WO2008015575A2

WO2008015575A2 - Melanogenesis stimulant compositions derived from extracts of plants of the genus nigella

Info

Publication number: WO2008015575A2
Application number: PCT/IB2007/003240
Authority: WO
Inventors: Michinori Kubo; Hideaki Matsuda
Original assignee: Unisantis F Z E
Priority date: 2006-05-09
Filing date: 2007-05-09
Publication date: 2008-02-07
Also published as: WO2008015575A3

Abstract

Disclosed are extracts and compositions containing the extracts useful for stimulating melanogenesis; the extracts are obtained from the plants of the genus Nigella (Ranunculaceae). The extracts and compositions are useful in personal care and pharmaceutical compositions for preventing, improving, or treating hair conditions and/or leukotrichia resulting from a reduction in or failure of melanin production. Such compositions may be applied topically or taken orally. The compositions stimulate melanin production as well as inhibit testosterone 5a-reductase, an indirect method to increase melanin production.

Description

Melanogenesis Stimulant Compositions Derived from Extracts of

Plants of the Genus Nigella

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates generally to a melanogenesis stimulant, and more particularly to a pharmaceutical or cosmetic preparation for stimulating activity to promote melanogenesis. The invention also relates to methods for treating and/or preventing conditions related to reduced melanogenesis, particularly in the hair.

Description of the Related Art

Melanin is produced by melanocytes existing in the epidermis or hair, yielding the pigmentation of hair. Decreased melanin production causes the loss of pigmentation in the hair, leading to white and/or gray hair. This effect can range from a local circumscribed area, to all the hair on a subject. Generally, the condition of reduced melanin production is referred to as leukotrichia, achromotrichia, or hypochromotrichia . Related conditions include grayness or whiteness of the scalp hair associated with aging (canities) or as a result of a pathologic condition (poliosis) .

The previously described conditions can be cosmetically treated through application of appropriate hair dyes to return color to hair. However, such methods do not treat the cause of decreased pigmentation, but instead only disguise the effect of reduced pigmentation.

Melanin produced by a melanocyte in the epidermis or hair is transported to a keratinocyte, where it is dispersed and provided to tissues. The melanin also causes proliferation and differentiation of the keratinocyte. The colour of each tissue is determined by the quantitative and qualitative difference of the melanin which is provided to the tissue. Skin spots, freckles, skin-darkening, and pigmentation disorders such as skin-blackening caused by steroids are conditions resulting from an excess accumulation of melanin.

Melanin is distributed widely in the animal and plant kingdoms. However, it is known that melanin is biosynthesized from tyrosine through complex pathways in a vertebrate, wherein tyrosine is oxidized by tyrosinase in a cytoplasmic granule of melanosome in the melanocyte, biosynthesizing dopa and dopaquinone, which turns into indolquinone through auto-oxidation by ultraviolet light.

Leukotrichia is mainly caused by a reduction of melanogenesis following a failure of tyrosinase activity of melanocytes in a hair follicle. This condition is considered to detract from beauty and youthfulness in both men and women. However, gradually whitening of the hair with aging is usually considered to be mainly caused by the reduction of melanogenesis . Methods to promote melanogenesis by the use of natural extracts include use of extract of Chrysanthemum leaves (US 6,726,940), extract of a rosacea of Sanguisorba officinalis (US 6,187,325), extract of Marrubium vulgare (US 5,653,983), extract of Coleus esquirolii, Coleus scutellarioides, or Coleus xanthantus (US 5,505,934); and Cyperus extract (US 5,476,651) .

Methods to promote melanogenesis by treatment with other chemical agents or mixtures include use of 2 , 3-R-pinanediol and an alpha-hydroxy acid (US 6,267,948), diols of norbornane derivatives (US 6,214,888), peptone-metal complexes (US 5,888,522), and cyanocarboxylic acid derivatives (US 5, 767, 152) . An additional target which has been identified as affecting melanin production is testosterone 5a-reductase . From testosterone, the reductase produces 5a- dihydrotestosterone, an androgenic hormone which depresses the activity of melanocytes through receptor binding. The resulting complex synthesizes a protein through expression of mRNA after interacting with a specific DNA. The synthesized protein is known to cause irregular hair cycles or hair growth based on the interaction with follicle epidermal cells, induce hair loss due to atrophy of follicle cells, and depress melanocyte activity. Therefore, inhibition of testosterone 5a-reductase will stimulate melanogenesis and promotes hair growth.

There are about 20 species of annual grasses, (e.g. Nigella damascena, Nigella sativa, Nigella hispanica, Nigella orientalis) that form the genus Nigella in the Ranunculaceae family. These grasses have hard upright stems growing in clumps and are found on slopes of the quay, wastelands or fallow in the Mediterranean area, Eurasia and North Africa. These grasses usually bloom in the summer season and are utilized as cut flowers, pot plants and/or in flower beds for ornamental purpose. The seed of the genus Nigella is commonly known as Black Seed or Black Cumin, and has traditionally been used as a spice in India, Turkey, Greece and Middle East (especially, Egypt and Tunisia) . In the Mediterranean basin it has also been used for the treatment of colds, asthma, conjunctivitis, polio, eczema etc. Since the ancient Egyptian era, various activities including hypoglycemic, anti-tumor, bronchodilating, hypotensive, antimicrobial, and antifungal effects have been reported.

Recent examples of the use of Nigella extracts include, herbal compositions comprising an extract of Nigella sativa seed for treatment of viral infections (US 6,841,174), black cumin oil for treatment of psoriasis (US 6,531,164), herbal compositions comprising powdered Nigella sativa seeds for the treatment of diabetes (US 6,042,834), an extract of Nigella sativa for inhibition of cancer cell growth (US 5,653,981), and an extract from Nigella sativa to increase immune function (US 5,482,711).

With respect to effects on hair, a stimulating effect on hair regrowth of topically applied compositions containing Nigella sativa oil is described in FR 2,728,168. As of yet, there is no satisfactory agent which is widely used as a fundamental remedy for white hair, though various medicines and/or cosmetics for treating hair are reported. Therefore a need exists for remedies for white hair caused by conditions such as leukotrichia, poliosis, and canities, a general graying and/or whitening of hair as a result of aging.

SUMMARY OF THE INVENTION

The invention provides extracts of a plant of genus Nigella having melanogenesis stimulating activity, where the extracts are prepared by a process comprising

(a) contacting one or more plant parts of a plant of genus Nigella with at least one solvent to form a solvent/plant mixture,- (b) removing solid material from the mixture of step

(a) ; and

(c) optionally removing the solvent.

Further, the invention provides pharmaceutical or personal care preparations having melanogenesis stimulating activity comprising extracts of plants of the genus Nigella.

In addition, methods are provided for preventing, improving, or treating hair conditions related to decreased melanin production in patients in need of such treatment. Such conditions include, but are not limited to leukotrichia, poliosis, and canities, a general graying and/or whitening of hair as a result of aging.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that extracts of plants of the genus Nigella (Ranunculaceae) have melanogenesis stimulating activity in cultured melanoma cells. Without wishing to be bound by any particular theory, it is believed that the stimulating activity is based on an indirect mechanism that involves inhibition of testosterone 5a-reductase, and a direct mechanism of acting on melanoma cells. These mechanisms have been identified using in vitro assays of melanin production and testosterone 5a-reductase inhibition. Therefore, the extracts and compositions of the invention are useful as a medicine or cosmetic for preventing, improving and/or treating hair conditions or leukotrichia resulting from a failure of melanin-production.

The invention further provides an extract having melanogenesis stimulating activity comprising an extract or plant of the genus Nigella. Preferably, the plant is Nigella arvensis, Nigella ciliaris, Nigella coerulea, Nigella confuse, Nigella damascena, Nigella divaricata, Nigella gallica, Nigella hispanica, Nigella indica, Nigella integrifolia, Nigella latifolia, Nigella multifida, Nigella nigellastrum, Nigella orientalis, Nigella sativa, Nigella tenuiflora, Nigella truncate, or mixtures thereof. More preferably, the plant is Nigella damascena or Nigella sativa. Particularly preferred extracts of the invention are derived from Nigella damascena.

The extracts of the invention are obtained from one or more plant parts including seeds, leaves, roots, rhizomes, or stems. Preferably the extract is obtained from the seeds of the Nigella plant. Such extracts are readily obtained using well known methods in the art, and preferably by use a solvent . The invention provides extracts to stimulate melanogenesis, wherein the extract is obtained with a solvent. Solvents include but are not limited to, a lower alcohols, water, or mixtures thereof. Examples of suitable lower alcohols include, but are not limited to methanol, ethanol, n- propanol, i-propanol, n-butanol, s-butanol, and t-butanol, water, or mixtures thereof. Preferably such extracts are obtained with methanol, ethanol, water, or mixtures thereof. Preferred solvents are methanol, mixtures of ethanol and water, or water alone. Even more preferably, the invention provides dried extracts, i.e., wherein the solvent has been removed .

In another embodiment, the invention provides personal care compositions useful for stimulating melanogenesis comprising an extract of a plant of genus Nigella and a dermatologically acceptable carrier. Preferably, the extract is obtained from seeds using, as the extraction solvent, methanol, a mixture of ethanol and water, or water alone. Preferably, the extract is obtained from Nigella damascene. Preferably, the dermatological carrier is chosen so that the composition is in the form of a shampoo, hair tonic, conditioner, pomade, ointment, lotion, cream, skin lotion, emulsion, foundation, oil-based cosmetic, or face pack.

The invention also provides methods for the prevention or treatment of white hair in a patient in need of such treatment, comprising the step of topically applying to the skin, e.g., the scalp, or hair the personal care composition to stimulate melanogenesis comprising an extract of a plant of the genus Nigella, wherein the extract is obtained from the seeds of the plant with methanol, a mixture of ethanol and water, or water alone,- a dermatologically acceptable carrier, and wherein the white hair is a result of canities, aging, poliosis, or leukotrichia . Preferably, the extract is obtained from Nigella damascene. Preferably, such carriers are in the form of a shampoo, hair tonic, conditioner, pomade, ointment, lotion, cream, skin lotion, emulsion, foundation, oil-based cosmetic, or face pack. In another embodiment, the invention provides pharmaceutical compositions to stimulate melanogenesis comprising an extract of a plant of the genus Nigella, wherein the extract is obtained with methanol, a mixture of ethanol and water, or water alone,- and a pharmaceutically carrier or excipient. Preferably, the extract is obtained from Nigella damascene.

Preferably, the pharmaceutical composition as previously described is in the form of a tablet, pill, capsule, syrup or elixir. Such compositions are useful for treating or preventing white hair is a result of canities, aging, poliosis, or leukotrichia .

In another embodiment, the invention provides methods for the prevention or treatment of white hair comprising the step of orally administering to a patient in need of such treatment, a pharmaceutical composition to stimulate melanogenesis comprising an extract of a plant of genus Nigella, wherein the extract is obtained from seeds with methanol, a mixture of ethanol and water, or water alone,- and a carrier or excipient. Preferably, the extract is obtained from Nigella damascene.

Preferably, the pharmaceutical composition as previously described is in the form of a tablet, pill, capsule, syrup or elixir. Such methods are useful for treating or preventing white hair is a result of canities, aging, poliosis, or leukotrichia.

In an alternate embodiment, the invention provides an extract of a plant of the Genus Nigella, where the extract is obtained using a solvent selected from lower alcohols, water, and mixtures thereof .

In another embodiment, the invention provides an extract of a plant of the Genus Nigella, where the extract is obtained using a solvent selected from lower alcohols, water, and mixtures thereof; and the plant is Nigella arvensis, Nigella ciliaris, Nigella coerulea, Nigella confuse, Nigella damascena, Nigella divaricata, Nigella gallica, Nigella hispanica, Nigella indica, Nigella integrifolia, Nigella latifolia, Nigella multifida, Nigella nigellastrum, Nigella orientalis, Nigella sativa, Nigella tenuiflora, Nigella truncate, or mixtures thereof. Preferably, the plant is Nigella damascene. More preferably, the extract is obtained from plant parts of Nigella damascene which are seeds, leaves, roots, rhizomes, stems, or mixtures thereof. Even more preferably, the plant is Nigella damascene and the plant parts are seeds.

In another embodiment, the invention provides an extract of a plant of the Genus Nigella, where the extract is obtained using a solvent selected from methanol, ethanol, n- propanol, i-propanol, n-butanol, s-butanol, t-butanol, and mixtures thereof, water, or mixtures thereof; and the plant is Nigella arvensis, Nigella ciliaris, Nigella coerulea, Nigella confuse, Nigella damascena, Nigella divaricata, Nigella gallica, Nigella hispanica, Nigella indica, Nigella integrifolia, Nigella latifolia, Nigella multifida, Nigella nigellastrum, Nigella orientalis, Nigella sativa, Nigella tenuiflora, Nigella truncate, or mixtures thereof. Preferably, the plant is Nigella damascene. More preferably, the extract is obtained from plant parts of Nigella damascene wherein the plant parts are seeds, leaves, roots, rhizomes, stems, or mixtures thereof. Even more preferably, the plant is Nigella damascene and the plant parts are seeds. In another embodiment, the invention provides an extract of a plant of the Genus Nigella, where the extract is obtained using a solvent selected from methanol, ethanol, and mixtures thereof water; and the plant is Nigella arvensis, Nigella ciliaris, Nigella coerulea, Nigella confuse, Nigella damascena, Nigella divaricata, Nigella gallica, Nigella hispanica, Nigella indica, Nigella integrifolia, Nigella latifolia, Nigella multifida, Nigella nigellastrum, Nigella orientalis, Nigella sativa, Nigella tenuiflora, Nigella truncate, or mixtures thereof. Preferably, the plant is Nigella damascene. More preferably, the extract is obtained from plant parts of Nigella damascene wherein the plant parts are seeds, leaves, roots, rhizomes, stems, or mixtures thereof. Even more preferably, the plant is Nigella damascene and the plant parts are seeds. Even more preferably, the plant is Nigella damascene; the plant parts are seeds,- and the solvent is ethanol and water. Even more preferably, the plant is Nigella damascene; the plant parts are seeds,- and the solvent is water.

Definitions

The term "alkyl" as used herein, means a straight or branched chain hydrocarbon containing from 1-6, preferably 1-4, and more preferably 1-3, carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl , iso-propyl, n-butyl, sec-butyl, iso-butyl, and tert- butyl .

The term "lower alcohol" as used herein, means a straight or branched alkyl as defined herein, substituted with a single -OH at any carbon in the alkyl chain. Examples of lower alcohols include, but are not limited to, methanol, ethanol, n-propanol, i-propanol, n-butanol, and t-butanol. Preferred lower alcohols are those having from 1 to 6, preferably 1-4, and more preferably 1-3, carbon atoms. Particularly preferred lower alcohols are methanol and ethanol . The term "pharmaceutically acceptable carrier" or "excipient" as used herein, refers to any inactive ingredient added to a drug to dilute it or to give it form or consistency, provide stability, and produce a drug dosage form as are familiar to those skilled in the art. Examples of carriers or excipients include, but are not limited to, binders, fillers, disintegrants, wetting agents, emulsifying and suspending agents, preservatives, lubricants, coatings, sweeteners, flavorings, or colors.

The term "dermatologically acceptable carrier" as used herein, refers to any non-toxic substance which may be used for topical application to the skin and/or hair wherein the carrier does not damage or negatively affect the aesthetics of hair or cause irritation to the underlying skin as are familiar to those skilled in the art. The term "white hair" as used herein refers to any condition affecting melanin production in the hair, resulting in decreased melanin production and therefore, decreased or no pigmentation of the hair. Hair can appear white, grey, silver, and the like, and combinations thereof, as a result of decreased melanin production. The condition may be a local or general effect on the hair of a subject. Examples of such conditions include, but are not limited to, greying hair due to aging, leukotrichia, poliosis, and canities.

Methods of preparation

Extract from the plants of the genus Nigella in the invention are prepared by known extraction methods. It is possible to use fresh plants or dried plants in the compositions of the invention, but dry powder, or an extract obtained with a solvent or pressure extract is preferable from the aspect of formulation and/or usability. For example, the extract may be obtained from seeds, leaves, roots, rhizomes, stems of the plants with water or organic solvents using well known methods in the art for extraction. Also powders are available from these extracts by removing the extraction such as by lyophilization, spray drying, or evaporation under reduced pressure. Suitable organic solvents include Ci-C₆alcohols, preferably Ci-C₄ alcohols such as methanol, ethanol, propanol and butanol ; C₂- C₄ ethers such as ethyl ether and methyl ether; and C₃-C₄ ketones such as acetone and methyl ethyl ketone. The most preferred solvent is ethanol. Each solvent may be used alone or in combination with one or more other solvents. For example, mixed solvents containing water and organic solvent, e.g., ethanol, methanol, or propanol, are useful.

Suitable extraction solvents include aqueous alcohol, and aqueous ethanol is most preferred. The ethanol content is preferably about at least 20% on a volume basis (v/v) , more preferably from 40% to 60 %, and most preferably about 50%. Thus, the most preferred extraction solvent is 50% (v/v) aqueous ethanol (50% EtOH) . The amount of the solvent used in the extraction is not critical. The specified method for extraction includes cold infusion, hot infusion and percolation etc., which are used to prepare extracts and tinctures, for example. The resulting extracts may be used without further modification, or after being condensed, diluted or purified. In addition, a single component prepared from the extract or powder through the purification of column chromatography etc. may be used in the invention. The extract of the invention may be used by itself, or in combination with other ingredients which are usually contained in cosmetics=or medicines. The content of the plants and/or its extract described above is usually from 0.0001 to 20 weight%, preferably 0.01 to 10 weight%, of the total weight in terms of dry weight of the plant (in a case of extraction, the dry weight of the solid plant used for extraction) .

The content of the plants and/or extracts described above of these external preparations is usually from 0.0001 to 20 weight%, preferably 0.01 to 10 weight%, of the total weight in terms of dry weight of the plant (in a case of extraction, the dry weight of the solid plant used for extraction) , and the said preparations can be produced by a conventional method. It may include components such as oils, surfactants, moisturizers, medical components, alcohols, preservatives, thickeners, coloring agent and flavor etc., which are commonly used in medicinal external preparations or hair cosmetics, as well as the plants and/or extracts described above.

Personal Care Formulations

The extract of the invention prepared by the methods described above may be used orally or topically, and an external preparation is convenient and preferable, which is directly applied for the scalp or hair.

Personal care compositions, especially compositions for topical application to the hair and/or skin, can be formulated into a wide variety of product types, including hair sprays, mousses, gels, lotions, creams, ointments, tonics, sprays, shampoos, conditioners, rinses, hand and body lotions, facial moisturisers, sunscreens, anti-acne preparations, topical analgesics, pomades, emulsions, foundations, oil-based cosmetics, face packs, and the like. Personal care compositions of the invention comprise a dermatologically acceptable diluent or carrier.

Personal care compositions of the invention preferably contain the melanogenesis stimulant extract in an amount of from 0.0001% to 30%, more preferably from 0.01 to 20%, even more preferably from 0.1 to 10% by weight. Compositions of the invention may, optionally, comprise a fragrance or perfume and/or one or more of the optional additional components described hereinafter, but are not limited to the following .

The carriers and additional components required to formulate such products vary with product type and can be routinely chosen by one skilled in the art. The following is a description of some of these carriers and additional components .

Extracts of the invention can comprise a dermatologically acceptable carrier, or a mixture of such carriers, which are suitable for application to the hair and/or skin. The carriers are at from about 0.5% to about 99.5%, preferably from about 5.0% to about 99.5%, more preferably from about 50.0% to about 99.0%, of the composition.

The choice of appropriate carrier will also depend on whether the product formulated is meant to be left on the surface to which it is applied (e.g., hair spray, mousse, tonic, or gel) or rinsed off after use (e.g., shampoo, conditioner, rinse) .

The carriers used herein can include a wide range of components conventionally used in hair care compositions. The carriers can contain a solvent to dissolve or disperse the particular extract being used, with water, the Ci-C₆ alcohols, lower alkyl acetate and mixtures thereof being preferred. The carriers can also contain a wide variety of additional materials such as acetone, hydrocarbons (such as isobutane, hexane, decene) , halogenated hydrocarbons (such as Freons) and volatile silicones such as cyclomethicone . When the hair care composition is a hair spray, tonic, gel, or mousse the preferred solvents include water, ethanol, volatile silicone derivatives, and mixtures thereof. The solvents used in such mixtures may be miscible or immiscible with each other. Mousses and aerosol hair sprays can also utilise any of the conventional propellants to deliver the material as a foam (in the case of a mousse) or as a fine, uniform spray (in the case of an aerosol hair spray) . Suitable propellants for use are volatile hydrocarbon propellants which can include liquefied lower hydrocarbons of 3 to 4 carbon atoms such as propane, butane and isobutane. Other suitable propellants are hydrofluorocarbons such as 1 , 2-difluoroethane (Hydrofluorocarbon 152A) supplied as Dymel 152A by DuPont . Other examples of propellants are dimethylether, N-butane, isobutane, propanes, nitrogen, carbon dioxide, nitrous oxide and atmospheric gas and mixtures thereof. A tonic or hair spray product having a low viscosity may also utilise an emulsifying agent. Examples of suitable emulsifying agents include nonionic, cationic, anionic surfactants, or mixtures thereof. If such an emulsifying agent is used, it is preferably at a level of from about 0.01% to about 7.5% by weight based on total weight of the composition. The level of propellant can be adjusted as desired but is generally from about 3% to about 30% by weight based on total weight for mousse compositions and from about 15% to about 50% by weight based on total weight for aerosol hair spray compositions.

Suitable spray containers are well known in the art and include conventional, non-aerosol pump sprays i.e., "atomisers", aerosol containers or cans having propellant, as described above, and also pump aerosol containers utilising compressed air as the propellant.

Where the hair care compositions are conditioners and rinses, the carrier can include a wide variety of conditioning materials. Where the hair care compositions are shampoos, the carrier can include, for example, surfactants, suspending agents, and thickeners. Hair styling creams or gels also typically contain a structurant or thickener, typically in an amount of from 0.01% to 10% by weight.

The carrier can be in a wide variety of forms . For example, emulsion carriers, including oil-in-water, water-in- oil, water-in-oil-in-water, and oil-in-water-in-silicone emulsions, are useful herein. These emulsions can cover a broad range of viscosities, e.g., from about 100 cps to about 200,000 cps. These emulsions can also be delivered in the form of sprays using either mechanical pump containers or pressurised aerosol containers using conventional propellants. These carriers can also be delivered in the form of a mousse. Other suitable topical carriers include anhydrous liquid solvents such as oils, alcohols, and silicones (e.g., mineral oil, ethanol, isopropanol, dimethicone, cyclomethicone, and the like) ; aqueous-based single phase liquid solvents (e.g., hydro-alcoholic solvent systems) ; and thickened versions of these anhydrous and aqueous-based single phase solvents (e.g., where the viscosity of the solvent has been increased to form a solid or semi -solid by the addition of appropriate gums, resins, waxes, polymers, salts, and the like) . Cream bases are, for example, beeswax, cetyl alcohol, stearic acid, glycerine, propylene glycol, propylene glycol monostearate, polyoxyethylene cetyl ether and the like. Lotion bases include, for example, oleyl alcohol, ethanol, propylene glycol, glycerine, lauryl ether, sorbitan monolaurate and the like.

Additional topical compositions useful in the invention can be formulated as solutions. Solutions typically include an aqueous solvent (e.g., from about 50% to about 99.99%, such as from about 90% to about 99%, by weight of a pharmaceutically acceptable aqueous solvent) .

Topical compositions useful in the subject invention may be formulated as a solution comprising an emollient. Such compositions preferably contain from about 2% to about 50% of an emollient (s) . As used herein, "emollients" refer to materials used for the prevention or relief of dryness, as well as for the protection of the skin. A wide variety of suitable emollients are known and may be used herein. See the International Cosmetic Ingredient Dictionary and Handbook, eds . Wenninger and McEwen, pp. 1656-61, 1626, and 1654-55 (The Cosmetic, Toiletry, and Fragrance Assoc., Washington, D. C, 7^th Edition, 1997) (hereinafter "ICI Handbook") contains numerous examples of suitable materials. A lotion can be made from such a solution. Lotions typically comprise from about 1% to about 20% (e.g., from about 5% to about 10%) of an emollient (s) and from about 50% to about 90% (e.g., from about 60% to about 80%) of water.

Another type of product that may be formulated from a solution is a cream. A cream typically comprises from about 5% to about 50% (e.g., from about 10% to about 20%) of an emollient (s) and from about 45% to about 85% (e.g., from about 50% to about 75%) of water.

Yet another type of product that may be formulated from a solution is an ointment. An ointment may comprise a simple base of animal or vegetable oils or semi-solid hydrocarbons. An ointment may comprise from about 2% to about 10% of an emollient (s) plus from about 0.1% to about 2% of a thickening agent (s). A more complete disclosure of thickening agents or viscosity increasing agents useful herein can be found in the ICI Handbook pp. 1693-1697.

The topical compositions useful in the invention can also be formulated as emulsions. If the carrier is an emulsion, from about 1% to about 10% (e.g., from about 2% to about 5%) of the carrier comprises an emulsifier (s) . Emulsifiers may be nonionic, anionic or cationic. Suitable emulsifiers are disclosed in the ICI Handbook, pp.1673-1686. Lotions and creams can be formulated as emulsions. Typically, such lotions comprise from 0.5% to about 5% of an emulsifier (s) . Such creams would typically comprise from about 1% to about 20% (e.g., from about 5% to about 10%) of an emollient (s) ; from about 20% to about 80% (e.g., from 30% to about 70%) of water; and from about 1% to about 10% (e.g., from about 2% to about 5%) of an emulsifier (s) .

Single emulsion skin care preparations, such as lotions and creams, of the oil-in-water type and water- in-oil type are well-known in the cosmetic art and are useful in the subject invention. Multiphase emulsion compositions, such as the water-in-oil-in-water type are also useful in the subject invention. In general, such single or multiphase emulsions contain water, emollients, and emulsifiers as essential ingredients . The topical compositions of this invention can also be formulated as a gel (e.g., an aqueous gel using a suitable gelling agent (s) ) . Suitable gelling agents for aqueous gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethyl cellulose and hydroxypropyl cellulose) . Suitable gelling agents for oils (such as mineral oil) include, but are not limited to, hydrogenated butylene/ethylene/styrene copolymer and hydrogenated ethylene/propylene/styrene copolymer. Such gels typically comprises between about 0.1% and 5%, by weight, of such gelling agents.

The personal care compositions of the invention can contain one or more humectant or moisturizing materials. A variety of these materials can be employed and each can be at a level of from about 0.1% to about 20%, more preferably from about 1% to about 10% and most preferably from about 2% to about 5%. These materials include urea,- guanidine,- glycolic acid and glycol ate salts (e.g. ammonium and quaternary alkyl ammonium); lactic acid and lactate salts (e.g. ammonium and quaternary alkyl ammonium) ; aloe vera in any of its variety of forms (e.g., aloe vera gel); polyhydroxy alcohols such as sorbitol, glycerol, hexanetriol, propylene glycol, butylene glycol, hexylene glycol and the like,- polyethylene glycols,- sugars and starches,- sugar and starch derivatives (e.g., alkoxylated glucose) ,- hyaluronic acid; lactamide monoethanolamine,- acetamide monoethanolamine,- and mixtures thereof Preferred humectants and moisturizers are glycerol, butylene glycol, hexylene glycol, and mixtures thereof.

The topical compositions of the invention can also be formulated into a solid formulation (e.g., a wax-based stick, soap bar composition, powder, or a wipe containing powder) .

Micelle formulations are also useful compositions of the subject invention. Such compositions can be prepared using single chain surfactants and lipids. Micelles typically have size from about 1 nm to about 100 nm, such as from about 10 nm to about 50 nm. The micelle preparation may then incorporated into one of the above carriers (e.g., a gel or a solution) in order to produce the micelle formulation.

The topical compositions useful in the subject invention may contain, in addition to the aforementioned components, a wide variety of additional oil-soluble materials and/or water-soluble materials conventionally used in compositions for use on hair and/or skin.

Pharmaceutical Formulations

The extracts of the invention may be administered orally, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising an extract of the inventions and a pharmaceutically acceptable carrier. One or more extracts of the invention may be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing extracts of the invention may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs . Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate,- granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques . In some cases such coatings may be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Formulations for oral use may also be presented as lozenges .

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,- dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol . Sweetening agents and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti -oxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

Pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring, and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3- butanediol . Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or diglycerides . In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of the present invention may also be administered in the form of suppositories, e.g., for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non- irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols. Compounds of the present invention may be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above- indicated conditions (about 0.5 mg to about 7 g per patient per day) . The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient. The daily dose can be administered in one to four doses per day. In the case of skin conditions, it may be preferable to apply a topical preparation of compounds of this invention to the affected area two to four times a day. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For administration to non-human animals, the composition may also be added to the animal feed or drinking water. It may be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It may also be convenient to present the composition as a premix for addition to the feed or drinking water. Preferred non-human animals include domesticated animals .

Examples

The invention will be explained in greater detail with reference to the following examples, but the scope of the invention is not limited to them.

Example 1 Preparation of extract of seeds of Nigella Damascena

Exemplary Extract-A was obtained according to the following steps:

(i) 10 g of seeds of Nigella Damascena were powdered and extracted with 100 ml of hot 50% aqueous EtOH for 2 hrs .

(ii) The extraction was repeated and the combined extracts were filtered.

(iii) The filtrate was evaporated in vacuo and the residue was freeze-dried to afford the extract (Extract-A, 17.9% yield).

Example 2

Alternative preparation of extract of seeds of Nigella

Damascena

Exemplary Extract-B was obtained in a similar manner to that described in Example 1 by extracting with methanol, followed by filteration and evaporation of the methanol in vacuo (11.8% yield). Example 3

Test for melanogenesis stimulation in cultured melanoma cells

Cell culture B16 (cultured melanoma mouse cell line) was adjusted to a concentration of 2 x 10⁴ cells/well (in a 6- well plate) /1.9 mL using a D-MEM medium (Invitrogen Corp.) containing 10% fetal bovine serum (FBS, Invitrogen Corp.)

(hereinafter "D-MEM medium"), and subjected to preliminary culture for 24h at 37⁰C in a CO₂ incubator (5% CO₂) . The

Nigella Damascena extract was dissolved in a 1:1 solvent mixture of dimethylsulfoxide (DMSO) -phosphate buffer saline

(pBS) to concentrations of 10 μg/tnl and 50 μg/ml . The 1:1 mixture of DMSO and PBS was used as the control . 4 μL of each of these solutions was diluted using 96 μL of D-MEM medium and the 100 μL of the solution thus obtained was added to the culturing plate. The final concentration of DMSO was adjusted to 0.1%. After culturing the cells for 3 more days, the melanin content inside and outside was determined as described below.

Example 4

Measurement of intra- and extracellular melanin

Intracellular: Cells collected by trypsin treatment were washed with Ca- and Mg-free PBS. 400μL of IN NaOH was added to the washed cells and the cells disrupted by keeping at 8O⁰C for 30 min. The intracellular melanin content was then determined by measuring the absorbance of this solution at 475 nm.

Extracellular: The pH of the medium was adjusted by adding an equivalent amount of 0.4M HEPES buffer (pH 6.8) /EtOH (9:1, v/v) . The supernatant of the medium obtained after centrifugation at 700 x g, 4⁰C for 10 min. was collected and the extracellular melanin content was determined by measuring the absorbance of this solution at 475 nm.

Example 5

Results of in vitro testing of Extract -A on melanin production

The results of the experiment according to Examples 3 and 4 are given in Table 1. It shows that when the cells are cultured in the presence of the extract of the seeds of Nigella damascena, the intracellular melanin content in the B16 melanoma cells and its content in the culture medium increased. This demonstrates that an aqueous ethanol extract of the seeds of Nigella damascena have a melanogenesis promoting activity.

Table 1 below shows the melanogenesis promoting effect of 50% Ethanol Extract of Nigella damascena in a B16 melanoma cell culture system:

The melanin content is given in terms of mean absorbance ± standard error. Significant differences (p < 0.01) are marked with a "*" . Example 6

Test for inhibiton of testosterone 5a-reductase activity in vitro

Preparation of crude enzyme solution (S-9) :

The liver of a 5 week-old Slc/SD male rat fasted for 24h was perfused with ice-cooled Kreps-Ringer solution. To this was added 5 times the amount of ice-cooled tris-HCl buffer (10 ttiM, pH 7.2), stirred and centrifuged at 900 x g for 10 min. The supernatant obtained was then centrifuged at 5000 x g for 10 min, and the supernatant obtained in this step was further centrifuged at 9000 x g for 10 min. The supernatant obtained here was used as the 5a-reductase-containing crude enzyme solution (S-9) . After adjusting the protein content of S-9 to 14 mg/ml , it was cryopreserved at -8O⁰C.

Inhibition of testosterone 5a-reductase :

1.0 ml of tris-HCl (10 mM, pH 7.2), 0.3 ml of testosterone

(1.1 g/ml) , 0.2 ml of a test solution in Table 1 (dissolved in 50% EtOH) and 1.0 ml of the crude enzyme solution (S-9) were mixed. 0.5 ml of the coenzyme, nicotinamide adenine dinucleotide phosphate (reduced form) -tetrasodium salt

(NADPH, 0.77 mg/ml) was added as a reaction accelerator to the mixture and the mixture was held at 37⁰C for 30 min. The reaction was quenched by adding 5 ml of dichloromethane . 0.5 ml of the internal standard (n-hexyl p-hydroxybenzoate, 0.1 mg/ml) was then added. The mixture was shaken for 10 min. and centrifuged at 900 x g for 10 min. After removing the supernatant, the dichloromethane was distilled off by topping from the remaining dichloromethane layer. The residue was dissolved in 5 ml of methanol to prepare the sample for High Performance Liquid Chromatography (HPLC) analysis. The testosterone content was determined by HPLC. The measurement conditions were, YMC-pack ODS-AM for the column, a 65/35 mixture of methanol -water for the mobile phase solvent, a flow rate of 1 ml/min, UV absorption beam of 254 nm, and a column temperature of 4O⁰C. The internal standard method was used for the measurement, and the conversion (%) and the inhibition (%) were calculated using the following formulas;

(i) Conversion (%) = [(Testosterone content of 0 min. control) - (Testosterone content after adding test solution)] x 100 /(Testosterone content of 0 min. control)

(ii) Inhibition (%) = [(Testosterone content of 0 min. control) - (Testosterone content after adding test solution)] x 100 / [(Testosterone content of 0 min. control) - (Testosterone content of 30 min. control)] (iii) Testosterone content of 0 min. control: The testosterone content after mixing the tris-HCl buffer, testosterone, test solution and the enzyme solution when dichloromethane is added to prevent the reaction, before adding the NADPH. (iv) Testosterone content of 30 min. control: The testosterone content after the reaction using 50% EtOH in place of the test solution.

Example 7 Results of tests of Extracts A & B for inhibiton of testosterone 5a-reductase activity

In vitro inhibition of teststerone 5a-reductase by the two extracts prepared in Examples 1 and 2 were assayed according to Example 6. The results of the experiment are given in Table 2 below. It shows that aqueous ethanol and aqueous methanol extracts of the seeds of Nigella Damascena have inhibitory activity on testosterone 5α-reductase . For comparison, activity of a known teststerone 5a-reductase inhibitor, ethinyl estradiol, was included.

Table 2 In vitro inhibition of testosterone 5α-reductase by Extracts A & B:

Final cone . Conversion Inhibition

Test Substance (mg/ml) (%) (%)

Control - 81.8 + 0.3 -

Extract-A 0.25 81.6 + 1.3 0.2 ± 1.6

(50% EtOH) 0.5 82.8 + 1.3 -1.3 ± 1.6

1.0 74.3 + 0.8** 9.2 ± 0.9

2.0 53.5 + 0.3** 34.5 ± 0.3

Extract-B 0.25 80.6 + 0.3 1.4 ± 0.4

(MeOH) 0.5 77.2 + 1.3** 5.6 ± 1.5

1.0 63.0 + 0.8** 23.0 ± 1.0

2.0 45.6 + 2.6** 44.2 ± 3.1

Ethinyl 1 (mM) 38.9 + 2.2** 52.4 ± 2.7 estradiol

The values are expressed as the mean absorbance ± standard deviation. ** Significant difference (p < 0.01)

Although the foregoing discussion provides details of particular aspects and features of the invention in connection with a selected one or more embodiments, numerous equivalents, alternatives and modifications to the aspects, features and embodiments of the invention will be readily apparent to a person of the ordinary skill in the relevant technical field upon reference to this disclosure.

Furthermore, while a particular feature or aspect of the invention may have been described with reference to only one of the exemplary and illustrative embodiments provided in the foregoing discussion, it should be understood that such an aspect or feature may be combined with such other aspects or features of the alternative embodiments as may desired or advantageous for any given or particular application. The foregoing disclosure should not be construed in a limiting sense, but should be interpreted as including all possible modifications and combinations of the illustrative embodiments, as well as other embodiments of the invention that may become apparent to persons skilled in the art upon reference to the foregoing description.

Claims

What is claimed is:

1. An extract of a plant of genus Nigella having melanogenesis stimulating activity prepared by a process comprising (a) contacting one or more plant parts of a plant of genus Nigella with at least one solvent to form a solvent/plant mixture,-

(b) removing solid material from the mixture of (a) ,- and (c) optionally removing the solvent.

2. The extract according to claim 1, wherein the plant is Nigella arvensis, Nigella ciliaris, Nigella coerulea, Nigella confuse, Nigella damascena, Nigella divaricata, Nigella gallica, Nigella hispanica, Nigella indica, Nigella integrifolia, Nigella latifolia, Nigella multifida, Nigella nigellastrum, Nigella orientalis, Nigella sativa, Nigella tenuiflora, Nigella truncate, or mixtures thereof .

3. The extract according to claim 2, wherein the plant is Nigella damascene.

4. The extract according to claim 2, wherein the extract is obtained from one or more plant parts which are a seed, leaf, root, rhizome, or stem.

5. The extract according to claim 4, wherein the extract obtained from seeds.

6. The extract according to claim 4, wherein the solvent is a lower alcohol, water, or mixtures thereof.

7. The extract according to claim 6, wherein the lower alcohol is methanol, ethanol, n-propanol, i-propanol, n- butanol, s-butanol, t-butanol, or mixtures thereof.

8. The extract according to claim 7, wherein the lower alcohol is methanol, ethanol, or mixtures thereof.

9. The extract according to claim 8, wherein the lower alcohol is methanol.

10. The extract according to claim 8, wherein the solvent is a mixture of ethanol and water.

11. The extract according to claim 6, wherein the solvent is water.

12. A personal care composition having melanogenesis stimulating activity comprising an extract according to Claim 9 or 10 and a dermatologically acceptable carrier.

13. A personal care composition according to claim 12, wherein the composition is in the form of a shampoo, hair tonic, conditioner, pomade, ointment, lotion, cream, skin lotion, emulsion, foundation, oil-based cosmetic, or face pack.

14. A method for the prevention or treatment of white hair in a person in need of such treatment, comprising the step of topically applying to the skin or hair the composition according to Claim 12.

15. The method according to Claim 14 wherein the white hair is the result of a condition selected from canities, aging, poliosis, and leukotrichia .

16. The method according to Claim 15 wherein the condition is leukotrichia.

17. A pharmaceutical composition having melanogenesis stimulating activity comprising the extract of Claim 9 or 10 and a pharmaceutically acceptable carrier or excipient .

18. The pharmaceutical composition according to Claim 17 wherein the composition is the form of a tablet, pill, capsule, syrup or elixir.

19. A method for the prevention or treatment of white hair comprising the step of orally administering to a patient in need of such treatment a pharmaceutical composition according to Claim 17.

20. The method according to Claim 19 wherein the white hair is the result of canities, aging, poliosis, or leukotrichia .

21. The method according to Claim 20 wherein the white hair is the result of leukotrichia.

22. The extract of a plant of the Genus Nigella which is soluble in a solvent selected from lower alcohols, water, and mixtures thereof.

23. The extract according to claim 22, wherein the plant is Nigella arvensis, Nigella ciliaris, Nigella coerulea, Nigella confuse, Nigella damascena, Nigella divaricata, Nigella gallica, Nigella hispanica, Nigella indica, Nigella integrifolia, Nigella latifolia, Nigella multifida, Nigella nigellastrum, Nigella orientalis, Nigella sativa, Nigella tenuiflora, Nigella truncate, or mixtures thereof .

24. The extract according to claim 23, wherein the plant is Nigella damascene.

25. The extract according to claim 24, wherein the extract is obtained from plant parts which are seeds, leaves, roots, rhizomes, stems, or mixtures thereof.

26. The extract according to claim 25, wherein the plant parts are seeds.

27. The extract according to claim 26, wherein the lower alcohol is methanol, ethanol, n-propanol, i-propanol, n- butanol, s-butanol, t-butanol, or mixtures thereof.

28. The extract according to claim 26, wherein the lower alcohol is methanol, ethanol, or mixtures thereof.

29. The extract according to claim 26, wherein the lower alcohol is methanol.

30. The extract according to claim 26, wherein the solvent is a mixture of ethanol and water.

1. The extract according to claim 26, wherein the solvent is water.