WO2007004229A2 - Methods of using pomegranate fractions for skin repair - Google Patents

Methods of using pomegranate fractions for skin repair Download PDF

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Publication number
WO2007004229A2
WO2007004229A2 PCT/IL2006/000787 IL2006000787W WO2007004229A2 WO 2007004229 A2 WO2007004229 A2 WO 2007004229A2 IL 2006000787 W IL2006000787 W IL 2006000787W WO 2007004229 A2 WO2007004229 A2 WO 2007004229A2
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pomegranate
juice
composition
peel
group consisting
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PCT/IL2006/000787
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French (fr)
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WO2007004229A3 (en )
Inventor
Ephraim Lansky
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Rimonest Ltd.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K8/00Cosmetics or similar toilet preparations
    • A61K8/18Cosmetics or similar toilet preparations characterised by the composition
    • A61K8/92Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/922Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K8/00Cosmetics or similar toilet preparations
    • A61K8/18Cosmetics or similar toilet preparations characterised by the composition
    • A61K8/96Cosmetics or similar toilet preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97Cosmetics or similar toilet preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILET PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/08Anti-ageing preparations
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0014Skin, i.e. galenical aspects of topical compositions

Abstract

Methods of using pomegranate fractions of the present invention for promoting skin repair are provided. The methods comprise the steps of providing and administering to a subject a composition comprising a therapeutically effective amount of at least one fraction from pomegranate fruit selected from the group consisting of pomegranate seed oil, a pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof.

Description

METHODS OF USING POMEGRANATE FRACTIONS FOR SKIN REPAIR

FIELD OF THE INVENTION

The invention relates generally to pomegranate fractions and methods of using same, and specifically to methods of using one or more pomegranate fractions to promote skin repair and regeneration of human skin. BACKGROUND OF THE INVENTION

Skin is typically characterized as consisting of two major layers. The stratum corneum or epidermis is the top layer and forms a protective covering for the skin. The epidermis contains mostly keratinocyte or epithelial cells, some melanocytes and Langerhans cells, and several Merkel cells. The lower level of the skin is known as the dermis, which provides strength, elasticity and thickness to the skin. Fibroblasts are the main cell type of the dermis and are responsible for synthesis and secretion of all the dermal matrix components such as collagen, elastin and glycosaminoglycans. Blood vessels, nerves, sensory organs, sweat glands, sebaceous glands, and hair follicles are present in the dermis.

Damage to the skin occurs as a consequence of the natural aging process and is exacerbated in chronically sun-exposed skin (photoaging) (Smith et al. 1962; Lavker 1979; Lovell et al. 1987; Kligman and Balin 1989; Schwartz et al. 1989; Oikrinen and Kallioinen 1989; Schwartz et al. 1993; Lavker 1995). Skin damage is also seen as a consequence of diabetes (Prakash, Pundit and Sharman 1974; Klenerman et al. 1996; Reiber 1998; Laing 1998), in other diseases that compromise the vasculature of the skin (Strigini and Ryan 1996; Stadelmann, Digenis and Tobin 1998; Mendez et al. 1998) and following long-term corticosteroid use (McMichael et al. 1996). Skin damage is a cosmetic issue, but it is also a medical problem. Severely damaged skin is prone to bruising, and often the bruises go on to form non-healing ulcers. Ulcer formation in the lower limbs of diabetic patients, for example, is the major cause of limb amputations (Reiber 1998; Margolis et al. 1999; Margolis and Hoffstad 2002).

Topical treatment of skin with biologically-active retinoids including all-trans retinoic acid (RA) and its parent compound, all-trans retinol (vitamin A) has been shown to repair damage in aged/photoaged skin (Kligman et al. 1986; Weiss et al. 1988). RA-induced repair is presumed to reflect retinoid capacity to influence connective tissue metabolism - i.e., suppress matrix metalloproteinase (MMP) generation (Fisher et al. 1996; Fisher et al. 1997) and stimulate matrix synthesis (Griffiths et al. 1993; Talwar et al. 1995). In addition to their dermal effect, the same biologically active retinoids also stimulate proliferation in the epidermis, leading to epidermal thickening. The epidermal effects of retinoids may contribute to improved appearance. Retinoid effects in the epidermis may also be important for improved wound repair since closure of superficial wounds relies on migration and proliferation of keratinocytes at the wound margin (Pilcher et al. 1997). In addition to potentially beneficial effects, retinoid epidermal hyperplasia with concomitant disruption of skin barrier function is also tied to the irritation response that occurs in many individuals on topical retinoid therapy (Griffiths et al. 1995; Varani et al. 2001).

It is difficult to separate the relative importance of dermal verses epidermal effects of retinoids since these agents have broad activity in both compartments of the skin. As a way to address this issue, a number of natural compounds for "partial" retinoid activities have been examined. In a recent study (Aslam et al. 2005), it was demonstrated that PADMA 28, a mixture of herbs formulated according to an ancient Tibetan recipe, had similar effects as RA in the dermis but was without epidermal effects.

The present study continues efforts to identify natural products derived from pomegranates having skin-repair potential.

While diverse biological activities have been attributed to pomegranate-derived compounds, it has not heretofore been recognized that pomegranate fractions, separately or in combination with each other, when used to treat the skin, would have the significant biological activity of promoting regeneration of the dermis and/or epidermis. Moreover, there are no known acceptable products available which incorporate extracts, fractions, or components of pomegranates for combating dermatological aging, repairing damaged skin and/or restoring skin to a more youthful appearance.

Consequently, there is a need for therapeutic and cosmetic compositions and methods to repair skin, treat wounds, improve the appearance and condition of skin, and, in particular, reverse, reduce, delay, or prevent the visible signs of wrinkled, aged and/or photodamaged skin. In particular, there is consumer demand for effective skin repair treatments based on "natural" products. This is due to the perception that natural products are pure and mild and superior to chemically synthesized products, which are perceived as harsh and environmentally unsafe.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected discovery that different fractions or extracts of pomegranate fruit (Punica granatum) have potentially useful dermal and epidermal activities. Aqueous fractions prepared from pomegranate peel and juice and lipophilic fractions prepared from pomegranate seeds were examined for effects on human epidermal keratinocyte and human dermal fibroblast function. Pomegranate seed oil, but not aqueous extracts of juice, peel or seed cake, was shown to stimulate keratinocyte proliferation in monolayer culture. In parallel, a mild thickening of the epidermis (without the loss of ordered differentiation) was observed in skin organ culture. The same pomegranate seed oil that stimulated keratinocyte proliferation was without effect on fibroblast function. In contrast, a pomegranate peel product or extract (and to a lesser extent, both the pomegranate juice product and seed cake extracts) stimulated type I procollagen synthesis and inhibited matrix metalloproteinase-1 (MMP-I; interstitial collagenase) production by dermal fibroblasts, but had no growth-supporting effect on keratinocytes. These results suggest the potential of pomegranate fractions for facilitating skin repair in a polar manner, namely aqueous extracts (especially of pomegranate peel) promoting regeneration of dermis, and pomegranate seed oil promoting regeneration of epidermis.

It is an object of the invention to provide a general method for prevention or alleviation of damage to the skin associated with damage and aging through the administration of at least one pomegranate fraction, alone or in combination with a suitable carrier or vehicle.

Another object is to add at least one pomegranate product or fraction to existing cosmetic preparations or to use it by itself to repair skin damage and improve the appearance of the skin.

These and other objects are achieved by the present invention, which is directed in one aspect, to methods of promoting skin regeneration in a subject and compositions therefor. The method comprises the step of providing pomegranate fractions and administering to the subject a composition comprising a therapeutically effective amount of at least one extract or IL2006/000787

product from pomegranate fruit selected from the group consisting of pomegranate seed oil, a pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof.

Another aspect of the present invention provides a method of promoting skin regeneration in a subject comprising providing pomegranate seed oil and administering to the subject a composition comprising a therapeutically effective amount of the pomegranate seed oil. In a further aspect of the invention, the composition further comprises a pharmaceutically acceptable carrier. The step of administering includes administration via an administration route selected from the group consisting of topical administration, oral administration, and injection. The composition is administered in a form selected from a group consisting of tablets, suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams, pastes, gels, and the like.

In another aspect of the invention, there is provided a method of promoting skin regeneration in a subject comprising providing at least one pomegranate fraction selected from the group consisting of a pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof and administering to the subject a composition comprising a therapeutically effective amount of said at least one pomegranate fraction.

In yet a further aspect of the invention, there is provided a method of promoting skin regeneration in a subject comprising (a) providing pomegranate seed oil; (b) providing at least one pomegranate fraction selected from the group consisting of a pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof; and (c) administering to the subject a composition comprising a therapeutically effective amount of a combination of pomegranate seed oil and at least one pomegranate fraction selected from the group consisting of a pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof. In yet another aspect, the composition comprises a combination of pomegranate seed oil and a pomegranate peel product.

In yet another feature of the invention, the pomegranate seed oil is the result of a process selected from the group consisting of cold pressing, expeller pressing, supercritical fluid extraction with carbon dioxide, and lyophilization. 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. 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 an alcohol, and a supercritical fluid extract of pomegranate peel.

Li another aspect of the invention, there is provided a composition for treating skin, the composition comprising a therapeutically effective amount of a pomegranate product. The pomegranate product includes at least one pomegranate fraction selected from the group consisting of pomegranate seed oil, a pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof.

Li yet a further aspect of the invention, the pomegranate seed oil has an epidermal regenerating activity and the remaining pomegranate fractions have dermal regenerating activity. Specifically, the pomegranate seed oil stimulates keratinocyte proliferation in the epidermis, whereas the remaining pomegranate fractions stimulate type I procollagen synthesis and inhibit MMP-I production by dermal fibroblasts.

The above features and advantages of the present invention will be better understood with reference to the accompanying figures, detailed description, and examples. It should also be understood that the particular methods and formulations illustrating the present invention are exemplary only and not to be regarded as limitations of the present invention. The invention may be practiced in various other ways and is capable of other embodiments. Also, it is contemplated that the phraseology and terminology used herein are for purposes of description and should not be regarded as limiting.

BRIEF DESCRIPTION OF THE FIGURES

The invention is herein described, by way of example only, with reference to the accompanying figures and the following detailed description, it being understood that the particulars shown are by way of example and illustrative discussion only, and are presented to provide what is believed to be the most useful and readily understood description of the embodiments of the invention.

In the drawings:

Fig. 1 is a graphical representation showing the effects of pomegranate seed oil on keratinocyte proliferation.

Fig. 2A is a graphical representation showing the effects of pomegranate juice on keratinocyte proliferation.

Fig. 2B is a graphical representation showing the effects of pomegranate peel on keratinocyte proliferation.

Fig. 2C is a graphical representation showing the effects of pomegranate seed cake extract on keratinocyte proliferation.

Fig. 3 are histological sections of human skin after incubation for 8 days in organ culture in the absence of pomegranate seed oil (panel A=control) and in the presence of pomegranate seed oil (panel B).

Fig. 4 A is a graphical representation showing the effects of pomegranate peel extract on fibroblast proliferation.

Fig. 4B is a graphical representation showing the effects of pomegranate peel extract on Type I procollagen synthesis.

Fig. 4C is a graphical representation showing the effects of pomegranate peel extract on MMP-I elaboration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention provides a method of promoting skin regeneration in a subject. The method comprises the step of administering to the subject a composition comprising a therapeutically effective amount of a pomegranate fruit product or extract or fraction selected from the group consisting of pomegranate seed oil, a pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof.

Pomegranate products or fractions used in preparing a composition according to the present invention are preferably prepared from Wonderful cultivar pomegranates. More preferably, these pomegranates are organically grown, still more preferably, they are grown at Kibbutz Sde Elialiu in Israel.

Methods of preparing the pomegranate seed oil, a pomegranate peel product, a pomegranate juice product, and pomegranate seed cake are commonly known in the art, and preferred methods are referenced in the Materials and Methods section below. In general, any methods that may produce pomegranate seed oil, a pomegranate peel product, a pomegranate juice product, and pomegranate seed cake may be used, as disclosed in my co-pending applications, Serial No. 09/859,431; 10/217,430; and 11/028,656, the disclosures of which are incorporated herein by reference.

Thus, as disclosed in my prior applications, the term "pomegranate seed oil" includes the results of a process such as, for example, cold pressing, expeller pressing, and supercritical fluid extraction with carbon dioxide. The supercritical fluid extraction is conducted with at least one fluid selected from the group consisting of CO2, ethanol, methanol and water. The term "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 CO2 extract of pomegranate peel or any combination thereof. A "pomegranate juice product" may comprise unprocessed or 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, and partially reduced pomegranate juice.

Compositions of the present invention may be of a variety of kinds, including, but not limited to, nutritional supplements, pharmaceutical preparations, vitamin supplements, food additives, or foods supplements. Compositions of the present invention may be in convenient dosage forms, including, but not limited to, tablets, suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams, pastes, gels, or the like.

Compositions of the present invention may include a carrier. Depending on the kind of composition, a carrier may be a dietary-suitable carrier or a pharmaceutically/cosmetically acceptable carrier or a dermatologically acceptable carrier, as long as it is compatible with the particular kind of composition of the present invention. The term "pharmaceutically / cosmetically acceptable carrier" refers to a vehicle for either pharmaceutical or cosmetic use which delivers the active components to the intended target and which will not cause harm. The term "dermatologically-acceptable," as used herein, means that the compositions or components thereof so described are suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic response, etc. Examples of a dietary- suitable carrier include, but are not limited to, dietary suitable excipients, diluents, and carriers. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, dispersants, carriers and diluents for the active ingredient(s) to achieve a composition usable as a dosage form. As used herein, the terms "pharmaceutically/cosmetically acceptable," "physiologically acceptable", "dermatologically acceptable", and variations thereof, as they refer to compositions, carriers, diluents, and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological side effects.

A pharmaceutically/cosmetically acceptable carrier may comprise materials commonly employed in skin care products, including but not limited to, water, liquid or solid emollients, silicone oils, acetone, ethanol, ethylene glycol, propylene glycol, butane- 1, 3 -diol, isopropyl myristate, isopropyl palmitate, mineral oil, emulsiiϊers, solvents, humectants, thickeners, powders, propellants and the like. The carrier will usually form from 5.0% to 99.9%, preferably from 20% to 99.9% and most preferably from 50% to 95% by weight of the composition and can, in the absence of other cosmetic adjuncts, form the balance of the composition. Techniques for formulation and administration of cosmetic and pharmaceutical compositions are well known, and can be found, for example, in Remington's Pharmaceutical Sciences, Eighteenth Edition, A. R. Gennaro, Ed., Mack Publishing Co. Easton Pa., 1990, which is incorporated herein by reference.

The therapeutic / cosmetic uses of the present compositions are numerous, namely treatment or prevention of any condition in which the skin's epidermis or dermal layer is at risk, deficient or damaged. For example, the pomegranate-fraction containing compositions can be used in prevention or treatment of dry skin conditions generally, or specific dry skin conditions, such as result from regular exposure to detergents, soaps and hot water; seasonal 006/000787

exposure to harsh weather conditions, e.g., cold, wind and/or sun; occupational exposure to harsh chemicals or other drying or damaging agents; or pathological conditions such as eczematous dermatides, psoriasis, ichthyoses, xerosis and the like. It is also well known that dry skin is commonly associated with aging (both intrinsic and photoaging). Pomegranate fractions and compositions comprising same can be used in prevention of further damage to aging skin, or treatment and/or reversal of already present damage. In addition, defective skin as a result of injuries such as burns, wounds, blisters, stasis ulcers and bedsores can be expected to benefit from application of the compositions of the invention.

Compositions of the present invention may also contain additives and adjuncts such as water, alcohols, oils (mineral vegetable, animal and synthetics), glycols, colorants, preservatives, emulsifiers, gelling agents, gums, esters, hormones, steroids, antioxidants, silicones, polymers, fragrances, flavors, sunscreens, other active ingredients, acids, bases, buffers, vitamins, minerals, salts, polyols, proteins and their derivative essential oils, other enzymes, co-enzymes and extracts, surfactants, detergents, soaps, anionics, non-ionics, ionics, waxes, lipids, UV filters, stabilizers, fillers, celluloses, glycans, amines, solubilizers, thickeners, sugars and sugar derivatives, ceramides, sweeteners, and opacifiers, as necessary or desirable to enhance the efficacy, storage, utility, or marketability of the skin care composition, so long as such additives do not defeat the objectives of the present invention. For orally-ingested forms of the invention, excipients or other materials may be added. The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. The term "active ingredient" refers to the at least one pomegranate fraction accountable for the biological or physiologic effect.

For instance, the formulation, in addition to the carrier and the active pomegranate component(s), can comprise other components including, but not limited to, water soluble colorants (such as FD&C Blue #1); oil soluble colorants (such as D&C Green #6); water soluble sunscreens (such as Eusolex 232); oil soluble sunscreens (such as Octyl Methoxycinnamate); particulate sunscreens (such as Zinc Oxide); antioxidants (such as BHT); chelating agents (such as Disodium EDTA); "emulsion stabilizers (such as carbomer); preservatives (such as Methyl Paraben); fragrances (such as pinene); flavoring agents (such as sorbitol); humectants (such as glycerine); waterproofing agents (such as PVP/Eicosene Copolymer); water soluble film-formers (such as Hydroxypropyl methylcellulose); oil-soluble film formers (such as Hydrogenated C-9 Resin); cationic polymers (such as Polyquaternium 10); anionic polymers (such as xanthan gum); vitamins; and the like. The amount of each additive to be used in any given formulation is readily determined in accordance with its usual dosage.

The compositions of the present invention may be used alone or in combination with other biologically active ingredients, including but not limited to, those that improve or eradicate age spots, keratoses and wrinkles, analgesics, anesthetics, anti-acne agents, antibacterials, antiyeast agents, antifungal agents, antiviral agents, antidermatitis agents, antiinflammatory agents, antihyperkeratolytic agents, anti-dry skin agents, antiperspirants, antipsoriatic agents, antiseborrheic agents, antiaging agents, antiwrinkle agents, sunscreen agents, antihistamine agents, skin lightening agents, depigmenting agents, wound-healing agents, vitamins, corticosteroids, tanning agents, or hormones.

A composition of the present invention, alone or in combination with other active ingredients, may be administered to a subject in a single dose or multiple doses over a period of time, generally by oral or topical administration. Various administration patterns will be apparent to those skilled in the art. The dosage ranges for the administration of the compositions of the present invention are those large enough to produce the desired effect but not so large as to cause any adverse side effects, such as unwanted cross-reactions and the like. Generally, the dosage will vary with the age, weight, sex, condition, and extent of a condition in a subject, and the intended purpose. The dosage can be determined by one of skill in the art without undue experimentation. The dosage can be adjusted in the event of any counter indications, tolerance, or similar conditions. Those of skill in the art can readily evaluate such factors and, based on this information, determine the particular effective concentration of a composition of the present invention to be used for an intended purpose.

Each pomegranate fraction to be employed in accordance with the present invention is present in the composition in an effective amount calculated to produce the desired therapeutic effect, preferably in association with the required diluent, e.g., a carrier or vehicle. Normally the therapeutically effective amount of the active material is present in an amount T/IL2006/000787

between 0.5% and 100% by weight of the composition. More preferably the amount is from 0.5% to 10% and most preferably from 1.0% to 5% in order to maximize benefits at a minimum cost. The term "therapeutically effective amount" as used herein means that the amount of the fraction or extract of the present invention administered is of sufficient quantity to achieve the intended purpose, such as, in this case, to promote regeneration of epidermal and/or dermal layers of the skin. By determining the appearance of the skin, one skilled in the art can readily determine whether the amount of the fraction of the present invention is therapeutically effective in view of the disclosure of the present invention without undue experimentation.

The terms "fraction", "extract" and "component" are used interchangeably herein to describe the active material derived from pomegranate fruit having skin repair potential.

The term "treating" as used herein includes within its scope reducing, delaying and/or preventing normal, but cosmetically undesirable, skin conditions caused by the normal aging process. The visible signs of aging, such as wrinkles, lines and/or sagging are delayed or reduced. The compositions, methods and uses according to the invention may be useful for treating skin that is already in a wrinkled, aged, and/or photodamaged condition or for treating youthful skin to prevent or reduce those aforementioned undesirable changes due to the normal aging/photoaging process.

A topical composition according to the present invention may be in any of the conventional cosmetic or pharmaceutical forms which are generally used for topical application such as liquids (both aqueous and non-aqueous solutions), creams (both oil-in- water and water-in-oil, O/W & W/O emulsions), gels (both aqueous and non-aqueous), lotions, serums, spray, ointments, paste, powders, liposomes, laminates, microspheres, capsules, and tablets, cosmetics, or the like. The active component(s) may be generally incorporated in a dermatologically/cosmetically acceptable vehicle or carrier in conventional manner. The active components can suitably first be dissolved or dispersed in a portion of the water or another solvent or liquid to be incorporated in the composition. The preferred compositions are oil-in-water or water-in-oil or water-in-oil-in-water emulsions.

The composition may packaged in any suitable manner such as in a jar, a bottle, tube, stick, roller-ball applicator, aerosol spray device or the like, in the conventional manner. It is also envisaged that the inventive compositions could be packaged as a kit of two separate compositions, one containing a first pomegranate fraction and the second containing at least one second pomegranate fraction of the present invention, to be applied to the skin simultaneously or consecutively.

The composition according to the invention may also be formulated into a form suitable for oral ingestion such as a capsule, tablet or the like. Such compositions may be prepared by conventional pharmaceutical means including the step of bringing into association the carrier with the active ingredient. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally, with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Each unit dose may be divided between one or more tablets, cachets or capsules. In a preferred embodiment, a safe and effective daily dose of the active ingredients is contained within two or less tablets, cachets or capsules.

In other embodiments, wherein more dramatic or more immediate results are desired, the method of the present invention may be carried out through intradermal injection of the skin care composition, in a suitably sterile form, by a licensed practitioner such as a deπnatologist, directly to the area to be treated.

The method and frequency of application of the compositions will vary depending upon the form of the composition, the nature of the condition to be treated or prevented, the concentration of the active components and the amount of composition used. As to method for application, the composition will generally be applied in the same manner as one would apply other compositions of the same type and form, e.g., as a cream or lotion to be applied for moisturizing the skin. As to frequency, for treatment of existing dry skin conditions or other conditions associated with damaged skin, the composition can be applied, for example, on an as-needed basis until the condition is improved. The compositions can also be applied daily to prevent the occurrence of dry skin. When used in combination with other active agents, as outlined above, the application frequency will be determined according to the usual pattern for topical application of the other active. A rinsing step may optionally follow depending on whether the composition is formulated as a "leave-on" or a "rinse-off product.

The following examples are intended to illustrate, but not to limit, the scope of the invention. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation.

EXAMPLES Materials and Methods Pomegranate Processing

We examined aqueous extracts of pomegranate juice, peel and seed. The extract of pomegranate juice was prepared according to the method previously described (Schubert et al. 1999). Briefly, fresh pomegranate juice obtained by pressing the arils of organically grown "Wonderful" pomegranates from Kibbutz Sde Eliahu, Israel, was combined with wine yeast (Lalvin EC- 1118, Lallemand, Montreal, Canada, in a ratio of 18,000 : 1 parts of juice:yeast, i.e., 5 g. yeast per 20 liters of juice, and allowed to ferment to completion (about two weeks). The alcohol was then removed by distillation, and the remaining liquor concentrated over an open fire to 50% total dissolved solids (TDS). The concentrate was layered upon ethyl acetate (EA) for 15 hours, after which the extract was removed from the EA phase by evaporating the EA with nitrogen gas.

To obtain the peel extract, dried pomegranate peels from the same pomegranate source noted above were combined with an equal amount of water and boiled in a covered steel vessel for one hour. The liquor was then strained, concentrated to 50% TDS, and extracted with EA as described above.

Three different fractions were prepared from pomegranate seeds — i.e., from the remaining small dried white pithy kernels following juice extraction and removal of the pulp. To prepare cold-pressed pomegranate seed oil, the seeds were forced through a mechanical extrusion device according to the method previously reported (Schubert et al. 1999) generating an oil (approximately 10% of the seed product) and the remaining pomegranate seed "cake" (90% of the initial seed). This cake was then exhaustively extracted with 95% food-grade ethanol to yield an oily phase (75%) that was discarded, and a dark heavier tarry phase (25%) that was tested for skin-repair potential. An additional sample of cold-pressed seed oil was prepared according to the method described above from a different seed source, specifically "Mixed Wildcrafted Cultivars" obtained from the southeastern region (Adana) of Turkey. Major constituents of seed oil and peel extract from (Wonderful) pomegranate are presented in Tables I and II, respectively.

TAJSLE I Major Chemical Constituents of Pomegranate Seed Oil

Fatty acids (as triglycerides -88%)

Fatty acids (as diacylglycersols -7%)

Fatty acids (as phospholipids -3%)

Punicic acid (-65%)

Linoleic acid (7%)

22:0 Docosanoic acid (1.3%)

24:0 Tetracosanoic acid (1.4%)

Campesterol (0.1%)

Stigmasterol (0.1%) β-Sitosterol (1%) γ-Tocopherol (1%)

17-α-estradiol (0.3%)

TABLE II Major Chemical Constituents of Aqueous Peel Extract

Punicalagin Punicalin Ellagic acid Gallic acid Quercetin Luteolin Kaempferol Naringenin

Example 1

Human skin organ cultures

Organ cultures of human skin were prepared as described previously (Varani et al. 1993; Varani et al. 1994). Briefly, replicate 2 mm full-thickness punch biopsies of sun- protected hip skin were obtained from young adult volunteers who provided written informed- consent prior to their inclusion in the study. Immediately upon biopsy, the tissue was immersed in culture medium consisting of Keratinocyte Basal Medium (KBM) (Cambrex Bioscience, Walkersville, MD). KBM is a Iow-Ca2+(0.15 mM) serum-free modification of MCDB- 153 medium optimized for high-density keratinocyte growth. For use in organ culture, it was supplemented with CaCl2 to bring the final Ca2+ concentration to 1.4 mM. This was done because previous studies had demonstrated that survival of both the epidermis and dermis in organ-cultured skin required conditions optimized for fibroblasts (Varani et al. 1993; Varani et al. 1994). After transport to the laboratory on ice, the biopsies were incubated in a 24-well dish containing 0.5 ml of Ca2+-supplemented KBM with varying amounts of the different pomegranate fractions. Cultures were incubated at 37°C in an atmosphere of 95% air and 5% CO2. Other than to incubate the tissue in a minimal volume of medium, nothing further was done to maintain a strict air-liquid interface. Incubation was for 8 days, with change of medium every second or third day. At the end of the incubation period, tissue was fixed in 10% buffered formalin and examined histologically after staining with hematoxylin and eosin. Example 2 Human epidermal keratinocytes and dermal fibroblasts in monolayer culture

Normal human epidermal keratinocytes and dermal fibroblasts were isolated from skin as described previously (Varani et al. 1994). Keratinocytes were maintained in Keratinocyte Growth Medium (KGK-I) (Cambrex Bioscience) and fibroblasts were maintained in Dulbecco's Modified Minimal Essential Medium supplemented with nonessential amino acids and 10% fetal bovine serum (DMEM-FBS). In some experiments, the HaCaT line of immortalized human epidermal keratinocytes (Boukamp et al. 1988) was used in place of normal keratinocytes. Both keratinocytes and fibroblasts were maintained at 37°C in an atmosphere of 95% air and 5% CO2. Cells were sub-cultured by exposure to trypsin/ethylenediamine tetraacetic acid (EDTA) and used at passage 2-3. Example 3 Survival and proliferation assays

Keratinocytes and fibroblasts were seeded at 4x104 cells per well in 24-well plates using their respective growth media. The cells were allowed to attach overnight. The next day, they were washed and then incubated in KBM with or without Ca2+ supplementation and with different concentrations of the various pomegranate fractions as indicated in the Results Section. Cell numbers were determined three days later by releasing the cells with trypsin/EDTA and enumerating them using a particle counter (Coulter Electronics, Hialeah,

FL).

Example 4

Type I Procollagen Assay

Fibroblast-conditioned medium was assayed for type I procollagen by enzyme-linked immunosorbant assay (ELISA) (Pan Vera Corp., Madison, WI) as described previously (Varani et al. 2000). The procollagen assay uses an antibody to the C-terminal propeptide region that is part of the collagen molecule as it is synthesized and secreted (before being proteolytically cleaved). As such, this assay is a measure of newly synthesized collagen. Example 5 MMP assays

The same fibroblast-conditioned medium was assayed for MMP-I (interstitial collagenase) and MMP-2 (72-kD gelatinase) by casein and gelatin zymography, respectively. Assays were carried out as described in a previous report (Gibbs et al. 1999). Briefly, SDS- PAGE gels were prepared from 30:1 acrylamide/bis with the incorporation of either β-casein or gelatin (lmg/ml) before casting. The gels were routinely 7.5 % acrylamide. Culture fluid samples and molecular weight standards were electrophoresed at constant voltage of 150V under non-reducing conditions. After electrophoresis, gels were removed and washed twice for 15 minutes in 50 mM Tris buffer containing 1 mM Ca2+ and 0.5 mM Zn2+ with 2.5% Triton X-100. The gels were then incubated overnight in Tris buffer with 1% Triton X- 100 and stained with Brilliant Blue R Concentrate the following day. After destaining, zones of hydrolysis were detected as clear areas against a dark background. Zymographic images were digitized. Negative images were created and quantified by scanning densitometry. Using the digitized images, quantitative values for MMP-I and MMP-2 were obtained. Example 6 Statistical analysis and Results

Differences between groups in experiments with multiple groups were analyzed for statistical significance by ANOVA (Analysis of Variance) followed by paired-group comparisons. Where there were only two groups, the Student t-test was used. Data were analyzed as paired or unpaired as appropriate. P<0.05 was considered significant. Results: Effects of pomegranate seed oil and extracts of pomegranate peel, pomegranate juice and seed cake on epidermal proliferation

In the first series of experiments, various pomegranate fractions were examined for ability to influence keratinocyte proliferation in monolayer culture.

Reference is made to Fig. I5 a graph showing the effects of pomegranate seed oil on keratinocyte proliferation, as well as to Figs. 2A-2C, graphs showing the effects of pomegranate juice (Fig. 2A)5 pomegranate peel (Fig. 2B) and pomegranate seed cake extract (Fig. 2C) on keratinocyte proliferation.. Proliferation assays were carried out as described in the Materials and Methods. Values shown represent the arithmetical means + standard errors based on triplicate experiments with duplicate data points in each. Statistical significance of the differences between control and pomegranate-treated groups was determined using ANOVA followed by paired-group comparisons. *indicates significance at the p<0.05 level.

As seen in Figure I5 cold-pressed pomegranate seed oil stimulated keratinocyte proliferation in a concentration-dependent manner. Under conditions in which optimal stimulatory activity was observed (0.5μl of pomegranate seed oil in 1 ml of serum-free, growth factor-free culture medium), keratinocyte proliferation was increased by 60% (proliferation index = 1.56 for no seed oil verses 2.04 in the presence of seed oil (0.5μl/ml; n=6; p<0.05). A second seed oil preparation (from a different pomegranate source) also stimulated keratinocyte proliferation under the same conditions (data not shown). In contrast to the effects observed with the pomegranate seed oil preparations, there was no keratinocyte stimulation with water-soluble extracts of pomegranate peel, fermented juice or seed cake. Rather, at high concentrations, these three fractions were all slightly inhibitory (Figures 2A- 2C).

Figure 3 demonstrates histological features of human skin after incubation for 8 days in organ culture (hematoxylin and eosin - stained; Xl 80) in the absence (Fig. 3A) or presence (Fig. 3B) of cold-pressed pomegranate seed oil. Consistent with findings in monolayer cell culture, treatment of human skin with pomegranate seed oil (0.5μl/ml) induced a mild thickening of the epidermis (Fig. 3B) as compared to control conditions (Fig. 3A). It can be seen from Fig. 3B that normal epidermal differentiation was maintained in the pomegranate seed oil-treated skin with basal, suprabasal and granular layers represented. In regard to these features of differentiation, the activity of pomegranate seed oil is significantly different from that of RA, which induces a much more intense hyperplasia but also interferes with normal differentiation (Eli as et al. 1981; Jetten et al. 1989; Floyd and Jetten, 1989). Results: Effects of pomegranate seed oil and extracts of pomegranate peel, pomegranate juice, and seed cake on dermal function

Referring now to Figs. 4A - 4C, the effects of pomegranate peel extract on fibroblast proliferation, type I procollagen synthesis and MMP-I elaboration are shown.

In Fig. 4 A, the effect of pomegranate peel extract on fibroblast proliferation is graphically illustrated. Values shown represent the arithmetical means + standard errors based on triplicate experiments with duplicate data points in each. Statistical significance of the differences between control and pomegranate-treated groups was determined using ANOVA followed by paired-group comparisons. *indicates significance at the p<0.05 level.

Human dermal fibroblasts were incubated in KBM and treated with various amounts of pomegranate peel extract. In the absence of the extract, there was no growth in this serum- free, growth factor-free medium and approximately half of the cells died over the three-day incubation period. As shown in Figure 4A, pomegranate peel extracts prevented cell death and allowed proliferation to occur. Concentrations between 0.05 and 0.5 μl of extract per ml of culture medium were effective. Consistent with past reports (Varani et al. 1990a; 1990b), when KBM was supplemented with extracellular Ca2+ to a final concentration of 1.4 mM, fibroblast survival and growth also occurred (Figure 4A). Two other extracts, (i.e., fermented juice and seed cake) were also protective, but were less effective than peel extract. In contrast, no protective activity was observed when pomegranate seed oil was examined (data not shown).

Results: Ability of pomegranate peel extract to alter type I procollagen production by dermal fibroblasts

In Fig. 4B, the effect of pomegranate peel extract on type I procollagen synthesis is shown. Values are means + standard deviations based on two separate experiments with duplicate data points per experiment. Statistical significance of the differences between control and pomegranate-treated groups was determined using ANOVA followed by paired- group comparisons, indicates significance at the p<0.05 level.

Fig. 4B demonstrates that type I procollagen synthesis was increased in the presence of pomegranate peel extract. Interestingly, dose-response studies indicated that type I procollagen production was stimulated at concentrations below those optimal for proliferation, with procollagen elaboration declining at higher concentrations. When KBM was supplemented with Ca2+ (1.4 mM final concentration), there was also a stimulation of procollagen synthesis. However, the level of increase with Ca2+ stimulation was not as great as that achieved with pomegranate peel extract.

In Fig. 4C, the effect of pomegranate peel extract on MMP-I elaboration is shown. Values are means and standard deviations based on four separate experiments with a single data point per experiment. Statistical significance of the differences between control and pomegranate-treated groups was determined using ANOVA followed by paired-group comparisons. *indicates significance at the p<0.05 level.

Fig. 4C demonstrates the effects of pomegranate peel extract on MMP-I production. It can be seen that MMP-I accumulation in the fibroblast-conditioned medium was dramatically reduced in the presence of the pomegranate peel extract. The dose-response for MMP-I inhibition mirrored (inversely) the dose response for proliferation — i.e., the same extract concentrations that stimulated proliferation inhibited MN-IP-I accumulation. It can also be observed from this figure that the level of MMP-I in Ca2+ - supplemented KBM was similar to the level in non-supplemented KBM. When pomegranate peel extract was included in Ca2+ - supplemented KBM, inhibition of MMP-I elaboration was observed over the same dose-range as was seen in the non-Ca2+ - supplemented medium (data not shown). In contrast to these results, when we assessed MMP -2 (72-kD gelatinase A) production in the pomegranate peel extract-treated fibroblasts, there was no effect (data not shown). This is of interest since our past studies have shown that unlike MMP-I, MMP-2 is not subject to regulation by agents that act through AP-I -mediated gene transcription (Fisher et al. 1996). Example 7

Typical Composition for Pomegranate Seed Oil Cream

The formulation below describes an oil in water cream suitable for the methods and uses according to the present invention. The percentages indicated are by weight of the composition.

Pomegranate seed oil extracted as described in the Methods and Materials section above in an amount between about 1.0% and 5.0% percent by weight; a carrier, such as Eucerin® hydrophilic lanolin, in an amount between about 20% and 80 percent by weight; an antioxidant in an amount between about 0.001% and 1.0 percent by weight; water in an amount between about 60% and 80 percent by weight; a sun screen agent in amount between about 0.001% and 1 percent by weight; an emollient in an amount between about 5% and 30 percent by weight; an emulsifier in an amount between about 0.001% and 30 percent by weight; a humectant in an amount between about 0.001% and 30 percent by weight; and a thickener in an amount between about 0.1% to 0.2 percent by weight. Instead of Eucerin® hydrophilic lanolin as the carrier, one or more vegetable oils, such as sesame oil, olive oil, apricot kernel oil, hemp seed oil or pumpkin seed oil, in an amount between about 20% and

80% by weight and an emulsifier such as beeswax in an amount between about 7% and 12% by weight, and preferably about 9% by weight, may be used.

Example 8

Typical Composition for Pomegranate Peel Cream

The formulation below describes an oil in water cream suitable for the methods and uses according to the present invention. The percentages indicated are by weight of the composition.

Pomegranate peel extracted as described in the Methods and Materials section above in an amount between about 1.0% and 5.0% percent by weight; a carrier, such as Eucerin® hydrophilic lanolin, in an amount between about 20% and 80 percent by weight; an antioxidant in an amount between about 0.001% and 1.0 percent by weight; water in an amount between about 60% and 80 percent by weight; a sun screen agent in amount between about 0.001% and 1 percent by weight; an emollient in an amount between about 5% and 30 percent by weight; an emulsifier in an amount between about 0.001% and 30 percent by weight; a humectant in an amount between about 0.001% and 30 percent by weight; and a thickener in an amount between about

0.1% to 0.2 percent by weight. Instead of Eucerin® hydrophilic lanolin as the carrier, one or more vegetable oils, such as sesame oil, olive oil, apricot kernel oil, hemp seed oil or pumpkin seed oil, in an amount between about 20% and 80% by weight and an emulsifier such as beeswax in an amount between about 7% and 12% by weight, and preferably about 9% by weight, may be used.

Example 9

Typical Composition for Combination Pomegranate Seed Oil and Pomegranate Peel Cream

The formulation below describes an oil in water cream suitable for the methods and uses according to the present invention. The percentages indicated are by weight of the composition.

Pomegranate seed oil extracted as described in the Methods and Materials section above in an amount between about 1.0% and 5.0% percent by weight; pomegranate peel extracted as described in the Methods and Materials section above in an amount between about 1.0% and 5.0% percent by weight; a carrier, such as Eucerin® hydrophilic lanolin, in an amount between about 20% and 80 percent by weight; an antioxidant in an amount between about 0.001% and 1.0 percent by weight; water in an amount between about 60% and 80 percent by weight; a sun screen agent in amount between about 0.001% and 1 percent by weight; an emollient in an amount between about 5% and 30 percent by weight; an emulsifier in an amount between about 0.001% and 30 percent by weight; a humectant in an amount between about 0.001% and 30 percent by weight; and a thickener in an amount between about 0.1% to 0.2 percent by weight. Instead of Eucerin® hydrophilic lanolin as the carrier, one or more vegetable oils, such as sesame oil, olive oil, apricot kernel oil, hemp seed oil or pumpkin seed oil, in an amount between about 20% and 80% by weight and an emulsifier such as beeswax in an amount between about 7% and 12% by weight, and preferably about 9% by weight, may be used. Discussion

The pomegranate (Punica gi-anatum) is a small tree cultivated in various parts of the world for its fruit. In addition to its value as a table fruit, pomegranate preparations have been used for ages in various folk remedies (reviewed in Ayensu 1981; Boulos 1983; Duke and Ayensu 1985). The pomegranate is a highly celebrated medicinal food plant chosen as the symbol of medicine for the 2000 UK Millenial Festival of Medicine (Langley 2000). Pomegranate peel (pericarp) is well regarded for its astringent properties, the seeds for conferring invulnerability in combat and stimulating beauty and fertility. Current interest focuses largely on the pomegranate's anti-oxidant and anti-inflammatory activities (Schubert, Lansky and Neeman 1999; Aviram et al. 2000; Gil et al. 2000; Singh, Murthy and Jayaprakasha 2002) and on growth-inhibiting activities (Murthy, Singh and Jayaprakasha 2002; Hora et al. 2003; Albrecht et al. 2004; Lansky et al. 2005). Pomegranate fruit has wide- ranging heuristic applications against (for example) erectile dysfunction (Azadzoi et al. 2005), carotid occlusion (Aviram et al. 2004) and neonatal ischemia (Loren et al. 2005).

In recent years, efforts have been made to identify bioactive moieties in pomegranate preparations and to characterize their effects at the cellular and biochemical levels. Pomegranate seed oil is a rich source of conjugated fatty acids (of which punicic acid is the most common) mainly in the form of mono-, di- and especially tri glycerols (Hora et al. 2003). Polyphenols compounds are also present in the seed oil of the pomegranate (Schubert et al. 1999). Polyphenols compounds originating in pomegranate seed oil, juice and peel have anti-oxidant activity and inhibit pro-inflammatory enzymes including the cyclooxygenases and lipoxygenases (Schubert et al 1999; Gil et al. 2000; Aviram et al. 2000; Murthy et al. 2002; Singh et al. 2002). As a result, pomegranate preparations containing these components are being evaluated for potential anti-inflammatory activity. Pomegranate seed oil has also been shown in experimental studies to suppress proliferation of several different tumor cell types (Kim et al. 2002; Albrecht et al. 2004; Kawaii and Lansky 2004; Lansky et al. 2005) and to reduce skin carcinogenesis in mice (Hora et al. 2003) and mammary carcinogenesis in a mouse mammary organ culture model (Mehta and Lansky 2004).

While most of the current interest in the pomegranate relates to its anti-oxidant, antiinflammatory and anti-carcinogenic activities, the focus of the invention is on the potential for one or more pomegranate fractions to promote skin repair. Aging of the skin is accompanied by thinning of the epidermis and a concomitant loss of dermal connective tissue (Smith et al. 1962; Lavker 1979; Lovell et al. 1987; Kligman and Balin 1989; Schwartz et al. 1989; Oikrinen and Kallioinen 1989; Schwartz et al. 1993; Lavker 1995). The loss of connective tissue elements including types I and III collagen in aged skin reflects progressive MMP- mediated connective tissue destruction (occurring over decades) (Fisher et al. 1996; Fisher et al. 1997; Varani et al. 2000) and a late-stage sharp decline in synthesis of replacement collagen (Griffiths et al. 1993; Talwar et al. 1995; Varani et al. 2001).

Skin damage is a cosmetic problem, but it is also a cause of significant morbidity. Aged/photo-aged skin bruises easily (e.g., senile purpura) and the chronic bruises often go on to form slow-healing ulcers. Similar detrimental changes are also observed in diabetic skin (Loots et al. 1999; Hehenberger et al. 1999) and in other conditions where the vasculature of the skin is compromised (Strigini and Ryan 1996; Stadelmann, Digenis and Tobin 1998; IL2006/000787

Mendez et al. 1998) as well as following long-term corticosteroid use (McMichael et al. 1996). If the connective tissue of damaged skin can be repaired, it might be possible to delay or prevent the serious consequences that occur in the damaged tissue (Lateef et al. 2005).

The present study clearly demonstrates, for the first time, that a water-soluble extract made from pomegranate peel inhibits elaboration of the major enzyme (MMP-I) responsible for collagen destruction in aged/photoaged skin (Brennan et al., 2003). Concomitantly, pomegranate peel extract induced type I procollagen production by dermal fibroblasts. In contrast, the same pomegranate peel extract, as well as two other water-soluble extracts (i.e., from the fermented juice and from the remaining seed cake following removal of the seed oil) had no apparent stimulatory effect on keratinocyte function. On the other hand, pomegranate seed oil stimulated keratinocyte proliferation in both cell culture and organ culture, but had little effect on fibroblast function. Combined, these epidermal and dermal activities of the pomegranate resemble activities associated with the biologically active retinoids. The biologically-active retinoids including RA and its parent compound, all-trans retinol have proven skin-repair capacity (Kligman et al. 1986; Weiss, Ellis and Voorhees 1988). It is generally believed that rebuilding damaged collagen is essential for skin repair (Griffiths et al. 1993; Talwar et al. 1995), but epidermal effects are also likely to be important. While it is difficult to separate epidermal and dermal effects with retinoids, it appears from the present studies that the epidermal and dermal regenerative activities of the pomegranate reside in different fractions. Thus, it is possible to use these fractions singly or in combination to identify and manipulate the biological events most important for specific skin repair activity. We have found that seed oil derived from pomegranates has a retinoid-like stimulatory effect on keratinocyte proliferation but does not stimulate dermal fibroblast function. In contrast, an aqueous extract of pomegranate peel has potent retinoid-like dermal effects - stimulating dermal fibroblast proliferation and collagen synthesis while inhibiting the major collagen-degrading enzyme in skin - but does not stimulate keratinocyte proliferation in the epidermis.

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.

Full bibliographic citation for references referred to within parentheses throughout this application is provided below. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

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Claims

What is claimed is:
1. A method of promoting skin regeneration in a subject comprising (a) providing pomegranate seed oil and (b) administering to said subject a composition comprising a therapeutically effective amount of said pomegranate seed oil.
2. The method of claim 1, wherein said composition further comprises a pharmaceutically acceptable carrier.
3. The method of claim 1, wherein said pomegranate seed oil is the result of a process selected from the group consisting of cold pressing, expeller pressing, and supercritical fluid extraction with carbon dioxide.
4. The method of claim 1, wherein said step of administering includes administration via an administration route selected from the group consisting of topical administration, oral administration, and injection.
5. The method of claim 1, wherein said composition is administered in a form selected from a group consisting of tablets, suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams, pastes, gels, and the like.
6. A method of promoting skin regeneration in a subject comprising (a) providing at least one pomegranate fraction selected from the group consisting of pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof and (b) administering to said subject a composition comprising a therapeutically effective amount of said at least one pomegranate fraction.
7. The method of claim 6, 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, and partially reduced pomegranate juice.
8. The method of claim 6, 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 an alcohol, and a supercritical fluid extract of pomegranate peel.
9. The method of claim 6 wherein said composition further comprises a pharmaceutically acceptable carrier.
10. The method of claim 6, wherein said step of administering includes administration via an administration route selected from the group consisting of topical administration, oral administration, and injection.
11. The method of claim 6, wherein said composition is administered in a form selected from a group consisting of tablets, suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams, pastes, gels, and the like.
12. A method of promoting skin regeneration in a subject comprising (a) providing pomegranate seed oil; (b) providing at least one pomegranate fraction selected from the group consisting of a pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof; and (c) administering to said subject a composition comprising a therapeutically effective amount of a combination of pomegranate seed oil and at least one pomegranate fraction selected from the group consisting of a pomegranate peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof.
13. The method of claim 12, wherein said composition comprises a combination of pomegranate seed oil and a pomegranate peel product.
14. The method of claim 12, wherein said pomegranate seed oil is the result of a process selected from the group consisting of cold pressing, expeller pressing, and supercritical fluid extraction with carbon dioxide.
15. The method of claim 12, 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, and partially reduced pomegranate juice.
16. The method of 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 an alcohol, and a supercritical fluid extract of pomegranate peel.
17. The method of claim 12, wherein said composition further comprises a pharmaceutically acceptable carrier.
18. The method of claim 12, wherein said step of administering includes administration via an administration route selected from the group consisting of topical administration, oral administration, and injection.
19. The method of claim 12, wherein said composition is administered in a form selected from a group consisting of tablets, suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams, pastes, gels, and the like.
20. A composition for treating skin, the composition comprising a therapeutically effective amount of a pomegranate product.
21. The composition of claim 20, wherein said pomegranate product includes at least one pomegranate fraction selected from the group consisting of pomegranate seed oil, a pomegranate .peel product, a pomegranate juice product, pomegranate seed cake, and mixtures thereof.
22. The composition of claim 20 wherein said pomegranate seed oil has an epidermal regenerating activity and wherein the remaining pomegranate fractions have dermal regenerating activity.
23. The composition of claim 22 wherein said pomegranate seed oil stimulates keratinocyte proliferation in the epidermis.
24. The composition of claim 22 wherein said remaining pomegranate fractions stimulate type I procollagen synthesis and inhibit MMP-I production by deπnal fibroblasts.
25. The composition of claim 20, wherein said pomegranate seed oil is the result of a process selected from the group consisting of cold pressing, expeller pressing, and supercritical fluid extraction with carbon dioxide.
26. The composition of claim 20, wherein said pomegranate juice product includes 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, and partially reduced pomegranate juice.
27. The composition of claim 20, 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 CO2 extract of pomegranate peel.
28. The composition of claim 20 further comprising a pharmaceutically acceptable carrier.
29. The composition of claim 20, wherein said composition is administered in a form selected from the group consisting of tablets, gel-caps, suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams, pastes, sticks, sprays, gels, and the like.
30. A method for treating skin in a mammalian subject comprising administering to said subject a therapeutic amount of the composition of claim 20.
PCT/IL2006/000787 2005-07-06 2006-07-06 Methods of using pomegranate fractions for skin repair WO2007004229A3 (en)

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Cited By (12)

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WO2009031153A2 (en) * 2007-09-06 2009-03-12 Ori Software Pomegranate-derived products for the treatment of skin sores and lesions
WO2009098019A2 (en) * 2008-02-07 2009-08-13 Beiersdorf Ag Skin care preparation for soothing stressed skin
WO2009098029A2 (en) * 2008-02-06 2009-08-13 Beiersdorf Ag Wound dressing articles
EP2132994A1 (en) * 2008-06-11 2009-12-16 Ludwig Manfred Jacob Fermentation of solutions containing pomegranate juice with sacchatomyces boulardii and lactobacilli, resulting fermented products and application thereof
WO2010055300A2 (en) * 2008-11-14 2010-05-20 Aston University Pomegranate oil
WO2010013015A3 (en) * 2008-08-01 2011-03-17 E.S.L.I. Limited Composition for accelerated production of collagen
WO2010089566A3 (en) * 2009-02-06 2011-03-31 Seeds Lp Composition for treatment of skin
WO2011051633A2 (en) 2009-10-30 2011-05-05 L'oreal Use of a punica granatum extract to fight against the whitening of the hair
FR2967063A1 (en) * 2010-11-09 2012-05-11 Oreal Non-therapeutic cosmetic use of at least one pomegranate extract, as agent for firming skin of a subject, who has weight modification prior to and/or after an aesthetic surgery and to prevent and/or treat sagging skin
CN104645209A (en) * 2015-03-20 2015-05-27 倪春仙 Chinese medicine composition for treating ichthyosis and preparation method and application of Chinese medicine composition
ES2582285A1 (en) * 2015-03-10 2016-09-12 Vitalgrana Pomegranate, S.L. Composite elements made from pomegranate fruit and its
EP3212288A4 (en) * 2014-10-31 2018-06-20 Pomega, Inc. Formulations containing pomegranate seed oil, rosa canina fruit oil and inula viscosa oleoresin or extract

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009031153A2 (en) * 2007-09-06 2009-03-12 Ori Software Pomegranate-derived products for the treatment of skin sores and lesions
WO2009031153A3 (en) * 2007-09-06 2009-09-03 Ori Software Pomegranate-derived products for the treatment of skin sores and lesions
WO2009098029A3 (en) * 2008-02-06 2010-07-01 Beiersdorf Ag Wound dressing articles
WO2009098029A2 (en) * 2008-02-06 2009-08-13 Beiersdorf Ag Wound dressing articles
WO2009098019A3 (en) * 2008-02-07 2009-11-05 Beiersdorf Ag Skin care preparation for soothing stressed skin
WO2009098019A2 (en) * 2008-02-07 2009-08-13 Beiersdorf Ag Skin care preparation for soothing stressed skin
EP2132994A1 (en) * 2008-06-11 2009-12-16 Ludwig Manfred Jacob Fermentation of solutions containing pomegranate juice with sacchatomyces boulardii and lactobacilli, resulting fermented products and application thereof
US8552063B2 (en) 2008-08-01 2013-10-08 E.S.L.I. Limited Composition for accelerated production of collagen
WO2010013015A3 (en) * 2008-08-01 2011-03-17 E.S.L.I. Limited Composition for accelerated production of collagen
WO2010055300A3 (en) * 2008-11-14 2010-07-08 Aston University Pomegranate oil
WO2010055300A2 (en) * 2008-11-14 2010-05-20 Aston University Pomegranate oil
WO2010089566A3 (en) * 2009-02-06 2011-03-31 Seeds Lp Composition for treatment of skin
US9403042B2 (en) 2009-02-06 2016-08-02 Seeds Group, Lp Composition for treatment of skin
WO2011051633A2 (en) 2009-10-30 2011-05-05 L'oreal Use of a punica granatum extract to fight against the whitening of the hair
FR2967063A1 (en) * 2010-11-09 2012-05-11 Oreal Non-therapeutic cosmetic use of at least one pomegranate extract, as agent for firming skin of a subject, who has weight modification prior to and/or after an aesthetic surgery and to prevent and/or treat sagging skin
EP3212288A4 (en) * 2014-10-31 2018-06-20 Pomega, Inc. Formulations containing pomegranate seed oil, rosa canina fruit oil and inula viscosa oleoresin or extract
ES2582285A1 (en) * 2015-03-10 2016-09-12 Vitalgrana Pomegranate, S.L. Composite elements made from pomegranate fruit and its
CN104645209A (en) * 2015-03-20 2015-05-27 倪春仙 Chinese medicine composition for treating ichthyosis and preparation method and application of Chinese medicine composition

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