FR2887772A1 - USE OF YEAST EXTRACT AS AN ACTIVE AGENT INDUCING THE SYNTHESIS OF SIRT PROTEINS IN SKIN CELLS. - Google Patents

Abstract

The present invention relates to the use of a yeast extract as an active agent inducing the endogenous synthesis of SIRT proteins in skin cells, in or for the preparation of a cosmetic or pharmaceutical composition.

Description

The invention relates to the field of cosmetics and pharmaceuticals,

  especially the field of dermatology. The present invention relates to the use of a yeast extract as an active agent inducing the endogenous synthesis of SIRT proteins in skin cells, in or for the preparation

  of a cosmetic or pharmaceutical composition.

  SIRT proteins are part of the Sirtuine family, they are nuclear proteins, NAD + dependent, playing an important role in the deacetylation of histones. SIR (Silent Information Regulators) genes, encoding SIR proteins, were first described in S. cerevisiae in 1979 (Rine Jet Al., Genetics 1979). Later, it was shown that overexpression of the SIR2P protein in C. elegans increased the lifespan of this organism (Tissenbaum et al., Nature 2001). This study thus emits the hypothesis that these proteins are linked to longevity.

  The SIRT1 protein is the most well-characterized human sirtuin, interacting with many transcriptional regulators. The human protein SIRT1 has been described as being involved in the regulation of p53 (Cheng HL et al. Proc Natl Acad Sci U S A. 2003); and, more recently, as a modulator of cellular senescence (Langley E et al., EMBO 1 2002). Other Sir human proteins have been discovered (SIRT2, SIRT3, SIRT4-7). The human protein SIRT2 has been little studied, but some studies have demonstrated its role in controlling mitotic activity (Dryden SC et al., Mol Cell Biol 2003) and its involvement in the regulation of p53 protein (Vaziri H et al., Cell 2001). To date, sirtuin deacetylases are considered to be an enzyme family that plays an important role in the regulation of cell death and its life cycle (Porcu M and Chiarugi, Trends Pharmacol Sci., 2005).

  It has recently been shown that this protein is significantly present in the skin and plays a very important role. It has been demonstrated that the SIRT protein, and more specifically the SIRT1 protein, is expressed in skin cells and that its expression is related to the different stresses that cutaneous cells encounter. In particular, it has been demonstrated that the induction of the expression of this protein by various agents makes it possible to protect the cells and better help them to fight against the stress and the intrinsic aging thereof.

  The SIRT1 protein thus occupies a very important function in aging and cellular protection. Thus, an increase in its expression allows the skin to better withstand the stress that surrounds it, that is to say, to better fight against oxidation and therefore, more generally, better fight against aging. More generally, the induction of the expression of this protein in skin cells provides a general improvement of cell protection mechanisms and would increase the cell life.

  The presence of this protein in the skin, as well as the benefits of its overexpression, puts into perspective new modes of action and new ways to improve cell survival, including cell survival of skin cells; but also, more generally means to improve the properties of the skin. By improving the properties of the skin means any means intended to maintain or restore a good functioning of the skin or any means that serves to preserve or improve its appearance and / or appearance.

  Thus, an increase in the synthesis of SIRT proteins will enable the skin to prevent the skin manifestations of aging, to be better protected against external aggressions and against the stresses to which it is subjected. Moreover, and according to more a more global view of the invention, an induction of SIRT will have anti-inflammatory and anti-irritant effects as well as a positive action on tissue regeneration and healing by an action, by example, on the increase of the synthesis of proteins constituting the extracellular matrix.

  The present invention thus relates to the use of an effective amount of at least one yeast extract as an active agent inducing the endogenous synthesis of SIRT proteins in skin cells. This active agent may be used alone or in combination with at least one other active agent, in or for the preparation of a cosmetic and / or dermopharmaceutical and / or dermatological composition.

  Indeed, by their inducing activity of SIRT protein synthesis, these yeast extracts have specific biological activities that make them directly usable in cosmetic, dermatological or pharmaceutical preparations or compositions.

  The use of a yeast extract, in the field of cosmetics, has already been described in numerous patents, particularly in skin care, as described in patent FR 2 826 576. However, its use as activating agent of SIRT protein synthesis has not yet been exposed.

  To date, very few uses of protein synthesis inducing compounds of the SIRT family in skin cells have been described. Among these compounds capable of activating the synthesis of SIRT proteins in the skin, mention may be made of various molecules including, for example, polyphenols. More particularly, mention may be made of: trans-stilbene derivatives (resveratrol, piceatannol), chalcone derivatives (isoliquiritigenine, butene), flavone derivatives (fisteine, luteoline, quercetin).

  Thus, the main object of the present invention relates to the use of a new compound capable of inducing the synthesis of SIRT proteins. The invention relates to the use of an effective amount of at least one yeast extract, as an active agent inducing the endogenous synthesis of SIRT proteins in skin cells, in or for the preparation of a cosmetic or pharmaceutical composition. Preferably, according to the invention, the compounds will activate a particular class of SIRT proteins, the SIRT1 proteins.

  By active agent inducing or activating the synthesis of SIRT proteins is meant an active agent capable of inducing the synthesis of SIRT proteins in the cells of the skin. That is to say, able to increase the amount of these proteins in the cells, but more particularly, is meant essentially designate all the active agents that can promote the endogenous production of SIRT proteins; and most particularly the molecules involved in the positive control of precursors such as DNA or RNA.

  The term "extract" refers to any isolated substance obtained from the raw material that yeasts constitute. The active agent can be defined as being a molecule or a set of molecules capable of modifying or modulating the functioning of a biological system.

  According to an advantageous embodiment of the invention, the yeast extract is a yeast extract of the genus Saccharomyces; preferably, the extract according to the invention is prepared from yeasts of the Saccharomyces cerevisiae species.

  The yeast extract of the genus Saccharomyces must be understood as a yeast extract belonging to the genus Saccharomyces. Of course, the extract can be prepared from yeasts of at least one of the many varieties and species belonging to the genus Saccharomyces.

  According to an embodiment of the presently preferred invention, the yeast extract is an aqueous extract. By aqueous extract is meant any combination of compounds soluble in water or in any solvent consisting wholly or partially of water. The extracts according to the invention are in particular purified aqueous extracts. In particular, aqueous solvents may be mentioned. By aqueous solvent is meant any solvent consisting wholly or partially of water. We can mention water itself, hydroalcoholic solvents in all proportions or solvents consisting of water and a compound such as propylene glycol or butylene glycol in all proportions. This extract can be obtained by dissolution in water, alcohol or ether, then by concentration of this solution involving evaporation or distillation.

  According to an advantageous embodiment of the invention, the yeast extract is an extract composed for the most part of compounds of peptide nature.

  By compounds of peptide nature is meant all compounds of protein nature, such as protein fragments, peptides but also free amino acids. The peptides, amino acids and protein fragments are determined according to conventional techniques, which are well known to those skilled in the art.

  Furthermore, according to another characteristic, the yeast extract contains compounds of low molecular weight.

  According to another advantageous embodiment of the invention, the yeast extract contains a quantity of peptide-like compounds representing between 30 and 70% of the total weight of the dry extract, more particularly this amount is between 40 and 50 % of the total weight of the dry extract.

  Any method of extraction or purification known to those skilled in the art can be used to prepare the extract according to the invention.

  Thus, in a first step, the yeasts are cultured in a conventional manner in a medium adapted to their development, preferably in the presence of lactose. They are harvested by centrifugation and then suspended in a buffer solution, preferably a phosphate buffer. In a second step, these cells are exploded using a French press or using a ball mill, the majority of the insoluble membrane components being removed by centrifugation or by filtration.

  A high protein filtrate can then be collected and redissolved. A fraction, rich in peptide-like compounds, can then be isolated by precipitation in an alcoholic medium or with a saline solution. The soluble components and the nucleic acids are thus discarded. According to a variant of the process for obtaining the extract according to the invention, a dialysis step and a hydrolysis step by a cocktail of proteases can be added in order to obtain a fraction rich in peptide-like compounds.

  An additional purification step by a chromatographic type method can be envisaged.

  According to another embodiment of the yeast extract according to the invention, the hydrolysis step may be carried out directly on the yeasts harvested after centrifugation or on the fractions obtained after cell bursting. Filtration and sterilization steps are subsequently performed.

  The use of the extract according to the invention capable of activating the synthesis of SIRT1 proteins in the cells of the skin will preferably be in the form of a composition adapted for external topical administration, mainly on the skin. , mucous membranes and / or integuments, comprising a cosmetically or dermatologically acceptable medium.

  The effective amount of active ingredient is the amount needed to achieve the desired result. Thus, according to an advantageous embodiment of the invention, the above active ingredient is used in the compositions at a concentration of between 0.00001% and 20% approximately, and preferably at a concentration of between 0.001% and 10%. approximately by weight relative to the total weight of the final composition.

  These extracts, capable of activating the endogenous synthesis of SIRT proteins, may be used, more generally, to treat dermatological conditions.

  Dermatological conditions are all diseases affecting the skin and having or not visible consequences. As such, there may be mentioned, for example: disorders related to cell differentiation and proliferation problems, problems related to keratinization, inflammatory or allergic disorders, disorders related to sebaceous functions, dermal or epidermal proliferations (benign or malignant); cutaneous disorders due to exposure to U.V. radiation, pathologies associated with chronological or actinic aging.

  Thus according to another aspect, the compounds according to the invention, as described above, intended to activate the endogenous synthesis of SIRT proteins in skin cells, may be used for the manufacture of a medicament for the treatment of dermal conditions. .

  According to an advantageous embodiment of the invention, the aforesaid active agent is first solubilized in one or more cosmetically or pharmaceutically acceptable solvents such as water, ethanol, propanol or isopropanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols or any mixture of these solvents.

  According to yet another advantageous embodiment of the invention, the aforesaid active agent is previously solubilized in a cosmetic or pharmaceutical vector such as liposomes or adsorbed on powdery organic polymers, mineral supports such as talcs and bentonites, and more generally solubilized in or attached to any cosmetically or pharmaceutically acceptable carrier.

  Whatever the form of the invention, the composition according to the invention can be ingested, injected or applied to the skin (on any cutaneous area of the body), hair, nails or mucous membranes. Depending on the mode of administration, the composition according to the invention may be in any of the galenical forms normally used. Preferably, the compositions according to the present invention will be in a dosage form suitable for topical administration to the skin. They cover all cosmetic or dermatological forms. These compositions must therefore contain a cosmetically acceptable medium, that is to say, compatible with the skin, hair or hair.

  According to another aspect, the present invention relates to a cosmetic treatment method for skin care intended to increase the endogenous synthesis of SIRT proteins in the cells of the skin, characterized in that a composition is applied to the skin. containing the active agent as defined above, in order to obtain the desired action.

  Particular embodiments of this cosmetic treatment method also result from the foregoing description.

  The cosmetic treatment process of the invention may be carried out in particular by applying the cosmetic compositions as defined above, according to the technique of usual use of these compositions, for example: application of creams, gels, serums , lotions, milks, shampoos or sunscreens, on the skin or on the hair, or application of toothpaste on the gums.

  Other advantages and characteristics of the invention will appear better on reading the examples given by way of illustration and not limitation.

  EXAMPLE 1 Demonstration of the Effect of the Extract According to the Invention on Fibroblasts

  The aim of the study is to determine the influence of yeast extract on the synthesis of SIRT1 proteins by fibroblasts, by the immunofluorescence technique and by the technique of western-blotting. Immunofluorescence and Westernblot techniques are semi-quantitative techniques that can be used to assess the level of proteins present in cells.

  Human fibroblasts are maintained in culture at 37 ° C. in a humidified atmosphere containing 5% CO 2. For immunofluorescence studies, fibroblasts were grown on 8-well slides; for the western blot, the cells were grown in boxes 100 mm in diameter. The cells are then incubated for 24 or 48 hours with the extract according to the invention dissolved in 0.5%; a control condition containing no extract is carried out.

  Immunofluorescence studies After removal of the supernatants and rinsing of the cultures, the cells are fixed with methanol for 4 minutes at 4 ° C. and then rinsed with PBS buffer. Anti-human SIRT1 rabbit antibodies, diluted 1/100, are then added (Santa Cruz Biotechnology). The incubation lasts 3 hours at room temperature; the supernatants are then removed and the cells are rinsed with PBS. Then a secondary antibody (anti-rabbit, Molecular Probes), coupled to a fluorescent marker (Alexa 488) is added. After 1 hour of incubation at room temperature, the supernatants are removed and the cells are rinsed with PBS. The slides are then mounted and examined under an Epi-fluorescence microscope (Nikon Eclipse E600 microscope).

  The amount of SIRT1 proteins, synthesized by the cells, is proportional to the intensity of the fluorescence. The results thus obtained demonstrate a significant increase in the expression of SIRT1 proteins at the nucleus of the cell after application of the extract according to the invention for 24 and 48 hours. Furthermore, the higher the concentration of extract applied at the cell level, the higher the intensity.

  Studies by Western-blot The cells are washed and scraped with extraction buffer (20mM Tris, 150mM NaCl, 10mM EDTA, 0.2% Triton X100) and a protease inhibitor cocktail. (Sigma). The proteins thus extracted are centrifuged at 4 ° C. at 10,000 rpm for 10 minutes and the samples thus obtained are stored at -80 ° C.

  After standardizing the samples with a BCA protein assay kit (Pierce), the cell lysates are electrophoretically separated on a 4-12% NuPAGE gel (Invitrogen) and transferred to a nitrocellulose membrane (PAL corporation). The membranes are incubated overnight with a human anti-SIRT1 rabbit antibody (Santa Cruz Biotechnology), diluted to 1/200, followed by incubation with an anti-rabbit IgG-peroxidase antibody, diluted to 1: 5000, (Beckman Coulter). The revelation is carried out with a chemiluminescent substrate.

  In order to quantitatively evaluate the proteins expressed in the cells, the bands present in the gel are quantified using ChimiImager software (Alpha Innotech Corporation, USA).

  The amount of protein is expressed as a percentage of light intensity, compared to the control condition, that is to say compared to cells that have not received the extract according to the invention.

  intensity% Incubation during 24 hours Incubation for 48 hours Control 100 100 Extract at 0.5% 114.1 271 The results, presented in the table above, allow us to conclude that the addition of the extract according to the invention in the fibroblast culture medium has the effect of increasing the synthesis of SIRT1 proteins by the cells. This stimulation has been observed significantly. Indeed, when the cells are incubated in the presence of the composition containing the extract according to the invention, there is an increase in the intensity of the fluorescence after 24 or 48 hours, compared to the untreated cells, thus expressing a stimulation of SIRT1 protein synthesis, this increase being likely dose-dependent.

  Example 2 Ex Vivo Detection of the Effect According to the Extract According to the Invention

  Ex vivo studies have made it possible, by immunostaining, to demonstrate the expression of SIRT1 proteins on skin samples.

  Human skin samples are cultured at the air / liquid interface. The extract according to the invention is applied topically to these samples, at the level of the epithelium, then the samples are incubated for 24 hours.

  These skin samples are then fixed with formaldehyde and then embedded in paraffin. Sections of 2 to 3 m are then made. Immunolabeling is performed after various stages of washing and incubation of these sections. Immunolabeling itself was performed with a human anti-SIRTI rabbit polyclonal antibody (IBL Co), diluted 1:50, and then with a secondary antibody (antilapin, Molecular Probes), coupled with a fluorescent marker. are then examined under a microscope at Epi-fluorescence (Nikon Eclipse E600 microscope).

  The results obtained demonstrate that the skins treated with the extract according to the invention express a significantly higher amount of SIRT1 proteins than the skins not treated with the extract. Thus, the extract according to the invention allows, at the cutaneous level, to increase the expression of SIRT1 proteins.

Claims (1)

11 CLAIMS   Use of an effective amount of at least one yeast extract, as an active agent inducing the endogenous synthesis of SIRT proteins in skin cells, in or for the preparation of a cosmetic or pharmaceutical composition .   2. Use according to claim 1 characterized in that the yeast extract is a yeast extract of the genus Saccharomyces.   3. Use according to claim 1 or 2 characterized in that the yeast extract is prepared from yeasts of the species Saccharomyces cerevisiae.   4. Use according to any one of the preceding claims, characterized in that the yeast extract is an extract of a peptide nature.   5. Use according to claim 4 characterized in that the yeast extract contains a quantity of compounds of peptide nature representing between 30 and 70% of the total weight of the dry extract, more particularly this amount is between 40 and 50% total weight of the dry extract.   6. Use according to any one of the preceding claims, characterized in that the SIRT proteins are SIRT1 proteins.   7. Use according to any one of the preceding claims characterized in that the composition contains cosmetically or pharmaceutically acceptable excipients.   8. Use according to claim 7, characterized in that the excipients are suitable for external topical administration.   9. Process for the cosmetic treatment of the skin intended to increase the endogenous synthesis of SIRT proteins in skin cells, characterized in that a composition containing the active agent as defined in the claims is applied to the skin. 1 to 8.