FR2829390A1 - Use of plant, yeast or fungal lipid extracts containing ergosterol in cosmetic compositions for enhancing the barrier function of the skin - Google Patents

Abstract

Plant, yeast or fungal lipid extracts containing ergosterol are used in cosmetic compositions for enhancing the barrier function of the skin. Independent claims are also included for: (1) a shiitake extract with an ergosterol content of about 20 wt.%; (2) a cosmetic composition comprising the extract of (1).

Description

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 USE OF A SHIITAKE EXTRACT IN COSMETIC COMPOSITIONS FOR SKIN CARE.

 The present invention relates to the use in a cosmetic composition or the preparation thereof, of an effective amount of a shiitake extract containing ergosterol to enhance the cutaneous barrier function of the skin.

 In general, few active agents have been described as acting on the one hand on proliferation and on the other hand on the differentiation of normal human keratinocytes. In fact, these active agents are: either inducing cell proliferation, and then promoting the expression of proliferation markers at the level of the epidermis or the synthesis of DNA, inducing cell differentiation, and then promoting the expression markers of differentiation.

 Thus, until now, anti-aging cosmetic active ingredients claim an effect on the increase of cell renewal and epidermal thickening thanks to the presence of proliferation-inducing active agents. This is the case of AHAs which by inducing surface mechanical desquamation cause an increase in the cellular renewal of the epidermis. Retinoids, widely used as anti-aging actives, are also known for their effectiveness on increasing the proliferative power of cells.

 By acting on the proliferation of keratinocytes, these active agents produce a homeostatic imbalance of the epidermis which is exerted to the detriment of a barrier function of the skin which is altered. This results in a decrease in the barrier function of the skin that can cause irritative reactions.

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 Thus today, AHAs are much less successful because of their irritative action on the skin. The retinoid family knows limits in its use in cosmetics because of the well-known teratogenic side effects of these molecules.

 It is known from French Patent Application No. 9711655, that an extract of shiitake containing ergosterol is capable of acting on increasing the proliferation of keratinocytes in culture. More particularly, this extract acts on the renewal of the epidermal cells by increasing their mitotic activity, resulting in a thickening of the epidermis.

Shiitake, also known as Lentinus anodes, is a mushroom of Asian origin known for its medicinal and nutritional properties, of which a lipid fraction has been found to be rich in ergosterol.

 The Applicant has now demonstrated that an extract of shiitake containing ergosterol is capable of stimulating the expression of differentiation markers and thus finds a new cosmetic utility to enhance the barrier function of the skin.

 Thus, this active ingredient, already known to stimulate the proliferation of epidermal keratinocytes and thus to increase cell renewal, also makes it possible, unexpectedly, to maintain a cutaneous barrier that would tend to be altered during aging.

 An active with this dual activity is beneficial to the skin because it gives it a younger appearance with a reduction of wrinkles while providing moisture and protection vis-à-vis external agents.

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 Few assets offer this dual activity. However, mention may be made of tricholine citrate, the efficacy of which has been reported to stimulate the growth of keratinocytes and the synthesis of phospholipids in these cells.

 The present invention therefore relates to the use in a cosmetic composition or for the manufacture of a cosmetic composition of a plant, yeast or mushroom lipid extract containing ergosterol to enhance the barrier function of the skin. The invention particularly relates to a shiitake extract.

Such an extract is therefore remarkable because it not only promotes the proliferation of epidermal cells (epidermal renewal) but also shows in addition a pro-differentiating activity (reinforcement of the skin barrier function). Both activities have been demonstrated through cell and skin model tests:
The increase in the proliferation of epidermal cells is demonstrated on epidermal cells cultured for 6 days and revealed by an MTT test on the one hand. Moreover, this activity is verified on a second protocol in which the epidermal cells are counted after 48H of culture.

 The demonstration of a pro-differentiating activity is evaluated by counting the horny envelopes formed after 48H of cell culture and by assaying a keratinocyte differentiation marker, the membrane transglutaminase after 5 days of cell culture.

 These results are confirmed on a "Skinetic" reconstructed skin model in which the effect of the extract on the expression of genes encoding

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 proteins of potential interest in cutaneous physiology such as proliferative marker proteins and marker proteins of a pro-differentiating state.

The lipid extract of shiitake used in the cosmetic compositions of the invention can be obtained from crushed mushrooms, for example in a supercritical type plant in the presence of CO 2, as described in French Patent No. 9711655. After extraction, the ergosterol concentration can be adjusted, and it is preferred for the use according to the invention, a shiitake extract containing from 3 to 50%, preferably from 15 to 25% and most preferably of the order of 20% by weight. weight of ergosterol. A shiitake extract according to the invention preferably comprises the following compounds, the amounts of which are expressed by weight relative to the weight of the extract: - ergosterol: 3 to 50% - triglycerides: 5 to 95% - free fatty acids: 0 to 30% - Tocopherol: 0.1 to 2% - Water: 1 to 30%
An example of shiitake extract according to the invention comprises: - 22% ergosterol - 40% triglycerides - 25% water - 15% free fatty acids - 0, 5% tocopherol
Such a lipid extract of shiitake comprising of the order of 20% of ergosterol has never been described in the prior art. This extract can be obtained by supercritical CO2 extraction from ground fungus under the following conditions: - pure supercritical carbon dioxide

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 - pressure = 500 bar - temperature = 50 oC - gas flow = 30 kg CO2 per kg of raw material.

 The invention therefore also relates to a shiitake extract comprising of the order of 20% by weight of ergosterol and the compositions, including cosmetic, the container.

 The cosmetic composition according to the invention comprises an effective amount of the lipid extract containing ergosterol to enhance the barrier function of the skin. This amount represents from 0.001 to 1% of the total weight of the composition and preferably from 0.005 to 0.01 of the total weight of the composition.

 The cosmetic composition according to the invention may comprise other active agents such as, for example, non-limiting agents: antiradical agents such as extracts of plants rich in flavonoids or pure flavonoids, plant extracts rich in nutrients and trace elements promoting growth. skin cells.

 The composition also includes one or more formulating agents, such as antioxidants, surfactants, preservatives, oils, glycols, perfumes etc.

 A composition according to the invention may be in any form known to those skilled in the field of cosmetics, such as, for example, a cream, a milk, a lotion, a gel or a mask, without any particular pharmaceutical restriction. other than those for application to the skin.

 Other advantages and characteristics of the invention will appear on reading the following examples concerning the preparation and the activity of

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 the shiitake extract according to the invention, and where reference will be made to the appended drawings in which: FIG. 1 represents the proliferation of normal human keratinocytes in the presence of 20% ergosterol shiitake extract.

 FIG. 2 represents the keratinocyte growth gain relative to the negative control after 6 days of culture. FIG. 3 represents the effect of shiitake at 20% ergosterol on cell proliferation after 48H of culture.

 FIG. 4 represents the effect of 20% ergosterol shiitake on the 48H cell cornification of culture.

 - Figure 5 shows the effect of shiitake 20% ergosterol on cell proliferation after 5 days of culture.

 - Figure 6 shows the effect of shiitake 20% ergosterol on the expression of transglutaminase after 5 days of culture.

 EXAMPLE 1 Preparation of the shiitake extract with 20% ergosterol

 As indicated above, the lipid extract of shiitake is prepared from ground fungus in a supercritical type plant in the presence of CO2. After extraction, the ergosterol concentration can be adjusted, ideally at the level of about 20%.

 1) Extraction conditions: - pure supercritical carbon dioxide - pressure = 500 bar - temperature = 50 oc - gas flow = 30 kg CO2 per kg of raw material.

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2) Separation in two stages to separate a maximum of the oily part:
120 and 60 bar / 30 oc
3) Separation of water: - by decantation - centrifugation - vacuum distillation
Obtaining an extract containing between 35 and 45% ergosterol and less than 3% water.

The presence of water is possible and the extract then has an emulsified appearance
4) Composition of the extract: - ergosterol: 3 to 50% - triglycerides: 5 to 95% - free fatty acids: 0 to 30% - tocopherol: 0.1 to 2% - 1 to 30%
Presence of aromatic and coloring compounds quantitatively not determined
5) Characterization of the classes of compounds present in the mushroom extract:
Thin layer chromatography provides a global composition profile. TLC of the different classes of lipids: - Mobile phase: hexane / ethyl ether / acetic acid (80/20/1).

 - Plate CCM: If 60 0, 25mm.

 - Revelation reagent: 5% phosphomolybdic acid in ethanol.

 - Well saturate the tank.

 - 0, 1 g of extract are suspended in 1 ml of hexane.

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 - Deposit 10 to 20 jll on 1,5 cm. To migrate on 16-17 cm - Spray the reagent after drying and heat to 110-1200C until optimum appearance of the spots.

. Caroténoïdes, Rf=l . Triglycérides-tocophérols : 0,60 env . Acides gras libres : 0,22 . Alcools triterpéniques : 0,12 . Stérols : 0,07 . Xanthophylles : 0
6) Calcul du pourcentage des fractions relatives : - % d'acides gras libres calculé à partir de l'indice d'acide (selon Norme AFNOR T60-204). - Results:

. Carotenoids, Rf = 1. Triglycerides-tocopherols: 0.60 approx. Free fatty acids: 0.22. Triterpenic alcohols: 0.12. Sterols: 0.07. Xanthophylls: 0
6) Calculation of the percentage of relative fractions: -% of free fatty acids calculated from the acid number (according to AFNOR Standard T60-204).

 -% H20: Karl fischer method according to standard NF T 60-225.

7) Percentage of tocopherols: HPLC method:
The unsaponifiable material is isolated according to the NF T60-205-1 standard. This is taken up in 100 ml of chloroform for HPLC analysis. Chromatographic conditions:
Mobile phase methanol / acetonitrile / water (60/37/3).

 - Mobile phase flow: lml / min.

 - Column lichrospher RP 18 5m.

 - UV detection: 290 nm.

 - Controls: use isomeric standards (X, ss, 8. Dilute Mg of each isomer in 100 ml of chloroform.

Approximate retention times:
Tr alpha tocopherol: 17 min approx.

 Tr beta tocopherol: approx.

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 Tr gamma tocopherol: 22 min approx.

 S = peak area of each tocopherol isomer control.

 Sl = area of the peak of each tocopherol isomer unknown.

% of each isomeric tocopherol in the unsaponifiable: 81/8 * m / M
Triglyceride content is calculated by approximate deduction
8) Composition of the extract used in the evaluation examples below: - 22% ergosterol - 40% triglycerides - 25% water - 15% free fatty acids - 0.5% tocopherol
EXAMPLE 2 Determination of the Effect of 20% Shiitake Extract on Ergosterol on the Proliferation of Epidermal Cells Cultured for 6 Days

 The cells used in Examples 2 to 4 are neonatal human keratinocytes purchased from Biowhittaker. They are grown in defined medium (Biowhittaker) from thawing until the end of the test. To allow cell growth, this 0.15 mM calcium base medium is supplemented with Hydrocortisone (0.5 μg / ml), Insulin (5 μg / ml), EGF (10 μg / ml), Bovine Pituitary Extract ( 30 mg / ml), penicillin-streptomycin (100 g / ml) and Fungizone (0.5 g / ml).

 1) Protocol.

 - Cellular culture.

 At pre-confluence, the keratinocytes are trypsinized and then seeded into 24-well plates, at the rate of

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 15,000 cells / well. in the above culture medium from which the EGF was removed.

After 24 hours of culture, the cells are treated with shiitake extract (1. 5 ng / ml). (OJ)
In parallel are tested an internal negative control (culture medium without EGF) and a positive internal control (culture medium + 5 ng / ml of EGF).

 - Evaluation of cell proliferation.

 The proliferation of keratinocytes is evaluated at J1, J2, J3 and J6 after treatment by quantification of the metabolic activity of the cells (mitochondrial dehydrogenases) by measurement of hydrolysis of MTT.

 2) Results.

 The results concerning the effect of shiitake 20% on cell proliferation for 6 days are shown in the graph of Figure 1 in the appendix. The cell growth gains obtained after 6 days of culture appear on the histogram of Figure 2 in the appendix.

 During 6 days of cell culture, the shiitake extract causes a gradual and continuous increase in the proliferation of keratinocytes. After 6 days of culture, the shiitake extract increases cell growth by 28% compared to the basic control.

 Ergosterol (Sigma E6510) tested at the same amount as that present in the shiitake extract increased cell growth by 43% compared to the basic control (not shown).

 EXAMPLE 3 Determination of the Effect of 20% Shitake Extract on Ergosterol on Cellular Proliferation and Differentiation: Evaluation by Cell Counting and Dosage of Corneal Envelopes

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 The horny envelopes are structures that appear very late during the phenomenon of differentiation and participate in the barrier effect of the stratum corneum. It is this property that is highlighted in this test.

 1) Protocol.

 - Cellular culture.

 When the cells have reached pre-confluence, trypsination is performed and the cells are seeded in a 12-well plate, at a rate of 12,200 cells / well.

 At pre-confluence, the cells are brought into contact with the 20% Shitake extract of ergosterol at a concentration of 1.5 ng / ml in the culture medium. Two internal references are included in the test: a basic control (cells maintained in the proliferation medium at 0.15 mM calcium) and a positive differentiation control (cells cultured in a differentiation-inducing medium containing 1.5 mM calcium and 1 mM sodium butyrate).

 - Counting epidermal cells.

 After 48 hours of treatment, the cells contained in each well are recovered by trypsination. In order to evaluate cell proliferation, cells are diluted half in trypan blue, a dye that enters dead cells, and living cells are counted on Malassez cell.

 - Counting horny envelopes from differentiated epidermal cells.

 To demonstrate cell differentiation, the cell pellets are subjected to a drastic treatment based on Dithiothreitol (20 mM) and Sodium Dodecyl Sulfate (1% PBS) and are placed at 900C for 10 minutes. After treatment, the envelopes

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 corneas, very resistant, can then be counted on Malassez cell.

 2) Results.

 The results concerning the effect of Shitake 20% on ergosterol on proliferation are presented in Table 1 below and in the graph of Figure 3 in the appendix.

 The results concerning the effect of Shitake 20% on ergosterol on differentiation are presented in Table 2 below and in Figure 2 of Figure 4 in the appendix. Each test was repeated three times.

Table 1

<Tb>
<tb> Number <SEP> of <SEP><SEP> Control of <SEP> Shitake <SEP><SEP> Control of
<tb> cells <SEP> base <SEP> 20% <SEP> differentiation
<tb> Assay <SEP> 1 <SEP> 620 <SEP> 000 <SEP> 690 <SEP> 000 <SEP> 530 <SEP> 000
<tb> Assay <SEP> 2 <SEP> 680 <SEP> 000 <SEP> 850 <SEP> 000 <SEP> 440 <SEP> 000
<tb> Assay <SEP> 3 <SEP> 580 <SEP> 000 <SEP> 720 <SEP> 000 <SEP> 480 <SEP> 000
<tb> Medium <SEP> 626 <SEP> 667 <SEP> 753 <SEP> 334 <SEP> 483 <SEP> 334
<tb> Standard Deviation <SEP> 41 <SEP> 096 <SEP> 69 <SEP> 442 <SEP> 36 <SEP> 817
<tb> Gain <SEP> of <SEP> 100 <SEP>% <SEP> 120.21% <SEP> 77.13%
<tb> growth
<Tb>
Table 2

<Tb>
<tb> Number <SEP><SEP> Indicator for <SEP> Shitake <SEP> Control
<tb> of envelopes <SEP> base <SEP> 20% <SEP> positive <SEP> of
<tb> corneas / 100 <SEP> differentiation
<tb> cells
<tb> Assay <SEP> 10, <SEP> 16129 <SEP> 0, <SEP> 07246 <SEP> 0, <SEP> 37736
<tb> Assay <SEP> 20, <SEP> 07353 <SEP> 0, <SEQ> 23529 <SEP> 0, <SEP> 795454
<tb> Assay <SEP> 30, <SEP> 08620 <SEP> 0, <SEP> 347222 <SEP> 0.8334
<tb> Average <SEP> 0, <SEP> 1064 <SEP> 0, <SEP> 221230, <SEP> 6551
<tb> Standard Deviation <SEP> 0, <SEP> 0387 <SEP> 0, <SEP> 0, <SEP> 20666
<tb> Gain <SEP> of <SEP> 0% <SEP> 107, <SEP> 96% <SEP> 515.86%
<tb> differentiation /
<tb> Witness
<Tb>

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The number of counted corneal envelopes is reduced to the number of cells in order to obtain a number of horned envelopes per 100 cells also called percentage of cornification.

 In the results shown below, the base control is considered to induce 100% proliferation and 0% cornification. Indeed, this basic control corresponds to the optimal culture medium to induce proliferation, but the factors necessary for the induction of cell differentiation are not present in this culture medium.

 After 48 hours of treatment, the Shitake extract shows a stimulation of the proliferation of keratinocytes. Indeed, tested at a concentration of 1.5 ng / ml, Shitake allows a growth gain of 20% compared to the basic control.

 The results obtained by this technique show that Shiitake acts relatively importantly on the formation of horny envelopes. This results in a 107% increase in cornification compared to the negative control (0%).

 Ergosterol (Sigma E6510) tested in parallel (not shown) seems to act on the proliferation of keratinocytes but does not seem to affect their differentiation.

 EXAMPLE 4 Determination of the Effect of 20% Shitake Extract on Ergosterol on Proliferation and Cell Differentiation: Evaluation by MTT Assay and Elisa-Transglutaminase Assay

 During differentiation, an enzyme is involved in the formation of the corneal envelopes, transglutaminase 1. This enzyme, which is a late marker of differentiation, makes it possible to evaluate the degree of

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 differentiation of the cells under the different conditions of cultures produced.

 1) Protocol.

 - Cellular culture.

 When the cells have reached pre-confluence, trypsination is performed and the cells are seeded in a 96-well plate, at a rate of 6000 cells / well (OJ). Cell culture treatments (active, basic and differentiation controls) are performed on D3 and D5. Shiitake is tested on this model at a concentration of 1.5 ng / ml.

 - MTT test.

 This test makes it possible to evaluate cell proliferation through the activity of a mitochondrial enzyme, succinate dehydrogenase. In our protocol, this test is performed after 6 days of culture.

nm. The cells are then put in contact with a solution of MTT at 1 mg / ml in DMEM for 3 hours at 37 ° C. Then 2 cycles of freezing / thawing are carried out and the crystals formed by the MTT are then dissolved in an ethanol-ethanol mixture. acetone (60/40 w / w). The coloration thus obtained is read 540 nm by removing the background noise at 650

nm.

 - Elisa-transglutaminase test.

 The level of cellular expression of transglutaminase 1 is evaluated using a specific monoclonal antibody directed against this protein (Tebu, Harbor Bio-products). The binding of this antibody is revealed by a second antibody, recognizing it and coupled to an enzyme, peroxidase (Beckman Coulter, Jackson Immunoresearch).

 Aspecific fixation reactions of the plates are first blocked with a solution of PBS-BSA 4% for 1 hour at 37 C. Then the cells are

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incubées avec le premier anticorps au 1/2000ième pendant 1 heure à 37 C. Après trois rinçages au PBS-tween 0, 05%, les cellules sont incubées avec le second anticorps au 1/5000ième pendant 1 heure à 37 C. Trois rinçages au PBStween 0,05% sont à nouveau effectués puis les cellules sont mises en contact pendant 30 min à l'obscurité avec le substrat de la peroxydase : l'O-phenylenediamine. La réaction colorimétrique est stabilisée avec une solution d'acide sulfurique 2N. La lecture des plaques est effectuée à 490 nm (une contre-lecture est effectuée à 650 nm afin d'éliminer les bruits de fond).

incubated with the first antibody at 1 / 2000th for 1 hour at 37 C. After three rinses with PBS-tween 0, 05%, the cells are incubated with the second antibody at 1 / 5000th for 1 hour at 37 C. Three rinses with PBStween 0.05% are again performed and then the cells are contacted for 30 min in the dark with the peroxidase substrate: O-phenylenediamine. The colorimetric reaction is stabilized with a 2N sulfuric acid solution. The plates are read at 490 nm (a counter reading is performed at 650 nm to eliminate background noise).

 2) Results.

 The results concerning the effect of Shitake 20% on ergosterol on cell proliferation are presented in Table 3 below in the graph of Figure 5 in the appendix. The results concerning the effect of Shitake 20% on ergosterol on cell differentiation are presented in Table 4 below and in the graph of Figure 6 in the appendix.

 A differentiation coefficient CD is calculated by dividing the Optical Density results obtained by the Elisa test by those obtained with the MTT test.

 In the results shown below, the base control is considered to induce 100% proliferation and 0% cornification. Indeed, this basic control corresponds to the optimal culture medium to induce proliferation, but the factors necessary for the induction of cell differentiation are not present in this culture medium.

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Table 3

<Tb>
<tb> DO <SEP><SEP> Control of <SEP> Shitake <SEP><SEP> Control of
<tb> base <SEP> 20% <SEP> differentiation
<tb> Assay <SEP> 1 <SEP> 0.65098 <SEP> 0.80804 <SEQ> 0.37747
<tb> Assay <SEP> 2 <SEP> 0, <SEQ> 74613 <SEP> * <SEP> 0, <SEP> 42925
<tb> Assay <SEP> 3 <SEP> 0, <SEP> 5913 <SEP> 0.92305 <SEQ> 0.23941
<tb> Assay <SEP> 4 <SEP> 0, <SEP> 7115 <SEP> 0.65775 <SEP> 0.51967
<tb> Average <SEP> 0, <SEP> 675 <SEP> 0.79628 <SEP> 0.39144
<tb> Standard Deviation <SEP> 0, <SEP> 06823 <SEP> 0.08133 <SEQ> 0.11716
<tb> Gain <SEP> of <SEP> 100% <SEP> 118% <SEP> 58%
<tb> growth
<Tb>
* Non-representative result
Table 4

<Tb>
<tb> DO <SEP> Elisa / DO <SEP> MTT <SEP><SEP> Control of <SEP> Shitake <SEP> Positive <SEP> Control
<tb> base <SEP> 20% <SEP> of
<tb> differentiation
<tb> Assay <SEP> 1 <SEP> 0, <SEP> 054754 <SEP> 0.091 <SEP> 0.19223
<tb> Assay <SEP> 2 <SEP> 0, <SEP> 08414 <SEP> 0, <SEQ> 153 <SEP> 0, <SEQ> 4272
<tb> Assay <SEP> 3 <SEP> 0, <SEP> 02374 <SEP> 0, <SEP> 146 <SEP> *
<tb> Assay <SEP> 4 <SEP> 0, <SEP> 083782 <SEQ> 0.26582 <SEQ> 0.438388
<tb> Average <SEP> 0, <SEP> 0616 <SEP> 0, <SEQ> 1639 <SEP> 0, <SEP> 3505
<tb> Standard deviation <SEP> 0, <SEQ> 02875 <SEP> 0, <SEQ> 07339 <SEP> 0, <SEQ> 1373
<tb> Gain <SEP> of <SEP> 0% <SEP> 166% <SEP> 468%
<tb> differentiation /
<tb> Witness
<Tb>
* Non-representative result
In view of the results, it appears that shitake stimulates the proliferation of keratinocytes. Indeed, for the treated wells, there is an increase in proliferation of 18% compared to the basic control.

 Shitake acts relatively importantly on the expression of transglutaminase. This results in a 166% increase in the expression of this enzyme compared to the negative control (0%).

 EXAMPLE 5 EFFECT OF SHIITAKE 20% ERGOSTEROL ON THE EXPRESSION OF GENES ENCODING STRUCTURAL AND REGULATORY PROTEINS

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1) Protocol.

In this example, the "cDNA macroarrays" technique is used to study the effects of Shiitake 20% on the expression of genes encoding 475 structural and regulatory proteins including proliferative marker proteins and the proteins represented in the differentiation. keratinocyte.

 The model used here for the study is a model of reconstructed epidermis (SkinEthic) in contact or not with shiitake 20% for a period of 18H.

 We then look at the expression of genes significantly suppressed or overexpressed in the presence of the active compared to the control.

 The methodology used is that recommended by Clontech (Palo Alto, USA) except extraction / purification of total RNA (TriReagent protocol, Sigma).

 2) Result.

 Among all the results obtained, the study showed in this model of reconstructed epidermis in contact with the active: - Overexpression of genes coding for calgranulin A.

 - Less significant overexpression of genes coding for calgranulin B.

 These 2 proteins are representative of a proliferative state of the epidermis.

 - Overexpression of genes coding for keratin Kl, a late marker of keratinocyte differentiation.

 - Less significant overexpression of the genes encoding transglutaminase 1, differentiation marker.

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 The results obtained make it possible to confirm at a genetic level on a model of reconstructed epidermis, the results described in examples 2,3 and 4 at the cellular level on keratinocyte cultures.

 If we compare the results obtained in this model of study with those obtained in the same model in the presence of retinoic acid (10-6 M), we can see: - A pro-proliferative effect in the presence of shiitake but less than that described in the presence of retinoic acid.

 An important pro-differentiating effect in the presence of shiitake while the opposite result is described with regard to retinoic acid (decrease in the expression of keratinocyte differentiation markers).

 EXAMPLE 6: FORMULAS INCLUDING SHITAKE CO 2 EXTRACT AT ABOUT 20% ERGOSTEROL

 1) Example of composition in emulsion form Water QSP CO2 extract of Shiitake at approx. 20% ergosterol 0.001% to 0.1% Blend of Preservatives 1.5% Propylene Glycol 5.00% Xanthan Gum 0.30% Copolymer Acrylate / Acrylate 0.50% Stearic Acid 100 EO * 3.00% Stearate sorbitan 2.00% Sorbitan laurate 20 OE 3.00% Cetylstearic alcohol 1.50% Beeswax 1.00% Wheat germ oil 5.00% Dimethicone 2.00% Cyclomethicone 5.00% Polyarcramide gel 2 , 00%

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Perfume 0, 10% ** stearic acid with 100 moles of EO
2) Example of composition in the form of water cream QSP CO2 extract of Shiitake at approx. 20% ergosterol 0.001% to 0.1% Xanthan gum 0.30% Sequestering agent (eg EDTA) 0.05% Preservatives 1, 50% C18 Acid 2, 50% C16 Acid 2, 50% Trilaurin 1 , 00% Shea butter 3, 00% Topopherol acetate 0, 05% ss-bisabolol 0, 05% Vegetable oil (wheat germ) 5, 00% Dimetheticone 3, 00% Polyacrylic acid 0, 30% TEA (Triethanolamine) 1, 50% Perfume 0, 10%
3) Example of composition in the form of lotion CO2 extract of Shiitake at approx. 20% ergosterol 0, 001 to 0, 1% Water QSP Sequestering agent 0, 05% Propylene glycol 2, 00% Mixture Preservatives 1, 5% Alcohol 5 to 50% Oleic alcohol 20 EO 1, 00% Perfume 0, 05%

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BIBLIOGRAPHIC REFERENCES - Bittiner B., Bleehen S. and Macneil S.

1991. 1α, 25 (OH) O3 vitamin Do increases intracellular calcium in human keratinocytes. British Journal of Dermatology. 124.230 to 235.

 - Denning M.F., Guy S.G., Ellerbroek S.M., Norvell S.M., Kowalczyk A.P. and Green K.J. 1998. The expression of desmoglein isoforms in cultured human keratinocytes is regulated by calcium, serum and protein kinase C. Experimental cell research. 239.50 to 59.

 - Iwatsuki K. Harada H., Zhang J. Z., Maruyama K. and Kaneko F. 1994. Regulation of pemphigus and desmosomal antigen expression by keratinocyte differentiation. Dermatology. 189.67 to 71.

 - Lubrano C., Poirier F. and Robin J. R. 1997: New use of ergosterol and its related compounds, in cosmetics. Patent No. 97 11655.

 - Michel S. 1995. Transglutaminases and precursor proteins of the horny envelope. Biology of the skin. 25-32.

 - Michel S., Courseaux A., Miquel C., Bernardon J.M., Schmidt R., Shroot B., Thacher S.M. and Reichert U.

1991. Immunosorbent assay. Anal. Biochem. 192.232 to 236.

 - Saintigny G., Schmidt R., Shroot B., Juhlin L., Reichert U. and Michel S. 1992. Differential expression of calgranulin A and B in various epithelial cell lines and reconstructed epidermis. J Invest Dermatol. 99.639-644.

 - Swendsen M.L., Daneels G., Geysen J., Binderup L. and Kragballe K. 1997. Proliferation and differentiation of cultured human keratinocytes is modulated by 1.25 (OH) 03D &> synthetic vitamin DZ-e.)

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 analogues in a cell-density, calcium-and-serum-dependent manner. Pharmacology & Toxicology. 80.49 to 56.

Claims (9)

1) Use in a cosmetic composition or for the manufacture of a cosmetic composition of a lipid extract of plant, yeast or mushroom containing ergosterol to enhance the barrier function of the skin.  2) Use according to claim 1, characterized in that the lipid extract of mushroom containing ergosterol is a shitakee extract.  3) Use according to one of claims 1 or 2, characterized in that the shiitake extract contains from 3 to 50%, preferably from 15 to 25% and most preferably of the order of 20% by weight of ergosterol.  4) Use according to one of claims 1 to 3, characterized in that the shiitake extract comprises the following compounds, the amounts are expressed by weight relative to the weight of the extract: - ergosterol: 3 to 50% - triglycerides: 5 to 95% - free fatty acids: 0 to 30% - tocopherol: 0.1 to 2% - Water: 1 to 30% 5) Use according to any one of claims 1 to 4, characterized in that the shiitake extract comprises the following compounds, the amounts are expressed by weight relative to the weight of the extract: - 22% ergosterol - 40% triglycerides - 25% water - 15% free fatty acids <Desc / Clms Page number 23>  - 0, 5% of tocopherol 6) Use according to any one of claims 1 to 5, characterized in that in the cosmetic composition the lipidic extract containing ergosterol is 0.001 to 1% of the total weight of the composition and preferably 0.05 at 0.1 of the total weight of the composition.  7) An extract of shiitake characterized in that it contains about 20% by weight of ergosterol.  8) A shiitake extract according to claim 7, characterized in that it comprises the following compounds, the amounts of which are expressed by weight relative to the weight of the extract: - 22% ergosterol - 40% triglycerides - 25% water - 15% free fatty acids - 0, 5% tocopherol 9) A cosmetic composition comprising a shiitake extract according to one of claims 7 or 8.