AU774619B2 - Mixture of pomegranate seed oil and juice products - Google Patents

Description

WO 01/37848 PCT/IL00/00800 "MIXTURE OF POMEGRANATE SEED OIL AND JUICE PRODUCTS" FIELD AND BACKGROUND OF THE INVENTION 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, climacteria, benign prostatic hyperplasia and estrogen deficiency.

Pomegranate (Punica granatum) has long been recognized as a fruit with many benefits for health.' 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 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 juice 4 and pericarp.

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.

6 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 1 Frawley, D and Lad, V. The Yoga ofHerbs: An Ayurvedic Guide to Herbal Medicine, Lotus Press, Twin Lakes, WI 1986.

2 Moneam, El Sharaky, and Badreldin, M.M. Oestrogen content of pomegranate seeds. Journal of Chromotography 438: 438-442, 1988.

SAbd El Wahab, El Fiki, Mostafa, S.F. and Hassan, A.E.B. Characterization of certain steroid hormones in Punica granatum L. seeds. Bulletin of the Faculty of Pharmacy of Cairo University 36(1): 11-15, 1998.

4 Artik, Cemeroglu, Burkami, and Mori, T. Determination of phenolic compounds in pomegranate juice by HPLC. Fruit Process 8 492499, 1998.

SBen Nasr, Ayed, and Metche, M. Quantitative determination of the polyphenolic content of pomegranate peel. ZLebensm Unters Forsch 203 374-378, 1996.

Schubert, Lansky, and Neeman, I. Antioxidant and eicosanoid enzyme inhibition properties of pomegranate seed oil and fermented juice flavonoids. Journal ofEthnopharmacology 66 11-17, 1999.

I

2 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 would 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, 7 8 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 these capabilities.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in Australia as at the priority date of any of the claims.

Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

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 acceptable carrier.

'Bonnefont, Munoz, and Inestrosa, N.C. Estrogen protects neuronal cells from the cytotoxicity induced by acetylcholinesterase-amyloid complexes. FEBS Letters 441: 220-224, 1998.

8 Xu, Gouras, Greenfield, Vincent, Naslund, Mazzarelli, Fried, Jovanovic, Seeger, M., Relkin, Liao, Checler, Buxbaum, JK.D., Chait, Thinakaran, Sisodia, Wang, Greengard, P.

and Gandy, S. Estrogen reduces neuronal generation of Alzheimer beta-amyloid peptides. Nature Medicine 4: 447-451, 1998.

W:\ciska\nki\species\17282a.doc 2a 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.

o o o W:\ciska\nki\species\17282a.doc WO 01/37848 PCT/ILOO/00800 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.

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 CO 2 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 treatment is selected from the group consisting of a prophylactic treatment, a palliative treatment and a therapeutic treatment.

WO 01/37848 PCT/ILOO/00800 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 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 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 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 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 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: WO 01/37848 PCT/IL00/00800 FIG. 1 is a histogram illustrating estrogenic activity of pomegranate juice (PJ) in 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 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 Cancerous lines murine melanocytic melanocytes [B16], human lung carcinoma [A549], human T cell leukemia [CCRF-HSB-2], human gastric lymph node metastasis [TGBC11 TKB]..; 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 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 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 4. WO 01/37848 PCT/IL00/00800 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; 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 ofhPCPs (stromal benign prostatic hypertrophy) cells; FIG. 24 is a histogram illustrating the effect of pomegranate fermented juice polyphenols 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; 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.; WO 01/37848 PCT/IL00/00800 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 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 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 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 GI 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 G1 phase of arrest; FIG. 35 is a histogram illustrating the effect of pomegranate fermented juice and pericarp polyphenols on the G2 stage of B16 melanin pigment producing mouse melanoma cells. The y axis denotes of cells at G2 at time of measurement.

WO 01/37848 PCT/IL00/00800 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 B16 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 murine melanin pigment producing melanoma cells by pomegranate fermented 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 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. 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; WO 01/37848 I PCT/IL00/00800 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

CO

2 EM-251 positive control (16-alpha-bromopropyl-estradiol)]; FIG. 44 is a graph of the effect of pomegranate fermented juice extract on proliferation of human multiple myeloma cell line HS-Sultan (HSS); FIG. 45 is a graph of the effect of pomegranate fermented juice extract and pomegranate pericarp extract on proliferation of human multiple myeloma cell line MM.IS; 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 CO 2 extracted seed oil, SEEE ethanolic extract of seed cake (following oil extrusion)]; FIG. 47 is a histogram comparing pomegranate fermented juice extract to a known Vitamin D differentiation inducing agent (cont-D) with respect to the prevention of carcinogenesis in a murine mammary gland organ culture; FIG. 48 is a histogram illustrating dose-dependent inhibition of HT-29 human colon adenocarcinoma cells by pomegranate fermented juice and pericarp 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 and pericarp FIG. 50 is a histogram illustrating dose-dependent inhibition on HPB-ALL human thymoma cells of pomegranate fermented juice and pericarp extracts relative to quercetin WO 01/37848 PCT/IL00/00800 FIG. 51 is a histogram illustrating a comparison of the anti-proliferative effect of pomegranate pericarp and fermented juice 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 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 dependent cancer.

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 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 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 WO 01/37848 PCT/ILOO/00800 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 CO 2 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, alzheimcr's disease, climacteria, benign prostatic hyperplasia and estrogen 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 WO 01/37848 PCT/IL00/00800 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 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 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 aqueous extract of pomegranate pericarp, and approximately 60% seed cake extract.

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 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: WO 01/37848 PCT/IL00/00800 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 af 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 I.M. One set of estradiol samples was also incubated with freeze dried pomegranate juice (Img 100l l MeOH). Another 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 oftritiated water after aromatization of androstenedione, consistent with the method described by Rabe et al.

10 The coded samples 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 pomegranate polyphenol fractions was according to the method described by Schubert et a 6 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 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 Na 2

SO

4 and again dried in a vacuum evaporator at 40 degrees. The resultant polyphcnols 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 9 Arnold, Robinson, Notides, 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 104 544-548, 1996.

'o Rabe, Rabe, D. and Runnebaum, B. New aromatase assay and its application for inhibitory studies of aminoglutethimide on microsomes of human term placenta. Journal ofSteroidBiochemistry 17: 305-309, 1982.

13 WO 01/37848 PCT/IL00/00800 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 each a third of the water phase volume. The EA fraction was collected and dried with anhydrous Na 2

SO

4 The EA was dried in a vacuum evaporator and the polyphenols resuspended in methanol.

A non-saponifiable fraction (NSF) of pomegranate 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 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 comification of vaginal epithelium. 1 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 Tomono Katase Ogawa K. andYano M. (1999) "Effect of citrus flavonoids on HL-60 cell differentiation" Anticancer Res. 19(2A): 1261-9.

Non-specific esterase activity was measured by the method of Rovera 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 Tomono Y., Katase Ogawa K, and Yano M. (2000)"Effect of coumarins on HL-60 cell differentiation" Anticancer Res. 20(4):2505-12.

Phagocytic activity was measured by the method of Kawaii Tomono Katase E., Ogawa K. and Yano M. (1999)"Isolation of furocoumarins from bergamot fruits as differentiation-inducing compounds" J. Agric. Food Chem. 47(10):4073-8.

Cellular proliferation in human promyelocytic leukemia cells was measured by the method of Kawaii Tomono Katase Ogawa Yano Takemura Ju-ichi M., Sharaf, A. and Nigm, S.A.R. The oestrogenic activity of pomegranate seed oil. Journal ofEndocrinology 29: 91-92, 1964.

WO 01/37848 PCT/ILOO/00800 Ito C. and Furukawa H. (1999)"Acridones as inducers of HL-60 cell differentiation" Leuk Res. 23(3):263-9.

EXAMPLE 1: 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 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 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.

1 2 EXAMPLE 2: ASSAY OF AROMATASE ACTIVITY 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 50% actually 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, inhibition at 50% dilution and 10%, and 88% inhibition at These results confirm an corroborate those of example I in suggesting potential medical utility for the assayed preparations of pomegranate. Aromatase inhibition and suppression of endogenous estrogenic activity are important factors in the control of breast cancer growth.' 3 1 2 Miksicek, RJ. Commonly occurring plant flavonoids have estrogenic activity. Molecular Pharmacology 44: 37-43, 1993.

1 Dowsett, Macaulay, Gledhill, Ryde, Nicholls, Ashworth, McKinna, A. and Smith, I.E. 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.

WO 01/37848 PCT/IL00/00800 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 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 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 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 WO 01/37848 PCT/IL00/00800 (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-TGBCllTKB) 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, the human foreskin 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 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 murine 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 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 normal cell lines are not affected.

WO 01/37848 PCT/IL00/00800 Example 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 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 5micrograms ml of PSO in the cell medium. The effect increased linearly until about I micrograms/ml, gradually increasing until 200 micrograms ml and then leveling off. An antiproliferative effect of the PSO employing a standard MIT 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 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%, 1% and 0.1% dilutions. Inhibition was recorded as the percentage of inhibition achieved relative WO 01/37848 PCT/IL00/00800 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 5% 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 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 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 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 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 pomegranate compounds in more aggressive, more poorly differentiated, androgen-independent prostate cancer lines 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 P) but not with pomegranate seed oil. These results demonstrate an WO 01/37848 PCT/IL00/00800 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.

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 to interrupt specific stages of the cell cycle in murine B16 (F 10) melanocytic melanoma and human HL-60 promyelocytic leukemia cells. Cells were grown in monolayer culture (35 x mm flasks) in 3 mL of RPMI 1640 medium supplemented with 10% fetal bovine serum and mg/L of gentamycin. Cultures, seeded with 3.3 x 107 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 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, [cells that excluded 0.4% oftrypan blue], were counted with a hemocytometer. Pomegranate fermented juice polyphenols were tested at 25, 50 and 100 micrograms solid material per ml of medium, while polyphenols derived from the pericarps 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 GI while treatment with W also showed a steady lengthening of 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 WO 01/37848 PCT/IL00/00800 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 GI, G2 and S stages. This material appears to inhibit cancer cell DNA synthesis In general, the effect of the pericarp fraction of polyphenols was similar to that of the fermented juice polyphenol fraction though the effect of W is greater. For example, inhibition of glycosylation of hemoglobin was observed with W but not P, implying that W has an additional antioxidant activity. The effects at GI 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 and pomegranate pericarp 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 CO 2 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 ofestrone (El) 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 WO 01/37848 PCT/IL00/00800 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 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.IS and U266 as described in Gooding et al., (1999) JHaematol 106(3):669-81] were incubated for 24 hours with each of four different pomegranate fractions. The fractions were pomegranate fermented juice extract pomegranate pericarp extract supercritical CO 2 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 proliferation was noted in the U266 and HSS cell lines from between 10 micromolar to 100 micromolar concentrations, but not in the MMIS 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) 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 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 WO 01/37848 PCT/IL00/00800 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.

Utilizing the Folin-Ciocalteu method 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 Ethnopharmacol., 66: 11, 1999) was quantified as millimolar equivalents of the flavonoid quercetin. The concentrations obtained were: cold pressed pomegranate seed oil 4mM eq. Quercetin; pomegranate pericarip extract 56 mM eq. Quercetin; and pomegranate fermented juice extract 133 mM eq. quercetin.

Selected concentrations of the three materials were incubated with cells, at 200,000 cells per well, for 24 h at 370 C in the presence of a 5% COz atmosphere. Cell lines employed were: 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 Plenum Press, New York, 1975); WI38 human diploid cell line from normal embryonic lung tissue (Exp. Cell Res. 25: 585, 1961); 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.

WO 01/37848 PCT/IL00/00800 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 safe and effective cancer chemopreventive agents.

Table 1: Toxicity of pomegranate pericarp and fermented juice extracts on selected cell lines expressed in mM equivalents ofquercetin.

Cell type IC50 (micro Molar) P W Oil HT29 <140 66-133 WI38 +140 +66 HPB-ALL 5.6-14 <13.2

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 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.

WO 01/37848 PCT/ILOO/00800 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.

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.

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 CO2 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 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 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 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.

Claims (34)

1. A cancer chemo-preventive mixture, the mixture comprising: a pomegranate seed oil product; and a pomegranate juice product.

2. A mixture according to claim 1, further comprising: a pomegranate peel product.

3. A mixture according to claim 1 or 2, wherein said 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.

4. A mixture according to any one of the preceding claims wherein said pomegranate seed oil product is produced from a material selected from the group consisting of pomegranate seeds and pomegranate seed cake. A mixture according to any one of the preceding claims wherein said pomegranate seed oil product is selected from the group consisting of pomegranate seed oil and a non saponifiable fraction thereof.

6. A mixture according to any one of the preceding claims wherein said 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. *0*0 0' W:\ciska\nki\species\1 7282a.doc 27

7. A mixture according to any one of the preceding claims wherein the mixture is provided in a form selected from the group consisting 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.

8. A mixture according to any one of the preceding claims, wherein the cancer is a hormone dependent cancer.

9. A mixture according to claim 8, wherein the hormone dependent cancer is selected from the group consisting of breast cancer and prostate cancer. A mixture according to claim 2, wherein said 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 C02 extract of pomegranate peel.

11. A pharmaceutical composition, the composition comprising physiologically active amounts of: a pomegranate seed oil product; a pomegranate juice product; and a pharmaceutically acceptable carrier.

12. A composition according to claim 11, further comprising: a pomegranate peel product.

13. A composition according to claim 11 or 12, wherein said pomegranate seed oil product is the result of a process selected from the group consisting of 30 expeller pressing, supercritical fluid extraction with carbon dioxide, and S.lyqphilization. W:\ciska\nki\species\1 7282a.doc 28

14. A composition according to any one of claims 11 to 13, wherein said pomegranate seed oil product is produced from a material selected from the group consisting of pomegranate seeds and pomegranate seed cake.

15. A composition according to any one of claims 11 to 14, wherein said pomegranate seed oil product is selected from the group consisting of pomegranate seed oil and a non saponifiable fraction thereof.

16. A composition according to any one of claims 11 to 15, wherein said 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, pomegranate peel residue and lyophylysates thereof.

17. A composition according to any one of claims 11 to 16, wherein the composition is provided in a form selected from the group consisting 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.

18. A composition according to claim 12, wherein said 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 CO 2 extract of pomegranate peel.

19. A pharmaceutical composition according to any one of the preceding 30 claims, wherein the pharmaceutical composition is efficaciously employed for S. treatment of a medical condition. W:\ciska\nki\species\1 7282a.doc 29 A pharmaceutical composition according to claim 19, wherein said medical condition is selected from the group consisting of cancer, alzheimer's disease, climacteria, benign prostatic hyperplasia and estrogen deficiency.

21. A composition according to claim 20, wherein the cancer is a hormone dependent cancer.

22. A composition according to claim 21, wherein the hormone dependent cancer is selected from the group consisting of breast cancer and prostate cancer.

23. A composition according to claim 21, wherein said treatment is selected from the group consisting of a prophylactic treatment, a palliative treatment and a therapeutic treatment.

24. A composition according to any one of claims 11 to 23, wherein the physiologic activity results from inhibition of an enzyme selected from the group consisting of aromatase and 17-beta-hydroxysteroid dehydrogenase (HSD) type 1. r A composition according to physiologically active ingredients pomegranate wine, aqueous extract extract. any one of claims 11 to 23, wherein comprise dealcoholized concentrated of pomegranate pericarp, and seed cake

26. A composition according to claim 25, wherein physiologically active ingredients comprise approximately 70% dealcoholized concentrated pomegranate wine, approximately 10% aqueous extract of pomegranate pericarp, and approximately 20% seed cake extract. 0 W:\ciska\nki\species\17282a.doc

27. A composition according to claim 25, wherein physiologically active ingredients comprise approximately 30% dealcoholized concentrated pomegranate wine, approximately 10% aqueous extract of pomegranate pericarp, and approximately 60% seed cake extract.

28. A method of prophylaxis or treatment of a medical condition comprising administration of a composition comprising: a pomegranate seed oil product; and a pomegranate juice product.

29. A method according to claim 28, wherein said composition further comprises: a pomegranate peel product.

30. A method according to claim 28 or 29, wherein said 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.

31. A method according to any one of claims 28 to 30, wherein said pomegranate seed oil product is produced from a material selected from the *"group consisting of pomegranate seeds and pomegranate seed cake.

32. A method according to any one of claims 28 to 31, wherein said pomegranate seed oil product is selected from the group consisting of pomegranate seed oil and a non saponifiable fraction thereof.

33. A method according to any one of claims 28 to 32, wherein said 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 S5.. pomegranate juice, partially dried pomegranate juice, partially fermented S. partially dried pomegranate juice, reduced pomegranate juice, partially reduced pomegranate juice, pomegranate peel residue and lyophylysates thereof. W:\ciska\nki\species\1 7282a.doc 31

34. A method according to any one of claims 28 to 33, wherein the composition is provided in a form selected from the group consisting 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. A method according to claim 28, wherein said 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 CO 2 extract of pomegranate peel.

36. A method according to any one of claims 28 to 35, wherein said medical condition is selected from the group consisting of cancer, alzheimer's disease, climacteria, benign prostatic hyperplasia and estrogen deficiency.

37. A method according to claim 36, wherein the cancer is a hormone dependent cancer.

38. A method according to claim 38, wherein the hormone dependent cancer is selected from the group consisting of breast cancer and prostate cancer.

39. A mixture according to claim 1, substantially as hereinbefore described with reference to any of the examples. A composition according to claim 11, substantially as hereinbefore described with reference to any of the examples. DATED: 5 August, 2003 30 PHILLIPS ORMONDE FITZPATRICK Attorneys for: RIMONEST LTD. W:\ciska\nki\species\17282a.doc