WO2016110276A1

WO2016110276A1 - Topical composition containing obakunone and skin-whitening method using same

Info

Publication number: WO2016110276A1
Application number: PCT/CN2016/070544
Authority: WO
Inventors: 韩强; 张翌; 李廷钊; 杜军; 李波
Original assignee: 捷通国际有限公司
Priority date: 2015-01-09
Filing date: 2016-01-11
Publication date: 2016-07-14
Also published as: TW201630589A; CN105816342A

Abstract

A topical composition capable of suppressing melanin synthesis and tyrosinase activity comprises obakunone having a purity of approximately 75-100%, a cosmetically acceptable carrier, and one or more optional cosmetic ingredients and/or skin-whitening agents. In the composition, obakunone is present in an amount that can effectively reduce melanin synthesis and/or suppress tyrosinase activity. The composition is used for skin whitening.

Description

Composition comprising corkone and its use in skin whitening applications

Technical field

Compositions comprising cedarone are described, and their use in methods of inhibiting tyrosinase activity and melanin synthesis in the skin and methods of skin whitening.

Background technique

Skin color depends mainly on the amount of melanin in the skin. Melanin is a brownish black pigment present in the skin. Since the pigment is dark, the lower the amount of melanin, the lighter the skin tone, and the higher the amount, the deeper the skin tone. Melanin is formed by oxidizing amino acid tyrosine to dihydroxyphenylalanine in melanocytes. This reaction is catalyzed by tyrosinase.

Excessive skin pigmentation may be caused by hormonal abnormalities in humans unrelated to ultraviolet light, genetic diseases, etc.; or excessive melanin production and uneven distribution of melanin due to excessive ultraviolet radiation. The proper amount of melanin in the skin has a positive effect of maintaining skin health and absorbing ultraviolet rays. However, excessive melanin causes negative effects such as darkening of the skin and uneven skin tone. Therefore, many scientists have studied the inhibition of melanin production.

Due to this critical role of tyrosinase in melanin formation, efforts to develop effective skin lightening compositions have focused on agents that inhibit the function and activity of tyrosinase. For example, various compositions have been proposed which include various known tyrosinase inhibitors, especially such as hydroquinone, vitamin C and its derivatives, kojic acid, arbutin, glutathione. , cysteine and mulberry extract.

One problem with synthetic skin lightening agents such as hydroquinone or kojic acid is that they may cause skin irritation or acute dermatitis. Therefore, there is an increasing need to introduce natural sources into the composition in order to address some of the undesirable aspects of the synthetic product.

Summary of the invention

One embodiment relates to a topical composition having activity to reduce melanin synthesis, consisting essentially of: about 75-100% pure phellodone in an amount effective to reduce melanin synthesis; An acceptable carrier; and optionally, one or more cosmetic ingredients selected from the group consisting of alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers , emulsifier, stabilizer, coloring And perfumes; and/or skin lightening agents selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.

Another embodiment is directed to a topical composition having activity to reduce melanin synthesis, consisting of about 75-100% pure pareonone in an amount effective to reduce melanin synthesis; a cosmetically acceptable carrier; Optionally, 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, colorants, and perfumes; And/or a skin lightening agent selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.

In the composition, the cedarone is purified from at least one extract of a plant selected from the group consisting of Evodiarutaecarpia, Dictamnicortex, Phellodendriamurensis cortex, musk Citrisarcodactylis fructus, Aurantiifrusctus immaturus and Citrifructus. The Evodia extract is normalized to the content of at least one of evodiamine, rutaecarpine, and limonine. In this composition, the cedarone is about 99.99% pure paphosone active. The composition is a formulation selected from the group consisting of creams, ointments, foams, lotions, plasters, tablets, granules or emulsions. In the composition, the weight ratio of the corkone to the skin lightening agent is from about 100:1 to about 1:100. In the composition, the effective amount to reduce melanin synthesis is from about 10 to 1000 mg. In the composition, the ketone ketone constitutes from about 1 to 99% by weight of the composition. Alternatively, the corkone constitutes from about 1 to about 75 wt% of the composition. Alternatively, the cedarone constitutes from about 50% to about 70% by weight of the composition.

A further embodiment is directed to a topical composition for inhibiting the activity of tyrosinase consisting essentially of at least about 90% pure phellodone in an amount effective to inhibit tyrosinase activity; a cosmetically acceptable carrier; and optionally, one or more cosmetic ingredients selected from the group consisting of alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stable Agents, colorants and fragrances; and/or skin lightening agents selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.

Another embodiment is directed to a topical composition for inhibiting the activity of tyrosinase consisting of at least about 90% pure phellodone in an amount effective to inhibit tyrosinase activity; An acceptable carrier; and optionally, 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, Colorants and fragrances; and/or skin lightening agents, selected from tyrosinase inhibitors, free radical scavengers And a mixture of them.

In the composition, the cedarone is purified from an extract of at least one of the following plants selected from the group consisting of the genus Ruthenium, the white peony, the yarrow, the bergamot, the citron, and the citron. The extract of Evodia rutaecarpa is normalized to the content of at least one of evodiamine, rutaecarpine, and citrate. In this composition, the cedarone is about 99.99% pure paphosone active. The composition is a formulation selected from the group consisting of creams, ointments, foams, lotions, plasters, tablets, granules or emulsions. In the composition, the weight ratio of the corkone to the skin lightening agent is from about 100:1 to about 1:100. In the composition, an effective amount for inhibiting the activity of tyrosinase is from about 10 to 1000 mg. In the composition, the ketone ketone constitutes from about 1 to 99% by weight of the composition. Alternatively, the corkone constitutes from about 1 to about 75 wt% of the composition. Alternatively, the cedarone constitutes from about 50% to about 70% by weight of the composition.

A further embodiment is directed to a topical composition comprising a cosmetically acceptable carrier and about 75-100% pure phbodone, wherein the cedarone comprises from about 1 to 99% by weight of the composition.

Another embodiment is directed to a method of whitening skin comprising topically applying a composition described herein to the skin, wherein the composition is applied in an amount and for a duration sufficient to significantly whiten the skin.

In still another embodiment, the topical skin whitening cosmetic composition comprises one or more selected from the group consisting of alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers. The cosmetic ingredients in stabilizers, colorants and perfumes are modified to consist of an effective amount of about 75-100% pure cedarone for whitening skin.

A further embodiment relates to the use of a topical composition as described herein in the manufacture of a cosmetic agent having activity to reduce melanin synthesis.

A further embodiment relates to the use of a topical composition as described herein in the manufacture of a cosmetic agent having enzyme inhibitory activity of tyrosinase.

DRAWINGS

Figure 1 depicts a graph showing the cytotoxicity-CTG of phellodone in B16F10 cells.

Figure 2 depicts a graph showing inhibition of tyrosinase enzymatic activity by corkone in B16F10 cells.

Figure 3 depicts a graph showing inhibition of melanin synthesis by corkone in B16F10 cells.

detailed description

It is to be understood that the invention is not limited to the particular compositions, methods or protocols described herein. In addition, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. It is also understood that the terminology used herein is for the purpose of describing the particular embodiments, and is not intended to limit the scope of the invention.

The present invention is based on the surprising discovery that corkone has a positive effect as a skin lightening agent and is non-toxic (Fig. 1) and provides an inhibitory effect on melanin production and tyrosinase activity. As shown in Fig. 2, the activity of tyrosinase was inhibited as the dose of cedarone was increased; as shown in Fig. 3, melanin production was gradually decreased or suppressed as the dose of phellodone was increased.

Thus, compositions comprising purified corkone (in an amount sufficient to achieve a reduction in melanin production and/or a decrease in tyrosinase activity), and their use in skin whitening or whitening methods are described.

definition

The term "composition" refers to a product that treats, ameliorates, promotes, enhances, manages, controls, maintains, optimizes, improves, reduces, inhibits, or prevents a particular state associated with a natural state, biological process, or disease or condition. The term composition includes, but is not limited to, a drug (ie, a drug) comprising an effective amount of a purified compound, an extract, at least one component thereof, or a mixture thereof, an over-the-counter (OTC), a cosmetic, a food, a food ingredient. Or a dietary supplement composition. Exemplary compositions include creams, cosmetic lotions, packs or powders, or as emulsions, lotions, tincture foams, tablets, plasters, granules or ointments.

The term "extract" as used herein refers to a solid, semi-fluid or liquid substance or formulation comprising the active ingredient(s) of a plant material such as Juss. in concentrated form. The term "extract" is intended to include not only the use of a solvent (selected from water, a lower alcohol of 1 to 4 carbon atoms such as methanol, ethanol, butanol, etc., ethylene, acetone, hexane, ether, chloroform, acetic acid B). Ester, butyl acetate, dichloromethane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butanediol, propylene glycol and combinations thereof) from plants The crude extract is prepared, and also includes a portion of the crude extract in the solvent. Any extraction method can be utilized as long as the extraction method ensures the extraction and storage of the active ingredient(s).

As used herein, the term "pure (i.e., purity by weight)" associated with corkone refers to a cedarone prepared by physically separating the flavonoids from the remaining plant extract components. Various Purification methods are known and can be used to obtain pure phellodone. For example, the method used can be as fractional or affinity purification. As a result of the purification process, the cedarone may be at least about 10% pure (ie, at least about 10% by weight pure); or, at least about 20% pure; or, at least about 30% pure; or, at least about 40% pure; or, At least about 50% pure; or, at least about 60% pure; or, at least about 70% pure; or, at least about 80% pure; or, at least about 90% pure; or, at least about 95% pure; or, at least about 99% pure; and preferably about 75% to 100% pure.

The term "effective amount" or "therapeutically effective amount" of the composition, extract, extract mixture, extract component and/or active agent or ingredient as used herein means sufficient to achieve the desired dosage and duration. The effective amount of results. For example, "effective amount" or "therapeutically effective amount" means: the amount of the composition, extract, extract mixture, extract component, and/or active agent or ingredient of the present invention, when administered to a subject (eg, a mammal, such as a human), sufficient to achieve treatment in a subject such as inhibiting melanin synthesis (ie, melanin production), inhibiting tyrosinase activity, skin whitening and/or whitening, reducing or reducing Dark spots and so on. The amount of the composition, extract, extract mixture, components of the extract, and/or active agent or ingredient of the present disclosure that constitute an "effective amount" or "therapeutically effective treatment" varies according to the following factors: The active agent or compound, the condition to be treated and its severity, the mode of administration, the duration of treatment or the age of the subject to be treated, although it is also conventional to consider his own knowledge and knowledge by those skilled in the art. Open to determine.

The term "pharmaceutically acceptable" means that the drug, agent, extract or inert ingredient is suitable for contact with tissues of humans and lower animals without excessive toxicity, incompatibility, instability. , irritating, etc., which is commensurate with a reasonable benefit/risk ratio.

The terms "administer", "administered", "administers" and "administering" are defined to provide a composition to a subject by routes known in the art. Administration routes include, but are not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal or intraperitoneal routes. In a preferred embodiment, topical route administration of the composition is suitable.

The term "subject" or "individual" as used herein includes a mammal to which a composition can 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 a human.

Corkone

The fruit of Evodiarutaecarpa BENTHAM (Russaceae) can be used EtOH is extracted to obtain an extract of Evodia rutae ("Wuhan extract") which can be used as a source of a phbodone compound which is useful in the unique composition of the present invention.

An exemplary structure of corkone (C ₂₆ H ₃₀ O ₇ ) is shown below:

Commercially, corkone is available from a variety of sources including ChemStrong Scientific Co., Ltd or Chengdu Biopurify Phytochemicals Ltd. In addition, cedarone can be purified from plant extracts, including plants such as: dried root bark of Dictamnusdasycarpus Turcz., Phellodendron amurense Rupr. (Family: Rutaceae) Dry bark of the bark, Phellodendronchinense Schneid., citrus species, Fortunella margarita, Casimiroaedulis, Citrisarcodactylis fructus, Aurantiifrusctusimmaturus ) and Citrifructus. In some aspects, the flowers, flower heads, or fruits or leaves of the plant are used as a source of extract. When extracted, the extract can be normalized to the active labeling compound. For example, the extract of Cortex phellodendriamurensis can be normalized to the content of at least one of berberine, palmatine, limonin and corkone.

Various purification and extraction techniques are discussed more fully below or are known in the art.

Exemplary extraction process

In one example, extracts and/or active compounds useful in the unique compositions of the present invention can be obtained by using organic solvent extraction techniques.

In another example, solvent sequential fractionation using H ₂ O, H ₂ O-EtOH, H ₂ O-MeOH or H ₂ O-acetone, etc. as a solvent can be used to obtain a unique composition in the present invention. Useful extracts and/or active compounds.

The aqueous hydro-ethanolic extraction technique can also be used to obtain extracts and/or active compounds that are useful in the unique compositions of the present invention. Usually this is called total extraction. The extract produced in this process will contain a variety of plantings present in the extracted material. Learn substances, including fats and water solubles. After collecting the extract solution, the solvent was evaporated to obtain an extract.

Total ethanol extraction can also be used in the present invention. This technique uses ethanol instead of aqueous ethanol as a solvent. The extract produced by the extraction technique may include, in addition to the water-soluble compound, a fat-soluble and/or lipophilic compound.

Another example of an extraction technique that can be used to obtain extracts and/or active compounds for use in the present invention is supercritical fluid carbon dioxide extraction (SFE). The material to be extracted during this extraction is not exposed to any organic solvent. More precisely, the extraction solvent is carbon dioxide in supercritical conditions (>31.3 ° C. and >73.8 bar), with or without modifiers. Those skilled in the art will recognize that temperature and pressure conditions can be varied to provide the best yield of the extract. This technique produces extracts of fat-soluble and/or lipophilic compounds similar to the total extraction techniques of hexane and ethyl acetate described above.

Those skilled in the art will recognize that there are many other methods of extraction and purification, both of which are known in the art and described in various patents and publications, for obtaining the practice of the present invention. Extract and / or active compound. For example, the extraction process described in the following references (which are incorporated herein by reference) can be used in the practice of 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. August 2000: 48(8): 3408-12; Hong et al., “Microwave-assisted extraction of phenolic compounds from grape seed.” Nat Prod Lett. 15(3): 197-204; Ashraf-Khorassani et al., "Sequential fractionation of grape seeds into oils, polyphenols, and procyanidins via a single system employing CO ₂ -based fluids." J. Agric Food Chem., 2004 5 Month 5; 52 (9): 2440-4.

According to one embodiment of the invention, the corkone extract can be prepared by extracting the fruit of Juss. with 70% EtOH. The final extract has a moisture content of > 5.0% so that the plant extract produced has a botanical: the crude extraction rate of the finished product is 5-7:1.

The ketone extract can then be analyzed and normalized to the active label content (eg, evodiamine, rutaecarpine, berberine, palmatine, and citrate).

The corkone active can then be purified by solvent-water separation such as dichloromethane-water, ethyl acetate-water, n-butanol-water system.

Alternatively, the corkone active substance can be passed through different chromatography methods such as macroporous resin, silicone tree Purification with lipids, MCI resins, gels, and the like.

Alternatively, the corkone active substance can be purified by preparative chromatography, semi-preparative chromatography such as preparative HPLC, semi-preparative HPLC, and the like.

Alternatively, the corkone active material can be purified by high speed countercurrent chromatography.

combination

The ketone can be formulated into a composition. In particular, certain embodiments relate to a composition having melanin synthesis-inhibiting activity, which comprises an amount of corkone that is effective to reduce melanin synthesis. Certain other embodiments are directed to compositions for inhibiting the enzymatic activity of tyrosinase, which comprise an amount of corkone that is effective to inhibit the enzymatic activity of tyrosinase. Certain other embodiments are directed to compositions for skin whitening and/or skin whitening comprising an amount of cedarone effective to whiten or whiten the skin.

Certain compositions relate to compositions for reducing melanin synthesis and/or inhibiting the enzymatic activity of tyrosinase, which comprise an amount of corkone that is effective to reduce melanin synthesis and/or inhibit tyrosinase enzymatic activity, wherein The ketone is at least about 75% pure. Alternatively, in the composition, the ketone is at least about 80% pure; or, at least about 85% pure; or, at least about 90% pure; or, at least about 95% pure; or, at least about 96% pure; or, At least about 97% pure; or, at least about 98% pure; or, at least about 99% pure; or, in the composition, the ketone is about 99.99% pure.

The corkone constitutes at least about 50% by weight of the composition, more preferably at least about 55% by weight of the composition, at least about 60% by weight of the composition, at least about 65% by weight of the composition, at least about 70% by weight of the composition, at least About 75 wt% of the composition, at least about 80 wt% of the composition, at least about 85 wt% of the composition, at least about 90 wt% of the composition, at least about 95 wt% of the composition, at least about 99 wt% of the composition. Alternatively, the ketone ketone constitutes from about 1 to 99% by weight of the composition; alternatively, the cedar ketone constitutes from about 1 to 90% by weight of the composition; or, the ketone ketone constitutes from about 1 to 75% by weight of the composition Or, the ketone ketone constitutes from about 50 to 70% by weight of the composition.

In the composition, the ketone can be present in an amount from about 0.001% to about 10%.

Of course, those skilled in the art will recognize that the amount of cedarone introduced into the composition may depend on the normalized level of the label. For example, for the cedarone prepared from the extract of Evodia rutae, the marker may be evodiamine, rutaecarpine and/or citrate. Thus, if the cedarone is normalized to about 40% of the labeled compound, the above amount of cedarone will be reduced by a factor of two. For example, the ketone extract can 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%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90% or 95% of the labeled compound content.

The pH of the composition may range from about 6.0 to about 8.0, or the pH of the composition may be substantially neutral. In certain embodiments, the composition using cedarone has a substantially neutral pH.

Certain embodiments are directed to compositions for increasing skin whitening or whitening. The composition includes an effective amount of cedarone to reduce melanin production and/or inhibit tyrosinase enzymatic activity. The composition can include a cosmetically acceptable carrier.

Certain additional embodiments are directed to methods of increasing skin whitening or whitening comprising administering to a subject a composition comprising a cedarone extract, wherein the composition reduces or reduces melanin production, pigmentation, or both Both.

The compositions of the invention may be administered topically.

The compositions of the present invention may be formulated in acceptable carriers, vehicles or excipients and may be prepared, packaged, and labeled for cosmetic or pharmaceutical use in the skin whitening or whitening of the subject. In certain embodiments, the paclitaxel comprises from about 50% to about 70% by weight of the composition, and the carrier, vehicle or excipient constitutes from about 50% to about 30% by weight of the composition.

Typically, the compositions include a cosmetically acceptable carrier. Examples of cosmetically acceptable carriers suitable for use in all embodiments of the invention include, but are not limited to, water, glycerin, various alcohols such as ethanol, propanol, vegetable oils, mineral oils, silicone oils, fatty ethers, fatty esters, fatty alcohols , diol, polyglycol or any combination 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.

In certain embodiments, the composition may further comprise one or more whitening agents to enhance the whitening effect of the composition. Exemplary whitening agents are considered to include: all known whitening agents and whitening agents that may be developed in the future. Preferably, the whitening agent is selected from the group consisting of 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 (Rosemary) extract and its derivatives as well as superoxide dismutase. Preferred whitening agents are selected from the group consisting of kojic acid, derivatives of kojic acid, arbutin, and arbutin Derivatives, bearberry extract, lemon extract, cucumber extract, vitamin C and its derivatives, extract of acerola cherry and ferment of acerola cherry.

Non-exclusive examples of vitamin C derivatives are, for example, alkyl esters of L-ascorbic acid such as L-ascorbyl palmitate, L-ascorbyl isopalmitate, L-ascorbyl dipalmitate, L-ascorbic acid Isoisostearate, L-ascorbyl distearate, L-ascorbyl diisostearate, L-ascorbyl myristate, L-ascorbyl isomalt, L-ascorbic acid 2-ethylhexanoate, L-ascorbyldi-2-ethylhexanoate, L-ascorbyl oleate and L-ascorbyl dioleate; phosphate of L-ascorbic acid such as L-ascorbic acid a base-2-phosphate and L-ascorbyl-3-phosphate; a sulfate of L-ascorbic acid such as L-ascorbyl-2-sulfate and L-ascorbyl-3-sulfate; they are mixed with an alkaline earth metal such as calcium And magnesium salts. They may be used singly or in combination of two or more.

When the ketone is mixed with any known whitening agent, the ratio of the ketone 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 about 1:10. It is about 10:1. Most preferably, the ratio of corkone to known brighteners is from about 1:5 to about 5:1.

The compositions of the present invention can be prepared in a variety of forms. For example, they may be in the form of a cosmetic preparation such as a cream, lotion, pack or powder, or as an emulsion, lotion, tincture foam, tablet, plaster, granule, aerosol spray, gel , mousse, pomade or ointment. In each of the formulations, various known conventional cosmetic ingredients can be introduced. For example, cosmetic ingredients such as alcohols, fats and oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, colorants and perfumes may be included. The compositions of certain further embodiments may be formulated in any convenient form suitable for topical application to the skin. Suitable emulsions include oil-in-water, water-in-oil, and silicone water-in-water emulsions.

Provided in certain embodiments is an improved skin whitening composition of this type comprising a skin lightening agent, wherein the improvement comprises the addition of cedarone. The composition can include a cosmetically acceptable carrier.

The composition can be administered as needed, daily, several times daily or any suitable regimen to achieve the desired result. The composition can be administered on a continuous or batch basis.

method

The compositions described herein can be used in a variety of cosmetic and/or pharmaceutical applications, including inhibiting or reducing melanin production, inhibiting or reducing tyrosinase enzymatic activity and/or skin whitening, whitening, and whitening.

Certain embodiments are directed to a method of whitening skin comprising topically applying to any combination of any of the compositions described above, and any combination thereof.

Certain specific embodiments are directed to a method of whitening skin comprising topically applying to a skin a composition comprising a cedarone in an amount effective to reduce melanin synthesis and/or inhibit tyrosinase enzymatic activity. The composition is applied in an amount and for a duration sufficient to significantly whiten the skin.

The topical skin whitening cosmetic composition comprises one or more cosmetic ingredients selected from the group consisting of alcohols, fats, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, and stabilizers. Agents, colorants and fragrances, the improvement includes an effective amount of cedarone to whiten the skin.

In this method, the frequency of local application will depend on several factors including the desired level of inhibition of melanin production and/or the desired level of inhibition of the tyrosinase enzyme activity and the amount of corkone in the composition. One suitable exemplary solution includes applying to the skin twice daily, once in the morning, and once in the evening.

Example

Example 1: Passing in B16F10 cells

Cytotoxicity of obacunone (CTG) Luminescent Cell Viability test (Luminescent Cell Viability Assay) carried out / safety evaluation

In order to determine the cell safety of phellodone, the following experiment was conducted.

The

(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 the quantification of ATP present as an indicator of metabolically active cells. 99.99% pure cedarone (100.00 μg/mL, 33.333 μg/mL, 11.111 μg/mL, 3.704 μg/mL, 1.235 μg/mL, 0.412 μg/mL, 0.137 μg/mL, 0.046) was used in this experiment. Gg/mL, 0.015 μg/mL) and hydroquinone (used as a reference, at the same gradient concentration). This experiment was performed using B16F10 cells.

According to the cell-based safety assessment using CTG, cedarone did not exhibit cytotoxicity (see Table 1).

Table 1:

The result is shown in Fig. 1.

Example 2: An agent for enzymatic activity against tyrosinase by using cedarone in B16F10 cells Quantitative inhibition

In order to determine whether or not phellodone affects the enzymatic activity of tyrosinase, the following experiment was conducted.

On day 1, B16F10 cells were plated at a density of approximately 18,000 cells/ml into a 24-well assay plate. The assay plates were incubated at 37 ° C, 5% CO ₂ , under humid conditions for 24 hours.

On the second day, the reference substance (arbutin) and the test compound (corkone) were added (the compound on the assay plate was 5 μl (final concentration: 1 ×)) onto the plate and the plate was at 37. Incubate for 2 days at °C, 5% CO ₂ under humid conditions.

On day 5, the plates were imaged. The inverted plate was shaken on a medium waste receptacle and sucked with a clean paper towel. 80 μl of 0.05 mM PBS (0.1% Triton-X) was added and the plate was placed in a refrigerator at -80 ° C for 4 hours. The plate was then thawed at room temperature and the washing and freezing steps were repeated twice. Samples were then transferred to a 96-well V bottom plate and spun at 3000 RPM for 10 minutes. 60 μl of the supernatant sample was transferred to a UV plate and 140 μl of fresh 0.1% L-DOPA was added to each well. The plates were incubated for 1 hour at 37 °C. Absorbance values were read at 475 nm after incubation. 5 μl of the supernatant sample was transferred to another UV plate to determine the protein concentration using BCA.

The percent inhibition of tyrosinase activity is calculated as follows:

Inhibition rate%=[(A-B)/A]*100

Where A is the difference in absorbance of the control sample (between samples with and without tyrosinase), and B is the absorbance of the test sample (between samples with and without tyrosinase) Poor (Diwakar G, Rana J, Scholten J D. Inhibition of melanin production by a combination of Siberian larch and pomegranate fruit extracts. Fitoterapia, 2012; 83: 989-95).

The largest signal was obtained from cells treated with L-DOPA; the bottom signal was derived from cells not treated with L-DOPA.

B16F10 cells treated with cedarone showed increased inhibition of tyrosinase enzyme activity (in which the inhibition rate was 90.5% at 100 μg/mL) compared to the positive control arbutin. When it was at the same concentration as arbutin, it had a much stronger inhibition rate (Table 2 and Table 3).

The results are shown in Table 2 and Table 3 below.

Table 2. Corkone

Table 3. Arbutin

As shown in Figure 2, administration of cedarone resulted in a dose-dependent inhibition of the enzymatic activity of tyrosinase by cedarone.

Example 3: Corkone shows a dose-dependent decrease in melanin production

In order to determine whether or not corkone affects melanin production, the following experiment was conducted.

100 ug/mL of cedarone was tested. Arbutin (a well-known tyrosinase inhibitor) was used as a positive control inhibitor in the experiment.

The phbodone was dissolved in DMSO and added to B16F10 cells, which were seeded at a density of 18,000 cells/mL for 24 hours in a 24-well plate prior to treatment. The final concentration of the test compound was 1 x. The cytoplasmic pH of B16F10 cells in this experiment was approximately 5-7. Each dose of corkone was added to the cells in triplicate. The plates were then incubated at 37 ° C, 5% CO ₂ , under humid conditions for 72 hours.

On day 5, the medium was removed from the cells, 150 μl of 1 M NaOH was added to each well of the assay plate and the plates were incubated at 80 ° C for 30 minutes. After the incubation, transfer 140 μl of the solution Moved to the UV panel and tested with a 400 nm signal. 5 μl of the solution was transferred to a UV plate and 200 μl of BCA reagent was added to each well. The plates were incubated for 20 minutes at 37 °C. Absorbance values were read at 562 nm after incubation.

The maximum signal was obtained under the action of DMSO; the minimum signal was obtained in 200 ug/mL arbutin.

The inhibitory activity of the samples was reported as a percentage reduction in melanin production relative to 100% untreated control (UT).

As a result, it was found that the melanin inhibition rate in the B16F10 cultured cells after incubation with the cedarone (100 mg/mL) for 72 hours was 77.8% as compared with the arbutin positive control (see Tables 4 and 5). This indicates that cedarone has excellent efficacy for interrupting melanin synthesis in B16F10 cells.

Table 4. Corkone

Table 5. Arbutin

Figure 3 plots the results. It can be seen that the use of cedarone causes a dose-dependent decrease in melanin production.

The present invention is to be understood as being limited by the scope of the invention

Claims

A topical composition having activity to reduce melanin synthesis, which consists essentially of:

a) about 75-100% pure cedarone, in an amount effective to reduce melanin synthesis;

b) a cosmetically acceptable carrier; and

c) Optional:

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, colorants, and perfumes; /or

Ii) a skin lightening agent selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.
A topical composition having activity to reduce melanin synthesis, which consists of:

a) at least about 90% pure cedarone, in an amount effective to reduce melanin synthesis;

b) a cosmetically acceptable carrier; and

c) Optional:

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, colorants, and perfumes; /or

Ii) a skin lightening agent selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.
A composition according to any one of claims 1 to 2, wherein said cedarone is purified from an extract of at least one of the following plants selected from the group consisting of Evodiarutaecarpia, Dictamnicortex, Guan Phellodendriamurensis cortex, Citrisardactylis fructus, Aurantiifrusctus immaturus and Citrifructus.
The composition of claim 3 wherein said extract of Evodia rutae is normalized to the amount of at least one of evodiamine, rutaecarpine and citrate.
The composition of any one of claims 1 to 4 wherein said cedarone is about 99.99% pure phellodone active.
A composition according to any one of claims 1 to 5 which is a preparation selected from the group consisting of creams, ointments, foams, lotions, plasters, tablets, granules or emulsions.
The composition of any of claims 1-2, wherein the weight ratio of the phbodone to the skin lightening agent is from about 100:1 to about 1:100.
The composition of any of claims 1-7, wherein the effective amount to reduce melanin synthesis is from about 10 to 1000 mg.
The composition of any of claims 1-8 wherein said cedarone comprises from about 1 to 99% by weight of said composition.
The composition of any of claims 1-8 wherein said cedarone comprises from about 1% to about 75% by weight of said composition.
The composition of any of claims 1-8 wherein said cedarone comprises from about 50% to about 70% by weight of said composition.
A topical composition for inhibiting the activity of tyrosinase consisting essentially of:

a) about 75-100% pure cedarone, in an amount effective to inhibit tyrosinase activity;

b) a cosmetically acceptable carrier; and

c) Optional:

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, colorants, and perfumes; /or

Ii) a skin lightening agent selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.
A topical composition for inhibiting the activity of tyrosinase, which consists of:

a) at least about 90% pure ketone, in an amount effective to inhibit tyrosinase activity;

b) a cosmetically acceptable carrier; and

c) Optional:

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, colorants, and perfumes; /or

Ii) a skin lightening agent selected from the group consisting of tyrosinase inhibitors, free radical scavengers, and mixtures thereof.
The composition according to any one of claims 12 to 13, wherein said cedarone is purified from an extract of at least one of the following plants selected from the group consisting of Rutaceae, Chalk, Guanhuang, Rutaceae , citrus and fragrant plant citron.
The composition of claim 14 wherein said cedarone is normalized to the amount of at least one of evodiamine, evodiamine, and citrine.
The composition of any of claims 12-15 wherein said cedarone is about 99.99% pure phellodone active.
A composition according to any one of claims 12-16, which is a preparation selected from the group consisting of creams, ointments, foams, lotions, plasters, tablets, granules or emulsions.
The composition of any of claims 12-13, wherein the weight ratio of the phbodone to the skin lightening agent is from about 100:1 to about 1:100.
The composition according to any one of claims 12 to 18, wherein the amount effective to inhibit the activity of tyrosinase is about 10 to 1000 mg.
The composition of any of claims 12-19 wherein said cedarone comprises from about 1 to 99% by weight of said composition.
The composition of any of claims 12-19 wherein said cedarone comprises from about 1% to about 75% by weight of said composition.
The composition of any of claims 12-19 wherein said cedarone comprises from about 50% to about 70% by weight of said composition.
A topical composition comprising a cosmetically acceptable carrier and at least 90% pure cedarone, wherein said cedarone comprises from about 1% to about 99% by weight of said composition.
A method of whitening the skin comprising topically applying the composition of any of claims 1-23 to the skin, wherein the composition is applied in an amount and for a duration sufficient to significantly whiten the skin.
Use of a topical composition according to any one of claims 1 to 23 for the manufacture of a cosmetic agent having activity to reduce melanin synthesis.
Use of the topical composition according to any one of claims 1 to 23 for the manufacture of a cosmetic agent having an enzyme inhibitory activity of tyrosinase.