WO2016183203A1

WO2016183203A1 - Compositions including obacunone and methods of using the same in skin anti-aging applications

Info

Publication number: WO2016183203A1
Application number: PCT/US2016/031861
Authority: WO
Inventors: Qiang Han; Yi Zhang; Tingzhao Li; Jun Du; Bo Li
Original assignee: Access Business Group International Llc
Priority date: 2015-05-14
Filing date: 2016-05-11
Publication date: 2016-11-17
Also published as: CN106265113A; TW201705937A

Abstract

Compositions for topical use and having a protein carbonylation-inhibiting activity that include an amount of about 75-100% pure obacunone effective to reduce and/or inhibit protein carbonylation are described. Methods for inhibiting protein carbonylation in skin and for skin whitening and lightening are also described.

Description

COMPOSITIONS INCLUDING OBACUNONE AND METHODS OF USING THE SAME IN SKIN ANTI-AGING APPLICATIONS

RELATED APPLICATONS

[000 1 ] The present patent document claims priority to Chinese Patent Application No. 201 5 10244524.X, filed May 14, 201 5, which is hereby incorporated by reference.

TECHNICAL FIELD

[0002] Compositions that include obacunone and their use in the methods of inhibiting protein carbonylation in the skin, as well as methods of improving the appearance of skin or at least one sign of aging of the skin, such as skin yellowness, are described.

BACKGROUND ART

[0003] Oxidative stress is linked to the production of reactive oxygen species non- enzymatically in a reaction termed protein carbonylation. Protein carbonylation is a term used to describe a process, in which reactive aldehydes or ketones are introduced into proteins by oxidation. Protein carbonyls have been shown to be major products of protein oxidation, and may be formed via oxidative cleavage of proteins, direct oxidation of amino acid residues or covalent reaction with aldehydes derived from lipid peroxidation.

[0004] Protein carbonylation occurs because of the direct metal-catalyzed oxidation of amino acid side chains (primary protein carbonylation) or the addition of reactive aldehydes to amino acid side chains (secondary protein carbonylation). As such, reactive aldehydes of exogenous origin, such as acrolein, are well known to be derived from cigarette smoke.

However, reactive aldehydes can also be formed endogenously, for example, oxidative degradation of polyunsaturated fatty acids, which are ubiquitously found in human body, can lead to the formation of various aldehydes, such as acrolein and 4-hydroxynonenal (4-HNE).

[0005] It has been previously reported that primary protein carbonylation plays a role in the mechanism of reactive oxygen species (ROS) signaling. Specifically, primary protein carbonylation mediates cell signaling and primary protein carbonylation is reversible.

I [0006] Primary protein carbonylation involves the direct metal-catalyzed oxidation of amino acid side chains, particularly on four susceptible amino acid residues: proline, arginine, lysine, and threonine. The oxidation of the proline or arginine side chains results in the formation of glutamic semialdehyde, while lysine oxidation produces aminoadipic semialdehyde, thereby introducing carbonyl groups in the protein structure. Oxidation also converts the hydroxyl group of threonine side chains to carbonyl, forming 2-amino-3- ketobutyric acid. Secondary protein carbonylation mechanisms include the Michael addition reactions of reactive aldehydes with lysine, histidine, and cysteine residue side chains, as well as the formation of Schiff bases by reducing sugars with amino groups on lysine and arginine, resulting in an increase in advanced glycation end-products. Protein carbonyls occur in various oxidative stress and disease conditions. In addition to proteins being inactivated, carbonylated cellular proteins undergo proteasome-dependent degradation. Primary protein carbonylation is thought to play a role in labeling damaged proteins during oxidative stress in order to eliminate them from the biological system.

[0007] It has been well documented that the production of carbonyl protein is a hallmark of oxidative stress, e.g., as an extrinsic factor, such as that induced by ultraviolet radiation or an external application of oxidative chemicals, or as intrinsic factors, such as chemical attack by reactive carbonyls derived from the degradation of lipid peroxides.

[0008] The photodamaged facial skin is characterized by various unique features, such as dark spots, wrinkles, and sagging. Elderly people, particularly Asians, tend to show a yellowish skin color change with photo-aging. In Chinese, facial yellowness means not pinky or dull. Common clinical signs of photodamage or aging include a characteristic sallow or yellow appearance to the skin. It has been shown that carbonylation in the skin occurs in both, epidermis and dermis layers of the skin. Also, it often results in significant

photodamage to skin tissues and structures facing the oxidative stress, resulting in irreversible discoloration: facial yellowness, if without prevention or treatment for an extended period of time. [0009] Thus, there is a great need for anti-aging compositions and methods that can inhibit or prevent protein carbonylation to improve the appearance of skin or at least one sign of aging of the skin, such as skin yellowness, and enhance skin lightening of the facial skin.

SUMMARY OF INVENTION

[001 0] One embodiment relates to a topical composition having a protein

carbonylation-reducing or inhibiting activity, consisting essentially of: an amount of about 75- 100% pure obacunone effective to reduce or inhibit protein carbonylation; a cosmetically acceptable vehicle; and optionally, one or more cosmetic ingredients, such as alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, coloring agents, and perfumes; and/or a skin whitening agent, such as tyrosinase inhibitors, free radical scavengers, and mixtures thereof.

[001 I ] A further embodiment relates to a topical composition having protein

carbonylation-reducing or inhibiting activity, consisting of: an amount of about 75- 1 00% pure obacunone effective to reduce or inhibit protein carbonylation; a cosmetically acceptable vehicle; and optionally, one or more cosmetic ingredients, such as alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, coloring agents, and perfumes; and/or a skin whitening agent, such as tyrosinase inhibitors, free radical scavengers, and mixtures thereof.

[001 2] In the compositions, the obacunone is purified from an extract of at least one plant selected from the group consisting of Evodia rutaecarpa, Dictamni cortex, Phellodendri amurensis cortex, Citri sarcodactylis fructus, Aurantii frusctus immaturus, and Citri fructus. The Evodia extract is standardized with respect to content of at least one of evodiamine, rutaecarpine, and limonine. In the compositions, the obacunone is about 99.99% pure obacunone active. The composition is a preparation selected from cream, ointment, foam, lotion, plaster, tablets, granules, or emulsion. In the composition, the amount effective to reduce or inhibit protein carbonylation is about 10- 1000 mg. In the composition, the obacunone forms approximately I -99% w/w of the composition. Alternatively, the obacunone forms approximately I -75% w/w of the composition. Alternatively, the obacunone forms approximately 50-70% w/w of the composition.

[001 3] Another embodiment relates to a topical composition comprising a cosmetically acceptable vehicle and an amount of at least 90% pure obacunone, wherein the obacunone forms approximately I -99% w/w of the composition.

[001 ] A further embodiment relates to a method of improving the appearance of skin or at least one sign of aging of the skin, the method comprising topically applying to the skin a composition in a cosmetically effective amount sufficient to improve the appearance of the skin or the at least one sign of aging of skin, wherein the composition comprises an amount of about 75- 1 00% pure obacunone, a cosmetically acceptable vehicle, and, optionally, one or more cosmetic ingredients, such as alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, coloring agents, and perfumes; and/or a skin whitening agent, such as tyrosinase inhibitors, free radical scavengers, and mixtures thereof. In the method, the composition is in a product form, such as an aerosol, a cream, a emulsion, a solid, a liquid, a dispersion, a foam, a gel, a lotion, a mousse, an ointment, a powder, a patch, a pomade, a solution, a pump a spray, a stick, a towelette, and combinations thereof.

[001 5] Another embodiment relates to a method of whitening skin comprising topically applying to the skin the composition described herein, wherein the composition is applied in an amount and for a period of time sufficient to visibly whiten the skin.

[001 6] Yet another embodiment relates to a method of lightening skin comprising topically applying to the skin the composition described herein, wherein the composition is applied in an amount and for a period of time sufficient to visibly lighten the skin.

[001 7] A further embodiment relates to the use of a topical composition described herein in manufacturing a cosmetic agent having a protein carbonylation-reducing or inhibiting activity. [001 8] Another embodiment relates to a method for preventing, improving or reducing at least one sign of aging in the skin comprising applying to the skin the composition described herein, wherein the composition is applied in an amount and for a period of time sufficient to prevent, improve or reduce the at least one sign of aging in the skin.

BRIEF DESCRIPTION OF DRAWINGS

[001 9] Figure I is a graph showing low cell toxicity(CTG) of obacunone in HepG2 cells.

[0020] Figure 2 is a graph showing a dose-dependent inhibition on protein carbonylation by obacunone in HepG2 cells.

DETAILED DESCRIPTION

[002 I ] It is to be understood that this invention is not limited to the particular compositions, methodology, or protocols described herein. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the claims.

[0022] The present invention is based on the surprising discovery that obacunone has positive effects as a skin whitening agent without toxicity (FIG. I ) and provides an inhibitory effect on protein carbonylation (FIG. 2). As shown in FIG. 2, protein carbonylation is inhibited with an increase in the dose of obacunone.

[0023] Accordingly, compositions that include purified obacunone in sufficient amounts to achieve a reduction or inhibition in protein carbonylation, and their use in the methods of skin anti-aging, whitening or lightening are described. [0024] Definitions

[0025] The term "composition" refers to a product that treats, improves, promotes, increases, manages, controls, maintains, optimizes, modifies, reduces, inhibits, or prevents a particular condition associated with a natural state, biological process or disease or disorder. The term composition includes, but is not limited to, pharmaceutical (i.e., drug), over-the-counter (OTC), cosmetics, food, food ingredient or dietary supplement compositions that include an effective amount of a purified compound, an extract, a natural complex, at least one component thereof, or a mixture thereof. Exemplary compositions include cream, cosmetic lotion, pack or powder, or as an emulsion, lotion, liniment foam, tablets, plasters, granules, or ointment.

[0026] As used herein, the term "extract" refers to a solid, viscid, or liquid substance or preparation that includes an active ingredient(s) of a substance of plant, such as Evodia rutaecarpa in a concentrated form. The term "extract" is intended to include not only a crude extract produced from a plant by use of a solvent selected from among water, lower alcohols of I to 4 carbon atoms, such as methanol, ethanol, butanol, etc., ethylene, acetone, hexane, ether, chloroform, ethylacetate, butylacetate, dichloromethane, N,N- dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1 ,3-butylene glycol, propylene glycol and a combination thereof, but also a fraction of the crude extract in such a solvent. So long as it assures the extraction and preservation of the active ingredient(s), any extraction method may be employed.

[0027] As used herein, the term "purified" in connection with obacunone refers obacunone prepared by physically separating obacunone from remaining components of a plant extract. Various purification methods are known and could be used to obtain pure obacunone. For example, methods such as fractionation or affinity purification may be used. As a result of a purification process, obacunone may be at least about 1 0% pure; alternatively, at least about 20% pure; alternatively, at least about 30% pure; alternatively, at least about 40% pure; alternatively, at least about 50% pure; alternatively, at least about 60% pure; alternatively, at least about 70% pure; alternatively, at least about 80% pure; alternatively, at least about 90% pure; alternatively, at least about 95% pure; alternatively, at least about 99% pure; but preferably about 75%- 1 00% pure.

[0028] As used herein, the term "effective amount" or "therapeutically effective amount" of a composition, extract, extract mixture, component of the extract, and/or active agent or ingredient refers to an amount effective at dosages and for periods of time sufficient to achieve a desired result. For example, the "effective amount" or

"therapeutically effective amount" refers to that amount of a composition, extract, extract mixture, component of the extract, and/or active agent or ingredient of this invention which, when administered to a subject (e.g., mammal, such as a human), is sufficient to effect treatment, such as reducing or inhibiting protein carbonylation, skin anti-aging, enhancing or improving skin whitening and/or lightening, decreasing or lightening dark spots and the like in a subject. The amount of a composition, extract, extract mixture, component of the extract, and/or active agent or ingredient of this disclosure that constitutes an "effective amount" or "therapeutically effective treatment" will vary depending on the active agent or the compound, the condition being treated and its severity, the manner of administration, the duration of treatment, or the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0029] The terms "preventing, improving or reducing at least one sign of aging" and "preventing, improving or reducing a sign of aging" are used interchangeably herein to designate preventing, arresting, reversing, ameliorating, diminishing, and/or reducing a sign of aging. Representative signs of aging include, but are not limited to, yellowing skin, lines, fine lines, wrinkles, crow's feet, dark eye circles, blemishes, age spots, stretch marks, or combinations thereof.

[0030] "Improving the appearance of the skin" and "improving the aesthetic appearance of the skin" are used interchangeably herein to designate an aesthetic improvement in the appearance of skin. Representative improvements may include, but are not limited to, favorable characteristics and/or properties related skin color, tone, texture, radiance, lightness, luster, brightness, clarity, thickness, elasticity, resiliency, moisturization, smoothness, contour, firmness, tautness, suppleness, softness, sensitivity, pore size, or combinations thereof. These terms may also be used to designate an improvement in an adverse skin condition. Representative adverse conditions affecting by, resulting in or resulting from such an adverse skin condition include, but are not limited to, skin aging, psoriasis, eczema, seborrhea, dermatitis, sunburn, estrogen imbalance, hyperpigmentation, hypopigmentation, discoloration, yellowing, freckles, skin atrophy, skin breakout, skin fragility, dryness, tactile roughness, chapping, sagginess, thinning, hyperplasia, fibrosis, enlarged pores, cellulite formation, bruising, acne formation, apoptosis, cellular

differentiation, cellular de-differentiation, prevention of tumor induction or tumor progression, viral infections, fungal infections, bacterial infections, spider veins

(telangectasia), hirsutism, rosacea, pruritis, calluses, warts, corns, or combinations thereof.

[003 I ] The term "pharmaceutically acceptable" means those drugs, medicaments, extracts or inert ingredients, which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, incompatibility, instability, irritation, and the like, commensurate with a reasonable benefit/risk ratio.

[0032] The terms "administer," "administered," "administers" and "administering" are defined as providing a composition to a subject via a route known in the art, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration. In preferred embodiments, topical routes of administering a composition are suitable.

[0033] As used herein, the term "subject" or "individual" includes mammals to which a composition may be administered. Non-limiting examples of mammals include humans, non-human primates, rodents (including transgenic and non-transgenic mice) or the like. In some embodiments, the subject is a mammal, and in some embodiments, the subject is human. [0034] Obacunone

[0035] Obacunone (CAS: 75 1 -03- 1 ) is a chemical compound (i.e., plant ingredient), having molecular weight of 454.52, that can be extracted from Evodia rutaecarpa BENTHAM (Rutaceae). For example, the fruit of Rutaceae can be extracted with EtOH to yield an extract of Evodia ("Evodia extract"), which can be used a source of obacunone compound that is useful in unique compositions of the present invention. The term "obacunone" refers to the plant ingredient as well as plant extract or a natural complex.

[0036] An exemplary structure of obacunone (C₂₆H₅₀Ov) is shown below:

[0037] Obacunone may be commercially obtained from various sources including ChemStrong Scientific Co., Ltd or Chengdu Biopurify Phytochemicals Ltd. In addition, obacunone may be purified from extracts of plants including plants, such as Evodia rutaecarpa, dried root bark of Dictamnus dasycarpus Turcz. (Rutaceae), dried bark of

Phellodendron amurense Rupr. (Fam. Rutaceae), dried bark of Phellodendron chinense Schneid. (Fam. Rutaceae), Citrus sp. Fortunella margarita, Casimiroa edulis, Citri sarcodactylis fructus, Aurantii frusctus immaturus, and Citri fructus. In certain aspects, the flower, capitulum or the fruit, or the leaves of a plant is used as the source of the extract. When extracted, the extract can be standardized to an active marker compound. For example, the cortex

Phellodendri amurensis extract may be standardized with respect to a content of at least one of berberine, palmatine, limonin, and obacunone.

[0038] Various purification and extraction techniques are discussed more fully below or known in the art. [0039] Exemplary Extraction Processes

[0040] In one example, an extract and/or active compound useful in the unique compositions of the present invention might be obtained using an organic solvent extraction technique.

[004 I ] In another example, solvent sequential fractionation using H₂0, H₂0-EtOH, H₂0-MeOH, or H₂0-Acetone etc. as a solvent may be used to obtain an extract and/or active compound useful in the unique compositions of the present invention.

[0042] Total hydro-ethanolic extraction techniques may also be used to obtain an extract and/or active compound useful in the unique compositions of the present invention. Generally, this is referred to as a lump-sum extraction. The extract generated in this process will contain a broad variety of phytochemicals present in the extracted material including fat and water solubles. Following collection of the extract solution, the solvent will be evaporated, resulting in the extract.

[0043] Total ethanol extraction may also be used in the present invention. This technique uses ethanol, rather than hydro-ethanol, as the solvent. This extraction technique generates an extract that may include fat soluble and/or lipophilic compounds in addition to water soluble compounds.

[0044] Another example of an extraction technique that may be used to obtain an extract and/or active compound useful in the present invention is supercritical fluid carbon dioxide extraction (SFE). In this extraction procedure the material to be extracted is not exposed to any organic solvents. Rather, the extraction solvent is carbon dioxide, with or without a modifier, in supercritical conditions (>3 1 .3° C. and >73.8 bar). Those of skill in the art will appreciate that temperature and pressure conditions can be varied to obtain the best yield of extract. This technique generates an extract of fat soluble and/or lipophilic compounds, similar to the total hexane and ethyl acetate extraction technique described above.

[0045] Those of skill in the art will appreciate that there are many other extraction and purification processes, both known in the art and described in various patents and publications that can be used to obtain the extracts and/or active compound to be used in practicing the present invention. For example, the extraction procedures described in the following references, which are incorporated herein by reference, could be used in practicing the present invention: Murga et al., "Extraction of natural complex phenols and tannins from grape seeds by using supercritical mixtures of carbon dioxide and alcohol." J. Agric Food Chem. 2000 August:48(8):3408- 12; Hong et al., "Microwave-assisted extraction of phenolic compounds from grape seed." Nat Prod Lett. 2001 ; 1 5(3): 197-204; Ashraf- Khorassani et al., "Sequential fractionation of grape seeds into oils, polyphenols, and procyanidins via a single system employing C0₂-based fluids." J. Agric Food Chem., 2004 May 5; 52(9):2440-4.

[0046] According to one example of the present invention, obacunone extract may be prepared by extracting Evodia rutaecarpa fruit with 70% EtOH. The final extract has a moisture content of > 5.0% to produce a plant extract with native extraction ratio of botanical: finished product 5-7: 1 .

[0047] The obacunone extract can then be assayed and standardized with respect to active marker content, such as content of evodiamine, rutaecarpine, berberine, palmatine, and limonine.

[0048] The obacunone active can then be purified by solvent-water partition, such as dichloromethane-water, ethyl acetate-water, n-butanol-water system.

[0049] Alternatively, the obacunone active can be purified by different chromatography methods, such as macroporous resin, silicone resin, MCI resins, gel etc.

[0050] Also, the obacunone active can be purified by preparative chromatography, semi- preparative chromatography, such as preparative HPLC, semi-preparative HPLC, etc.

[005 I ] Also, the obacunone active can be purified by high-speed countercurrent chromatography.

I I [0052] Compositions

[0053] Obacunone may be formulated into a composition. Specifically, certain embodiments relate to a composition having a protein carbonylation reducing or inhibiting activity that includes an amount of obacunone effective to reduce or inhibit protein carbonylation. Certain other embodiments relate to a composition for skin whitening and/or skin lightening that includes an amount of obacunone effective to whiten or lighten the skin.

[0054] Certain other embodiments relate to a skin anti-aging composition that includes an amount of obacunone effective to prevent or treat skin aging.

[0055] Certain embodiments relate to compositions for reducing or inhibiting protein carbonylation that include an amount of obacunone effective to reduce or inhibit protein carbonylation, where obacunone is at least about 75% pure. Alternatively, in the composition, obacunone is at least about 80% pure; alternatively, at least about 85% pure; alternatively, at least about 90% pure; alternatively, at least about 95% pure; alternatively, at least about 96% pure; alternatively, at least about 97% pure; alternatively, at least about 98% pure; alternatively, at least about 99% pure; or alternatively, in the composition, obacunone is about 99.99% pure.

[0056] The obacunone forms approximately at least 50% w/w of the composition, more preferably, at least approximately 55% w/w composition, at least approximately 60% w/w composition, at least approximately 65% w/w composition, at least approximately 70% w/w composition, at least approximately 75% w/w composition, at least approximately 80% w/w composition, at least approximately 85% w/w composition, at least approximately 90% w/w composition, at least approximately 95% w/w composition, at least approximately 99% w/w composition. Alternatively, obacunone forms approximately I -99% w/w of the

composition; alternatively, obacunone forms approximately 1 -90% w/w of the composition; alternatively, obacunone forms approximately 1 -75% w/w of the composition; alternatively, obacunone forms approximately 50-70% w/w of the composition. [0057] In the composition, obacunone may be present in an amount from about 0.001 % to about 1 0%.

[0058] Of course, one of skill in the art will appreciate that the amount of obacunone incorporated into the composition may depend on the standardized content of a marker. For example, for obacunone prepared from Evodia extract a marker may be evodiamine, rutaecarpine, and/or limonine. Thus, if obacunone is standardized to about 40% of a marker compound, the above described amounts of the obacunone will be reduced by one-half. For example, the obacunone extract could be standardized to about 0.5%, 1 .0%, 1 .5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 1 0%, 1 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% marker compound content.

[0059] The compositions may have a pH between about 6.0 to about 8.0, or

alternatively the composition may have a pH that is substantially neutral. In certain embodiments, the compositions in which the obacunone is used have a generally neutral pH.

[0060] Certain embodiments relate to a composition for increasing skin whitening or lightening. The composition includes an effective amount of obacunone to increase skin whitening or lightening. The composition may include a cosmetically acceptable vehicle.

[006 I ] Certain further embodiments relate to a method of increasing skin whitening or lightening that includes administering to the subject a composition that includes an obacunone extract, wherein the composition reduces or inhibits protein carbonylation in the skin, thereby resulting in increased skin whitening or lightening.

[0062] Compositions of the present invention may be formulated in an acceptable carrier, vehicle or excipient and may be prepared, packaged, and labeled for cosmetic or pharmaceutical use in skin whitening or lightening in a subject. In certain embodiments, the obacunone forms approximately 50-70% w/w of the composition, and the carrier, vehicle or excipient forms approximately 50-30% w/w of the composition. [0063] In general, the compositions include a cosmetically acceptable vehicle. Examples of cosmetically acceptable vehicles suitable for all embodiments of the present invention include, but are not limited to, water, glycerin, various alcohols such as ethanol, propyl alcohol, vegetable oil, mineral oil, silicone oils, fatty ethers, fatty esters, fatty alcohols, glycols, polyglycols or any combinations thereof. Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, Gennaro, Mack Publishing Co., Easton Pa., 1990, which is incorporated herein by reference.

[0064] In certain embodiments, the compositions may further include one or more whitening agents to enhance the whitening effect of the composition. Exemplary whitening agents are believed to include all the known whitening agents and those that may be developed in the future. Preferably, the whitening agents are selected from tyrosinase inhibitors, free radical scavengers, and mixtures thereof. Examples of suitable tyrosinase inhibitors include, but are not limited to, kojic acid and its derivatives, arbutin and its derivatives, licorice extract and its derivatives, ascorbic acid and its derivatives, and hydroquinone and its derivatives. Examples of suitable free radical scavengers include, but are not limited to, licorice extract and its derivatives, vitamin E and its derivatives, vitamin A and its derivatives, vitamin C and its derivatives, Rosemary extract and its derivatives and superoxide dismutase. Preferred whitening agents are kojic acid, derivatives of kojic acid, arbutin, derivatives of arbutin, bearberry extract, lemon extract, cucumber extract, vitamin C and its derivatives, extracts of acerola cherry and fermentates of acerola cherry.

[0065] Non-exclusive examples of the vitamin C derivatives are, for instance, alkyl esters of L-ascorbic acid such as L-ascorbyl palmitrate, L-ascorbyl isopalmitate, L-ascorbyl dipalmitate, L-ascorbyl isostearate, L-ascorbyl distearate, L-ascorbyl diisostearate, L- ascorbyl myristate, L-ascorbyl isomyristate, L-ascorbyl 2-ethylhexanoate, L-ascorbyl di-2- ethylhexanoate, L-ascorbyl oleate and L-ascorbyl dioleate; phosphates of L-ascorbic acid such as L-ascorbyl-2-phosphate and L-ascorbyl-3-phosphate; sulfates of L-ascorbic acid such as L-ascorbyl-2-sulfate and L-acorbyl-3-sulfate; their salts with alkaline earth metals such as calcium and magnesium. They can be used alone or in a mixture of two or more. [0066] When the obacunone is mixed with any of the known whitening agents, the ratio of the obacunone to the known whitening agent is from about 1 : 100 to about 100: 1 , preferably from about 1 :50 to about 50: 1 , more preferably from about 1 : 10 to about 1 0: 1 . Most preferably, the ratio of obacunone to a known whitening agent is from about 1 :5 to about 5: 1 .

[0067] The compositions of the present invention may be prepared in various forms. For example, they may be in the form of a cosmetic preparation such as cream, cosmetic lotion, pack or powder, or as an emulsion, lotion, liniment foam, tablets, plasters, granules, aerosol spray, gel, mousse, pomade, or ointment. In each formulation, various known conventional cosmetic ingredients may be incorporated. For example, cosmetic ingredients such as alcohols, fats and oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, coloring agents, and perfumes may be included. Certain further embodiments relate to compositions that may be formulated in any convenient form suitable for topical application to the skin. Suitable emulsions include oil- in-water, water-in-oil, and water-in-silicone emulsions.

[0068] In certain embodiments there is provided an improved skin whitening

composition of the type containing skin whitening agents wherein the improvement comprises adding obacunone. The composition may include a cosmetically acceptable vehicle.

[0069] The compositions of the invention may be administered topically.

[0070] The compositions may be administered as needed, daily, several times per day or in any suitable regimen such that the desired outcome is achieved. The compositions may be administered on a continuous basis or intermittent basis.

[007 1 ] Methods

[0072] The compositions described herein may be used for various cosmetic and/or pharmaceutical applications, including inhibiting or reducing protein carbonylation, and/or increasing or improving skin whitening, lightening and brightening. [0073] Certain embodiments relate to a method of whitening skin that includes topically applying to the skin any composition described above and any combinations thereof.

[0074] Certain specific embodiments relate to a method of whitening skin that includes topically applying to the skin a composition that includes an amount of obacunone effective to reduce or inhibit protein carbonylation in the skin. The composition is applied in an amount and for a period of time sufficient to visibly whiten the skin.

[0075] In a topical skin whitening cosmetic composition containing one or more cosmetic ingredients, such as alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, coloring agents, and perfumes, the improvement comprising an effective amount of obacunone to whiten the skin.

[0076] In the method, the frequency of topical applications will depend on several factors, including the desired level of suppression of protein carbonylation and the content of obacunone in the composition. A suitable exemplary regimen includes application to the skin twice daily, with one application in the morning and one in the evening.

[0077] A further embodiment relates to a method of improving the appearance of skin or at least one sign of aging of the skin, the method comprising topically applying to the skin a composition in a cosmetically effective amount sufficient to improve the appearance of the skin or the at least one sign of aging of the skin, wherein the composition comprises an amount of about 75- 100% pure obacunone; a cosmetically acceptable vehicle; and optionally, one or more cosmetic ingredients, such as alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, coloring agents, and perfumes; and/or a skin whitening agent selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof. In the method, the composition is in a product form selected from an aerosol, a cream, a emulsion, a solid, a liquid, a dispersion, a foam, a gel, a lotion, a mousse, an ointment, a powder, a patch, a pomade, a solution, a pump a spray, a stick, a towelette, and

combinations thereof. [0078] Certain embodiments relate to the use of a topical composition described herein in manufacturing a cosmetic agent having a protein carbonylation-reducing or inhibiting activity.

[0079] Certain further embodiments relate to a method for preventing, improving or reducing at least one sign of aging in the skin comprising applying to the skin any

composition described above, wherein the composition is applied in an amount and for a period of time sufficient to prevent, improve or reduce the at least one sign of aging in the skin. Representative signs of aging include, but are not limited to, yellowing skin, lines, fine lines, wrinkles, crow's feet, dark eye circles, blemishes, age spots, stretch marks, or combinations thereof.

EXAMPLES

[0080] Example I : Cell Toxi city/Safety Assessment of Obacunone by CellTiter-Glo® (CTG) Luminescent Cell Viability Assay in HepG2 Cells

[008 I ] To determine cell safety of obacunone, the following assay was performed.

[0082] The CellTiter-Glo® (CTG) Luminescent Cell Viability Assay (from Promega) was performed according to the manufacturer's instructions to determine the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. A 99.99% pure obacunone and hydroquinone (used as a reference) at same gradient concentrations ( 100, 33.3, I I . I , 3.7, 1 .23, 0.41 , 0.14, 0.05, 0.02 (ug/mL)) was used in the assay. The assay was performed using HepG2 cells.

[0083] According to cell-based safety assessment by CTG, obacunone showed no cell toxicity (see Table I ). [0084] Table I .

[0085] The results are illustrated in FIG. I .

[0086] Example 2: Obacunone Demonstrated Dose Dependent Reduction of Protein Carbonylation in HepG2 cells

[0087] To determine if obacunone affected protein carbonylation, the following cell- based assay using OxiSelect™ Protein Carbonyl ELISA Kit (Cell Biology #STA-3 10) was performed.

[0088] HepG2 cells (ATCC) were used for the testing. Cells were plated at 100,000 cells per well and the assay plates (96 well cell plates from Costa) were incubated for 24 hours at 37 degrees C, 5% C0₂ under humidified conditions.

[0089] Obacunone at 0 ug/mL, 33 ug/mL, 1 00 ug/mL, and 300 ug/mL was tested.

Vitamin C, a well-known protein carbonylation inhibitor, at 0 ug/mL, 33 ug/mL, 1 00 ug/mL, and 300 ug/mL, was used as positive inhibitor control in the assay.

[0090] The reference and test compound solution (200x) was prepared according to the platemap. Obacunone was solubilized in MEM and added to HepG2 cells that were seeded 24 hours prior to the treatment as noted above. The final concentration of the test compound was I x (Table 2). The plate was then incubated at 37 °C, 5% C0₂ under humidified conditions for 72 hours. [009 1 ] Table 2.

[0092] On day 3, the medium was removed from the cells. The wells were washed twice with HBSS. 100 μΜ 8-OH Quinoline and 100 μΜ FeCI₃ was added to the wells and the plates were incubated for I hour. The cells were then washed with HBSS once. Fresh medium was added to each well following the washing step. The plates were then incubated at 37°C, 5% C0₂ under humidified conditions for 72 hours.

[0093] On day 4, the medium was removed from the cells and cells were washed twice with HBSS. 100 μΙ_ of HBSS (containing the cocktail (commercially available protease inhibitor cocktail tablets (Roche#04 693 124 001 )) was added to each well. The plates were then placed in -80°C for 30 minutes, thawed and the freezing/thawing steps were repeated at least 3 times. The cell lysis was then mixed and 25 μΙ_ of cell lysate was tested to determine protein concentration. Table 3 shows the preparation for the BCA Kit to determine protein concentration on day 4.

[0094] Table 3.

[0095] The protein concentration was then adjusted. 100 μΙ_ of the sample (induced BSA dilution was used) was then added to the 96-well Protein Binding Plate and incubated at 4°C overnight. [0096] On day 5, ELISA assay was performed. The wells were washed 3 times with 250 μΙ_ I XPBS per well. The excess of the wash solution was then removed from the wells. 100 μΙ_ of the DNPH Working Solution was added to each well (Table 4) and the plates were incubated for 45 minutes at room temperature in the dark.

[0097] Table 4.

[0098] The wells were then washed 5 times with 250 μΙ_ I XPBS/Ethanol ( I : I ) with incubation on an orbital shaker for 5 minutes (Table 5).

[0099] Table 5.

[001 00] The wells were then washed 2 times with 250 μΙ_ I XPBS. 200 μΙ_ of Blocking Solution was added per well (Table 6) and the pates were incubated for 1 -2 hours at room temperature on an orbital shaker.

[001 01 ] Table 6.

[001 02] The wells were then washed 3 times with 250 μΙ_ of I X Wash Buffer (Table 7) with thorough aspiration between each wash. [001 03] Table 7.

[001 04] Next, 100 μΙ_ of anti-DNP antibody was added per well (Table 8) and the plates were incubated for I hour at room temperature on an orbital shaker.

[001 05] Table 8.

[001 06] Next the strip wells were washed 3 times ( l *wash buffer). 100 μΙ_ of the diluted HRP conjugated secondary antibody was added to all wells and the plates were incubated for I hour at room temperature on an orbital shaker. See Table 9. [001 07] Table 9.

[001 08] Next, the strip wells were washed 5 times ( l *wash buffer). The Substrate

Solution was warmed to room temperature and 1 00 μΙ_ of the Substrate Solution was added to each well. The strip wells were then incubated for 1 5 minutes. The enzyme reaction was stopped by adding 1 00 μΙ_ of Stop Solution to each well. Results were read immediately as the color fades over time.

[001 09] The results are shown in Table 1 0 and in FIG. 2.

[001 10] Table 1 0.

[001 I I ] The average protein carbonylation inhibition rates of obacunone were 1 .6%, 23.6%, 37.75%, 39.1 5%; the carbonylation inhibition rates of Vitamin C were -3.05%, 3 1 .05%, 55.35%, and 62.65%. [001 12] This suggests that obacunone has excellent efficacy to inhibit protein carbonylation as compared to the known positive control, Vitamin C in HepG2 cells. This further suggests the possibility of use of products containing obacunone for skin lightening and anti-aging.

[001 13] It is therefore intended that the foregoing detailed description be regarded as illustrative rather than limiting, and that it be understood that it is the following claims, including all equivalents, that are intended to define the spirit and scope of this invention.

Claims

1 . A topical composition having a protein carbonylation-reducing or inhibiting activity, consisting essentially of: a) an amount of about 75- 1 00% pure obacunone effective to reduce or inhibit protein carbonylation; b) a cosmetically acceptable vehicle; and c) optionally, i) one or more cosmetic ingredients selected from the group consisting of alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, coloring agents, and perfumes; and/or ii) a skin whitening agent selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.

2. A topical composition having a protein carbonylation-reducing or inhibiting activity, consisting of: a) an amount of at least about 90% pure obacunone effective to reduce or inhibit protein carbonylation; b) a cosmetically acceptable vehicle; and c) optionally, i) one or more cosmetic ingredients selected from the group consisting of alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, coloring agents, and perfumes; and/or ii) a skin whitening agent selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.

3. The composition of any of claims I -2, wherein the obacunone is purified from an extract of at least one plant selected from the group consisting of Evodia rutaecarpa, Dictamni cortex, Phellodendri amurensis cortex, Citri sarcodactylis fructus, Aurantii frusctus immaturus, and Citri fructus.

4. The composition of claim 3, wherein the Evodia extract is standardized with respect to content of at least one of evodiamine, rutaecarpine, and limonine.

5. The composition of any of claims I -4, wherein the obacunone is about 99.99% pure active obacunone.

6. The composition of any of claims I -5, the composition being a preparation selected from the group consisting of cream, ointment, foam, lotion, plaster, tablets, granules, or emulsion.

7. The composition of any of claims I -2, wherein the ratio of the obacunone to the skin whitening agent is from about 1 00: 1 to about 1 : 1 00.

8. The composition of any of claims I -7, wherein the amount effective to reduce or inhibit protein carbonylation is about 1 0- 1 000 mg.

9. The composition of any of claims I -8, wherein the obacunone forms approximately I -99% w/w of the composition.

1 0. The composition of any of claims I -8, wherein the obacunone forms approximately I -75% w/w of the composition.

I I . The composition of any of claims I -8, wherein the obacunone forms approximately 50-70% w/w of the composition.

12. A topical composition comprising a cosmetically acceptable vehicle and an amount of at least 90% pure obacunone, wherein the obacunone forms approximately 1 -99% w/w of the composition.

1 3. A method of improving the appearance of skin or at least one sign of aging of the skin, the method comprising topically applying to the skin a composition in a cosmetically effective amount sufficient to improve the appearance of the skin or the at least one sign of aging of the skin, wherein the composition comprises a. an amount of about 75- 100% pure obacunone; b. a cosmetically acceptable vehicle; and c. optionally, i) one or more cosmetic ingredients selected from the group consisting of alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, coloring agents, and perfumes; and/or ii) a skin whitening agent selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.

14. The method of claim I 3, wherein the composition is in a product form selected from the group consisting of an aerosol, a cream, a emulsion, a solid, a liquid, a dispersion, a foam, a gel, a lotion, a mousse, an ointment, a powder, a patch, a pomade, a solution, a pump a spray, a stick, a towelette, and combinations thereof.

1 5. A method of whitening skin comprising topically applying to the skin the composition of any of claims 1 - 12, wherein the composition is applied in an amount and for a period of time sufficient to visibly whiten the skin.

16. A method of lightening skin comprising topically applying to the skin the composition of any of claims 1 - 12, wherein the composition is applied in an amount and for a period of time sufficient to visibly lighten the skin.

17. A use of the topical composition claimed in any of claims I - 1 2 in manufacturing a cosmetic agent having a protein carbonylation-reducing or inhibiting activity.

18. A method for preventing, improving or reducing at least one sign of aging of the skin comprising applying to the skin the composition of any of claims 1 - 12, wherein the composition is applied in an amount and for a period of time sufficient to prevent, improve or reduce the at least one sign of aging of the skin.