EA043647B1 - METHOD OF DARKENING THE SKIN - Google Patents

Description

Field of technology to which the invention relates

The invention relates to a composition and method for darkening human skin.

Prerequisites for creating the invention

Most people are concerned about some aspect of their skin. For example, consumers with age spots or freckles often want dark spots to be less pronounced. Some other people may be concerned about darkening of the skin caused by exposure to sunlight or, alternatively, may wish to lighten their natural skin color. These consumer desires are leading to the development of products that reduce or delay the production of melanin in melanocytes (ie, reduce melanogenesis).

On the other hand, there are people who want a darker skin tone or a tanned look.

Melanocytes transfer melanin to keratinocytes in their vicinity, where melanin serves to protect cellular DNA from UV damage due to its ability to absorb UV radiation. When the skin is exposed to UV light, melanin synthesis is enhanced and melanin transfer to keratinocytes is also enhanced. This results in a visible darkening of the skin color, which is known as tanning. Tanning due to overexposure to UV radiation is a known phenomenon. However, it is also known that overexposure to UV radiation leads to accelerated aging, which can lead to an increased incidence of skin cancer.

Melanin is a black pigment present in our hair and skin. It is synthesized by melanosomes from tyrosine. Melanosomes are organelles found in melanocytes, a type of cell present at the dermal-epidermal junction. Tyrosine is acted upon by the enzyme tyrosinase, which is a key step in melanogenesis.

In melanosomes, melanin is synthesized from monomers and transferred to neighboring cells called keratinocytes. Keratinocytes divide and differentiate and thus transfer the melanosome to the surface of the skin. The tone or shade of our skin color depends on the number and size of melanocytes, melanin content and the rate of formation and migration/transfer of melanosomes into keratinocytes.

Cases of melanoma in Caucasians are largely the result of a lack of melanin in their skin. UV easily penetrates the skin and causes cellular changes and leads to skin cancer. To avoid this, people use various tanning products such as chemicals or regulate their UV exposure. Such chemicals can be harsh and may act by depositing on the skin and then changing to a melanin-like color. They have the disadvantage of their tendency to wash off when in contact with water. In fact, some of these chemicals turn yellow rather than brown, and some of them cause unpleasant sensations. Regulated UV exposure can also be problematic because it is difficult to calculate the appropriate dose, which varies from individual to individual.

Thus, there is a need for a solution that can naturally increase the amount of melanin in the skin. The present inventors have solved this problem by discovering molecules that induce melanin naturally, i.e. through stimulating the melanin synthesis mechanism to synthesize more melanin. This additionally synthesized melanin can protect the skin by absorbing UV radiation.

There are some active substances that are known and used to affect skin pigmentation even without exposure to UV rays or sunlight. Such active substances are known as sunless tanning products. One such sunless tanning agent that is widely used in practice is dihydroxyacetone. However, DHA has some negative associations in the consumer's mind, and thus there is a need to develop new and potentially safer active ingredients that are still highly effective. In this regard, the inventors of the present invention have conducted extensive experiments with many active substances currently used in skin care products for various purposes and have discovered very specific benzoic acid derivatives that act as skin darkening agents.

Huang H.-N. et al., Inhibition of the activity of mushroom tyrosinase by alkylbenzoic acids. Food Chem., 94 (2006), pp. 1-6. In this article, some benzoic acid derivatives are mentioned as fungal tyrosinase inhibitors for skin lightening, beneficial effects against browning of fruits, vegetables and other frozen foods. This is in direct contrast to what was found in the present invention, where a more obvious methodology was used involving the use of human primary melanocytes, and where it was concluded that certain very specific (C ₇ -C ₁₂ )alkyl and alkyloxybenzoic acids exhibit a darkening effect skin.

WO 9617589 (Kao) discloses a method for promoting collagen synthesis and smoothing or removing wrinkles, which includes administering an effective amount of a benzoic acid derivative or a salt thereof, and using this compound in agents for promoting collagen synthesis and smoothing or removing wrinkles. This compound stimulates collagen synthesis in human dermal fibroblasts and therefore smoothes or removes wrinkles caused by aging and/or

- 1 043647 photoaging. There is no indication in this patent publication that these active substances can cause darkening of the skin.

Thus, the subject of the present invention is a composition for darkening the skin, the activity of which is ensured by the use of widely used and safe active substances.

The essence of the invention

The present invention relates to a method for darkening skin, comprising the step of applying a composition comprising (i) a compound of formula 1 o

where R1 represents R2 or OR2;

where R2 represents a C7-C ₁₂ alkyl group; and (ii) a cosmetically acceptable carrier.

Preferably, the compound of formula 1 is selected from 4-heptyloxybenzoic acid, 4-heptyloxybenzoic acid, 4-octyloxybenzoic acid, 4-nonyloxybenzoic acid, 4-undecyloxybenzoic acid or 4-decyloxybenzoic acid.

The composition includes 0.01-10% of the compound of formula 1 by weight of the composition, which induces cellular synthesis of melanin in the range from 110 to 150% of that synthesis in the control sample.

The cosmetically acceptable carrier is selected from an anhydrous base, gel, lotion, cream or emulsion.

This method is not therapeutic in nature.

In another aspect, the use of a composition as described above for darkening skin is disclosed.

Detailed Description of the Invention

These and other aspects, features and advantages will become apparent to those skilled in the art upon reading the following detailed description and the accompanying claims. For the avoidance of doubt, any feature of one aspect of the present invention may also be used in any other aspect of the invention. The word including is intended to mean containing, but not necessarily consisting of or composed of. In other words, the steps or options listed are not necessarily exhaustive. It is noted that the examples set forth in the description below are intended to illustrate the invention and are not intended to limit the invention to these examples per se. Likewise, all percentages are percentages w/w unless otherwise noted. Except in the Working Examples and Comparative Examples, or where expressly stated otherwise, all numbers in this specification indicating quantities of material or reaction conditions, physical properties of materials and/or applications thereof are to be understood as modified by the word about.

Numerical ranges expressed in the format x to y are understood to include x and y. When several preferred ranges are given for a particular characteristic in the format x to y, it should be understood that all ranges in which different cutoff values are combined are also assumed.

As used herein, the term including includes the terms consisting essentially of and consisting of. When the term including is used, the steps or options listed are not necessarily exhaustive. Unless specifically stated otherwise, numerical ranges expressed in the format x to y are understood to include x and y. When specifying any range of values or quantities, any upper limit value or quantity may be combined with any lower limit value or quantity. Except in examples and comparative experiments, or where expressly stated otherwise, all numbers herein are to be understood as modified by the word approximately. All percentages and ratios contained herein are by weight unless otherwise noted. When used herein, singular nouns mean at least one or one or more unless otherwise stated. Various features of the present invention mentioned in the individual sections above are applicable, where appropriate, to other sections with appropriate modifications. As a consequence, features described in one section can be suitably combined with features described in another section. Any section headings are added for convenience only and are not intended to limit disclosure in any way.

As used herein, the skin darkening composition comprises

- 2 043647 position for external application to any keratin substrate of mammals, especially humans. Such a composition may be of the leave-in or rinse-off type. Leave-in composition means that the composition is applied to the desired surface of the skin and left there for a period of time (say, one minute to 24 hours), after which it can be wiped off or washed off with water, typically during normal washing. A wash-off/rinse-off composition means a composition that is applied to the desired skin surface for a shorter period of time on the order of seconds or minutes, and which typically contains sufficient surfactant to assist in cleaning the surface, and which is then rinsed off with plenty of water. It is particularly preferred that the composition of the invention be formulated as a leave-in composition. The composition may also be included in a product that is applied to the human body to improve appearance, cleansing, odor control, or general aesthetics. The composition of the present invention may be in the form of a liquid, lotion, cream, foam, scrub, gel or stick, and may be applied using a roll-on device or using a propellant-containing aerosol can. As used herein, the term skin covers the skin of any part of the body, such as the neck, chest, back, shoulders, armpits, hands, legs, buttocks and head. It also includes keratin-containing fibers, like hair, which can be made darker using the composition of the invention.

Melanin is produced as a result of a complex set of reactions in melaninocytes, which basically include the enzyme tyrosinase and the amino acid L-tyrosine. It is known that tyrosinase is a necessary component for the synthesis of melanin. Tyrosinase catalyzes the conversion of L-tyrosine to dopaquinone via L-DOPA (L-3,4-dihydroxyphenylalanine) as an intermediate. Dopaquinone undergoes additional transformation to form melanin.

Increased amounts of the biological pigment melanin in melanocytes are usually associated with cytotoxicity and unregulated proliferation. Some known remedies are not as safe as they should be, and examples include forskolin and IBMX (3-isobutyl-1methylxanthine). Sunless tanning agents are introduced into two types of cosmetic products. Of these, the most traditional is self-tanning lotion. The beneficial effect imparted in this case is the achievement of a skin color equivalent to that achieved by sun tanning. Then the second type of products were approved. They add sunless tanning product in small quantities to your regular moisturizing lotion.

The most preferred non-sun tanning product is dihydroxyacetone (DHA, which is chemically 1,3-dihydroxy-2-propanone). DHA is believed to exert its effects through interactions between its hydroxyl groups and the amino groups of amino acids and peptides found naturally in the hydrolipid mantle and the first layers of the stratum corneum of the skin. It is believed that these Maillard reactions (see, for example, Bobin et al., J. Soc. Cosmet. Chem., 35:255 (1984)) lead to the formation of brown pigments in the skin, thereby obtaining an appearance similar to a type of tan obtained naturally.

The inventors have discovered that a select class of benzoic acid derivatives, which are known to have antimicrobial properties and are used as preservatives, also cause skin darkening. This is confirmed by a cell-based assay that is more representative of in vivo action compared to the fungal tyrosinase-based assays that have so far been used in the screening of skin darkening/lightening actives.

According to the invention, a composition for darkening skin is disclosed, comprising (i) a compound of formula 1

where R1 represents R2 or OR2;

where R2 represents a C ₇ -C ₁₂ alkyl group; and (ii) a cosmetically acceptable carrier.

Preferred compounds of Formula 1 for inclusion in the composition of the invention are selected from one or more of 4-heptyloxybenzoic acid, 4-heptyloxybenzoic acid, 4-octyloxybenzoic acid, 4-nonyloxybenzoic acid, 4-undecyloxybenzoic acid or 4decyloxybenzoic acid. Particularly preferred compounds of formula 1 are selected from one or more of 4-heptyloxybenzoic acid, 4-octyloxybenzoic acid, 4-nonyloxybenzoic acid and 4-undecyloxybenzoic acid.

The compounds mentioned above have the structures shown below.

- 3 043647

4-G eptylbenzoic acid

OaAn CH ₃ (CH ₂ ) ₅ CH ₂ . _o zM

4-(Heptyloxy)benzoic acid

ABOUT

CH ₃ (CH ₂ ) ₇ CH ₂ . _O D^

4-(Nonyloxy)benzoic acid

ABOUT

CH ₃ (CH ₂ ) _to CH ₂ . ₀ A^

4-(Octyloxy)benzoic acid

O pAon CH ₃ (CH _g ) ₉ CH ₂ . ₀ D^

4-(Undecyloxy)benzoic acid

4-(Dodecyloxy)benzoic acid

It was noted that the compounds of the application are effective in producing melanin, while other alkyl or alkyloxybenzoic acid compounds were found to be ineffective in producing melanin. The authors of the present invention tested alkyl and alkyloxy chains with a length less than C ₇ , i.e. C1-C6, as well as alkyl and alkyloxy chains longer than _C12 , and have found that such chains are relatively ineffective for the effect of the present invention. Moreover, it has been found that the present invention is limited to the alkyl or alkyloxy position at the 4-position and does not work when these groups are attached at any other position of the benzene ring.

It has been noted that the compounds of formula (I) are effective in vitro at concentrations of 1-50 micromoles, preferably at concentrations from 5 to 35 micromoles. This observation indicates that these molecules are likely to be effective at low dosage in cosmetic compositions, such as non-sun tanning products. Without wishing to be bound by any theory, the present inventors believe that benzoic acid derivatives in the para position (derivatized with C ₇ -C ₁₂ -alkyl or alkyloxy) induce melanin synthesis in human primary melanocytes through cell signaling without acting as substrate and without directly changing the activity of the enzyme as such. They induce melanin synthesis at the cellular level and can affect cell signal transmission.

It was also noted that, in accordance with the invention, cellular melanin synthesis ranges from 110 to 150% of the synthesis in the control sample. This is a significant increase, comparable to the increase typically caused by standard/known ingredients.

Typically, such compounds are associated with cytotoxicity, which is an inevitable side effect. However, it has been noted that preferably the cell viability in the present invention is greater than 75%, more preferably greater than 90%, and most preferably greater than 95%. In other words, the cytotoxicity of the molecules of formula (I) is less than 25%, preferably less than 10%, and most preferably less than 5%. These numbers occur under in vitro conditions.

Preferably, the compound of formula 1 in the composition of the invention is included in the composition in an amount of 0.01-10%, preferably 0.05-5%, more preferably 0.1-3% by weight of the composition.

The composition of the invention includes a cosmetically acceptable carrier, which is preferably selected from an anhydrous base, gel, lotion, cream or emulsion. When the composition of the invention is a cosmetic composition as described above, it preferably includes one or more of a fragrance, a surfactant, an organic sunscreen, an inorganic sunscreen, an extender pigment and a preservative.

- 4 043647

Sun filters include materials that block harmful ultraviolet radiation. Preferred sunscreens are paminobenzoic acid derivatives (PABA), cinnamate and salicylate. For example, avobenzophenone (Parsol® 1789), octyl methoxycinnamate, and 2-hydroxy-4-methoxybenzophenone (also known as oxybenzone) can be used.

Octyl methoxycinnamate and 2-hydroxy-4-methoxybenzophenone are commercially available under the trade names Parsol® MCX and benzophenone-3, respectively. You can also use Ecamsule® - a derivative of benzylidene camphor and drometrizole trisiloxane, benzotriazole. Other examples include octocrylene, phenylbenzimidazole sulfonic acid (also known as Ensulizole®), ethylhexyl salicylate, diethylhexyl naphthylate, bimotrizinol (under the trade name Tinosorb® S) and bisoctrizole (Tinosorb® M). Inorganic sunscreens include oxides like titanium dioxide and zinc oxide, which reflect or scatter the sun's rays. The number of sunscreens present in the compositions may vary depending on the desired degree of UV protection. Preferably the compositions include 0.01-15 wt.%, more preferably 0.1-10 and most preferably 0.5-7.5 wt.% sunscreen filter.

Illustrative examples of the types of fragrances that can be used include fragrances including terpenes and terpene derivatives, such as those described in Bauer K. et al., Common Fragrance and Flavor Materials, VCH Publishers (1990). Other examples include myrcene, dihydromyrcene, citral, tagetone, cis-geranic acid, citronellic acid, mixtures thereof.

The carrier acts as a diluent or dispersing agent for the ingredients of the compositions. The carrier may be aqueous, anhydrous, or an emulsion, whereby a water-in-oil or oil-in-water emulsion is generally preferred. If water is desired, it will typically account for the balance of the composition, most preferably from 40 to 80% by weight of the composition.

In addition to water, organic solvents may optionally be included as a carrier to assist any other carrier in the compositions of the present invention. Examples include alkanols like ethyl and isopropyl alcohol.

Other suitable organic solvents include ester oils like isopropyl myristate, cetyl myristate, 2-octyl dodecyl myristate, avocado oil, almond oil, olive oil and neopentyl glycol dicaprate. Typically, such ester oils assist in emulsifying the compositions, and typically use an amount effective to provide a stable emulsion, and most preferably, a water-in-oil emulsion.

Also, if desired, emollients can be used as a carrier. Alcohols like 1-hexadecanol (ie cetyl alcohol) are preferred. Other emollients include silicone oils and synthetic esters.

Silicone oils suitable for use include cyclic or linear polydimethylsiloxanes containing from 3 to 9, preferably from 4 to 5, silicon atoms. Fixed silicone oils useful as emollients include polyalkylsiloxanes, polyalkylarylsiloxanes, and polyether-siloxane copolymers. Suitable non-volatile polyalkylsiloxanes are polydimethylsiloxanes. Silicone elastomers can also be used. Ester emollients that may optionally be used are listed below:

(i) alkenyl or alkyl esters of fatty acids with 10-20 carbon atoms. Examples thereof include isoarachidyl neopentanoate, isononylisononanoate, oleyl myristate, oleyl stearate and oleyl oleate;

(ii) esters of ethers, such as esters of fatty acids and ethoxylated fatty alcohols;

(iii) esters of polyhydric alcohols. Suitable esters of polyhydric alcohols are mono- and diesters of fatty acids and ethylene glycol, mono- and diesters of fatty acids and diethylene glycol, mono- and diesters of fatty acids and polyethylene glycol (200-6000), mono- and diesters of fatty acids and propylene glycol, polypropylene glycol monooleate 2000 , polypropylene glycol monostearate 2000, mono- and diglyceryl esters of fatty acids, polyglyceryl esters of polyfatty acids, ethoxylated glyceryl monostearate, 1,3-butylene glycol monostearate, 1,3-butylene glycol distearate, polyoxyethylene polyol fatty acid ester, fatty acid esters of sorbitan and fatty acid esters and polyoxyethylene sorbitan ;

(iv) esters of waxes such as beeswax, spermaceti, stearyl stearate and arachidyl behenate; and (v) sterol esters, examples of which are cholesterol esters of fatty acids.

Emollients, when present, typically comprise from 0.1 to 50% by weight of the composition, including all ranges within these limits.

Fatty acids having from 10 to 30 carbon atoms may also be included as carriers. Examples of such fatty acids include pelargonic, lauric, myristic, palmitic, stearic, isostearic, oleic, linoleic, arachidic, behenic or erucic acid.

- 5 043647 acid and mixtures thereof.

Humectants such as polyhydric alcohols can also be used in the compositions. The humectant generally increases the effectiveness of the emollient, reduces the formation of flakes on the surface of the skin, stimulates the removal of flakes, and improves the feel of the skin. Typical polyhydric alcohols include glycerin, polyalkylene glycols and, more preferably, alkylene polyols and their derivatives, including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and their derivatives, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-butylene glycol, 1,2,6-hexanetriol, ethoxylated glycerin, propoxylated glycerin and mixtures thereof. For best results, the humectant is preferably polypropylene glycol or sodium hyaluronate. Other humectants that can be used include hydroxyethylurea. The amount of humectant may range from 0.2 to 25% by weight of the composition, and is preferably from 0.5 to 15% by weight.

Moisturization can be improved by applying petroleum jelly or paraffin wax. Thickeners can also be used as part of the carrier in the composition. Typical thickeners include crosslinked acrylates (eg, Carbopol® 982), hydrophobically modified acrylates (eg, Carbopol® 1382), cellulose derivatives and natural gums. Useful cellulose derivatives include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, ethylcellulose and hydroxymethylcellulose. Natural gums suitable for the present invention include guar gum, xanthan gum, sclerotia gum, carrageenan, pectin, and combinations of these gums. The amount of thickener can vary from 0.001 to 5 wt.%, optimally from 0.01 to 0.5 wt.% of the composition.

Surfactants may also be present. When present, the total amount of surfactants is optimally between 5 and 12% by weight of the composition. The surfactant is selected from the group consisting of anionic, nonionic, cationic and amphoteric agents. Particularly preferred nonionic surfactants are those with a hydrophobic C ₁₀ - ₂₀ fatty alcohol or acid condensed with 2100 moles of ethylene oxide or propylene oxide per mole of hydrophobic substance; mono- and diesters of fatty acids and ethylene glycol; monoglycerides of fatty acids; mono- and diesters of C ₈ -C ₂₀ fatty acids and sorbitan; block copolymers (ethylene oxide/propylene oxide) and polyoxyethylene sorbitan, as well as their combinations. Alkyl polysaccharides and amides of fatty acids and saccharides (eg, methyl gluconamides) are also suitable nonionic surfactants.

Preferred anionic surfactants include soaps, alkyl ether sulfates and sulfonates, alkyl sulfates and sulfonates, alkyl benzene sulfonates, alkyl and dialkyl sulfosuccinates, C ₈ -C ₂₀ acyl isethionates, acyl glutamates, C ₈ -C ₂₀ alkyl ether phosphates, and combinations thereof.

Various other ingredients may also be used in the compositions. Active ingredients are defined as skin-beneficial agents, other than emollients and ingredients that merely enhance the physical characteristics of the composition. While not limited to this category, common examples include extender pigments such as talcs and silicas, as well as alpha hydroxy acids, beta hydroxy acids and zinc salts.

Beta hydroxy acids include salicylic acid. Zinc oxide and zinc pyrithione are examples of suitable zinc salts.

Compositions, especially those containing water, require protection from harmful microorganisms. Antimicrobial compounds such as triclosan and preservatives may be necessary. Suitable preservatives include p-hydroxybenzoic acid alkyl esters, hydantoin derivatives, propionate salts and various quaternary ammonium compounds. Particularly preferred preservatives are methylparaben, propylparaben, phenoxyethanol and benzyl alcohol. Preservatives constitute from 0.1 to 2 wt.% of the composition.

The packaging may be a patch, vial, tube, roll-on applicator, propellant aerosol device, squeeze pack, or jar with lid.

The present invention relates to a method for darkening skin, comprising the step of applying a composition disclosed hereinabove to a desired surface of the skin. The method is preferably non-therapeutic in nature. Suitable skin surfaces include the face, hands and shoulders, feet and legs, and neck and chest. The area of interest is the skin of the face, including the forehead, around the mouth, chin, around the eyes, nose and/or cheeks. The composition can be applied and left on the desired surface for a sufficient time or can be reapplied a sufficient number of times. In some embodiments, the contact time is longer than about 1, 2, 6, 8, 12, or 24 hours. The contact time is the time from application of the composition to the time the composition is removed. In some embodiments, the composition can be removed by rinsing or washing the substrate. The composition can be removed by washing or rinsing the skin. The composition can be applied at least once a day. In other embodiments, the composition is applied at least

- 6 043647 at least twice a day. Multiple applications may occur over a period of at least about one week. On the other hand, the application period may last longer than about 4 weeks, or longer than about 8 weeks. In some embodiments, the processing period will extend to several months (ie, 3-12 months) or years. In the case of a cosmetic composition, such a composition can be applied daily for an extended period of time.

According to yet another aspect, the invention relates to the use of a composition disclosed hereinabove for darkening skin. The use is preferably cosmetic.

The invention will be explained in more detail using the following non-limiting examples.

Examples

The test compounds were purchased from SIGMA-ALDRICH in powder form. They were tested at various concentrations in cell culture (see below). Forskolin (SIGMA, cat. no. F6886) was used as a reference standard because it has been reported to increase melanin content. Primary human neonatal foreskin epidermal melanocytes were purchased from Cascade Biologicals (passage labeled P0). Melanocytes were maintained in 254CF medium (Cascade, Cat. No. M-254CF-500) supplemented with human melanocyte growth supplement (Cascade; Cat. No. S-002-5), hereinafter referred to as MGM. Cells were maintained at 37°C in a humidified incubator under an atmosphere of 5% CO ₂ .

Analysis of cell viability and melanin content.

Seed 50,000 cells in a 24-well plate in MGM; After 24 hours, the cultures are treated with the test materials at various concentrations and left without any exposure for a further period of 72 hours. At the same time, a parallel experiment is carried out for comparison with a control vehicle of 0.25% (v/v) DMSO. At the end of the incubation period, cell viability is determined using the calcein method.

Briefly, depleted cell culture media are removed and cells are washed once with 0.4 ml PBS-Ca-Mg 1x solution. Add fresh 1 µM calcein-AM (0.2 ml/well), including control wells without cells. The plates are covered with aluminum foil and incubated for 30 min at 37°C in a conventional CO2 incubator. Calcein fluorescence is then measured (λmax 490 nm and λmax 520 nm) in a TECAN® M1000 Infinite series microplate reader.

Analysis for melanin content.

After calcein fluorescence readings were obtained, cells were drained and freshly prepared 1N NaOH (in 10% DMSO) was added at 125 μL per well. Cells are lysed by resuspension and incubation (60°C/1 h). This lysate is then transferred to a new 386-well plate and OD _{405 nm} is measured in a TECAN M1000 microplate reader (melanin relative content estimate).

Computing.

Calcein fluorescence values were converted to a 9-100 scale (% survival), with 100 representing the value of the 0.25% DMSO sample. The melanin content, in %, is calculated as the ratio of the OD value of _{405 nm} of any sample to the OD value of _{405 nm} of the reference sample in DMSO. Then the normalized melanin content is calculated as 100x(melanin content, %)/(cell survival, %).

The percentage increase in melanin content is assessed as [(normalized melanin content)-100].

The following six molecules were tested:

example 1 - 4-heptylbenzoic acid;

example 2 - 4-heptyloxybenzoic acid;

example 3 - 4-octyloxybenzoic acid;

example 4 - 4-nonyloxybenzoic acid;

example 5 - 4-undecyloxybenzoic acid;

example 6 - 4-decyloxybenzoic acid.

Survival data, in %, for various compounds tested at several concentrations, together with data on melanin content, in %, compared to vehicle control are summarized in the table below.

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Claims (9)

Test material Concentration, micromoles Survival rate, % Melanin content, % Control 100 100 Example 1 25 93 from Example 1 12.5 102 129 Example 1 6.25 102 124 Example 2 25 96 137 Example 2 20 95 129 Example 2 15 96 126 Example 2 10 94 117 Example 3 25 86 86 Example 3 20 100 127 Example 3 15 103 144 Example 3 10 101 127 Example 4 25 103 137 Example 4 20 103 130 Example 4 15 109 136 Example 4 10 109 129 Example 4 5 102 126 Example 5 25 79 120 Example 5 20 90 134 Example 5 15 100 146 Example 5 10 97 136 Example 6 25 76 93 Example 6 20 82 101 Example 6 15 100 122 Example 6 10 102 129 Example 6 5 104 126 The results shown in the table above show that the compounds of the invention (examples 1-6) provide an increase in melanin content compared to the control. Cell survival for all samples at the tested concentrations is also quite good, as can be seen from the % survival rate, which was within acceptable limits. CLAIM 1. A method for darkening skin, comprising the step of applying to the desired surface of the skin a composition comprising (i) a compound of formula 1 o where R1 represents R2 or OR2; where R2 represents a C ₇ -C ₁₂ alkyl group, and (ii) a cosmetically acceptable carrier. 2. The method of claim 1, wherein the compound of formula 1 is selected from 4-heptyloxybenzoic acid, 4-heptyloxybenzoic acid, 4-octyloxybenzoic acid, 4-nonyloxybenzoic acid, 4-undecyloxybenzoic acid or 4-decyloxybenzoic acid. 3. The method of claim 2, wherein the compound of formula 1 is selected from 4-heptyloxybenzoic acid, 4-octyloxybenzoic acid, 4-nonyloxybenzoic acid and 4-undecyloxybenzoic acid. 4. The method according to any of the preceding claims, in which the composition includes 0.01-10% of the compound of formula 1 by weight of the composition. 5. The method of any one of the preceding claims, wherein the cosmetically acceptable carrier is selected from an anhydrous base, gel, lotion, cream or emulsion. 6. The method according to any of the preceding claims, in which the compound of formula 1 induces cellular synthesis of melanin in the range from 110 to 150% of that synthesis in the control sample. 7. A method according to any of the preceding claims, wherein said method is not therapeutic in nature. 8. Use of a composition comprising (i) a compound of formula 1 - 8 043647 where R1 represents R2 or OR2; where R2 represents a C7-C12 alkyl group; and (th) a cosmetically acceptable vehicle for darkening the skin. 9. Use according to claim 8, wherein said use is cosmetic. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2