WO2000059466A1

WO2000059466A1 - Cosmetic composition comprising a lipid skeletonema extract

Info

Publication number: WO2000059466A1
Application number: PCT/FR2000/000818
Authority: WO
Inventors: Carine Nizard; Nicolas Provost; Cécile VIRON; Valérie KRZYCH; Alex Saunois
Original assignee: Lvmh Recherche
Priority date: 1999-04-02
Filing date: 2000-03-31
Publication date: 2000-10-12
Also published as: JP2002541084A; FR2791568B1; FR2791568A1; EP1165036A1

Abstract

The present invention relates to cosmetic compositions comprising the following: an active ingredient such as at least one substance that promotes intercellular communication of skin cells, especially keratinocytes, and fibroblasts and pre-adipocytes of the skin; the use of at least one substance promoting intercellular communication of keratinocytes, fibroblasts and pre-adipocytes of the skin as a cosmetic agent, optionally in the presence of a cosmetically acceptable vehicle; a method for promoting and/or increasing the activity of a cosmetic agent acting directly in the cell or via second intracellular messengers, and a method for treating ageing of the skin using cosmetics.

Description

COSMETIC COMPOSITION COMPRISING A SKELETONEMA LIPID EXTRACT

The invention essentially relates to a cosmetic composition comprising at least one substance promoting intercellular communication of skin cells, in particular keratinocytes, fibroblasts, and pre-adipocytes of the skin, in particular promoting the formation of connexin, use and cosmetic treatment process. The search for means to fight against the aging of man mobilizes a large number of researchers today. This is in particular the case in the field of cosmetics where it is sought to combat, or at least slow down, the appearance of the aesthetic and physiological effects of skin aging, such as wrinkles, loss of elasticity of the skin. skin, color, etc. The aging of the human species is characterized by many disorders that affect living tissue. The marks of this natural process are easily detectable on an organ such as the skin (Montagna, W., & Carlisle, K. (1990). Structural changes in aging skin. British journal of dermatology, 122 (35), 61-70 ). Aging of the skin is a combination of two phenomena, an intrinsic cellular process (genetic) associated with so-called extrinsic aging which brings together environmental aggressions (Grove, GL (1989). Physiology changes in older skin. Clinics in geriatrics medicine , 5 (1), 115-125). The effects of senescence on the skin tissue are visible mainly by the formation of wrinkles. These reflect the significant changes that take place in the dermis and epidermis. Tissue homeostasis, important for the skin, is modified during aging. Communicating junctions participate in the regulation of cell homeostasis, their role in maintaining the physiological balance of the skin is therefore important. The inventors have highlighted an effect of aging on the functional capacity of cells to communicate with each other, in particular via communicating junctions (JC) or "gap junctions" (GJ), as well as an effect of aging on amount of connexin 43 present in cells. They have indeed demonstrated that the functional capacity of cells to communicate with each other, in particular via communicating junctions (JC) decreases with age. They also demonstrated that the amount of connexin 43 decreases with age The inventors have therefore shown the advantage of using substances which promote cell communication, in particular through JC, to combat the signs of skin aging, in particular to slow their appearance. This is a new way to fight against skin aging, in particular against πdes and loss of elasticity of the skin.

Communicating junctions (JC) or Gap Junctions (GJ), are transmembrane protein structures allowing the passage of small molecules (<1000 Da) between two cells (Watt, FM (1991). Intercellular communication via gap junctions In L. A Goldsmith (Eds), Physiology, biochemistry, molecular biology of the skin (pp 857-859) New York Oxford Oxford university press) These hexagonal structures are called Connexons which are themselves forms of Connexins

When intercellular contact is established, the Connexons of one cell align end to end with those of the neighboring cell, thereby establishing a junctional channel (Goodenough, D A., Goher. JA, & Paul, DL (1996) Connexms, Connexons, and cellular communication. Annual review of biochimistry, 65, 475-502) These communicating junctions (JC) allow the maintenance of cellular and tissue homeostasis. Connexins are part of a family of proteins, each with tissue specificities. Connexin 43 is the major protein of keratinocytes (Salomon, D., Saurat, J.-H., & P., M (1988). Cell-to-cell communication within intact human skin. Journal of clmical investigation, 82, 248-254). In the skin, JC are present in all the layers of the epidermis with the exception of the stratum corneum. On the other hand, it is known that the molecules acting on the functioning of cells can do so, either by penetrating into the cells, or without penetrating into the cells, but by attaching themselves to receptors present on the cellular plasma membrane, triggering thus a senna of reactions which will lead to the release of small molecules called "second messengers" in the cell cytoplasm. These second small messengers, such as for example AMPc (3 ', 5'-adenosine cyclic monophosphate), circulate from one cell to another through the JC, thus transmitting information from one cell to the other. It is the same for the molecules of small sizes penetrating in the cells, and able to circulate from a cell to the other through the JC. Thus, by increasing cell communication, especially through the JC, the inventors have put developed a new way to promote and / or increase the activity ^of a cosmetic agent.

In addition, the inventors have shown that, surprisingly, the use of boldine, or independently, the use of lipid extracts of the Skeletonema costatum alga makes it possible to restore intercellular communication, in particular through JC and allows also increase the level of connexin 43 present in skin cells, in particular in keratinocytes, fibroblasts and pre-adipocytes.

Thus, the main object of the present invention is to solve the new technical problem consisting in providing a new solution for improving the effectiveness of cosmetic compositions, in particular for combating, or at least slowing down, the appearance of aesthetic effects and physiological effects of skin aging, such as wrinkles, loss of skin elasticity, color, etc.

According to the present invention, this technical problem has been solved for the first time, in a surprising and not obvious manner, by the discovery that a substance which promotes intercellular communication of keratinocytes, fibroblasts and pre-adipocytes of the skin allows to provide a cosmetic composition having improved properties, in particular to combat, or at least slow down, the appearance of the aesthetic and physiological effects of skin aging, such as wrinkles, loss of elasticity of the skin, color, etc. .

On the other hand, the present invention has also made it possible to discover, unexpectedly and not evidently, that a substance promoting the formation of connexin, in particular connexin 43, makes it possible to prepare a cosmetic composition with increased effectiveness, in particular for combating, or at least slow down, the aforementioned appearance of the aesthetic and physiological effects of skin aging.

It has also been discovered, according to the present invention, unexpectedly and not evidently, on the one hand that boldine and on the other hand, independently, that a lipid extract of the Skeletonema alga, in particular of the Skeletonema alga costatum, in particular a total lipid extract of said alga, promotes intercellular communication through the communicating junctions of skin cells, in particular keratinocytes, fibroblasts, and pre-adipocytes of the skin, and thus makes it possible to prepare a cosmetic composition with increased effectiveness, in particular for combating or slowing down the appearance of the aesthetic and physiological effects of skin aging, or for combating adipose overloads. Thus, according to a first aspect, the present invention relates to a cosmetic composition, characterized in that it comprises, as active ingredient, at least one substance which favors the intercellular communication of skin cells, in particular keratinocytes , fibroblasts, and skin preadipocytes.

According to a particular embodiment of the invention, the composition is characterized in that said substance promoting intercellular communication, promotes mtercellular communication through the communicating junctions of skin cells, in particular keratinocytes, fibroblasts, and skin pre-adipocytes.

According to another advantageous embodiment of the invention, the composition is characterized in that the said substance promoting intercellular communication favons the formation of connexin, in particular connexin 43. According to yet another advantageous embodiment of the invention , the aforementioned composition is characterized in that said substance promoting cell communication comprises at least one lipid extract of the Skeletonema alga, in particular of the Skeletonema costatum alga, in particular a total lipid extract of said alga. It is recalled that the Skeletonema alga, in particular Skeletonema costatum (named SKC in the remainder of the document), is a well known unicellular alga, from the phylum of Chlorophytes, of the branch of Chrysophycophytes, from the class of Diatomophycees and from the 'order of Power Plants. Diatomophycees are very widespread in fresh water, manna or brackish. The life of species of this class can be planktonic or benthic. The protoplasm is enclosed in a siliceous frustule. Skeletonema costatum (SKC) is a cosmopolitan species, most often manna, which is frequently found associated with phytoplanktonic bloom in coastal waters.

According to an advantageous embodiment of the invention, this lipid extract is characterized in that it is an extract obtained by extraction of the Skeletonema alga with an alcoholic solvent chosen from the group consisting of isopropanol, ethanol, and methanol.

According to another advantageous embodiment, the extraction is carried out under reflux. According to yet another advantageous embodiment, the alga is frozen before being extracted with the alcoholic solvent, preferably freezing being performed at a temperature between - 40 ° C and - 20 ° C approximately and for a period preferably between 1 and 7 days approximately.

According to another advantageous embodiment, the frozen algae is immersed directly in the heated alcoholic solvent. Thermal shock makes it possible to facilitate the decantation of silica (from the skeleton of algae cells)

According to another advantageous embodiment, in the case of an extraction with an alcahnized alcohol, the extract of the abovementioned alga is obtained after the succession of the following steps a) the alcoholic solvent is alcahnized up to a pH of between 10 and 14, preferably at a pH equal to 13, for example with an aqueous solution of sodium hydroxide or with an aqueous solution of potassium hydroxide, b) the insolubles are eliminated from the hydroalcoholic phase, c) distilled water is added to the hydro-alcoholic phase, d) the solution obtained undergoes a liquid-liquid extraction with an apolar solvent immiscible with the hydro-alcoholic phase, such as for example heptane, hexane or cyclohexane, e) the phase containing the apolar solvent is eliminated, f) the hydroalcoholic phase recovered after elimination of the phase containing the apolar solvent, is acidified to a pH between 1 and 3, preferably at a pH equal to 2, e.g. example with an aqueous solution of sulfunque acid or with an aqueous solution of hydrochloric acid, g) the solution obtained after acidification undergoes a liquid-liquid extraction with an apolar solvent immiscible with the hydro-alcoholic phase, such as for example l heptane, hexane or cyclohexane, h) the hydro-alcoholic phase is eliminated, î) the phase containing the non-polar solvent recovered after elimination of the hydro-alcoholic phase undergoes evaporation in order to obtain an oil free of non-polar solvent , this oil being the desired extract.

The use of an alcahnized, then acidified alcohol makes it possible to obtain an extract with acceptable visual and olfactory characteristics in cosmetic compositions (yellow color, and acceptable odor). According to yet another advantageous alternative embodiment, the above-mentioned extract is obtained by extraction with supercritical CO ₂ .

According to yet another advantageous embodiment, before any other extraction operation, a maceration of the alga is carried out in the alcoholic solvent at room temperature and preferably for a period of between 5 minutes and 80 minutes approximately, and preferably again, for a period of between about 20 minutes and 40 minutes.

According to yet another advantageous alternative embodiment, the amount of alcoholic solvent used is between 0J liter and about 20 liters of solvent, preferably between 2 liters and about 10 liters of solvent, per 100 g of algae, expressed by weight dry seaweed.

According to yet another advantageous variant embodiment, the extraction can be carried out under an inert atmosphere, preferably under an atmosphere saturated with nitrogen. This makes it possible in particular to avoid a pronounced oxidative degradation of the active molecules

The packaging of this lipid extract is preferably carried out under an inert gas such as nitrogen in order to protect the active molecules.

According to yet another advantageous alternative embodiment, the aforementioned composition is characterized in that it comprises from 0.01% to 10% approximately, and in particular from 0.1% to 3% approximately by weight of said lipid extract of the seaweed

Skeletonema, in particular of the Skeletonema costatum seaweed relative to the total weight of the final composition.

According to a second aspect, the present invention also relates to the use of at least one substance promoting the intercellular communication of keratinocytes, fibroblasts, and pre-adipocytes of the skin, as cosmetic agent, possibly in the presence of a cosmetically vehicle acceptable.

According to a particular embodiment of the use, it is characterized in that said substance promoting mtercellular communication, promotes intercellular communication through the communicating junctions of keratinocytes, fibroblasts, and pre-adipocytes of the skin.

According to another particular embodiment of the invention, the substance promoting the cellular communication favonse the formation of connexin, in particular connexin 43. According to another embodiment of the invention, the substance comprises at least one lipid extract of Skeletonema seaweed, especially seaweed Skeletonema costatum, in particular a total lipid extract of said alga, in particular as previously defined. Advantageously, this abovementioned extract is an extract obtained by liquid-liquid extraction between an alkalinized and then acidified alcohol and an apolar solvent immiscible with the hydro-alcoholic phase, such as for example heptane, hexane or cyclohexane.

According to another embodiment of the invention, the substance promoting intercellular communication of the skin cells is boldine, a product which corresponds to the formula below:

advantageously the compositions of the invention which contain said boldine, contain about 0.001 to 10%, advantageously 0.01 to 1%, by weight.

According to a third aspect, the present invention also covers a process for promoting and / or increasing the activity of a cosmetic agent acting directly in the cell or through second intracellular messengers, characterized in that it comprises the simultaneous or preliminary application to that of said cosmetic agent, on the areas of the skin concerned of a person in need thereof, of an effective amount of at least one substance promoting intercellular communication, in particular a substance promoting intercellular communication such than previously defined.

According to a fourth aspect, the present invention also covers a method of anti-aging cosmetic treatment of the skin, characterized in that it comprises the application to the areas of the skin concerned of a person in need thereof, of a effective amount of at least one substance promoting intercellular communication, to obtain an anti-aging effect on said areas of the skin, in particular for improving skin firmness, improving the elasticity of the skin, delaying the appearance of ndes or reducing their depth, in particular a substance promoting cell communication as defined above. The examples which follow are given purely by way of illustration of the invention, with reference to the accompanying drawings in which.

- Figure 1 shows the modulation curve of intercellular communication as a function of the age of the donors. It is obtained from keratinocytes isolated from donors of different ages by a method known as scratch-loading. The curve is expressed as a function of the ratio of the fluorescent surface to the total surface, on the ordinate, and as a function of the donor age, in years, on the abscissa. "R" is the correlation coefficient here equal to 0.76. The measurements obtained with young donors are represented by solid squares with the standard deviation and the measurements noted with white circles were obtained with elderly donors, also with the standard deviation,

This figure should be read in conjunction with Table I (Example I),

similarly, FIG. 2 also represents a modulation curve of intercellular communication as a function of the age of the donors, obtained by a micro-injection method, but this time expressed as a function of the number of cells marked with keratinocytes, on the ordinate, and as a function of the age expressed in years, on the abscissa, r still represents the correlation coefficient here equal to 0.91 and the measurements obtained with young donors being noted with black squares, with their standard deviation, while the measurements obtained with elderly donors are noted with white circles, with their standard deviation,

This figure should be read in conjunction with Table II (Example I),

FIG. 3 represents the evolution of the rate of connexin 43 measured in keratinocytes of donors of different ages, obtained by flow cytometry, expressed as a function of the percentage of labeling on the ordinate and as a function of the age of the donors (in years), on the abscissa, with a correlation coefficient r equal here to 0.82. The measurements obtained with young donors are noted with black squares with their standard deviation, and the measurements obtained with elderly donors are noted with white circles, with their standard deviation,

This figure is to be read in conjunction with Table III (Example I), - Figure 4 represents the results of modulation of intercellular communication in normal human keratinocytes, noted KHN, in different donors treated or not with a lipid extract of l alga Skeletonema costatum, abbreviated SKC, represented in the form of histograms, expressed according to relative unit of diffusion on the ordinate, the white stick being obtained with control KHNs and the gnsé stick being obtained with KHNs treated with a lipid extract of the Skeletonema costatum alga in a proportion of 2.5 μg / ml / 24h, in vitro,

This figure should be read in conjunction with the NI table (Example III),

FIG. 5 represents the measurement by the micro-injection technique, of the modulation of intercellular communication in normal human keratinocytes (abbreviated as KHΝ) in different donors treated or not with a lipid extract of the Skeletonema costatum alga, abbreviated SKC, with on the ordinate the number of labeled cells, the results with the control KHΝ, untreated, being shown with a white stick called "control", and the KHΝ treated with a lipid extract of the alga Skeletonema costatum or SKC at the dose of 2.5 μg / ml / 24h being represented by a black stick, known as treated,

This figure should be read in conjunction with Table V (Example III),

- Figure 6 also shows a histogram, obtained from results measured in flow cytometry, of the modulation of the amount of connexin 43 relative to their respective controls of donors of different ages treated with the lipid extract of the alga Skeletonema costatum, abbreviated to SKC, at a dose of 2.5 μg / ml / 24h, with the percentage increase on the ordinate and the age of the donors, in years, on the abscissa.

This figure is to be read in conjunction with Table VI (Example III). Other advantages of the invention will become apparent from the description and the examples which follow.

Unless otherwise indicated, the proportions given in the example compositions are expressed as a percentage by weight

Example I - Demonstration of the decrease in the functionality of intercellular communication by Communicating Junctions (CIJC) with age and the decrease in the amount of connexin 43 with age.

IJ- Materials and methods

I 1 1 - Culture of normal human keratinocytes

Normal human keratinocyte (KHΝ) cultures are produced from skin samples.

The sample is initially taken 4 times in PB S (Phosphate Buffered Saline - Sigma) (50 ml tubes). It is then decontaminated by soaking it in two successive baths of 70% ethanol for 30 seconds. When the sample is decontaminated, it is placed in a petri dish containing PBS. Strips 1 mm wide are then cut, taking care to eliminate a maximum of adipose tissue and dermis. The strips are placed progressively in a petn box containing PBS.

To be able to recover the keratinocytes from the epidermis, the strips are placed for 4 h at 37 ° C. in a 0.25% trypsin solution in PBS

The dermis of the epidermis is then dissociated by scraping the strips with a scalpel, the epidermal cells obtained are suspended in a tube containing DMEM medium (Dulbecco Modified Eagle Medmm - Gibco) + 10% of SVF (Fetal Calf Serum - Eurobio) After homogenization of the suspension, the surface part consisting of cells of the stratum corneum is removed and filtered through a sieve. The filtered part is centrifuged for 5 minutes at 176 g. The pellet is taken up with KHN-D medium (DMEM + 10% FCS + hydrocortisone 0.4 μg / ml + EGF 10 ng / ml + cholenic toxin 10 ^-9 M). The cells are counted and then seeded at 15 × 10 ⁶ cells / bottle.

After 24 hours of culture, the medium is changed, the cells are rinsed with PBS and the culture medium K-SFM (Gibco) is used for the rest of the culture.

The keratinocytes are transplanted in a completely conventional manner, but it is necessary to transplant them at 60-70% of the confluence so that they keep their capacity to proliferate. Thus, when the cells are at 60-70% of the confluence, the maintenance medium is eliminated and the cell carpet is rinsed with PBS. The cells are placed in the presence of 3 ml of a trypsin-EDTA solution, then when the cells detach, the trypsin is inhibited by medium with 10% FCS (Eurobio). The cell suspension is homogenized, recovered and then centrifuged at 20 g for 5 minutes at room temperature. The pellet thus obtained is repns with medium alone. For maintenance, the seeding is done as before at 10 ⁶ cells / 75 cm ² in ventilated bottles and stored under the conditions mentioned above. The confluence is obtained after ten days and the cells can be amplified for 6 to 7 passages.

In the case where it is desired to test the activity of a substance on these keratinocytes, the tests will be carried out from the culture medium with keratinocytes, at the time of the decent confluence above. 1.1 2- Scratch-Loading or in English "Scrape loadmg"

This technique was developed in 1987 by the Trosko team (El-Fouly, MH, Trosko, J. E, & Chang, C.-C. (1987). Scrape loading and dye transfert a rapid and simple technique to study gap junctional intercellular communication. Experimental cell research, 168, 422-430). It is based on the use of a dye, Lucifer Yellow (Lucifer Yellow - Sigma) of Molecular Weight (PM) = 457.2. This molecule (an aminophta mide) is highly fluorescent and diffuses only through the communicating junctions. This molecule does not pass through the plasma membrane of intact cells. The cell layer is injured using a scalpel, this allowing the dye to access the communicating junctions.

The confluence cells are rinsed with PBS (Sigma) before the addition of the dye. 2 ml of 0.05% Lucifer Yellow (Sigma) diluted in PBS are deposited on the cells and the "scratch" is er at room temperature. Six '"scratches" are made in 60 mm boxes. The cells are left in the presence of the dye for 5 minutes, then it is removed and recovered. The cells are then rinsed with PBS, in order to eliminate the excess fluorescence. They are fixed using 3% Paraformaldehyde (PFA) for 10 minutes. The cells are again rinsed with PBS, and after rinsing, 2 ml of PBS are left in the dishes.

Then, the boxes are observed using an inverted fluorescence microscope equipped with a camera which allows the acquisition of images. Six or eight photos are taken for each box and are then analyzed with image analysis software (Perfectimage, Iconix). We then quantify the fluorescence corresponding to the surface occupied by Lucifer Yellow in the cell layers. For this, there is a rectangle of predefined size at the level of the area to be studied. The computer calculates and gives the ratio of the surfaces: Fluorescent surface / Total surface. The higher the ratio, the greater the capacity of the cells to communicate with each other.

1.1.3- Micro-imection

The preparation of the cells is similar to that used by the scratch-loading technique or in English "scrape-loading"

Thanks to the precision of this technique, Lucifer Yellow (10% in 0.33 M LiCl) is injected directly into a cell, only using a capillary connected to a micromanipulator (Leitz). The injection is controlled by a m _j ector automatic (Tranjector 5246, Eppendorf), and lasts 1 second with an intensity of 10335 HPa. The injections are made under an inverted microscope (20X) in phase contrast (Leica microscope, Fluovert FU) Twenty injections are carried out in a 60 mm petn dish After fixing the cells with 3% paraformaldehyde, the cells are rinsed with PBS The observation and the counting of the number of labeled cells are then carried out

I 1 4- Flow rate When the confluence is reached, the culture medium is removed and the cells are rinsed 2 times with PBS buffer. Then, they are trypsinized using a mixture of 0% trypsin and 0.02% EDTA. The trypsin inhibition is carried out with culture medium adding 10% of FCS. A count of the number of cells to be had, in order to adjust this to 10 cells per tube. The cell suspension is centrifuged at 176 g for 5 minutes and the pellet is taken up in 100 ml of PBS. Cells are fixed with 3% PFA for 25 minutes at 4 ° C. To remove the PFA, the cells are again centrifuged at 176 g for 5 minutes and rinsed twice in PBS.

The buffer, in which the antibodies are prepared, consists of PBS, 1% FCS and 0.2% saponin (Sigma), a detergent which induces microporation of the membrane, but which does not damage the cell. This detergent is used to keep intact and to quantify the intracytoplasmic connexin 43 but also transmembrane (Personal communication, R. Mouawad, Hôpital Salpétnere - Pans) The primary antibody used is the anti-connexin 43 (Zymed, monoclonal AC made in the souns). The concentration of the primary antibody should be 1.3 μg / ml (l / 750 ^th ). The incubation is 45 minutes at 4 ° C. In order to remove the excess antibody, the cells are centrifuged and incubated with the buffer used for the preparation of the antibodies (PBS, saponin and SVF). They are then placed in the presence of the secondary anti-sun antibody coupled to fluorescein. at the dilution of l / 50 ^th (Jackson Immunotech). After incubation with the second antibody, the cells are centrifuged and nncnées, then repnses in 1 ml of buffer (PBS, saponin, SVF)

The fluorescence intensity is measured in the KHNs treated as above and in KHNs which have not been put in the presence of the secondary anti-sun antibody coupled to fluorescein, with a flow cytometer (Epies profile II from COULTRONIC), the measurements are processed and compared by software (Phoenix flow system soft from COULTRONIC), which gives us a value measured in an arbitrary unit given by the machine, which we call: marking rate of the Connexin 43 in KHN, which reflects the amount of Connexin 43 per KHN (on average).

1.2- Results

1.2 J- Evaluation of the functionality of Intercellular Communication by Communicating Junctions (CIJC) or Gap Junctional Intercellular Communication (GJIC) according to the age of the donors.

.2.1.1- Scratch-Loading or "Scrape-Loading" (Figure 1) The study was carried out on 9 donors aged 40 to 75 years.

Table I

Modulation of intercellular communication according to the age of the donors.

It can be observed in the results shown in Table I, and in FIG. 1 (in the appendix), that intercellular communication decreases as a function of the age of the donors, the capacity of keratinocytes to communicate seems to be inversely proportional to age donors.

The correlation coefficient is 0.76. I.2J.2- Micro injections (Figure 2)

The study was conducted on the same donors as for the scratch-loading or "scrape-loading", except the donor of 43 years.

Table II

Modulation of intercellular communication according to the age of the donors

Reading the results shown in Table II and in Figure 2 (in the appendix), we observe a CIJC inversely proportional to age, but with this technique more precise than scratch-loading or "Scrape-Loading", the correlation coefficient is 0.91.

The two techniques, scratch-loading or "Scrape-Loading" and Micro-injections, give similar results: the functionality of the CIJC decreases with age very significantly.

1.2.2- Evaluation of the effect of age on the quantity of connexin 43 (Figure 3)

Flow cytometry:

The study was carried out on the same donors as for the scratch-loading or "scrape-loading", except the donor of 43 years and the donor of 75 years. Table III

Connexin 43 labeling rate in KHN, measured in keratinocytes of donors of different ages.

Table III and FIG. 3 (in the appendix) show a decrease in the rate of connexin 43 as a function of age, the amount of connexin 43 appearing to be inversely proportional to the age of the donors.

The correlation coefficient is 0.82.

These results highlight an effect of aging on the functional capacity of cells to communicate with each other, in particular via communicating junctions (JC) or "gap junctions" (GJ), as well as an effect of aging on the quantity Connexin 43 found in cells. They show, in fact, that the functional capacity of cells to communicate with each other, in particular via communicating junctions (JC) decreases with age. They also show that the amount of Connexin 43 decreases with age.

The inventors have therefore shown the advantage of using substances which promote intercellular communication, in particular through JC, to combat the signs of skin aging, in particular to slow their appearance. They have therefore developed a new way to fight against skin aging, in particular against wrinkles and loss of elasticity of the skin.

The inventors have therefore also shown the advantage of using substances promoting intercellular communication, in particular through JC, to promote and / or increase the activity of a cosmetic agent. They have therefore developed a new means to promote and / or increase the activity of a cosmetic agent. Example II - Obtaining lipid extracts from the Skeletonema costatum alga.

II J - Isopropanol extraction. according to a first process Preferably, all of the extraction will be carried out under an inert atmosphere (nitrogen saturation) in order to avoid pronounced degradation of the active molecules.

In this preparation, we use 250 kg of biomass (Skeletonema costatumλ The algae, which have been frozen at -20 ° C, are immersed in isopropanol (IPA) under reflux at 80-83 ° C, and this with stirring. The thermal shock makes it easier to decant the silica (from the skeleton of the algae cells).

The amount of solvent used is 10 liters of API for 1 liter of water contained in the biomass. In this preparation, for a percentage of dry matter of 30%, the 250 kg of biomass represent an amount of dry matter of 75 kg and 175 kg of water. The amount of API engaged here is

1925 kg.

The whole (biomass + IPA) is brought to reflux for half an hour with stirring at approximately 80 ° C., before being cooled to around 50 ° C.

After the reaction mass has cooled to around 50 ° C., the extract is transferred to a GUEDU type filter in order to carry out the spent biomass / lipid extract separation under IPA.

The lipid extract is concentrated in a batch reactor (concentration factor = 71.5).

The yield relative to the dry weight of this first step is 28% in crude oil.

To start the second step, the lipid extract is taken up in cold 1TPA at the rate of 10 kg of solvent per 1 kg of oil. Agitation is continued for 20 minutes. The juice is then filtered (this eliminates the sticky residual mud).

The bleaching and deodorization treatment is carried out in two batches in an 80 liter schott reactor and lasts 30 minutes at room temperature. The amount of zeolite (ABSENT 2000, supplier UOP) added is 0.94 kg and that of activated carbon (CXV, supplier CECA) is 1.6 kg. The carbon to zeolite ratio is 1.7. The zeolite and the charcoal are then removed by filtration on paper.

The yield relative to the dry weight of this second step is 37%.

Thus, the overall oil yield for the whole process is 10% relative to the dry weight of biomass Antioxidants (DL α-tocopherol at 0.05% final by weight and ascorbyl palmitate at 0.05 % final by weight) are incorporated by a solution

The filtrate and the antioxidants are then concentrated in batch until a brown-colored oil is obtained. The packaging of this lipid extract (in the form of an oil) is carried out under inert gas such as nitrogen.

This oil will be named hereinafter the lipid extract El of SKC

II 2 - Extraction with ethanol according to a second process

The extraction begins with a dispersion of 49.8 kg of frozen plant biomass at 29% by dry mass in 539 kg of ethanol at 96%, alkalized with 9 kg of aqueous sodium solution at 30.5% After maceration of 30 minutes at 40 ° C under reflux and under a nitrogen atmosphere, the whole is cooled to 18 ° C. The insolubles are then separated by wringing under nitrogen and are eliminated.

To the 573.9 kg of filtrate are added 151 kg of distilled water. ^The mixture is stirred slowly for 10 minutes to then be confronted with 162 kg of heptane. The heptanic epiphase of the liquid / liquid partition is eliminated. Note that hypophase contains the fatty acid salt form ^The process is repeated two more times

The 756 kg of solution relating to the above-mentioned hypophase are acidified by adding 2.8 kg of sulfuric acid to obtain a pH of 2.2. The whole is stirred for 10 minutes under nitrogen and then faces 158 kg of heptane. The free fatty acids are extracted from the 147 kg constituting the first heptanic epiphase of this new liquid / liquid partition. The operation is repeated five more times to recover a total of 697 kg of heptanic phase. This phase evaporated to dryness on a rotary evaporator and then by molecular distillation provides the active extract, ie an amount representing 1J kg of oil. The oil produced is liquid, homogeneous and has a dark yellow color.

This oil will be named below: the lipid extract E2 from SKC.

Example III - Evaluation of the Effect of a Lipid Extract from the SKC Alga on Intercellular Communication by Communicating Junctions (CIJC) and the rate of Connexin 43 on KHN of elderly donors.

The lipid extract of the SKC alga used in this example is the lipid extract E2 of SKC obtained according to the ethanol extraction process described above.

The studies described below were also carried out with the lipid extract E1 of SKC (results not shown), with results comparable to those described below and obtained with the lipid extract E2 of SKC.

The studies are carried out on Normal Human Keratinocytes (KHN) obtained according to the protocol described in Example I (1.1.1 - Culture of Normal Human Keratinocytes). Unlike the controls, the KHNs treated with the lipid extract El or E2 of SKC (at the time when the culture is in confluence) are brought into contact with an amount "x" of said extract expressed in μg per milliliter of culture and for 24 hours (x μg / ml / 24 hours) before undergoing evaluations carried out with the Scratch-Loading, Micro-injections or Flow Cytometry techniques, according to the protocols described in Example I (respectively 1.1.2, 1.1.3, 1.1.4).

^• Scratch-Loading or "Scrape-Loading" (Figure 4) The study was carried out on Normal Human Keratinocytes (KHN) from 4 donors aged 60 to 79 years.

Table IV

Modulation of intercellular communication in NHKs from different donors treated with SKC lipid extracts (E2) measured by the scratch-load method

Where "R" is the ratio of the fluorescent surface to the total surface.

The results shown in Table IN and FIG. 4 (in the appendix) demonstrate that the lipid extracts of SKC induce an increase in the CIJC in elderly donors (p <0.0001-student test).

There is therefore a restoration of cellular communication by the lipid extracts of SKC, at about the same level as the KHΝ of young donors.

Micro-injections (Figure 5)

The study was carried out on 12 donors aged 49 to 79 years.

Table V

Modulation of intercellular communication in NHKs from different donors treated with SKC li-extracts (E2)

A 2-fold increase in the number of labeled cells is observed (p <0.0001-student test). The lipid extracts of SKC therefore induce a restoration of the CIJC of the cells of elderly donors approximately at the level of the CIJC of the cells of young donors.

• Flow cytometry (Figure 6)

The study was carried out on 6 donors aged 49 to 60.

Table VI

Modulation of the amount of connexin 43 after treatment with the lipid extract (E2) of SKC at 2.5 μg / ml / 24 h measured in Flow Cytometry.

There is an average increase of 25.6% in the labeling of the treated cells compared to the control cells.

SKC lipid extracts increase the amount of connexin 43.

The results obtained with the various means of evaluation of the effect of lipid extracts of the Skeletonema costatum alga on intercellular communication, in particular through JC and on the rate of connexin 43, carried out on KHN of elderly donors, show that the use of lipid extracts of the Skeletonema costatum algae makes it possible to restore intercellular communication, in particular through JC and also makes it possible to increase the rate of connexin 43 present in KHN.

These results show the advantage of using lipid extracts of the Skeletonema costatum alga in cosmetic compositions to combat the signs of skin aging, in particular to slow their appearance. The ~>.

The inventors have therefore developed a new means for combating skin aging, in particular against wrinkles and loss of elasticity of the skin.

In addition, the results obtained above show the advantage of using lipid extracts of the Skeletonema costatum alga in cosmetic compositions to promote and / or increase the activity of other cosmetic agents acting directly in the cell or via second intracellular messengers, and present or not, in the same cosmetic composition as the lipid extracts of SKC.

Example IV: Evaluation of the effect of boldine by the scratch-loading test or "scrape-loading"

The study was carried out on normal human keratinocytes (KHN). The concentrations used are: 12.5, 25, 50, 100 and 200 mM. The incubations are 24 hours in the culture medium. The table below summarizes the measured diffusion surface values.

Table VII

As can be seen in Table VII, the dose that seems most effective in increasing the functionality of the ICJC is 50 mM / 24h. Statistical analysis of the data shows that only this concentration makes it possible to obtain a truly significant difference in the increase in the CIJC compared to untreated KHN. The results in Table VII suggest a dose effect of boldine on the CIJC. Example V: Day cream for the face, anti-aging

Example VI: Emulsion-gel for the face, anti-wrinkle

Example VII: Firming Emulsion for the Body

Example VIII: Emulsion-gel for the face, anti-wrinkle

Claims

1. Cosmetic composition, characterized in that it comprises, as active ingredient, at least one substance promoting intercellular communication of skin cells, in particular keratinocytes, fibroblasts, and pre-adipocytes of the skin .

2. Composition according to claim 1, characterized in that said substance promoting intercellular communication, promotes intercellular communication through the communicating junctions of skin cells, in particular keratinocytes, fibroblasts, and pre-adipocytes of the skin .

3. Composition according to claim 1 or 2, characterized in that said substance promoting intercellular communication, favonse the formation of connexin, in particular connexin 43

4. Composition according to one of claims 1 to 3, characterized in that said substance promoting intercellular communication comprises at least one lipid extract of the Skeletonema alga, in particular of the Skeletonema costatum alga, in particular a total lipid extract of said alga.

5. Composition according to claim 4, characterized in that it is an extract obtained by extraction of the Skeletonema alga with an alcoholic solvent chosen from the group consisting of isopropanol, ethanol, and methanol.

6. Composition according to claim 5, caracténsée in that the above-mentioned extract is obtained by extraction of the alga with isopropanol.

7. Composition according to Claim 5, characterized in that the above-mentioned extract is obtained by extraction of the algae with ethanol.

8. Composition according to one of claims 4 to 7, characterized in that the extraction is carried out under reflux.

9. Composition according to one of claims 4 to 8, characterized in that the alga is frozen before being extracted with the alcoholic solvent, preferably the freezing being carried out at a temperature between -40 ° C and - 20 ° C approximately and for a period preferably between 1 and 7 days approximately.

10. Composition according to one of claims 4 to 9, characterized in that the frozen algae is immersed directly in the heated alcoholic solvent.

11. Composition according to one of claims 5 to 10, characterized in that the extract of the abovementioned alga is obtained after the succession of the following stages: a) the alcoholic solvent is alkalized up to a pH of between 10 and 14, preferably at a pH equal to 13, for example with an aqueous solution of sodium hydroxide or with an aqueous solution of potassium hydroxide, b) the insolubles are eliminated from the hydroalcoholic phase, c) from l distilled water is added to the hydro-alcoholic phase, d) the solution obtained undergoes a liquid-liquid extraction with an apolar solvent immiscible with the hydro-alcoholic phase, such as for example heptane, hexane or cyclohexane , e) the phase containing the apolar solvent is eliminated, f) the hydroalcoholic phase recovered after elimination of the phase containing the apolar solvent, is acidified to a pH between 1 and 3, preferably at a pH equal to 2, for example with a solutio n aqueous sulfuric acid or with an aqueous hydrochloric acid solution, g) the solution obtained after acidification undergoes liquid-liquid extraction with an apolar solvent immiscible with the hydroalcoholic phase, such as for example heptane, hexane or cyclohexane, h) the hydro-alcoholic phase is eliminated, i) the phase containing the non-polar solvent recovered after elimination of the hydro-alcoholic phase undergoes evaporation in order to obtain an oil free of non-polar solvent, this oil being l 'extract sought.

12. Composition according to claim 4, characterized in that the above-mentioned extract is obtained by extraction with supercritical CO.

13. Composition according to one of claims 5 to 11, characterized in that, before any extraction operation, a maceration of the alga is carried out in the alcoholic solvent at room temperature, preferably for a period of between 5 minutes and approximately 80 minutes, and more preferably, for a period of between 20 minutes and approximately 40 minutes.

14. Composition according to one of claims 5 to 10, or 13, characterized in that the amount of alcoholic solvent used is between 0.1 and approximately 20 liters of solvent, preferably between 2 liters and approximately 10 liters of solvent, per 100 g of algae, expressed in dry weight of algae.

15. Composition according to one of claims 4 to 14, characterized in that the extraction is carried out under an inert atmosphere, preferably under an atmosphere saturated with nitrogen.

16. Composition according to one of claims 4 to 15, characterized in that it comprises from 0.01% to 10% approximately, and in particular from 0.1% to 3% approximately by weight of said lipid extract of the Skeletonema seaweed, in particular Skeletonema costatum seaweed relative to the total weight of the final composition.

17. Composition according to one of claims 1 to 3, characterized in that said substance promoting intercellular communication of skin cells is boldine.

18. Composition according to claims 17, characterized in that it comprises from 0.001% to 10% approximately, and in particular from 0.01% to 1% approximately by weight of boldine relative to the total weight of the final composition.

19. Use of at least one substance promoting intercellular communication of keratinocytes, fibroblasts, and pre-adipocytes of the skin, as cosmetic agent, possibly in the presence of a cosmetically acceptable vehicle.

20. Use according to claim 19, characterized in that said substance promoting intercellular communication, promotes intercellular communication through the communicating junctions of keratinocytes, fibroblasts, and pre-adipocytes of the skin.

21. Use according to claim 19 or 20, characterized in that the substance promoting intercellular communication promotes the formation of connexin, in particular connexin 43.

22. Use according to one of claims 19 to 21, characterized in that said substance comprises at least one lipid extract of the Skeletonema alga, in particular of the Skeletonema costatum alga, in particular a total lipid extract of said alga, in particular as defined in any one of claims 5 to 16, advantageously an extract obtained by liquid-liquid extraction between an alcahnized and then acidified alcohol and an apolar solvent immiscible with the hydro-alcoholic phase, such as for example heptane, hexane or cyclohexane.

23. Use according to one of claims 19 to 21, characterized in that said substance is boldine.

24. Method for promoting and / or increasing the activity of a cosmetic agent acting directly in the cell or through second intracellular messengers, characterized in that it comprises the simultaneous or preliminary application to that of said cosmetic agent , on the areas of the skin concerned of a person in need thereof, of an effective amount of at least one substance promoting intercellular communication, in particular a substance promoting intercellular communication as defined in any one of claims 2 to 18.

25. Cosmetic anti-aging skin treatment method, characterized in that it comprises the application to the areas of the skin concerned of a person in need thereof, of an effective amount of at least one substance promoting intercellular communication, to obtain an anti-aging effect on said areas of the skin, in particular to improve skin firmness, improve the elasticity of the skin, delay the appearance of wrinkles or decrease their depth, in particular a substance promoting intercellular communication as defined in any one of claims 2 to 18.