertrophy. The observance of the anti-proliferative effect even in the most
poorly
differentiated cells (DU-145) illustrates the potency and significance of this
effect. Further
studies are necessary to elucidate the mechanism of action, which clearly
extends beyond
suppression of the hormonal influence, as evidenced by results with PC-3 and
DU-145.
l0 Example 8:
localization of antiproliferative effects of pomegranate fractions to specific
stages of
the cell cycle
Ethyl acetate extracted polyphenol fractions of fermented pomegranate juice
and an
aqueous extract of pomegranate pericarps were assessed for their ability to
suppress growth and
15 to interrupt specific stages of the cell cycle in marine B 16 (F 10)
melanocytic melanoma and
human HL-60 promyelocytic leukemia cells. Cells were grown in monolayer
culture (35 x 10
mm flasks) in 3 mL of RPMI 1640 medium supplemented with 10% fetal bovine
serum and 80
mg/L of gentamycin. Cultures, seeded with 3.3 x 10' cells/L, were incubated
for 24 h at 37 C in
a humidified atmosphere of 5% C02. The media were decanted and replaced with
fresh media
20 containing the pomegranate fractions, and incubations were continued for
additional time
commensurate with the stage of the cell cycle being assessed. The medium and
detached cells
were decanted from cells grown in monolayer culture, and then incubated with
trypsin-EDTA at
37 C for 2 min. Trypsin was inactivated by suspending the cells in medium
containing 10% of
FBS. The trypsinized cells were pelleted at 250 x and resuspended in HBBS.
Viable cells,
25 [cells that excluded 0.4% of trypan blue], were counted with a
hemocytometer. Pomegranate
fermented juice polyphenols (W) were tested at 25, 50 and 100 micrograms solid
material per
ml of medium, while polyphenols derived from the pericarps (P) were tested at
50 and 100
micrograms per ml only. Results are summarized in figures 34-42. Both W and P
clearly
caused inhibition of growth at G1 while treatment with W also showed a steady
lengthening of
30 G2, suggesting a possible G2/M arrest. This occurred in both cell lines
studied. Inhibition of the
S phase (DNA synthesis) was pronounced for the W fraction. Investigation of
the effects on
apoptosis showed a dramatic apoptosis stimulating effect from the pomegranate
seed oil, and
considerably less even from the highest doses of the fermented juice
polyphenols. These results
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uggest that the mechanisms underlying the antiprolifertive effects of
pomegranate fractions
observed in cancer cells include influence on the normal cell cycle. Of
special interest is that the
seed oil and the aqueous fractions derived from the fermented juice and
pericarp extracts seem to
exert their antiproliferative effects in different ways suggesting that
mixtures of these two
fractions might be expected to exhibit synergy in vivo. . The strongest degree
of inhibition was
observed with the polyphenol fraction of the fermented juice, and this effect
was most dramatic
at the G1, G2 and S stages. This material appears to inhibit cancer cell DNA
synthesis (S). In
general, the effect of the pericarp fraction of polyphenols (P) was similar to
that of the fermented
juice polyphenol fraction (W), though the effect of W is greater. For example,
inhibition of
1o glycosylation of hemoglobin was observed with W but not P, implying that W
has an additional
antioxidant activity. The effects at G1 and G2 are significant with both W and
P, with the oil
showing a much milder effect. The oil is apparently more active than the other
fractions in the
promotion of apoptosis, consistent with earlier published observations
observations that
gamma-tocopherol, in significant quantity in the oil, is also a strong
promoter of apoptosis.
Example 9:
Inhibition of 17-hydroxysteroid dehydrogenase Type 1 by pomegranate fermented
juice
and pericarp extracts and supercritical fluid extracted pomegranate seed oil.
Extracts of pomegranate fermented juice (W) and pomegranate pericarp (P) were
prepared according to the method of Schubert et al. ( (1999) J Ethnopharmacol.
66(1):11-7), and
pomegranate seed oil was obtained by supercritical fluid extraction (SCFO)
utilizing COZ as a
solvent. These three pomegranate fractions were tested at 1, 10, 100 and 1000
micrograms / ml
as potential inhibitors of the enzyme 17-beta-hydroxysteroid dehdrogenase
(HSD) type 1
utilizing the assay as previously reported by Luu-The et al ((1995) Biochem
Mol Biol.
55(5-6):581-7). Specifically, the enzyme 17-beta-HSD type 1 was transfected
into the cytosolic
fraction of sonicated HEK-293 cells. A known inhibitor of 17-beta-HSD type 1,
EM-251
(16-alpha-bromopropyl-estradiol) was employed as a positive control. The assay
measures the -
transformation of estrone (E1) to 17-beta-estradiol (E2) utilizing thin-layer
chromatography and
quantification of C-14 labeled El and E2 using a Phosphor Imager, allowing for
both the percent
of transformation and the percent of inhibition. Results are summarized in
figure 43. No
significant inhibition was noted from any of the compounds at 1 and 10
micrograms / ml. All
compounds caused inhibition at 1000 micrograms / ml, but only the
supercritically extracted oil
caused inhibition at 100 micrograms / ml. These results demonstrate an
additional mechanism
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by which pomegranate fractions inhibit the biosynthesis of active estrogen
(E2) in vivo. This
mechanism apparently complements the inhibition of aromatase, which leads to
E2 by a different
biosynthetic pathway. These results provide further evidence of the anti-
estrogenic properties of
pomegranate fractions and support the idea that a chemopreventive effect
against
estrogen-dependent breast cancer in vivo is likely. The differences between
oil and pericarp or
fermented juice extracts implies synergy between the aqueous and fatty phases
of the
pomegranate for cancer chemoprevention, especially estrogen-dependent cancer .
Example 10:
l0 Inhibition of proliferation of multiple myeloma cell lines by pomegranate
fermented juice
and pericarp extracts.
Three different human multiple myeloma cell lines[ HS-Sultan (HSS), MM.1 S and
U266
as described in Gooding et al., (1999) J Haematol 106(3):669-81 ] were
incubated for 24 hours
with each of four different pomegranate fractions. The fractions were
pomegranate fermented
15 juice extract (W), pomegranate pericarp extract (P), supercritical C02
extracted pomegranate
seed oil (SESCO) and ethanolic pomegranate seed cake extract (SEEE). The
fractions were
employed at mM quercetin equivalent concentrations as measured and described
previously
(Tedesco et al., (2000) J Nutr Biochem 11: 114-119). Inhibition of
proliferation was assessed
utilizing the MTT assay. Results are summarized in figures 44-46. Significant
inhibition of
2o proliferation was noted in the U266 and HSS cell lines from between 10
micromolar to 100
micromolar concentrations, but not in the MM1 S cells. These results
demonsrate that human
multiple myeloma cells are also subject to growth inhibition by pomegranate
fractions.
Solubility problems render the inhibition observed from the two oily fractions
(SEEE and
SESCO) somewhat equivocal. However, the inhibition from the aqueous fractions
(P and W)
25 was clear. Further studies will be needed to elucidate the specific
compounds within the extracts
responsible for the observed effects.
Example 11:
Promotion of differentiation in human breast cancer cells by a pomegranate
fermented
3o juice extract.
Mammary glands of BALB/c mice were placed in organ culture and treated with
the
carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to induce preneoplastic
lesions. Results
are summarized in figure 47. The experimental material (fermented pomegranate
juice
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polyphenols) was added to the culture medium for 5 days of growth, and the
number of
neoplastic lesions evaluated in 25 glands. A 42% reduction was noted in the
experimental group,
compared to only a 20% reduction from a Vitamin D analog known as a
differentiation inducer.
In previous studies reported in the literature, there is excellent correlation
between such
experimental lesions and the development of tumors in athymic mice in which
the cells from
neoplastic lesions produced in this way are injected. These results suggest
that the tested
pomegranate fraction is able to reverse the cancer-promoting effect of a known
carcinogen,
apparently by promoting cell differentiation. Therefore, the tested material
appears to be a novel
cancer chemopreventive agent. Because the tested pomegranate extract, unlike
most vitamin D
analogs, has no known undesirable hypercalcemic side effect, it is
particularly attractive.
Example 12:
Pomegranate fermented juice and pericarp extracts differentially inhibit
proliferation of
cancer cell lines and their normal counterparts.
t 5 Utilizing the Folin-Ciocalteu method (J. Nutr. Biochem., 11: 114, 2000),
the
concentration of total polyphenols in cold pressed pomegranate seed oil and in
ethyl acetate
extracts of pomegranate fermented juice and pericarp extracts (J.
Ethnopharmacol., 66: 11, 1999)
was quantified as millimolar equivalents of the flavonoid quercetin. The
concentrations obtained
were:
2o cold pressed pomegranate seed oil = 4mM eq. Quercetin;
pomegranate pericarip extract (P) = 56 mM eq. Quercetin; and
pomegranate fermented juice extract (W) = 133 mM eq. quercetin.
Selected concentrations of the three materials were incubated with cells, at
200,000 cells per
well, for 24 h at 37° C in the presence of a 5% C02 atmosphere. Cell
lines employed were:
25 HeLa derived from a human cervix epitheliod carcinoma (Cancer Res. 12: 264,
1952);
HT-29 derived from a human colon adenocarcinoma (In Human tumor cells in
vitro, pp.
115, J. Foght (ed.), Plenum Press, New York, 1975);
WI38 human diploid cell line from normal embryonic lung tissue (Exp. Cell Res.
25: 585,
1961 );
3o HPB-ALL from a human thymoma (Int. J. Cancer, 21: 166, 1978); and
PBL peripheral blood lymphocytes isolated from healthy volunteers.
Results are summarized in figures 48-51 and in table 1. The pericarp and
fermented juice
extracts significantly inhibited proliferation of all cancer cell lines in a
dose dependent manner.
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Most sensitive to these fractions were the HPB-ALL cells, which were three
times more sensitive
than their normal counterpart, PBL cells. These findings suggest that the
pomegranate extracts
are specifically toxic to cancer cells, but not to normal cells. Because of
solubility problems, the
cytotoxicity of the oil was not adequately assessed. These findings further
broaden the range of
cancer cells whose growth is inhibited by extracts from pomegranate pericarp
and fermented
juice. These results are the first which quantify the total polyphenol content
of assayed extracts
and compare their activities to that of quercetin in a dose-dependent manner.
In addition, the
difference between the toxicity of the assayed fractions to cancer cells as
opposed to normal cells
is clearly demonstrated, suggesting that pomegranate extracts can be
efficaciously employed as
l0 safe and effective cancer chemopreventive agents.
Table 1: Toxicity of pomegranate pericarp (P) and fermented juice (W) extracts
on
selected cell lines expressed in mM equivalents of quercetin.
Cell type IC50 (micro
Molar)
P W Oil
HT29 <140 66-133 > 80
WI38 +_ 140 _+ 66 > 80
HPB-ALL 5.6-14 < 13.2 > 80
PBL
Example 13:
Production of an elixir from a mixture of pomegranate products.
Figure 52 shows production steps inmanufacture of a pharmaceutical composition
including 30% dealcoholized concentrated pomegranate wine, 10% aqueous extract
of
pomegranate pericarp, and 60% seed cake extract. The 120 ml of elixir
represents 1000 doses of _
an elixir for womenwhich could be delivered, for example as gel-tabs . The
elixir is expected to
have beneficial effects in climacteria as well as to offer protection against
development of breast
cancer and to be beneficial in treating breast cancer. Raw materials are 1440
Kg of whole
pomegranates and 1099 Kg of pomegranate seeds. The pomegranates are initially
processed into
juice and pericarp. The juice is then fermented and distilled. The
dealcoholized wine is then
reduced to a 36 Kg. Concentrate containing approximately 20% total solids. The
pericarp is
subjected to an aqueous extraction which produces a pericarp extract
containing approximately
20% total solids. The seeds are "dry cleaned" or solvent extracted to produce
a seed cake which
is further extracted with ethanol. The resultant seed cake extract becomes a
component of the
elixir.
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Figure 53 shows production steps inmanufacture of a pharmaceutical composition
including 70% dealcoholized concentrated pomegranate wine, 10% aqueous extract
of
pomegranate pericarp, and 20% seed cake extract. The 120 ml of elixir
represents 1000 doses of
an elixir for men which could be delivered, for example as gel-tabs . The
elixir is expected to
have beneficial effects preventing beingn prostatic hyperplasia (BPH) and/or
prostate cancer.
The production process is essentially as described for the elixir for women.
Example 14:
Production gel-caps from pharmacutical compositions according to the present
invention.
to As detailed hereinabove, phamaceutical compositions of the present
invention may be
provided in a wide variety of physical forms. One of these forms is gel-caps.
Production of
gel-caps typically includes the following steps:
1) Obtaining concentrated fermented juice and concentrated aqueous pericarp
extracts.
t 5 2) Mixing the two components together, for example in a 9:1 ratio
(fermented juice
pericarp).
3) Submitting these component extracts ( together or individually) to
supercritical
fluid extraction using C02 as a solvent with an ethanol modifier to obtain a
polyphenol
fraction. Suspending this polyphenol fraction in pomegranate seed oil in a
ratio of, for
2o example, 1 : 100 (polyphenol fraction : seed oil) to prepare the
chemopreventive /
phytoestrogen supplement. The prepared elixir may be encapsulated, for example
in soft
gel capsules.
Although the invention has been described in conjunction with specific
embodiments
thereof, it is evident that many alternatives, modifications and variations
will be apparent to
2s those skilled in the art. Accordingly, it is intended to embrace all such
alternatives,
modifications and variations that fall within the spirit and broad scope of
the appended claims.
All publications, patents and patent applications mentioned in this
specification are herein
incorporated in their entirety by reference into the specification, to the
same extent as if each
individual publication, patent or patent application was specifically and
individually indicated to
30 be incorporated herein by reference. In addition, citation or
identification of any reference in this
application shall not be construed as an admission that such reference is
available as prior art to
the present invention.
