WO2012136930A2

WO2012136930A2 - Composition of chaulmoogra oil and tribulus terrestris for skin pigmentation

Info

Publication number: WO2012136930A2
Application number: PCT/FR2012/050723
Authority: WO
Inventors: Nadine Leconte; Jacques Leclere
Original assignee: Laboratoire Nuxe
Priority date: 2011-04-05
Filing date: 2012-04-03
Publication date: 2012-10-11
Also published as: WO2012136930A3; FR2973697A1; FR2973697B1; EP2694028A2

Abstract

The invention relates to a composition that can be used in dermatology and/or in cosmetics. The composition includes an effective amount of chaulmoogra oil and/or the components thereof as well as an extract of Tribulus terrestris, combined in a ratio of Tribulus terrestris to chaulmoogra oil of 1:1,000 to 1:5 by weight. The invention can be used in cosmetic and dermatological compositions for treating depigmented or non-pigmented areas of the skin.

Description

COMPOSITION BASED ON CHAULMOOGRA OIL AND

TRIBULUS TERRESTRIS FOR THE PIGMENTATION OF THE SKIN.

The present invention relates to a new composition based on chaulmoogra oil and Tribulus terrestris used in cosmetics, more particularly for the pigmentation of the skin.

The skin consists of superficial layers, namely the epidermis, and deeper layers, the dermis and hypodermis, and each has specific properties allowing the whole to react and adapt to the conditions of its environment. The epidermis, which is composed of three types of cells, namely keratinocytes (90% of epidermal cells), melanocytes (2 to 3% of epidermal cells) and Langerhans cells, constitutes the outer layer and plays a role. fundamental to ensure the protection and maintenance of good trophicity. The dermis serves as a support for the epidermis and is mainly composed of fibroblasts and an essential extracellular matrix ^¬ lement based on collagen and elastin. Collagen fibers contribute to the texture and tone of the skin and elastin is responsible for its elasticity. Other cells, such as macrophages and leucocytes, are also present in the dermis layer. The hypodermis, which is the deepest layer of the skin, contains lipid-producing fat cells so that the subcutaneous tissue makes a fat layer that protects the muscles, bones, and internal organs from shock.

The pigmentation of the skin results from the presence of melanin in the epidermis and the dermis. Melanin is a pigment produced by melanocytes located mainly in the basal layer, in the presence of tyrosinase (or monophenol monooxygenase), a cuproprotein enzyme that catalyzes the transformation of L-tyrosine into L-dihydroxyphenylalanine (L- dopa) which is then oxidized to dopaquinone, then

B10059wo dopachrome, to achieve melanin following a complex mechanism. This biosynthesis, or melanogenesis, is a complex process, nowadays relatively well known, in which tyrosinase plays an essential role.

Under normal conditions, the pigmentation of the skin is uniform. However, the appearance of hyperpigmentation, that is to say excessive pigmentation locally, which can be manifested by freckles, age spots, pigmentation due to excessive exposure to the sun, is frequently observed. post-inflammatory hyperpigmentation due to abrasion, etc. Conversely, it is sometimes desirable to promote the pigmentation of the skin for aesthetic reasons or to fight against the effects of certain diseases, such as for example vitiligo, depigmented lentigo, certain mycoses, traces of stretch marks, burns , etc., which result in a localized defect of pigmentation.

Many substances are known to promote melanogenesis and pigmentation of the skin, and for example carotenoids and certain vitamins such as vitamin E and vitamin C. Melanin synthesis compounds are also known hydroxy acids as in the patent application WO 99.51198, plant extracts, in particular Sanguisorba officinalis as in patent EP 993,826, or compounds promoting the expression of tyrosinase as psoralens. The patent application WO 03082227 describes a composition for regulating melanogenesis, which comprises both promoters and inhibitors of melanin synthesis.

Some polyphenols have also been proposed to enhance the pigmentation of the skin. Thus, patent application FR 2 851 916 describes the use of catechol polyphenols in order to promote the natural pigmentation of the skin, and for example cocoa extracts that can be used in compositions for oral administration. of the xanthine extracts can be used in cosmetic and dermatological composi ^¬ tions to promote pigmentation of the skin, such as those obtained from cocoa beans described in FR 2654935.

Chaulmoogra oil has long been used in traditional medicine in Asia, particularly in India and China, for the treatment of leprosy, before the emergence of sulfonated drugs, including diaminodiphenylsulfone, and anti-tuberculosis antibiotics. such as rifampicin and rifamycin. Hydnocarpic acid, one of the main constituents of chaulmoogra oil, has been shown to exhibit antimycobacterial activity by inhibiting the growth of certain mycobacteria, such as Mycobacterium leprae, as indicated by PL Jacobsen and L. Levy, Antimycobacterial Agents and Chemotherapy (1973) pp. 373-379.

Patent FR 2,706,304 describes the use of chaulmoogra oil as a component of cosmetic compositions intended to harmonize the pigmentation of the skin, thanks to the pigmentation effects of the achromic cutaneous zones, that is to say little or no pigmented areas. , by migration of pigmentation from pigmented areas. Patent FR 2,518,402 describes a composition containing chaulmoogra oil that can be used in cosmetics for the normalization of sebaceous secretions and cutaneous microbial flora. FR 2,876,908 shows that chaulmoogra oils also have lipolytic effects useful in compositions intended for the treatment of fat overloads.

Chaulmoogra oils are mainly obtained by extracting the seeds of woody plants of the family Flacourtiaceae, growing in tropical regions, including a tree species Hydnocarpus wightiana and Taraktogenos kurzii. These plants are mainly Asian ori ^¬ gine, including India, Vietnam and Philippines, as well as Central and South America, including Brazil.

The seeds from which the chaulmoogra oils are extracted contain a high proportion of lipids, between 30 and 50% depending on the species, 15 to 20% of proteins and 4 to 6% of mineral matter, as well as 1 to 3% of 'unsaponifiables, glycerides of unsaturated fatty acids to pentenoic cycle, consti ^¬ essentially killed by chaulmoogric acid, hydnocarpic acid and gorlique acid. It seems that these acids are responsible for the therapeutic antimycobacterial action in the traditional treatment of leprosy. It has been observed that the spatial structure of these acids is similar to the cyclopentanoperhydroxyphenantrene ring characteristic of sterols.

The acids chaulmoogric, hydnocarpic and gorlic can be represented by the following general formulas, respectively:

CH- <C¾4 - OOOH

These three acids include the same pentenoic cycloaliphatic ring having an acid group at the end of a carbon chain - (CH2) _n ^_ wherein n is 10 in the case of acid hydno ^¬ carpique and 12 in the case of chaulmoogric acid, this same chain having a double bond in the case of gorlic acid. The contents of each of these three acids in chaulmoogra oils vary according to the origin of the species. Chaulmoogra oils also contain fatty acids such as palmitic, oleic, palmitoleic, stearic, myristic, and traces of alepric and aleprylic acid.

Tribulus terrestris (terrestrial tribulus) is a plant of the zygophyllaceae family. It is a creeping little branched herb with a small yellow flower and very spiny fruit, a native species in southern France that is widespread in the arid and uncultivated regions of Africa and Asia. It contains one essence, steroidal saponosides whose genin may be diosgenin and trigogenin. Consumed as it is, it causes intoxication in sheep by clogging of the hepatic ducts as reported by R. R. Aslani et al., Vet. Res. Common. 27, 53-62 (2003). It is sometimes used as a sexual stimulant, and tests on the mouse, in oral administration, have shown an increase in testosterone and sperm volume, as indicated for example by K. Gauthaman, Phytomedicine, 15, 44-54 ( 2008).

The patent CN 101406511 describes a topical composition combining several substances of vegetable origin and in particular extracts of fruit of Malaytea scurfpea, fruit of Cnidium, Chaulmoogra and Tribulus terrestris or Astragalus complanatus, as well as metal derivatives (litharge and chloride mercureux), in substantially equivalent amounts, and this composition could be useful for the treatment of vitiligo.

KR 20090056778 discloses compositions of asso ^¬ ting medicinal herbs, in particular Hydnocarpi semen, Sesamum indicum, Dictamni radicis cortex, Mormodicae semen and Tribulus terrestris in substantially equal proportions, for the treatment of skin allergies, in particular atopic dermatitis and allergies to insect bites and drugs. The studies carried out by the applicant on human melanocytes and reconstituted epidermis have shown that tribulus terrestris, used topically in the form of hydroglycolic extracts, has significant but weak depigmenting effects.

On the other hand, the studies carried out by the Applicant have unexpectedly shown that the combination of Chaulmoogra oil and Tribulus terrestris in a weight ratio Tribulus terrestris / Chaulmoogra oil of between 1: 1,000 and 1: 5, presents a significant pigmenting effect capable of acting in all stages of pigmentation, and greater than that of chaulmoogra used alone.

The subject of the present invention is therefore a new cosmetic composition combining chaulmoogra oil and / or its components and an extract of Tribulus terrestris, in appropriate concentrations, and more particularly in a weight ratio extracted from Tribulus terrestris / chaulmoogra oil. from 1: 1,000 to 1: 5, for the treatment of depigmented or unpigmented areas of the skin.

The invention also relates to the use of chaulmoogra oil, or its essential components such as chaulmoogric, hydnocarpic and gorlic acids, as well as their salts and esters, and extracts of Tribulus terrestris for the preparation of a cosmetic composition for the treatment of depigmented or non-pigmented areas of the skin.

The subject of the invention is also the use in cosmetics of the combination of an extract of Tribulus terrestris and of chaulmoogra oil in a weight ratio Tribulus terrestris / chaulmoogra oil of between 1: 1,000 and 1: 5, for the pigmentation of the skin.

The invention further relates to a non-therapeutic cosmetic treatment process for the skin, more particuliè ^¬ surely repigment for non-pigmented areas or dépigmen- Tees skin by applying a composition based on of Chaulmoogra oil and Tribulus terrestris extract in appropriate concentration on the area of the skin requiring such treatment.

The components of the chaulmoogra oil are preferably the chaulmoogric, hydnocarpic and gorlic acids and their salts and esters, which may be chosen from methyl, ethyl or benzyl esters, as well as sodium or potassium.

The salts or esters of chaulmoogric, hydropyric and gorlic acids may be chosen from methyl, ethyl or benzyl esters, as well as sodium or potassium salts, and for example sodium or potassium chaulmoograte and Sodium hydnocarpate.

The pigmenting activity of chaulmoogra oil is mainly due to the chaulmoogric, hydnocarpic and gorlic acids it contains, and the experiments carried out have shown that the association with an extract of Tribulus terrestris unexpectedly provides an improvement in this activity. known from Chaulmoogra whereas Tribulus, used alone, has a depigmenting action.

Specifically, it was shown that the asso ^¬ ciation of Chaulmoogra oil and extract of Tribulus terrestris

• presents a perfect safety vis-à-vis the endothelial cells of the epidermis and human melanocytes in culture;

• induces a significant effect on the activity of tyrosinase even in the absence of UV irradiation;

• induces a significant effect on the uptake of L-tyrosine and L-phenylalanine as well as on the synthesis of L-tyrosine in the absence of UV irradiation while the effect is not significant in the presence of UV irradiation ;

• exhibits such effects in all stages of the pigmentation process, providing an improvement over the use of Chaulmoogra alone. Studies have shown that this effect is remarkable with a small proportion of Tribulus terrestris compared to Chaulmoogra (weight ratio between 1: 200 and 1:20), whereas trials show that Triibulus terrestris, in equivalent concentration, used alone, has a dépig ^¬ menting effect.

The chaulmoogra oils used in the present invention can be extracted from the seeds of plants of varieties such as Hydnocarpus wightiana, Taraktogenos kurzii, Hydnocarpus alpina, Hydnocarpus anthelmintica, Hydnocarpus cauliflora, Hydnocarpus dawnensis, Hydnocarpus heterophylla, Hydnocarpus hutchinsonii, Hydnocarpus ovoidea, Hydnocarpus subfalcata, Hydnocarpus venenata, Hydnocarpus verrucosa, Hydnocarpus woodii, Hydnocarpus calvipetala, Hydnocarpus ilicifolia, Hydnocarpus octandra, Gynocardia odorata, Oncoba echinata, Caloncoba glauca, Caloncoba welwitschii, Carpotroche brasiliensis, Carpotroche amazonica, Asteriastigma macrocarpa, Mayna odorata and Lindakeria dentata.

Extracts of Tribulus terrestris are prepared from dried plants, the extract may represent from 1 to 20% of the total weight of the plant.

For example, hydroalcoholic extracts may be prepared, for example, by way of the usual techniques, more particularly hydroglycolic or hydroethanolic extracts, or even hydroglycerinated extracts. After drying and spraying the plants, the extraction can be done for example by percolation or maceration at 100 g of plant per 500 g of water. The cake is expressed and the liquors combined and supplemented to 500 g by addition of glycol such as butylene glycol and propylene glycol.

The extracts of Tribulus terrestris used in the compositions according to the present invention are preferably in the form of hydroglycolic extracts, for example in butylene glycol, of the whole plant dried and reduced to powder or, preferably, seeds or fruits dried and reduced to powder.

According to an advantageous embodiment, the extract that can be used in the composition of the invention is obtained from the dried and powdered fruits, which are macerated in a mixture of glycol and water, for example a mixture of 50/50 of glycol and water, followed by decantation, clarification, if necessary, and filtration. Clarification is usually by centrifugation or addition of a microparticle-absorbing substance, such as cellulose, polyvinyl pyrrolidone or absorbing soil. The glycol may be chosen from butylene glycol-1,3, butylene glycol-1,4, and dipropylene glycol, and butylene glycol is preferably used.

The hydroglycolic extract of Tribulus terrestris used in the present invention is in the form of an amber-yellow liquid of characteristic odor, soluble in water and in alcohol, characterized by:

- dry matter) 0.1-1.0%

- pH 5.3-7.3

refractive index at 22 ° C 1,380-1,420

Density at 22 ° C. 1,000-1,050 In the composition according to the invention, the ratio by weight of the tribulus terrestris extract to the oil of chaulograogra is between 1: 1,000 and 1: 5 and preferably between 1: 200 and 1:20.

In particular, the extract content of Tribulus terrestris may be between 0.01 and 0.5%, and preferably between 0.05 and 0.2% by weight relative to the total weight of the composition, while the content in oil of chaulmoogra, or in acids or salts or esters is generally between 0.1 and 20%, and preferably between 2 and 10% relative to the total weight of the composition, to provide the best pigmenting effects. Chaulmoogra oil, and its acids or esters, can be used advantageously in a form encapsulated in liposomes.

According to a known technique in the manufacture of cosmetic compositions, the liposomes are constituted by small hollow spheres, generally less than 500 nm in diameter, whose wall is formed of a double layer of lipids such as glucolipids or phospholipids . They can be obtained for example by ultrasonic treatment of a mixture of an aqueous solute and lipids. Lipids (phospholipids or glucolipids) are reorganized in a configuration where the energy of the whole is minimal, therefore thermodynamically the most stable. Liposomes are used in the cosmetics industry to deliver compounds into cells when the vesicle fuses with the plasma membrane.

According to a preferred embodiment of the invention, at least part of the chaulmoogra oil is encapsulated in liposome-type vesicles.

The compositions according to the present invention are preferably topically administrable. In conventional terminology, topical administration refers to any method of applying the substance or composition directly to the skin on the area requiring treatment.

According to the present invention, the composition can be administered topically may advantageously contain, in addition to the basic components described above, one or more other substances known to exert beneficial complementary effects for the skin, and more particu ^¬ larly tocopherol, vitamin A (retinol), retinoic acid, bactericidal agents, theophylline, monomethylsilanol, proline, or shea butter, as well as plant extracts such as Camellia japonica. It is also possible to add to the composition extracts or compounds known to facilitate the penetration of the active ingredients of the composition through the epidermis, for example ethoxydiglycol and saponosides of the hederagenin type.

The cosmetic and pharmaceutical compositions in accordance with the present invention are preferably intended for topical administration, and therefore contain carriers and excipients commonly used in compositions of this type, such as O / W or W / O emulsions, creams, gels or lotions. In the case of emulsions, the fatty phase may represent between 10 and 60% of the weight of the composition, the aqueous phase between 10 and 80% and the emulsifier between 2 and 20%, the remainder being constituted by the components above and the other components listed below.

The composition may also contain various substances and excipients chosen according to their known properties and the intended dosage form. Thus, preservatives, emulsifiers, viscosifiers, thickeners, gelling agents, antioxidants, moisturizers, surfactants, perfumes, oils, lipids, a specific solvent and the like can be incorporated into the composition. only water and various additives to improve the physical properties of the composition.

The emulsifier may be selected from high molecular weight carboxyvinyl polymers (e.g.

® ®

Carbopol), polysorbates (eg Tween 60 or

Polysorbate 80®), sorbitan esters and in particular a monostearate such as Span ^60® , a tristearate, a monopalmitate, and a sorbitan laurate. It is also possible to use other emulsifying agents such as various stearic or palmitic acid derivatives, and for example stearate of

PEG 100®, mono- or diglycerides of stearic or palmitic acid, self-emulsifiable propylene glycol stearate, or polyglyceryl-2-sesquioleate, 1-ether polyoxyethylene cetyl, a siloxane polyglucoside, or an emulsifiable silicone. Nonionic emulsifying mixtures such as Protegin X may also be used.

The viscosifying agents used in the compositions of the invention may be chosen from various polymers of acrylic acid, a copolymer of acrylate and acryloyl laurate, a cellulose gum, a silica, carboxyvinyl polymers, an aluminum silicate and magnesium, and it is possible to use, for example, the colloidal silica sold under the trademark Aerosil 200 or a crosslinked polyacrylic acid such as Carbopol 940.

The gelling agents or thickeners may be chosen for example from polyacrylamides, acrylates such as Pemulen®, cellulose derivatives such as hydroxypropyl cellulose, or natural gums.

The moisturizing agents used may be chosen for example from a polyol, sorbitol, maltitol, pentaerythritol, glyceryl polyacrylates and polymethacrylates, glycerol or glycerol derivatives. It is also possible to add emollients such as alkyl malate, isohexadecane, capric or caprylic acid triglycerides and the like.

The usual preservatives of the technique of dermatological or cosmetological compositions can be used in the invention, and for example benzoic acid and an alkyl p-hydroxybenzoate such as methyl p-hydroxy-benzoate (Methylparaben), an alcohol such as phenoxyethanol or chlorphenesin or imidazolidinyl urea.

The constituents of the fatty phase, that is to say the oils and lipids, can be chosen from jojoba oil, corn oil, liquid petroleum jelly, sweet almond oil, hydrogenated coconut oil, safflower oil, saturated fatty acid glycerides, stearic acid, palmitic acid, octyl stearate, glyceryl palmitate, octyl palmitate, triglyceride caprylic and caprylic acids, 2-octyl-dodecanol, polyethylene glycol, 2-ethyl hexyl adipate, or silicone oils such as methyl phenyl polysiloxane, dimethicone, cyclomethicone and phenyl dimethicone .

The composition may also contain a solvent selected depending on the components used and the adminis tration form ^¬ envisaged. The solvent may be, for example, water, and preferably demineralized water, or a specific solvent such as propylene glycol, a diethylene glycol ether, or an alcohol such as ethanol.

Ultraviolet protection agents may also advantageously be incorporated in the compositions, and for example hydrophilic or lipophilic UV-A and UV-B sunscreens, chosen from benzophenone or a benzophenone derivative such as 2-hydroxybenzophenone. 4-methoxybenzophenone (Eusolex® 4360), or a cinnamic acid ester and more particularly octyl methoxycinnamate (Eusolex® 2292), ethyl-2-hexyl methoxycinnamate (Parsol MCX®), or a cyano-β, β-diphenylacrylate such as octo-crylene (Eusolex® OCR), 4-methylbenzylidene camphor (Eusolex 6300®), and dibenzoylmethane derivatives such as 4-isopropyl dibenzoylmethane (Eusolex 8020), t -butyl-methoxy dibenzoylmethane (Parsol 1789®), and 4-methoxydibenzoylmethane. It is also possible to use anti-ultraviolet screen pigments, such as, for example, titanium dioxide, zinc oxide, zirconium oxide or aluminum oxide.

The pH of the composition is preferably between 5.3 and 7.5, and may be adjusted, depending on the compositions, by the addition of an acid such as citric acid or a base such as sodium or potassium.

The composition according to the present invention may be presented in the forms conventionally used for topical application, that is to say in the form of gel, lotion, emulsion (in particular cream or milk), transdermal patches. mask or ointment containing compatible and pharmaceutically acceptable excipients and carriers. These forms of topical administration are prepared by the known techniques, and for example, in the case of a cream, by dispersion of a fatty phase in an aqueous phase to obtain an oil-in-water emulsion, or conversely to prepare a water-in-oil emulsion. In the case of creams, it is preferred to use lamellar structure emulsions containing little or no ethoxylated products. The chaulmoogra oil is preferably preheated before being incorporated into the fatty phase, while the Tribulus terrestris extract used in combination is introduced into the aqueous or alcoholic phase.

By way of example, it is possible to prepare topical compositions in accordance with the invention in the form of creams, milks or gels, which can be used in one or more daily applications, the duration of the treatment being generally of the order of one to four. three months.

The examples of compositions given below illustrate the invention without limiting its scope. Unless otherwise indicated, parts and percentages are by weight.

Example 1

An anti stretch mark cream based on chaulmoogra oil and Tribulus terrestris extract having the composition indicated below is prepared according to the usual techniques:

Phase A

Demineralized water q. s. p. 100.00

Glycerin 2.00 Pentylene Glycol 5.00

Bentonite 1.00

Ethyl Hexyl Glycerine 0.20 Sodium Dehydroacetate 0.10 Phase B

C12-C16 alcohols and palmitic acid

and hydrogenated lecithin 5.00

Capric / caprylic triglycerides 5.00 Squalane 3.00

Shea Butter 3.00

Glycerol ester of vitamin F 1.00

Behenyl alcohol 2.50

Beeswax 2, 00 Sunflower oil 5,00

Glyceryl undecylenate 0.10

Tocopherol 0.70

Chaulmoogra Oil 10.00 Phase C

Demineralized water 10.00 Xylityl glucoside and anhydro-xylitol

and xylitol 1.00

Monomethyl silanol 1.00

Proline 1.00 Extract of Camellia japonica 5.00

Tribulus terrestris extract 0.10 Phase D

Perfume 0,50

The aqueous phase A is heated to 70-75 ° C while the fat phase B is heated to about 75 ° C, and then the two phases are thoroughly mixed with stirring. Phase C, after homogenization, is added to the mixture at a temperature of about 35 ° C. The whole is mixed and gradually cooled.

This anti stretch mark cream is used topically, once to twice a day on the areas of the skin to be treated, for a period of 1 to 3 months depending on the importance of the depigmentation to be treated. The first significant effects can be observed as of the end of the 4th week, in the form of an attenuation of the contrast between depigmented zone and normally pigmented zone and these repigmenting effects are amplified in the continuation of the treatment.

Example 2

A sunscreen based on chaulmoogra oil and Tribulus terrestris extract having the composition indicated below is prepared according to the usual techniques:

Phase A

Tween 60 3.50 Span 60 2.50

Vaseline oil 5.00

Sweet almond oil 2.00

Chaulmoogra oil 10.00

Cetostearyl alcohol 1.00 Phenyl dimethicone 2.00

Tocopherol 0.70 Phase B

Zinc oxide and capric triglycerides

/ caprylic and Paraffinum liquidum and

polyhydroxystearic acid 10.00 Phase C

Xanthan Gum 0.50

Pentylene glycol 4.00

Ethyl hexyl glycerin 0.30 Anisic acid 0.10

Levulinic acid 0.20

Zinc Oxide and Water and Silica and PEG 2M 5.00

Demineralized water q.s. 100.00

Tribulus terrestris extract 0.10 Phase D

Perfume 0,50

Fat phase A is heated with stirring to about 75 ° C and fat phase B is added thereto and then aqueous phase C at about 75 ° C, and the three phases are thoroughly mixed. with stirring. Finally, phase D is added and the mixture is then mixed and cooled progressively.

This cream can be used per application on the depigmented areas of the skin to be treated, once or twice a day, for a period of one to three months. Positive results are observed in some cases, especially when treating an area affected by vitiligo, from the end of the 4th week of application. If the treatment is stopped after 1 month, there is a regression of the general pigmentation but the repigmented areas remain acquired.

Example 3

Evaluation of cytotoxicity

The cytotoxicity of the combination of Tribulus terrestris extract and chaulmoogra oil was evaluated by studying human melanocytes in culture, as follows.

To constitute epidermals, keratinocytes of human origin are seeded on polycarbonate filters of 0.5 cm ² in a defined medium (modified MCDB 153) and supplemented. The cells are cultured for 14 days at the air / liquid interface, the culture medium being changed every two days.

The thus formed epidermis (SKINETIC®) are used for the study from the i4 ^th day of culture.

Human melanocytes were established from human skin (Clonetics) and amplified in MGM medium (Melanocytes Growth Medium, Clonetics). The tests were carried out on melanocytes between the 2 ^nd and 4 ^th passage to ensure reproducibility between different experiments.

Cytotoxicity was assessed for co-culture human melanocytes / reconstructed epidermis, for deter ^¬ mination of cell viability by the reduction of formazan blue test (MTT). The melanocytes were distributed in 24-well plates at a rate of 2 × 10 ⁵ cells per well in 0.5 ml of culture medium for 12 hours. The treated and untreated epidermis was then deposited in the wells containing the melanocytes in culture.

The test was conducted after 24 hours of contact between the studied product and the reconstituted melanocyte / epidermal co-culture.

Two batches of co-cultures were set up for the tests.

Lot 1: negative control co-culture receiving no product.

Lot 2: co-cultivation treated by the combination of chaulmoogra oil (5%) / Tribulus terrestris (0.1%).

The viability of melanocytes is evaluated as follows.

After 24 hours of co-culture incubation, cell viability was determined by the Formazan blue reduction test (MTT test). After incubation of the cells of each batch, the epidermis are recovered and the wells containing the cells are emptied by inversion and the cell layer is rinsed with the culture medium, 200 μl of a solution of MTT (bromide of 3- (4, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium) 5 mg / ml are distributed in all wells. The plates are incubated at 37 ° C for 3 hours. The wells are again emptied by inversion, and the cellular mat is fixed for 1 minute with 200 μl of formo-calcium solution. The cells are then lysed and the Formazan blue crystals are dissolved with 200 μl of dimethylsulfoxide. The optical density (OD) of the plates is read, after homogenization of the staining by stirring, using a spectrophotometer set at 570 nm, thus making it possible to know the relative quantity of living and metabolically active cells.

The cell viability of the epidermis was evaluated as follows. The reconstituted epidermis was individually contacted with 150 μl of MTT and placed in the incubator for 1 hour. At the end of the incubation, the epidermis are recovered. The cells are then lysed and the crystals of Formazan blue dissolved, then 200 μl of dimethyl sulfoxide are added. After homogenization of the coloration by stirring, the plates are read by a spectrophotometer set at 570 nm.

The results are summarized in the tables below. Reconstituted Epiderms:

These results show that the combination according to the invention shows no cytotoxicity with respect to the reconstituted epidermis.

Example 4

Evaluation of the effect on pigmentation The pigmenting effect was studied through

· Evaluation of the capture of ^[3 H] L-tyrosine

• evaluation of [ ¹⁴ C] L-phenylalanine uptake

• evaluation of the synthesis of [ ¹⁴ C] tyrosine

• evaluation of tyrosinase activity

The study was done on the same batches as in Example 3 above. The test is conducted in triplicate after 3 days of contact.

Evaluation of the capture of L-tyrosine:

The evaluation of the [ ³ H] L-tyrosine uptake was carried out as indicated below.

The ability of the cells previously treated by the products under study to capture the radioactive precursors was evaluated by the method of withdrawal. The radioactive precursors are added to the cultures after incubation with the combination of the invention. The cells are then incubated for 20 minutes at 37 ° C. in the presence of [ ³ H] L-tyrosine (130 pmol., Specific activity 47 Ci / mmol).

The medium is removed by aspiration, then the cells are washed 3 times with serum-free medium to remove the radioactivity, then detached from the support and washed again with medium and finally centrifuged at 600 g for 5 minutes. The cell pellet is taken up in 500 μΐ of medium. To determine the radioactivity incorporated in the melanocytes, the cells are sonicated and then introduced into counting flasks. The radioactivity is measured in a scintillation counter with the presence of 5 ml of scintillation fluid (organic Ready, Beckman) containing 0.9% acetic acid.

The results are summarized in the table below.

These results show that the irradiation of melanocytes with ultraviolet B rays significantly increases (44%) the capture of [ ³ H] L-tyrosine. In the absence of irradiation, the combination of the invention induces a significant effect compared to the negative control on the capture of [ ³ H] L-tyrosine.

Evaluation of the capture of L-phenylalanine:

The same method as above was used replacing the [H] L-tyrosine with [C] L-phenylalanine (130 pmol, specific activity 474 mCi / mmol.).

The results are summarized in the table below.

These results show that the irradiation of the co-culture with ultraviolet B rays significantly increases (40%) the capture of [ ¹⁴ C] L-phenylalanine. In the absence of irra diation ^¬, the combination of the invention induced a significant (18%) compared to the negative control.

Evaluation of the synthesis of L-tyrosine:

The [ ¹⁴ C] L-tyrosine neosynthesis was evaluated by incorporation of [ ¹⁴ C] L-phenylalanine into human melanocytes in culture. Once captured, the phenylalanine is converted into a tyrosine effect, which results in the synthesis of melanin.

The transformation of [ ¹⁴ C] L-phenylalanine into [ ¹⁴ C] L-tyrosine is studied over a period of 20 minutes. The capture and transformation of phenylalanine is monitored after addition of 130 pmol of L-phenylalanine to melanocytes in monolayer.

The medium is removed and the cells are washed three times with Tris / HCl (0.05 M, pH 7.5) and then digested with trypsin (1 mg / ml). [ ¹⁴ C] L-tyrosine and [ ¹⁴ C] L-phenylalanine are studied by HPLC with a chloroform / methanol / water / ammonium hydroxide eluent (29: 15: 1: 4 v / v).

The results are summarized in the table below.

These results show that the irradiation of melanocytes with ultraviolet B rays significantly increases (35%) the synthesis of [ ¹⁴ C] L-tyrosine. In the absence of irra diation ^¬, the combination of the invention induced a significant (19%) compared to the negative control.

Evaluation of the activity of L-tyrosinase:

At the end of incubation, the culture medium is removed and the cells are then rinsed with PBS and placed in contact with Triton X-100 (Sima, France) at 1%, and then incubated for 10 minutes. The enzymatic reaction is initiated by addition of L-dopamine (Sigma, France) to 10 mM in PBS devoid of Ca ²⁺ and Mg ²⁺ .

After one hour of incubation at 37 ° C, protected from light, the tyrosinase activity is evaluated by measuring the absorption at 475 nm using a spectrophotometer.

The results are summarized in the table below.

Tyrosinase activity%

Witness 100

Positive control UVB 100mJ / cm ² 172

T. terrestris 0.1% + oil of 120

chaulmoogra 5%

T. terrestris 0.1% + 175 oil

chaulmoogra 5% UVB 100mJ / cm ² These results show that the irradiation of co-culture with ultraviolet B rays significantly increases the activity of tyrosinase. In the absence of irradiation, the combination of the invention induces a significant effect compared to the negative control.

These results confirm that the combination of Tribulus terrestris and chaulmoogra oil of the invention plays a significant pigmenting role in all stages of the pigmentation process. Example 5 (comparative)

Evaluation of the Depigmenting Effect of Tribulus Terrestris Cytotoxicity was verified by evaluation of cell viability on human melanocytes as in Example 4 above.

The study shows that the extract of Tribulus terrestris, at concentrations of 0.05% and 0.1%, exhibits no cyto ^¬ toxicity. The extract is cytotoxic from 0.2%.

Four batches were constructed to show the effect of Tribulus terrestris on epidermal / melanocyte co-cultures.

Lot 1: negative control co-culture receiving no product.

Lot 2: positive control co-culture receiving koic acid (2%).

Lot 3: treated coculture Tribulus terrestris (0.05%).

Lot 4: co-cultivation treated Tribulus terrestris (0.1%). The effect on pigmentation was studied by evaluating the activity of tyrosinase and by evaluating the synthesis of melanin.

The evaluation of the synthesis of melanin intracellu ^¬ lar is carried out by spectrophotometry at 475 nm after suspending and dissolving the cells in NaOH (IN) and dimethyl sulfoxide for 30 minutes. The results are summarized in the table below.

These results show that the extract of Tribulus terrestris, at the concentrations tested, results in a significant decreased ^¬ tion of melanin levels comparable to the positive control receiving the 2% kojic acid known depigmenting agent.

The evaluation of tyrosinase activity is performed as in Example 4 above.

The results are summarized in the table below.

These results show that Tribulus terrestris extract leads to a significant decrease in tyrosinase activity comparable to that of kojic acid.

All of these results show that tribulus terrestris extract, used alone, has a significant, although weak, depigmenting activity.

Claims

A cosmetic composition for skin pigmentation, comprising chaulmoogra oil and / or its components and an extract of Tribulus terrestris in a weight ratio Tribulus terrestris / chaulmoogra oil of between 1: 1,000 and 1: 5.

3. Composition according to claim 1, characterized in that the components of the oil of chaulmoogra are chaulmoogric, hydnocarpic and gorlic acids, as well as their salts and esters.

3. Composition according to claim 2, characterized in that it comprises the salts or esters of chaulmoogric, hydnocarpic and gorlic acids.

4. Composition according to Claim 3, characterized in that the salts and esters are chosen from methyl, ethyl or benzyl esters, as well as sodium or potassium salts.

5. Composition according to any one of the preceding revendica ^¬ tions, characterized in that the oil content of chaulmoogra, or acids or salts or esters is between 0.1 and 20% by weight relative to the total weight of the composition.

6. Composition according to claim 5, characterized in that the oil content of chaulmoogra, or acids or salts or esters is between 2 and 10% by weight relative to the total weight of the composition.

7. Composition according to claim 1, characterized in that the extract of Tribulus terrestris is obtained from seeds or fruits previously dried and crushed.

8. Composition according to claim 7, characterized in that the tribulus terrestris extract is a hydroglycolic extract.

9. Composition according to claim 1, characterized in that the extract content of Tribulus terrestris is between 0.01 and 0.5% by weight relative to the total weight of the composition.

10. Composition according to any one of the preceding revendi ^¬ cations, characterized in that the weight ratio of the tribulus terrestris extract to the oil of chaulmoogra in the composition is between 1: 200 and 1:20.

11. Composition according to any one of the preceding revendi ^¬ cations, characterized in that the oil content of chaulmoogra, or acids or salts or esters is between 2 and 10% by weight, and the extract content of Tribulus terrestris is between 0.05 and 0.2% by weight relative to the total weight of the composition.

12. Process for the cosmetic treatment of the skin, for the pigmentation of depigmented or non-pigmented areas, by application of a composition based on chaulmoogra oil and Tribulus terrestris extract on the area of the skin requiring such treatment .