EP0673237A1

EP0673237A1 - Use of a simaba extract to reduce patchy skin pigmentation.

Info

Publication number: EP0673237A1
Application number: EP94902015A
Authority: EP
Inventors: Frédéric Bonte; Alain Meybeck; Marc Dumas
Original assignee: LVMH Recherche GIE
Current assignee: LVMH Recherche GIE
Priority date: 1992-12-11
Filing date: 1993-12-10
Publication date: 1995-09-27
Also published as: JPH08507289A; EP0797982A1; FR2699081A1; US5676949A; WO1994013259A2; WO1994013259A3; FR2699081B1

Abstract

A simaba extract is used to produce a cosmetic or pharmaceutical, and particularly dermatological, composition or a skin cell culture medium. The resulting compositions are also disclosed. The simaba extract has a significant skin depigmentation activity and can enhance the protective function of the skin, particularly its water barrier function, as well as having a significant keratinocyte differentiation activity.

Description

Use of an extract of Simaba for the attenuation of skin pigment spots or to strengthen the protective function of the skin, or for the preparation of a culture medium for skin cells, and composition thus obtained.

The present invention essentially relates to the use of an extract of Simaba for the manufacture of a cosmetic or pharmaceutical composition, in particular dermatological, having in particular a depigmenting activity and an activity promoting the differentiation of keratinocytes, and intended in particular for attenuation skin pigmentation spots, especially spots of senescence, in the treatment of vitiligo, in strengthening the protective function of the skin, or in improving the appearance of the hair; or for the preparation of a culture medium for skin cells, and composition thus obtained.

More particularly the invention relates to the use of an extract of Simaba for the manufacture of a cosmetic or pharmaceutical composition, in particular dermatological, having in particular a depigmenting activity as well as an activity promoting the differentiation of keratinocytes, and intended for particular to treat skin pigment spots, especially spots of senescence, to treat vitiligo, skin disorders accompanied by dysregulations of the differentiation of keratinocytes, such as psoriasis, to restore, preserve and / or reinforce protective function of the skin, in particular the water barrier function, as well as the cohesion of the cells of the epidermis or even improving the quality of the hair; or for the preparation of a cell or tissue culture medium, in particular for the mass culture of skin cells, in particular of keratinocytes. The Simaba plant is a plant from the Simaroubaceae family.

Among the Simaba plants, we can cite Simaba cedron (Planchon), Simaba cuspidata (Sprucc), Simaba moretii, Simaba multiflora (Adr. Juss.) And Simaba guyanensis (Aublet Engl.).

These plants are known to have similar properties (see the works: "Traditional pharmacopoeias in Guyana" by Pierre GRENAND et al., Ed. ORSTOM, Paris 1987, pages 400-401 and 508-509; and "A Modem Hcrbal" by Mrs. M. GRIEVE, edited by Mrs. CF LEYEL, Ed. PENGUIN BOOKS, page 178).

In particular, Simaba cedron (Planch.) Is a tree from the forests of Central America, especially Guyana. The natives use the roots of this tree as a dewormer or as an antimalarial remedy. They use also the bark in decoction for the preparation of baths intended to treat rashes of various origins. Finally, the seeds of this tree have been used for a long time as a remedy against snake bites, both in local applications on the wound and in ingestion. However, the inventors of the present invention have been able to observe that the Simaba extracts are particularly active at the melanocyte level with regard to the inhibition of elanogenesis, this activity having been able to be demonstrated by in vitro tests on cultures. of melanocytes, which will be described later. In addition, it has been observed that the extracts of -Simaba are also particularly active in promoting the differentiation of keratinocytes and can thus be used to treat skin disorders accompanied by dysregulation of the differentiation of keratinocytes. This differentiation is reflected in particular, at the level of the epidermis, by greater cell cohesion, by regulation of the transformation of keratinocytes into comeocytes by loss of the nucleus and increase in cellular corneification, by an increase in the number of layers of comeocytes forming the stratum corneum, all of these phenomena contributing to the strengthening of the protective function of the skin vis-à-vis the external environment and to the strengthening of the water barrier preventing excessive loss of water by the epidemic; and, at the level of the hair follicle, a regulation of the processes of synthesis of keratins by keratinocytes, these proteins being the main constituent of the hair shaft and the quality of which is essential for a hair in good condition. In addition, the inventors have observed that these extracts make it possible to promote, accelerate and improve the differentiation of skin cells, in particular keratinocytes, during their culture in a culture medium.

Thus, the present invention aims to solve the new technical problem consisting in the supply of a new formulation of cosmetic or pharmaceutical compositions, in particular dermatological compositions having good depigmentation efficiency, thereby making it possible to use it for the treatment of pigment spots. skin, in particular spots of senescence, as well as, in the treatment of vitiligo, the attenuation of the contrast between the areas affected by vitiligo and their surroundings. It should be noted, in this regard, that the spots of senescence result from complex phenomena, generally comprising local hypercoloration linked to hyperpigmentation and sometimes accompanied by localized hyperkeratosis. The main object of the present invention is also to solve the technical problem consisting in the supply of a new formulation of cosmetic or pharmaceutical composition, especially dermatological, having an activity promoting the differentiation of keratinocytes and intended in particular for treat skin disorders accompanied by dysregulation of the differentiation of keratinocytes, such as psoriasis, to restore, preserve and / or strengthen the protective function of the skin, in particular the water barrier function, thus leading to a hydrating effect, by in particular by preventing excessive loss of water from the epidermis, an advantageous application of which is the treatment of ichthyotic skin as well as the treatment of psoriatic skin, and of improving the quality of the hair, thus enhancing the appearance of the hair .

The main object of the present invention is also to solve the new technical problem consisting in providing a solution making it possible to promote, accelerate and improve the differentiation of skin cells, in particular keratinocytes, in culture.

The present invention solves these technical problems satisfactorily for the first time and can be used on an industrial scale for the preparation of cosmetic or dermatological compositions, or for the preparation of culture media, in particular in mass of skin cells. Thus, according to a first aspect, the present invention relates to the use of an extract of Simaba for the manufacture of a cosmetic or pharmaceutical composition, in particular dermatological, having in particular a depigmenting activity, and intended in particular for treating spots skin pigments, especially age spots, to treat vitiligo, skin disorders accompanied by dysregulation of the differentiation of keratinocytes, such as psoriasis, to restore, preserve and / or strengthen the protective function of the skin, in particular the water barrier function, as well as the cohesion of the cells of the epidermis or to improve the quality of the hair; or for the preparation of a cell or tissue culture medium, in particular for the mass culture of skin cells, in particular of keratinocytes.

According to a particular characteristic, the abovementioned extract is obtained from Simaba cedron (Planchon), Simaba cuspidata (Spruce), Simaba moretii, Simaba multiflora (Adr. Juss.), Simaba guyanensis (Aublet Engl.), In particular from bark of trunks, stems or roots of these plants. According to another particular characteristic, the abovementioned extract is an extract obtained by extraction with at least one polar solvent, such as water, a alcohol, preferably a lower alcohol, such as methanol or ethanol, a glycol, in particular propylene glycol, or a hydro-alcoholic mixture in any propor¬ tion.

According to another particular characteristic, the above-mentioned extract is present in the composition at a concentration of between 0.001 and 5% by weight, expressed as dry extract, relative to the total weight of the composition, preferably between 0.01 and 1% and more preferably between 0.1 and 0.5% by weight.

According to another particular characteristic, the aforementioned composition contains, in addition, an active agent chosen from the group consisting of kojic acid and its salts or esters, caffeic acid and its salts or esters, hydroquinone and its derivatives, a mulberry extract, tretinoin, vitamin A or its derivatives or esters, a carotenoid, in particular beta-carotene, vitamin C, a corticosteroid, glutathione, cysteine, parahydroxycinnamic acid or its salts or esters, salts, esters and derivatives of the abovementioned substances being chosen from those which are cosmetically or pharmaceutically acceptable.

According to yet another particular characteristic, the aforementioned composition can also contain at least one moisturizing agent, such as hyaluronic acid.

According to another particular characteristic, the abovementioned mulberry extract is present in a proportion by weight of between 0.001 and 5%, preferably between 0.01 and 1%, more preferably between 0.1 and 0.8% by weight. relative to the total weight of the composition.

According to yet another particular characteristic, the kojic acid or its abovementioned salts or esters is present in the composition in a proportion by weight of between 0.001 and 5%, more preferably between 0.01 and 2%, by weight relative to the total weight of the composition.

According to another particular characteristic, the aforementioned extract of Simaba is at least partly incorporated in hydrated lipidic lamellar phases or in liposomes, either alone or combined with at least one other active substance present in the composition.

The preparation of liposomes containing at least part of the abovementioned Simaba extract can be carried out according to one of the known methods for incorporating active substances into liposomes.

According to a preferred embodiment, according to the present invention, a process is used for atomizing the constituents of the lipid phase, making it possible to obtain a lipid powder easily dispersible in an aqueous solution. to form liposomes, for example according to the method described in document US-A-4,508,703. The suspension of liposomes thus obtained can be homogenized using ultrasound, or in the case of mass production, at by means of pressure homogenization, in accordance with the process described in US-A-4,621,023.

The above-mentioned Simaba extract can be incorporated either in the lipid phase or in the aqueous phase of the liposomes in particular using the methods described above. In the case of incorporation into the lipid phase of the liposomes, the procedure can be as follows. The Simaba extract is dissolved with the constituents of the lipid phase, before atomization, in an organic solution containing at least one amphiphilic lipid, such as soy lecithin, optionally a hydrophobic liphophilic compound such as a stcrol, especially cholesterol or β-sitosterol. Preferably, the solvent is chosen from dichloromethane, chloroform or methanol, or one of their mixtures.

The organic solution can advantageously contain an antioxidant agent such as α-tocopherol.

The lipid powder obtained is dispersed in a suitable aqueous medium, for example a PBS buffer solution, a glucose solution or a sodium chloride solution. A suspension of liposomes is thus obtained.

According to an advantageous embodiment, and especially in the case of a liposome composition, after having optionally homogenized the composition obtained, the liposome compositions are gelled by mixing with a gel, such as a vinyl polymer gel, in particular marketed under the trade name Carbopol® 940. This gelation procedure is also described in US-A-4,508,703, in particular in the examples.

According to an advantageous embodiment of the invention, the concentration of the abovementioned Simaba extract is between 0.001 and 30% by weight, preferably between 0.01 and 10% by weight, of the lipid phase of said liposomes . According to a second aspect, the present invention also relates to a cosmetic, or pharmaceutical, in particular dermatological, composition preferably for topical application, having in particular a depigmenting activity, as well as an activity promoting the differentiation of keratinocytes, and intended in particular for treating skin pigment spots, in particular age spots, to treat vitiligo, skin disorders accompanied by dysregulation of the differentiation of keratinocytes, such as psoriasis, restore, preserve and / or strengthen the protective function of the skin, in particular the water barrier function, thus in particular making it possible to obtain a hydrating effect by preventing excessive loss of water by the epidermis, thus allowing use in particular for the treatment of dry skin, whatever the degree of dryness, including ichthyotic skin, and psoriatic skin, to improve the quality of the hair, thus leading to an improvement of the hair, characterized in that it comprises a cosmetically or pharmaceutically, in particular dcrmatologiqucmcnt, effective amount of a Simaba extract. Other particular characteristics of the cosmetic or pharmaceutical composition, in particular dermatological, appear clearly from the preceding description concerning the various particular characteristics of the use and also appear to the skilled person from the complete description of the invention, in particular illustrated by the examples which follow.

A composition according to the invention described above containing an extract of Simaba, optionally in form at least partially incorporated in hydrated lipidic lamellar phases or in liposomes, can be in different forms usable in cosmetics or in dermatology. For example, these compositions can be gels, creams, milks or lotions.

These compositions, applied to the areas to be treated of the skin, or of the scalp, have the effect of regulating the differentiation of keratinocytes, thereby promoting the formation and restoration of good quality epidermis, in particular at the layer cornea, to reinforce the protective skin barrier function of the epidemic, in particular of water barrier, and to obtain an embellishment of the hair, as was explained above as regards the effects and the advantages of this differentiation .

Thus, according to a particular embodiment, the cosmetic or dermatological composition according to the invention has a hydrating power, in particular by preventing excessive loss of water in the epidemic, and can be intended for the treatment of dry skin. , especially ichthyotic skins.

According to another particular embodiment, the cosmetic or dermatological composition according to the invention makes it possible to restore the normal differentiation of keratinocytes during their transfomiation into comeocytes, accom- worsened by the loss of the nucleus and cellular comification. Thus, this composition can be intended for the treatment of psoriatic skin.

In this context, usually the Simaba extract is incorporated into a cosmetically or dermatologically acceptable excipient. Also, the Simaba extract can be incorporated into hydrated lipid lamellar phases or liposomes, as described for the first previous aspect.

According to a third aspect, the present invention also relates to a cell or tissue culture medium, in particular for the culture of skin cells, in particular of keratinocytes, making it possible to promote, accelerate and improve their differentiation, characterized in that it includes an effective amount of a Simaba extract to achieve such differentiation.

According to a fourth aspect, the invention also relates to a method for promoting, accelerating or improving the differentiation of skin cells, in particular keratinocytes, in particular in the context of a mass culture of skin cells, for the production of skin. artificial or for the preparation of reconstituted skin models, characterized in that a culture medium is used as defined in the preceding description, or in the following description taken as a whole.

The process according to the invention called cell differentiation presents in particular a great industrial interest. We will quote for example:

- the production of biochemical mediators by treatment of mass culture of keratinocytes in bioreactors;

- the preparation of artificial skin in order to carry out skin grafts, the method of the invention being particularly advantageous in the case of self-grafts of burn victims, thanks to the time savings it provides in the preparation of artificial skin. In particular, the acceleration and improvement of the differentiation of keratinocytes results in the faster formation of a good quality horny layer;

- the production of skin models comprising reconstituted skin, intended for example for evaluating skin penetration or the toxicity of a substance or composition applied topically, when it is intended in particular for local treatment or for systemic treatment (transderme), in particular.

According to a particular variant embodiment, this culture method will generally comprise the preparation of a culture medium for the growth of human keratinocytes comprising a DMEM nutritive base medium (Gibco®), epidermal growth factor ("EGF"), 10% fetal calf serum, isoproterenol and / or forskolinc, as well as hydrocortisone. In the context of the invention, this medium also comprises an extract of Simaba as described in the preceding or following description, generally at a concentration of 0.01 to 0.5% by weight.

In this method, a mass culture of skin cells is carried out by inoculating keratinocytes in order to immobilize them on supports such as hollow fibers, microbeads, or microporous matrices and this using the culture medium above. . One can ensure an infusion of the medium in order to have a sufficient contribution to growth and differentiation even when the biomass is high.

The culture medium according to the invention can advantageously be used for the mass culture of skin cells, in particular keratinocytes, for the production of artificial skin or for the preparation of reconstituted skin models.

According to a fifth aspect, the present invention also covers a cosmetic or dermatological treatment process for the phenomena of cutaneous hyperpigmentation, comprising in particular spots of senescence and other cutaneous pigment spots, characterized in that one applies, at least to the skin pigment spots to be treated, an effective amount of a Simaba extract to obtain their attenuation. Advantageously, this extract is obtained from the plant of Simaba cedron (Planch.), In particular from the bark. Particular extracts have been described in the preceding description.

According to another embodiment, the present invention also relates to a cosmetic or dermatological treatment process for vitiligo, charac¬ terized in that it is applied, at least to the pigmented areas of the skin bordering those depigmented by vitiligo, an effective amount of a Simaba extract to obtain attenuation of the pigmentation of said pigmented areas, thereby attenuating the contrast between the pigmented areas affected by vitiligo and their surroundings, which improves the aesthetic appearance of the subject.

For one or other of the preceding aspects, the extract can be applied in the form of a composition containing a Simaba extract at a concentration of between 0.001 and 5%, expressed as dry extract, relative to the total weight of the composition, preferably between 0.01 and 1% and more preferably between 0.1 and 0.5% by weight. In the case of use in a culture medium, according to a particular embodiment, it is possible to use from 0.01 to 0.5% by weight of Simaba extract, relative to the total weight of the final culture medium .

On the other hand, according to a particularly advantageous embodiment, one can introduce into the cosmetic or dermatological composition, a complementary active principle, in particular a hydrating agent such as hyaluronic acid.

On the other hand, in the context of the invention, when the composition is a cosmetic composition, the abovementioned Simaba extract can be advantageously incorporated in a cosmetically acceptable excipient.

Likewise, when this composition is a pharmaceu¬ tic, in particular dermatological, composition, the aforementioned Simaba extract can be incorporated in a pharmaceutically acceptable, in particular dermatologically, excipient. Such excipients are well known to those skilled in the art and also result from the description of several examples of compositions which will follow.

According to a particular variant, the excipient of the cosmetic composition, or that of the pharmaceutical composition, is at least partly made up of very small solid particles, in particular of the order of 0.05 μm to 100 μm , and the Simaba extract, as active substance, is distributed, at least in part, on the surface of said particles, as described in French patent application FR-A-2 685 635.

Thus, other objects, characteristics and advantages of the invention will appear clearly in the light of the explanatory description below made with reference to several illustrative examples of the invention which therefore cannot in any way limit the scope of the invention. In the present description, including the examples, the percentages are given by weight unless otherwise indicated.

Example 1 of the invention Aqueous extract of Simaba bark

From an extract of the root bark of the Simaba cedron tree (Planch.) From Guyana, an extraction is carried out with water by Soxhlet extraction, that is to say at reflux. for several hours. The solvent-shell ratio is generally 10-1 by weight. After extraction, this extract is generally concentrated for cosmetic or pharmaceutical use. The concentrated extract is called product II.

Naturally, as is easily understood by those skilled in the art, the removal of the solvent, in this case water, can be continued until a dry extract is obtained, by evaporation under reduced pressure or by lyophilization.

EXAMPLE 2 OF THE INVENTION From 100 g of root bark of the Simaba cedron tree (Planch.) From Guyana, an extraction is carried out with 1 l of methanol according to the so-called Soxhlet method, for several hours. at reflux.

After extraction, the methanolic extract is concentrated until a product containing very little methanol called product 12 is obtained.

Example 3

Demonstration of the depigmenting activity of the extracts according to the invention

The depigmenting activity of the extracts according to the invention is determined at different non-cytotoxic concentrations on melanocyte cultures of the Cloudman S91 line originating from ATCC and bearing the reference CCL 53.1 clone M-3. Each concentration of extract is tested on three culture dishes. The culture dishes are prepared by introducing 200,000 S91 cells per dish into a culture medium consisting of complete EMEM medium containing 2% v / v of fetal calf serum and 0.08 μg / ml of mitomycinc C.

After 24 h of incubation at 37 ^° C under humid atmosphere, the medium is replaced with an identical medium containing the test products but now without mitomycin C.

5 days of incubation after renewal of the medium, the melano¬ cytes are counted with a counting device such as the "Counter Coulter®" and the melanin content of the cells is determined by measuring the absorbance at 405 nm then by transfoming this value in quantity of melanin by means of a standard curve established with synthetic melanin from the company Sigma which establishes a linear relationship between known quantities of melanin and the absorbance at 405 nm. The amount of melanin is then reduced to 10 ^ melano¬ cytes. In parallel, a control culture of melanocytes was carried out in three dishes, with the same cell line and under the same culture conditions. Turkish, except that the renewal medium introduced 24 h after incubation contains no product to be tested according to the invention.

The results obtained by calculating for each concentration of extract the average over the three boxes used, are listed in Tables I and II for each of the products II and 12 of Examples 1 and 2.

The significance of the results is determined by the Student t statistical test with a threshold of 0.05, which compares the cultures treated with the products, to the control cultures, not treated.

TABLE I

TABLE II

S = significant.

Depigmenting activity A is calculated as follows

T - P

A = x lOO

in which

T = melanin content in micrograms for 10 ^ cells of the control sample and P = melanin content in micrograms for 10 "cells of the test product II or

12. It appears, from table I, that product II produces a depigmenting activity of at least 19% compared to the control, even at the very low dose of 1 μg / ml, this activity increasing up to 54% for a dose of lϋ g / ml.

On the other hand, from Table II, it can also be seen that the product 12 provides a depigmenting activity of at least 24.3% at the low dose of 2.8 μg / ml. Thus, products II and 12 exhibit significant depigmenting activity.

Furthermore, the activity of products II and 12 is significant from the first tested dose of 0.1 μg / ml.

EXAMPLE 4 Activity Test of Simaba Cedron Extract on the Differentiation of Keratinocytes

For the present study, a skin sample taken during a breast plasty operation of a woman of twenty years is used. On day D = -2, 12 wells are inoculated with a multi-well culture plate with 300,000 cells per well of keratinocytes isolated from this sample in a conventional culture medium for the culture of keratinocytes, well known of those skilled in the art. Advantageously, this culture medium can be constituted by an M199 medium supplemented with 2 mM of L-glutamine, 10% of fetal calf serum, cholera toxin, hydrocortisonc, as well as the epidermal growth factor EGF.

On days D = 1, D = 5, D = 8 ct D = 11, the culture medium is renewed by an identical medium on 6 of the 12 wells which constitute untreated control wells, and, on the 6 remaining wells, constituting the treated wells are renewed with an identical medium, but in which 2.5 μg / ml of extract of Simaba cedron as obtained in Example 2, or product 12, have been dissolved and on which a sterilizing filtration is carried out on filter, for example at 0.22 μm.

On day D = 12, a cell count is carried out as well as inclusion of the cells in epoxy resin after fixing and labeling of the cell structures with uranylc acetate.

Sections of each of the resin blocks thus obtained are produced in the form of ultrafine sections, for example using a device known by the name of Microtome, and this is observed with a transmission electron microscope.

The sections of the six control cultures were then observed, on the one hand, and the sections of the six cultures treated with the extract of Simaba cedron. on the other hand. It has been observed that the cells treated with the Simaba Cedron extract according to the invention, for example the extract 12 obtained in Example 2, have a cell differentiation clearly superior to the control, namely:

- from 5 to 10 cell layers while the control has 3 to 6 cell layers on average;

- the upper layers of the treated cultures exhibit regular comification with well-formed long comocytes and little intercellular space, while the upper layers of the control cultures generally have undifferentiated comocytes and non-uniform stratification; - the desmosomes of the intermediate layers of the treated cultures are large and well differentiated, while the desmosomes of the control cultures are only at the beginning of a differentiation;

- In addition, the upper layers of the treated cultures have a density of keratin very much higher than the density of keratin observed in the same upper layers of the control cultures.

These clearly superior results on cell differentiation obtained with the Simaba cedron extract according to the invention makes it possible to envisage the application of Simaba cedron extracts to the preparation of cosmetic or pharmaceutical compositions, in particular dermatological compositions, in particular the following:

- compositions promoting the formation of a well-differentiated epidermis, thus giving "beautiful" skin with a pleasant texture and feel;

- compositions combating disorders of epidermal differentiation, occurring in particular during aging; - compositions reinforcing the skin barrier therefore protecting from external aggressions, for example by allergens or surfactants, on the one hand, and limiting the excessive loss of water by the epidemic, on the other hand;

- compositions making it possible to improve the quality of the hair, in particular by improving the quality of the keratin of the hair shaft originating from the activity of the keratinocytes of the follicles which differentiate from the base of the hair.

Also, it is possible to envisage the application of these extracts to the preparation of cell culture media, in particular intended for the mass culture of keratinocytes, or of compositions intended for treating keratinocyte cultures to promote the speed and / or the quality of the architecture of these cultures when they are, in particular, intended to make skins artificial, called equivalent for grafts, in particular on burns, or else for the preparation of models intended to evaluate the toxicity or the skin penetration of various tested substances.

Various examples of cosmetic or pharmaceutical compositions, in particular demiatological compositions according to the invention, using an extract of Simaba are given below.

Example 5 Cosmetic or dermatological depigmenting composition in the form of a gel

- lyophilized aqueous extract of Simaba cedron bark from Example 1 .... 0.3 g

- propylene glycol 4 g

- ethanol 3 g

- 2% Carbopol 940® gel + preservative q.s.p. 100g

This gel is used topically twice a day for 6 weeks on the areas of the skin to be depigmented.

Example 6 Cosmetic or dermatological composition containing an extract of Simaba in liposomes, in the form of a gel

0.3 g of product 12 of Example 2 is taken, which is dissolved in 50 ml of a dichloromethane / methanol mixture 8-2 (v / v) to which 9 g of soy lecithin and 1 are added. g of cholesterol. The solution obtained can be treated to obtain a suspension of liposomes by the well-known rotary container evaporation process which consists in depositing a lipid layer in the flask of the rotary evaporator by evaporation of the solvent, then adding water or a suitable aqueous solution containing preservatives, well known to those skilled in the art, to obtain 100 g of aqueous suspension of liposomes.

This suspension can be homogenized by means of ultrasound at the power of 150 W for 10 min at 4 ^* C.

The homogenized liposome suspension obtained is then gelled by addition of an equivalent weight of 2% Carbopol 940® gel, neutralized with triethanolamine. A gelled composition is thus obtained containing 0.15% by weight of product 12 incorporated at least in part into the lipid bilayer of the liposomes, relative to the total weight of the composition.

This composition can be applied locally, for example once a day for 4 weeks, in order to reduce age spots.

EXAMPLE 7 Depigmenting Gel

- aqueous extract of root bark of Simaba cedron (Planch.) according to example 1 0.2

- mulberry extract 0.35

- ascorbic acid 0.6

- Carbopol 940® 2,0 - water + preservatives q.s.p. 100g

This gel is used in local applications on stains.

EXAMPLE 8 Whitening Cream intended to lighten the skin

- methanolic extract of Simaba cedron root bark (Planch.) according to Example 2 0.3

- Héliocarotte® 1,5 - mineral oil 4,0

- kojic acid 0.5

- glyceryl stearate 4.0

- PEG 30 glyceryl stearate 3

- water + preservative q.s.p. 100g

HELIOCAROTTE® is in the form of an oil titrated at 0.05% in β-carotene. It is commercially available from BERTIN, Courbevoie - France.

This cream is used in local applications, twice a day for four-week courses. EXAMPLE 9 Depigmenting Dermatological Gel

- ethanolic extract from the root bark of

Simaba Cedron (Planch.) 0.45

- retinoic acid 0.02

- alcohol 30.0

- Carbopol 940® 2,5

- water + preservative and antioxidant q.s.p. 100g

The aforementioned ethanolic extracts were obtained according to the process described in Example 2, except that the methanol was replaced by ethanol.

This gel is used in application on dark areas, twice a day, for three weeks. Example 1

Skin lightening milk

- aqueous extract of Simaba guyanensis root bark (Aubl. Engl.) prepared according to the extraction method described in Example 1 1.5 - hydroquinone 1.5

- Héliocarotte® 2

- wheat germ oil 3

- tocopherol linoleate 0.5

- glycerin 2 - Carbopol 0.5

- glyceryl stearate 2.0

- PEG 30 glyceryl stearate 3.0

- perfumes + solubilizer 0.15

- water + preservative q.s.p. 100g

This milk is used in application after the shower on the parts of the body to be treated, once a day by cures of two to three weeks.

This cream is used in local applications, twice a day for four-week courses. EXAMPLE 1 Dermatological composition for the restoration of the hydrous barrier of the epidermis

- aqueous extract of root bark

Simaba Cedron (Planch.) From Example 1 0.5 g

- Cremophor RH 40® 1 g

- Carbomer 980® 0.2 g

- triethanolamine 0.18 g - PEG 6-30 of stearate 7 g

- Cetyl alcohol 2 g

- vegetable oils 25 g

- aqueous excipient scented q.s.p. 100g

The components are mixed in a conventional manner to obtain a treating emulsion applied morning and evening by lightly massaging the areas to be treated. This emulsion is used as a day and / or night cream.

Example 12 Dermatological composition for the treatment of psoriatic skin

- methanolic extract of Simaba cedron from Example 2 0.3 g

- 2% Carbopol 980® gel 35 g

- perfumed aqueous excipient q.s.p 100 g

The Simaba Cedron extract is introduced into the aqueous excipient to disperse it, then the Carbopol gel is added so as to obtain a gelled composition which is applied locally to the lesions for 6 weeks.

Example 13

Cosmetic composition for maintaining a satisfactory state of hydration of the epidemic

- methanolic extract of Simaba cedron from Example 2 0.18 g - α-bisabolol 0.1 g

- hyaluronic acid 1 g - urea 3 g

- cosmetic excipient acceptable in the form of a body milk q.s.p 100 g

Apply the milk on the areas to be treated, in particular on the legs after hair removal. This composition makes it possible in particular to reinforce the skin water barrier function of the epidemic by improving the epidermal inter-cellular cohesion. It thus allows the skin to maintain a satisfactory state of hydration.

EXAMPLE 14 Liposomal Cosmetic Composition for Balancing the Exfoliation of the Corneal Layer of the Epidemic and Giving a Smooth Epidemic

- methanolic extract of Simaba cedron from Example 2 0.1 g

- soy lecithin 2 g

- β-sitosterol 0.2 g

- cosmetic cream emulsion based on squalane of oil-in-cau type, q.s.p 100 g

In a first step, an aqueous suspension of liposomes is prepared encapsulating the methanolic extract of Simaba cedron of Example 2 in the lipid phase of said liposomes. To do this, we proceed as follows. 0.1 g of the methanolic extract of Simaba cedron from Example 2, 2 g of soy lecithin and 0.2 g of β-sitosterol are dissolved in 50 ml of a 4: 1 mixture of dichloromethane and methanol . This solution is evaporated under reduced pressure (200 mm Hg approximately) in a rotary flask heated at 45 ^° C. The lipid film obtained is taken up in 25 ml of an aqueous solution of monopotassium phosphate and 0.2 g 1 phosphate disodium at 1.44 g / 1, with stirring for 1 h.

This gives about 25 ml of a suspension of liposomes encapsulating the extract of Simaba cedron which is then subjected to homogenization with ultrasound (15 min, 150 W, 4 ^* C). In a second step, the suspension of liposomes obtained is conventionally incorporated into the emulsified excipient, in order to obtain the cream according to the invention.

The cream can be applied daily to the face in the event of rough skin flaking off.

This composition strengthens the cohesion of the stratum corneum and regulates the detachment of dead cells.

Example 15 Cosmetic composition containing an extract of Simaba cedron. intended for the preventive treatment of dry skin

- methanolic extract of Simaba cedron obtained in Example 2..0.5 g

- excipient emulsion cream of the oil-in-cau type, q.s.p ... 100 g

The methanolic extract of Simaba cedron of Example 2 is conventionally incorporated into the cream emulsion excipient, to obtain the cream according to the invention.

You can apply the cream daily to the parts of the body that you want to treat.

EXAMPLE 1 Cosmetic Composition Containing an Simaba Extract intended for the Treatment of Ichthyotic Skin

- glycerol 3 g

- hvaluronic acid 1 g

- aqueous extract of Simaba cedron from Example 1 0.2 g

- cream emulsion type oil-in-cau excipient, q.s.p. 100g

The aqueous extract of Simaba cedron obtained in Example 1, as well as glycerol and hvaluronic acid, is conventionally incorporated into the cream emulsion excipient to obtain the cream according to the invention.

This cream is applied daily to the desired areas of the skin, until a much smoother skin is obtained thanks to a regular renewal of the stratum corneum. Example 17 Preparation of a Culture Medium and its Use for the Mass Culture of Skin Cells

For the preparation of this culture medium as well as for its use for the mass culture of skin cells, those skilled in the art may refer in particular to the article by Philip C. Famillctti et al. in Biotcchnology, vol. 6, January 1988, pages 41 to 44 as well as to the references cited in this article, in particular Arathoon et al. in the journal Science (1986), 232. page 1390 et seq. the article by Ku, K. et al. in the journal Biotcchnology, Biocng. (1980), 23, pages 79 et seq.

Thus, an optimal culture medium is prepared for the growth of human keratinocytes comprising a nutritive base medium DMEM (Gibco®), growth factor EGF, 10% fetal calf serum, isoproterenol and / or forskolin, as well as hydrocortisonc. This medium will comprise an extract of Simaba according to the invention, preferably at a concentration of 0.01 to 0.5% by weight of extract of Simaba relative to the total weight of the final culture medium.

A culture based on skin cells is carried out by inoculating keratinocytes in order to immobilize them on supports such as hollow fibers, microbeads, or microporous matrices and this using the culture medium described above. An infusion in a medium in an infusion system of the type described by Sylvie GUICHARD-BALESTR1NI in the journal Biofutur, supplement no.56, April 1987, pages 2 to 14, will be provided in order to have an adequate contribution to the growth and differentiation even when the biomass is large.

At the end of the culture, the substances secreted by the keratinocytes are recovered, containing mainly lipids, sources of raw material for the formulation of cosmetic or pharmaceutical compositions for topical application to the epidemic or the scalp. Thanks to the product of the invention, it is possible to treat reconstituted skin cultures, in particular cultures of keratinocytes or other epidermal cells cultivated on an appropriate support, such as a collagen support (containing or not containing fibroblasts). , for example described in the document La Recherche, (1987), n \ 185 _. pages 149-159 and in Br. J. Dermatol. (1986), 114. pages 91-101, or a support constituted by half excised. Thanks to the use of the composition according to the invention, a faster and more complete epidermization will be obtained. This will allow in a shorter time to provide doctors with reconstituted skin, a kind of biological dressing for autografts, for example in the case of extensive burns. The invention will also allow industrial and competitive production of reconstituted skins of good quality to carry out penetration or tolerance tests.

The invention naturally includes all the means constituting the technical equivalents of the means described as well as their various combinations. The invention also covers any characteristic which appears to be new with respect to any prior art, from the present description taken as a whole.

Claims

1. Use of an extract of Simaba for the manufacture of a cosmetic or pharmaceutical composition, in particular a dermatological composition, exhibiting in particular a depigmenting activity as well as an activity promoting the differentiation of keratinocytes, and intended in particular for the treatment of skin pigment spots, especially spots of senescence, to treat vitiligo, skin disorders accompanied by dysregulation of the differentiation of keratinocytes, such as psoriasis; to restore, preserve and / or strengthen the protective function of the skin, in particular the water barrier function, as well as the cohesion of the cells of the epidermis or else to improve the quality of the hair; or for the preparation of a cell or tissue culture medium, in particular for the mass culture of skin cells, in particular of keratinocytes.

2. Use according to claim 1, characterized in that the aforementioned extract is obtained from Simaba cedron (Planchon), Simaba cuspidata (Spruce), Simaba moretii, Simaba multiflora (Adr. Juss.) Or Simaba guyanensis (Aublet Engl .), in particular from the trunks, stems or roots of these plants.

3. Use according to claim 1 or 2, characterized in that the abovementioned extract is an extract obtained by extraction with at least one polar solvent, such as water, an alcohol, preferably a lower alcohol, such as methanol or ethanol, a glycol, in particular propylene glycol, or a hydroalcoholic mixture in any proportion.

4. Use according to one of the preceding claims, characterized in that the above-mentioned extract is present in the composition at a concentration of between 0.001 and 5% by weight, expressed as dry extract, relative to the total weight of the composition, preferably between 0.01 and 1.% and more preferably between 0.1 and 0.5% by weight.

5. Use according to one of the preceding claims, characterized in that the aforementioned composition contains, in addition, an active agent chosen from the group consisting of kojic acid and its salts or esters, caffeic acid and its salts or esters, hydroquinone and its derivatives, a mulberry extract, tretinoin, vitamin A or its derivatives or esters, a carotenoid, in particular beta-carotene, vitamin C, a corticosteroid, glutathione, cysteine, parahydroxy acid cinnamique or its salts or esters, the salts, esters and derivatives of the aforementioned substances being chosen from those which are cosmetically or pharmaceutically acceptable.

6. Use according to one of the preceding claims, characterized in that the above composition can also contain at least one moisturizing agent, such as hyaluronic acid.

7. Use according to claim 5 or 6, characterized in that the abovementioned mulberry extract is present in a proportion by weight of between 0.001 and 5%, preferably between 0.01 and 1%, more preferably between 0, 1 and 0.8% by weight relative to the total weight of the composition.

8. Use according to claim 5 or 6, characterized in that the kojic acid or its aforementioned salts or esters is present in the composition in a proportion by weight of between 0.001 and 5%, more preferably between 0.01 and 2 %, by weight relative to the total weight of the composition.

9. Use according to one of the preceding claims, characterized in that the aforementioned extract of Simaba is at least partly incorporated in hydrated lipidic lamellar phases or in liposomes, either alone or combined with at least one other active substance present in the composition.

10. Use according to claim 9, characterized in that the concentration of the abovementioned Simaba extract is between 0.001 and 30% by weight, preferably between 0.01 and 10% by weight, of the lipid phase of said liposomes.

11. Cosmetic or pharmaceutical composition, in particular derma¬ tological, preferably for topical application, having in particular a depigmenting activity, as well as an activity promoting the differentiation of keratinocytes, and intended in particular for the treatment of skin pigment spots, especially spots senescence, to treat vitiligo, skin disorders accompanied by dysregulation of the differentiation of keratinocytes, such as psoriasis, to restore, preserve and / or strengthen the protective function of the skin, in particular the function of water barrier, in thus making it possible in particular to obtain a moisturizing effect by preventing excessive loss of water by the epidermis, thereby allowing use in particular for the treatment of dry skin, whatever the degree of dryness, including ichthyotic skin, and psoriatic skin, to improve the quality of the hair, thus leading to an beautifier of the hair, characterized in that it comprises a cosmetically or pharmaceutically, in particular demologologically, effective amount of an extract of Simaba, optionally incorporated in a cosmetically or pharmaceutically excipient, in particular demologologiquemcnt, acceptable.

12. Cosmetic composition according to claim 11, characterized in that the excipient is at least partially made up of very small solid particles, in particular of the order of 0.05 μm to 100 μm, and that the extract of Simaba, as active substance, is distributed, at least in part, on the surface of said particles.

13. Cosmetic composition according to claim 11 or 12, characterized in that the composition is as defined in any one of claims 2 to 10.

14. Culture medium for cells or tissues, in particular for the culture of skin cells, in particular keratinocytes, making it possible to promote, accelerate and improve their differentiation, characterized in that it comprises an effective amount of 'an extract from Simaba to obtain such a differentiation.

15. Culture medium according to claim 14, characterized in that it comprises from 0.01 to 0.5% by weight of Simaba extract relative to the total weight of the final culture medium.

16. Culture medium according to claim 14 or 15, characterized in that it is used for the mass culture of skin cells, in particular keratinocytes, for the production of artificial skin or for the preparation of reconstituted skin models .

17. Method for promoting, accelerating or improving the differentiation of skin cells, in particular keratinocytes, in particular within the framework of a mass culture of skin cells, for the production of artificial skin or for the preparation of skin models reconstituted, characterized in that a culture medium is used as defined in one of claims 14 to 16.

18. The method of claim 17, characterized in that a culture medium is prepared for the growth of human keratinocytes comprising a nutritive base medium DMEM, an epidermal growth factor EGF, 10% fetal calf serum, l isoproterenol and / or forskolinc, as well as hydro-cortisone.

19. Culture method according to claim 17 or 18, characterized in that a mass culture of skin cells is carried out by inoculating kerati¬ nocytes in order to immobilize them on supports such as hollow fibers, microbills, or microporous matrices, possibly ensuring a perfusion of the medium in order to have a sufficient contribution to growth and differentiation even when the biomass is high.

20. A method of cosmetic treatment of the phenomena of cutaneous hyperpigmentation, comprising in particular the spots of senescence and other cutaneous pigment spots, characterized in that an effective amount is applied, at least to the cutaneous pigment spots to be treated 'A Simaba extract to obtain their attenuation, this extract being advantageously obtained from the Simaba plant mentioned in claim 2, in particular from the bark.

21. Cosmetic treatment process for vitiligo, characterized in that at least on the pigmented areas of the skin neighboring those depigmented by vitiligo, an effective amount of a Simaba extract is applied to obtain the attenuation of the pigmentation of said pigs. pigmented areas, this Simaba extract advantageously being obtained from the plant as defined in claim 2, in particular from the bark.