Pichia stipitis extract and its cosmetic uses

Abstract

The present invention relates to a cosmetic active ingredient and its cosmetic uses, in particular its anti-aging effects. The cosmetic active ingredient comprises an extract of Pichia stipitis comprising peptides, in particular at least 70% peptides and aims to combat skin aging, in particular to reduce wrinkles, increase skin hydration, as well as increase the radiance of the complexion by acting on dermo-epidermal communication, the dermis and the epidermis.

Description

Title of the invention: Pichia stipitis extract and its cosmetic uses Technical field

[0001] The invention relates to the field of cosmetics and concerns a new cosmetic active ingredient for its anti-aging effects. The cosmetic active ingredient comprises an extract of Pichia stipitis comprising peptides, in particular at least 70% of peptides and aims to combat skin aging by relaunching communication between the dermis and the epidermis, in particular to smooth the microrelief, increase skin hydration, as well as increase the radiance of the complexion. State of the art

[0002] The majority of yeasts are mixed organisms, both aerobic and anaerobic. In the presence of oxygen, yeasts undergo aerobic respiration, that is, they transform carbohydrates and oxygen into carbon dioxide and water in order to multiply. Conversely, in the absence of oxygen, they ferment by transforming carbohydrates into carbon dioxide and alcohol and therefore do not multiply. This latter phenomenon allows in particular the production of biofuel or the production of beer or wine.

[0003] The yeast Pichia stipitis, also known as Scheffersomyces stipitis, has one of the highest native xylose fermentation capacities of any known microorganism. It has the unique ability to ferment xylose by converting it into ethanol.

[0004] Given the abundance of xylose and its potential for the bioconversion of lignocellulosic materials into renewable fuels, Pichia stipitis has been widely studied. Given this interest, it has also been genetically modified to optimize its natural capabilities. In particular, its genome has been completely sequenced. Thus, the yeast Pichia stipitis is already known as a biotechnology tool.

[0005] However, contrary to the state of the art and the majority of publications concerning Pichia stipitis, yeast is not known as a cosmetic active ingredient.

[0006] Thus, in the present invention, the inventor was directly interested in the yeast Pichia stipitis as such to develop a new extract constituting the cosmetic active principle of the invention.

[0007] Indeed, the inventor took advantage of the respiratory metabolism of Pichia stipitis in order to multiply the number of Pichia stipitis cells. The yeast culture was carried out in aerobic conditions, controlling the parameters so that it divides and reproduces as much as possible. Oxygen and the substrate (i.e. at least one source of carbon, oxygen, nitrogen, hydrogen, phosphorus, sulfur and magnesium) were thus provided in such a way as to optimize the production of Pichia stipitis biomass. In this context, the culture conditions do not allow fermentation, particularly of xylose.

[0008] The inventor then extracted the potential of this biomass to develop a new cosmetic active ingredient for anti-aging effects, in particular to combat skin aging. Indeed, in our modern societies, there is always a need for new natural anti-aging cosmetic solutions. This is the subject of the present invention. Summary of the invention

[0009] The invention thus relates to an active ingredient derived from the yeast of the species Pichia stipitis, more particularly an extract of the yeast Pichia stipitis, said extract comprising peptides. Advantageously, the invention relates to an active ingredient comprising an extract derived from Pichia stipitis, enriched in peptides, the latter comprising more than 70% of peptides, by weight of the total weight of the extract.

[0010] The high peptide content of this extract makes it possible to improve cellular communication, in particular from the dermis to the epidermis, which is altered during aging. Several biological pathways are involved in communication from the dermis to the epidermis. They thus regulate epidermal aging, such as the IGF-1 / IGF-1R pathway, growth factors, inflammation mediators as well as cellular interactions but also micro-RNAs via extracellular vesicles. By acting on these pathways, the active ingredient according to the invention makes it possible to regulate cutaneous biological functions and to combat skin aging.

[0011] This extract may also include other constituents directly derived from the yeast Pichia stipitis, in particular sugars and minerals.

[0012] The extract therefore preferably comprises at least 70% of peptides, by weight of the total weight of the extract, even more preferably, at least 80% of peptides.

[0013] According to another preferred object of the invention, at least 80% of the peptides consist of peptides having a molecular weight of less than 3500 Da, even more preferably a molecular weight of less than 2000 Da.

[0014] According to another preferred subject of the invention, the extract according to the invention is a hydrolyzate of Pichia stipitis, more preferably an enzymatic hydrolyzate of Pichia stipitis.

[0015] Also, the extract according to the invention is preferentially obtained by at least one hydrolysis step, in particular an enzymatic hydrolysis, for example by the action of a protease.

[0016] According to a particularly preferred embodiment, the active ingredient according to the invention is obtained by a process comprising the following steps: a. solubilization of the Pichia stipitis biomass in water, b. enzymatic hydrolysis, c. enzymatic inactivation by heat treatment, d. separation of soluble and insoluble phases by centrifugation, and recovery of the soluble phase, e. molecular sorting f. chromatographic purification.

[0017] The richness in peptides of the Pichia stipitis extract thus makes it possible to obtain a cosmetic active ingredient having a global anti-aging cosmetic efficacy, that is to say acting at least on the dermo-epidermal communication and / or on the epidermis and / or on the dermis. This global anti-aging cosmetic efficacy results in a cosmetic efficacy chosen from an anti-wrinkle action, an improvement in hydration, an increase in skin density, an improvement in the radiance of the complexion, an acceleration of cell renewal, a strengthening of the dermal matrix, and the combination of at least two of these actions.

[0018] This extract is therefore an active principle of particular interest in cosmetics as an ingredient derived from natural materials to combat skin aging.

[0019] Thus, the invention relates, according to another aspect, to the cosmetic use of the cosmetic active ingredient according to the invention comprising an extract of Pichia stipitis according to any of the preceding embodiments, for its anti-aging effects. In particular for its anti-aging effects at the level of the dermis and / or the epidermis, in particular to improve dermo-epidermal communication. Said cosmetic active ingredient according to the invention is used and applied topically.

[0020] According to a particularly preferred object, said cosmetic active ingredient according to the invention is used for its moisturizing and / or smoothing effects, and / or to improve the radiance of the complexion and / or to reduce wrinkles.

[0021] To achieve this, the cosmetic active ingredient advantageously regulates the IGF-1 / IGF-1R pathway, inflammatory mediators, interaction proteins or even miRNAs transported by extracellular vesicles and thus improves dermo-epidermal communication allowing a global anti-aging action.

[0022] According to a final aspect, the invention relates to a cosmetic composition comprising at least 0.1% of an active ingredient according to any of the embodiments previously described, by weight of the total weight of the composition.

[0023] Other characteristics and advantages will emerge from the detailed description of the invention and the examples which follow. Detailed description of the invention

[0024] Definition

[0025] For the purposes of the present invention, the term "Pichia stipitis extract" means at least one molecule, preferably a set of molecules obtained from a raw material, namely yeast of the species Pichia stipitis also known as Scheffersomyces stipitis, regardless of the extraction method of said molecules. It may be, for example, an extract obtained by extraction, in particular by aqueous extraction, in particular hydroalcoholic or hydroglycolic, or by hydrolysis, in particular enzymatic or acid. The Pichia stipitis extract therefore excludes molecules obtained directly by the fermentation of Pichia stipitis.

[0026] For the purposes of the invention, the term "Pichia stipitis hydrolysate" means an extract from the yeast Pichia stipitis, obtained by a process comprising at least one step of hydrolysis of the biomass of Pichia stipitis, regardless of the hydrolysis agent used (acid, enzyme, etc.). The term Pichia stipitis hydrolysate therefore excludes molecules extracted solely by fermentation of Pichia stipitis.

[0027] By "active ingredient" within the meaning of the invention, we mean an extract comprising at least one molecule, preferably a set of molecules having a cosmetic effect, in particular when applied topically, in particular the cosmetic effect is an anti-aging effect, or a smoothing moisturizing effect, or a boost to the radiance of the complexion.

[0028] For the purposes of the invention, the term “biomass” means the total mass of organisms present in a given biotope or location at a given time.

[0029] For the purposes of the invention, the term “Pichia stipitis biomass” means the total mass of yeasts produced in a bioreactor, recovered by centrifugation and used to produce the active ingredient. The biomass therefore corresponds to Pichia stipitis cells.

[0030] By "global anti-aging action" within the meaning of the invention, is meant at least one cosmetic effect chosen from an anti-wrinkle action, an improvement in hydration, an increase in skin density, an improvement in the radiance of the complexion, an acceleration of cell renewal, a strengthening of the dermal matrix, and their combinations.

[0031] Active ingredient

[0032] The yeast Pichia stipitis, also known as Scheffersomyces stipitis, is a yeast of the Saccharomycetaceae family, particularly recognized for its ability to directly ferment xylose, which makes it a biotechnological tool of choice for producing ethanol from lignocellulosic residues. Thus, this yeast has been particularly studied in the field of bioconversion of lignocellulosic materials into renewable fuels. However, no cosmetic active ingredient based on Pichia stipitis is known.

[0033] Thus, according to a first aspect, the present invention relates to an active principle cosmetic comprising at least one extract obtained from Pichia stipitis, said extract comprising peptides. In particular, said Pichia stipitis extract is enriched with peptides which allows it to restore communication from the dermis to the damaged epidermis and thus enable a global anti-aging action.

[0034] Advantageously, the extract according to the invention comprises at least 70% of peptides, by weight of the total weight of the extract, more preferably at least 80% of peptides. Said extract is therefore particularly enriched in peptides.

[0035] The peptide content can be determined by the KJELDHAL method (reference: Official method of analysis of the AOC 1975, 12th ed. W Horwitz, ED, New York, p. 15-60) or by the BIURET method.

[0036] Said peptides preferably have a molar mass less than or equal to 3,500 Da. More preferably, at least 80% of the peptides have a molecular weight less than 3,500 Da. Very preferably, said peptides have a size less than 2,000 Da.

[0037] The distribution and size of the peptides can be determined by FPLC chromatography followed by spectrophotometric determination according to the Lowry method (Lowry et al., Protein measurement with the Folin reagent, J. Biol. Chem., 193, 265-275, 1951).

[0038] The characterization and quantification of the amino acids present in the extract and therefore in the active principle according to the invention can be carried out by liquid chromatography after total hydrolysis of the sample.

[0039] Thus, the extract is preferentially rich in aspartic acid, glutamic acid and lysine.

[0040] The inventor continued the characterization of this extract and thus found that it also advantageously comprises sugars and minerals. He therefore determined and quantified the content of sugars, ash, and proteins forming the extract. The active ingredient according to the invention comprises less than 5% of sugars, less than 10% of ash and more than 70% of peptides by weight of the total weight of the extract.

[0041] Total sugars were measured using the DUBOIS method (Dubois M. et al., Analytical chemistry, 28, 3, 350-356, 1956) and the protein content was determined by the KJELDHAL method (reference: Official method of analysis of the AOC 1975, 12th ed. W Horwitz, ED, New York, p. 15-60).

[0042] The raw ash content can be determined by weighing the residues from incineration at 550°C in an electric muffle furnace.

[0043] As a result, the extract according to the invention is particularly enriched in peptides, and may comprise sugars and minerals. This enrichment in peptides is preferably obtained using a process combining hydrolysis, preferably enzymatic hydrolysis, molecular sorting and purification. Thus, the Pichia stipitis extract according to the invention is preferably a hydrolyzate, more preferably an enzymatic hydrolyzate.

[0044] According to another particularly preferred object, the extract is capable of being obtained by a process advantageously comprising at least the following steps: a. solubilization of the Pichia stipitis biomass in water, preferably at a rate of at least 100 g / L, b. enzymatic hydrolysis, preferably by at least one protease, c. enzymatic inactivation by heat treatment, d. separation of the soluble and insoluble phases by centrifugation, and recovery of the soluble phase, e. molecular sorting f. chromatographic purification.

[0045] Also, a first form of the active ingredient according to the invention is obtained comprising at least the extract of Pichia stipitis as described previously.

[0046] The cosmetic active ingredient according to the invention may also advantageously comprise at least one other ingredient such as a preservative, an antioxidant, a stabilizer, an atomization support, a mineral filler, a polymer, a plasticizer, a surfactant and / or their combination.

[0047] The extract according to the invention present in the cosmetic active ingredient can then be in liquid form or in solid form or in film form.

[0048] When it is in liquid form, the active ingredient according to the invention may consist of the extract of Pichia stipitis accompanied by a stabilizer and / or preservation system.

[0049] The extract in liquid form is preferably in the form of a clear liquid solution, with a weak odor and a light yellow to yellow color.

[0050] When it is in liquid form, the dry matter content of the extract can be determined by weighing the residues resulting from drying the extract according to the invention at 105°C in an oven until a constant weight is obtained. Preferably, the extract according to the invention in liquid form has a dry matter content of between 8 and 20 g / L, preferably between 10 and 16 g / L.

[0051] In the case where it is in solid form, the active ingredient according to the invention could consist of the extract of Pichia stipitis as previously described and of a support chosen from maltodextrin, gum arabic or soy lecithin. According to a particularly suitable embodiment, the extract represents at least 10% by weight of the active ingredient and the support at most 90% by weight of the active ingredient.

[0052] In the case of a solid form in which the active ingredient is associated with a carrier, the sugar and protein contents relative to the dry matter in the active ingredient can be modified since the carriers (maltodextrin, gum

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[0064] arabic) are exclusively saccharides. The active ingredient according to the invention can also be presented in the form of a film as described in patent FR3079145. In this case, the Pichia stipitis extract represents at least 0.1% by weight of the film. When presented in film form, the active ingredient includes: - at least the extract of Pichia stipitis according to the invention. - at least one mineral filler, and - at least one polymer of natural origin, and - at least one plasticizer, and - at least one surfactant. The naturally occurring polymer can be chosen from: pectin, tamarind gum, alginate, pullulan, psyllium, xanthan, guar, tara, carob, agar, gum arabic, gellan, dextran, carrageenan, cellulose, konjac and chitosan. The plasticizer can be chosen from: glycerol, sorbitol, sucrose, erythritol, urea, propylene glycol and butylene glycol. The mineral filler can be chosen from: calcium carbonate, green clay, kaolin, perlite, talc, magnesium silicate, mica, diatomaceous sericite, silica, calcium sulfate, calcium chloride, potassium chloride, iron oxide and zinc oxide. Finally, the active ingredient may also include a pigment to color the film. Process for preparing the extract according to the invention According to another aspect, the invention relates to an extraction method comprising at least one hydrolysis step from the biomass of the yeast Pichia stipitis, in particular an enzymatic hydrolysis, a molecular sorting step and purification by chromatography. Prior to the process of obtaining the extract as such, a production of the biomass of Pichia stipitis is carried out in the following manner: - The yeasts are cultured aerobically in a conventional manner in a medium suitable for their development, - Yeasts are preferentially harvested by centrifugation - The recovered fraction is the biomass of Pichia stipitis The medium used contains at least one source of carbon, oxygen, nitrogen, hydrogen, phosphorus, sulfur and magnesium. Pichia stipitis biomass production can be characterized by comparing the number of cells at the start and end of production. For example, production is started with a density of 1.1x106 CFU / mL (Colony Forming Unit). At the end of the culture, the bioreactor can contain a density of 1.56x1010 CFU / mL. According to a particularly suitable embodiment, the active ingredient according to the invention is obtained by implementing the following steps: a. solubilization of Pichia stipitis biomass in water, preferably at a rate of at least 100 g / L, b. enzymatic hydrolysis, preferably by at least one protease, c. enzymatic inactivation by heat treatment, d. separation of the soluble and insoluble phases by centrifugation, and recovery of the soluble phase, e. molecular sorting f. chromatographic purification.

[0065] The solubilization is carried out in water, preferably at a rate of at least 100 g / L. The hydrolysis is preferably an enzymatic hydrolysis, in particular by the action of at least one protease, making it possible to improve the enrichment in compounds of interest, namely the peptides. Such hydrolysis is followed by an enzymatic inactivation, in particular by heat treatment in order to stop the enzymatic reaction.

[0066] The separation of the soluble and insoluble phases is carried out by any means known to those skilled in the art, for example by centrifugation, filtration or decantation, preferably by centrifugation. The separation of the soluble and insoluble phases is carried out to recover the soluble phase containing the peptides of interest.

[0067] A molecular sorting step by filtration followed by a chromatographic purification step makes it possible to improve the concentration of natural biopeptides.

[0068] The extract obtained at this stage is an extract of Pichia stipitis, and constitutes a first form of the active ingredient according to the invention, then appearing in liquid form.

[0069] The steps of the processes described above, taken individually, are customary in the field of extractions of active ingredients from natural raw materials and those skilled in the art are able to adjust the reaction parameters on the basis of their general knowledge.

[0070] Cosmetic compositions according to the invention

[0071] The active ingredient according to the invention is intended to be integrated into a cosmetic composition, in particular a composition comprising at least 0.1% by weight of said active ingredient and a physiologically acceptable medium, preferably a cosmetically acceptable medium.

[0072] These compositions may be presented in particular in the form of oil-in-water emulsions, water-in-oil emulsions, multiple emulsions (Water / Oil / Water or Oil / Water / Oil) which may optionally be microemulsions or nanoemulsions, or in the form of solutions, suspensions, hydrodispersions, aqueous gels, powders, or in the form of a film. They may be more or less fluid and have the appearance of creams, emulsions, gels, masks or any other aspects known to those skilled in the art. job.

[0073] Preferably, these may be compositions comprising between 0.1 and 20% of the active ingredient according to the invention, in particular in liquid form, more preferably between 0.5 and 10%.

[0074] These compositions comprise, in addition to the active ingredient, a physiologically acceptable medium such as a cosmetically acceptable medium, that is to say which does not cause sensations of discomfort for the user such as redness, tightness or tingling.

[0075] The compositions according to the invention may also contain as an ingredient at least one compound chosen from: - oils, which may be chosen in particular from silicone oils, linear or cyclic, volatile or non-volatile, - waxes, such as ozokerite, polyethylene wax, beeswax or camauba wax, - silicone elastomers, - surfactants, preferably emulsifying surfactants, whether non-ionic, anionic, cationic or amphoteric, - co-surfactants, such as linear fatty alcohols, - thickeners and / or gelling agents, - humectants, such as polyols like glycerin, - colorants, preservatives, fillers, - tensors, - sequestrants, - perfumes, - and their mixtures, without this list being exhaustive.

[0076] Examples of such ingredients are cited in particular in the CTFA Dictionary (International Cosmetic Ingredient Dictionary and Handbook) published by the Personal Care Product Council.

[0077] Of course, those skilled in the art will take care to choose the possible complementary compounds, active or non-active, and their quantity, in such a way that the advantageous properties of the mixture are not, or not significantly, altered by the envisaged addition.

[0078] Cosmetic use

[0079] Finally, according to a last aspect of the present invention, the active ingredient according to any of the embodiments previously described, including when said active ingredient is included in a cosmetic composition, is intended to be used for a global anti-aging action.

[0080] Thus, the invention also aims at a cosmetic use of the active ingredient according to the invention or a composition according to the invention for its anti-aging effects, preferably to combat skin aging.

[0081] Due to its position at the interface with the environment, the epidermis is the skin layer most exposed to the various factors of aggression responsible for the alteration of its functions and its aging. This results in a thinning of the epidermis, a loss of its capacity for self-renewal, which results in an alteration of the microrelief, a reduction in the radiance of the complexion, and a reduction in skin hydration.

[0082] The active ingredient, by restoring communication from the dermis to the epidermis, which is altered during aging, restores epidermal functionalities. Indeed, it regulates the proteome, the expression of miRNAs present in extracellular vesicles and stimulates the IGF-1 / IGF-1R pathway, leading to stimulation of the synthesis of keratinocyte biological markers, an increase in the thickness of the living layers, an increase in skin density, an acceleration of cell renewal and the strengthening of the dermal matrix. Thus, the invention also relates to the use of the active ingredient as an anti-aging agent in the dermis and / or epidermis, as well as to improve dermo-epidermal communication.

[0083] These biological effects then result in: - an improvement in the water content of the epidermis, thus promoting hydration, - an improvement in skin microrelief and a reduction in wrinkles, resulting in a smoothing effect and a boost in the radiance of the complexion.

[0084] The cosmetic benefits of this cosmetic active ingredient and of the composition including it are therefore the following, namely a moisturizing effect, and / or a smoothing effect, and / or an improvement in the radiance of the complexion. Thus, according to a preferred object, the present invention aims at the cosmetic use of the active ingredient for its moisturizing, and / or smoothing effects, and / or for improving the radiance of the complexion.

[0085] According to another preferred object, the active ingredient according to the invention or the composition according to the invention is intended to be used on healthy skin in order to combat the signs of aging. Thus, the active ingredient according to the invention or the composition according to the invention is advantageously intended for a cosmetic treatment in topical application to the skin.

[0086] The invention is now illustrated by non-limiting examples of compositions according to the invention and by results. Examples

[0087] Example 1: Extract according to the invention

[0088] The active ingredient is obtained by implementing the following steps: - Solubilization of Pichia stipitis biomass in water, at a rate of at least 100 g / L. - Enzymatic hydrolysis - Enzymatic inactivation by heat treatment - Separation of soluble and insoluble phases by centrifugation - Recovery of the soluble phase - Molecular sorting - Chromatographic purification. - Deodorization - filtration and sterilizing filtration.

[0089] The active ingredient obtained has the following analytical characteristics: - a dry matter content: 14.9 g / L - a total sugar content (according to the Dubois method): 1% - an ash content: 4% - protein content (according to the Kjeldhal method): 95%

[0090] The characterization and quantification of the amino acids constituting the extract according to the invention were carried out by liquid chromatography after hydrolysis of the sample. The distribution of the different amino acids is presented in Table 1 below.

[0091] [Tables 1] Distribution (%) Name of the amino acid Active ingredient according to the invention Aspartic acid 11.0 Threonine 5.0 Serine 5.3 Glutamic acid 14.3 Proline 4.0 Glycine 4.0 Alanine 7.3 Cystine 1.5 Valine 5.9 Methionine 1.1 Isoleucine 5.0 Leucine 8.2 Tyrosine 2.9 Phenylalanine 3.7 Histidine 2.5 Lysine 11.6 Arginine 6.7

[0092] Evaluation of the effectiveness of the active ingredient according to the invention

[0093] Test 1 - Evaluation of the capacity of the active ingredient according to the invention to regulate the synthesis of proteins involved in communication from the dermis to the epidermis.

[0094] The objective of this study is to evaluate the capacity of the active ingredient according to the invention to regulate the synthesis of proteins linked to the dermis-epidermis communication pathways altered during aging. This study was carried out by proteomics on secretomes from young or aged human fibroblasts.

[0095] The protocol is as follows: human fibroblasts are seeded and incubated at 37°C in an atmosphere containing 5% CO2. Then, the fibroblasts are treated with the active ingredient according to the invention at 0.4% (V / V) for 48 hours and incubated at 37°C in an atmosphere containing 5% CO2. Subsequently, the secretomes of the fibroblasts are recovered and then stored and preserved at -80°C.

[0096] Secretomes (from young fibroblasts, aged fibroblasts or fibroblasts aged treated with the principle according to the invention) are then analyzed by proteomics. The proteome was determined by LC-MS / MS (nano-LC coupled with a mass spectrometer) via a quantitative "Shotgun label free" approach. The modulated proteins are analyzed using databases that allow the detection of biological pathways of interest and the impact of the active principle according to the invention on them.

[0097] The results are as follows. Proteomic analysis of the secretomes of young fibroblasts allowed the detection of 355 proteins, 137 of which are dedicated to an intercellular communication function.

[0098] These have made it possible to highlight 4 metabolic pathways linked to dermis-epidermis communication listed, namely the IGF regulation and transport pathway, the growth factor signaling pathway, the inflammation signaling pathway, and the cellular interaction pathway.

[0099] A comparison of the communication pathways between the secretomes from young and aged fibroblasts then revealed a deregulation of protein synthesis in the secretome of aged fibroblasts of the order of: - 54% in the IGF regulation and transport pathway; - 57% in the growth factor signaling pathway; - 51% in the inflammation signaling pathway; - 53% in the pathway of cellular interactions.

[0100] Tested at 0.4% on aged fibroblasts, the active ingredient according to the invention makes it possible to modulate the synthesis of certain proteins involved in the different pathways of dermis-epidermis communication, namely: - 68% of the proteins in the IGF regulatory and transport pathway are modulated by the active ingredient, - 60% of the proteins in the growth factor signaling pathway are modulated by the active ingredient, - 61% of proteins in the inflammation signaling pathway are modulated by the active ingredient, - 47% of proteins in the cellular interaction pathway are modulated by the active ingredient.

[0101] Thus, the active ingredient according to the invention makes it possible to limit the undesirable effects of aging on dermo-epidermal communication.

[0102] Test 2 - Evaluation of the capacity of the active ingredient according to the invention to regulate the expression of miRNAs in the extracellular vesicles involved in communication from the dermis to the epidermis,

[0103] The objective of this study is to evaluate the effect of the active ingredient (AI) according to the invention on the expression of miRNAs, present in the extracellular vesicles secreted by aged dermal fibroblasts.

[0104] Extracellular vesicles (EVs) act as biological messengers due to their ability to transport proteins, nucleic acids (e.g. miRNA) and lipids. Secreted by cells, they are internalized by neighboring cells in which they regulate various biological activities. In addition, they play a major role in cellular communication from the dermis to the epidermis and promote the adaptation of cells to their environment.

[0105] With age, the expression of many miRNAs is modified. Nine miRNAs were identified as being involved in the regulation of physiological events in the skin and were tested in the study.

[0106] The protocol is as follows. Human fibroblasts are seeded and incubated at 37°C in an atmosphere containing 5% CO2. Then, the fibroblasts are treated with the active ingredient according to the invention at 0.4% (V / V) for 48 hours and incubated at 37°C in an atmosphere containing 5% CO2. Then, the extracellular vesicles secreted by the fibroblasts are isolated from the secretomes and characterized before being stored and preserved at - 80°C.

[0107] The miRNAs present in the extracellular vesicles are extracted and then reverse-transcribed and the complementary DNAs obtained are analyzed by the quantitative PCR technique. The quantification of the incorporation of fluorescence (SYBR Green) is measured continuously using a thermocycler.

[0108] The results are presented in Table 2, below.

[0109] [Tables2] Epidermal biological pathways miRNA N VE from young fibroblasts VE from aged fibroblasts PA according to the invention 0.4% Capacity to restore the expression of miRNAs involved in skin aging (%) Proliferation miR-13 0a-3p 100 218 141 +65% miR-13 8-5p 100 365 223 +54% miR-18 la-5p 100 257 155 +65% Differentiation Barrier function miR-10 a-5p 100 235 157 +58% miR-30 a-3p 100 237 142 +69% miR-13 7-3p 100 241 137 +74% miR-14 0-3p 100 155 99 +102% Regeneration miR-19 l-5p 100 250 135 +77% miR-21 4-5p 100 179 107 +91%

[0110] Compared to young fibroblasts, miRNAs present in extracellular vesicles (EVs) secreted by aged fibroblasts have significantly increased expression.

[0111] Tested at 0.4%, the active ingredient according to the invention significantly increases the capacity of aged fibroblasts to reduce the expression of miRNAs involved in epidermal aging. Thus, by significantly regulating the expression of miRNAs in extracellular vesicles, the active ingredient according to the invention reestablishes communication from the dermis to the epidermis.

[0112] Test 3 - Evaluation of the capacity of the active ingredient according to the invention to stimulate the IGF-1 / IGF-1R pathway acting in the communication from the dermis to the epidermis.

[0113] The functional integrity of the skin depends on the numerous interactions between dermal fibroblasts and epidermal keratinocytes. A characteristic marker of dermal-epidermal communication, the IGF-1 / IGF-1R pathway is described as central to longevity processes. The growth factor IGF-1, synthesized by fibroblasts, activates its receptor (IGF-1R) expressed by keratinocytes in order to regulate the mechanisms of proliferation, differentiation or even epidermal adhesion. During aging, several studies have shown a decline in the secretion of IGF-1 by aged fibroblasts leading to a decrease in activation of the IGF-1 receptor and consequently, a deleterious effect on the skin.

[0114] The objective of this study is therefore to evaluate the effect of the active ingredient according to the invention on the activity of the IGF-1 / IGF-1R pathway. This study was carried out on human explants from young (< 35 years) and elderly (> 60 years) donors. The secretion of IGF-1 was studied by ELIS A assay and the activation of the IGF-1 receptor (phosphorylation) by immunohistofluorescence.

[0115] The protocol is as follows. Human skin explants were made and maintained in survival in culture medium at 37°C in an atmosphere containing 5% CO2. The explants are treated systemically in the presence or absence of the active ingredient according to the invention at 0.4% or 0.8% (V / V). Then, the secretomes are recovered and stored at -80°C pending ELISA dosing and the explants are fixed, dehydrated and embedded in paraffin. Sections are then made using a microtome.

[0116] The level of phosphorylated IGF-1 receptor is measured and this is proportional to the fluorescence intensity present on the explants. A quantitative analysis of the images was carried out using an image analysis script. The results are expressed in arbitrary units (AU).

[0117] The results of the capacity of the active ingredient according to the invention to stimulate the secretion of IGF-1 by fibroblasts in young and old human explants are presented in Table 3, below.

[0118] [Tables3] IGF-1 synthesis (pg / mL) Capacity to stimulate IGF-1 synthesis (%) Young explants Control 0.335 PA according to the invention 0.8% 0.452 Old explants Control 0.059 PA according to the invention 0.4% 0.090 +11% PA according to the invention 0.8% 0.141 +30%

[0119] The results of the capacity of the active ingredient according to the invention to stimulate the phosphorylation of IGF-1R in the epidermal compartment of young and old human explants are presented in Table 4, below.

[0120] [Tables4] Phosphorylated IGF-1R (xl04 UA) Capacity to stimulate IGF-1R phosphorylation (%) Young explants Control 353 PA according to the invention at 0.8% 444 Old explants Control 190 PA according to the invention at 0.4% 283 +57% PA according to the invention at 0.8% 328 +85%

[0121] Explants from aged donors show a significant decrease in the secretion of the growth factor IGF-1 and the phosphorylation of its receptor at the epidermal level. Tested at 0.8% on aged explants, the active ingredient according to the invention significantly stimulates the secretion of IGF-1 by 30% and the phosphorylation of its epidermal receptor by 85%.

[0122] Thus, by significantly stimulating the IGF-1 / IGF-1R biological pathway altered during aging, the active ingredient according to the invention reestablishes communication from the dermis to the epidermis.

[0123] Test 4 - Evaluation of the capacity of the active ingredient according to the invention on the dermal compartment

[0124] The objective of this study is to evaluate in vivo in volunteers the effect of the active ingredient according to the invention formulated at 1.7% in emulsion on the dermal compartment of the skin, in comparison with a placebo formula. The active ingredient according to the invention formulated at 1.7% and the placebo were applied to the half-face, twice daily, for 28 days. This study was carried out on 18 volunteers aged 62 to 77 years (mean age 68 + 4 years).

[0125] The effect of the active ingredient according to the invention on the dermal compartment of the skin was evaluated in comparison with the placebo after 28 days of twice-daily application to the half-face using the following methods: - Study of dermal density by high-frequency ultrasound - Study of the quality of the dermal matrix by LC-OCT - Study of wrinkles by projection of fringes.

[0126] The results corresponding to the effect of the active ingredient according to the invention formulated at 1.7% on the density of the dermis studied by high-frequency ultrasound in volunteers are presented in Table 5 below.

[0127] [Tables5] Density variation / D0 (%) Placebo -0.7 PA at 1.7% 8.5

[0128] After 28 days of twice-daily application and in comparison with the placebo, the active ingredient according to the invention formulated at 1.7% in emulsion significantly increases the density of the dermal matrix of Caucasian volunteers by 9.3%. This improvement was observed in 72% of the volunteers.

[0129] The results corresponding to the effect of the active ingredient according to the invention formulated at 1.7% on the quality of the fibers of the dermal matrix studied by LC-OCT are presented in Table 6 below.

[0130] [Tableauxô] Change in dermal matrix quality / D0 (%) Placebo 0.6 PA to 1.7% 11.3

[0131] After 28 days of twice-daily application and in comparison with the placebo, the active ingredient according to the invention formulated at 1.7% significantly improves the quality of the fibers of the dermal matrix of Caucasian volunteers (+ 10.7%). This improvement is visible in 89% of the volunteers.

[0132] The results corresponding to the anti-wrinkle effect of the active ingredient according to the invention formulated at 1.7% studied by fringe projection are presented in Table 7 below.

[0133] [Tables7] Variation of the Rz parameter / Placebo (%) D14 D28 Rz parameter -6.0 -9.8

[0134] From 14 days of twice-daily application by Caucasian volunteers and in comparison with the placebo, the active ingredient according to the invention formulated at 1.7% in emulsion reduces wrinkles around the eyes. This anti-wrinkle effect is accentuated after 28 days of treatment where a reduction in the Rz parameter (-9.8%) is measured in 69% of subjects.

[0135] The active ingredient according to the invention formulated at 1.7% in emulsion and in comparison with the placebo, presents significant effects on the matrix dynamics of the volunteers after 28 days of twice-daily treatment on the half-face.

[0136] Thus, the active ingredient according to the invention allows an increase in the density of the dermis. An improvement in the quality of the fibers of the dermal matrix and a reduction in wrinkles has also been observed.

[0137] Test 5 - Evaluation of the capacity of the active ingredient according to the invention to stimulate epidermal functions, in particular the synthesis of keratin-cell biological markers.

[0138] The objective of this study is to evaluate the impact of the defect in communication from the dermis to the epidermis appearing during aging on the functionalities epidermal.

[0139] To achieve this, several markers were analyzed, namely: - proliferation marker: Ki-67, key protein in epidermal cell proliferation; - epidermal differentiation marker: Filaggrin (FLG), a protein playing a major role in the formation of the stratum corneum; - cell cohesion marker: Desmoglein 1 (DSG1), desmosomal protein ensuring intercellular junctions; - hydration marker: Aquaporin3 (AQP3), a protein playing an essential role in water transport and maintaining hydration of the stratum corneum; - epidermis-dermis anchoring marker: Laminin 332 (LAMA3), a major protein of the dermo-epidermal junction playing a key role in the adhesion of epidermal cells by interacting with integrins.

[0140] These studies were carried out on human keratinocytes from young donors (< 30 years) or from elderly donors (> 60 years) by immunocytofluorescence (synthesis of Ki-67) or by quantitative PCR (expression of the markers FLG, DSG1, AQP3, LAMA3).

[0141] The protocol is as follows. Young and aged human keratinocytes are seeded and incubated at 37°C in an atmosphere containing 5% CO2. The aged keratinocytes are treated with an aged fibroblast secretome (SFV) treated or not with the active ingredient according to the invention at 0.2% and 0.4% (V / V). The cells are then incubated in an atmosphere containing 5% CO2 at 37°C for 48 hours.

[0142] The level of synthesized Ki-67 is proportional to the fluorescence intensity present in the cells. A quantitative analysis of the images was carried out using an image analysis script.

[0143] The cells were recovered and the total RNAs extracted. The RNAs were reverse-transcribed and the complementary DNAs obtained were analyzed by the quantitative PCR technique. The mRNAs of the internal reference control proteins were analyzed in parallel with the mRNAs of the markers involved in the epidermal biological pathways. The incorporation of fluorescence is measured continuously using a thermocycler.

[0144] The results showing the impact of the aged fibroblast secretome on aged keratinocytes are presented in Table 8, below.

[0145] [Tables8] Young keratinocytes (%) Old keratinocytes (%) Old keratinocytes + aged fibroblast secretome (SFV) (%) Impact of SFV on aged keratinocytes / old control keratinocytes (%) Proliferation Ki-67 100 60 41 -32% Differentiation Filaggrin (FLG) 100 39 30 -23% Cohesion Des-moglein 1 (DSG1) 100 52 23 -56% Hydration Aquaporin3 (AQP3) 100 70 47 -33% Epidermis-dermis anchoring Laminin 332 (LAMA3) 100 101 73 -28%

[0146] Markers of different epidermal biological pathways of aged human keratinocytes treated with the secretome of aged fibroblasts are significantly decreased.

[0147] The effect of the secretome of aged fibroblasts, pre-treated with the active ingredient according to the invention, was then studied on the proliferation of aged keratinocytes. The results are presented in Table 9 below.

[0148] [Tables9] Ki-67 positive cells (%) Capacity to stimulate Ki-67 synthesis (%) Young keratinocytes Control 57 Old keratinocytes + aged fibroblast secretome (SFV) Control 23 PA according to the invention at 0.2% 28 +15% PA according to the invention at 0.4% 30 +21%

[0149] Furthermore, the effect of the secretome of aged fibroblasts, pre-treated with the active ingredient according to the invention, on the expression of epidermal biological markers by aged keratinocytes is presented in Table 10, below.

[0150] [TableauxlO] Young keratinocytes Old keratinocytes + aged fibroblast secretome (SFV) Control Control PA according to the invention at 0.2% PA according to the invention at 0.4% Capacity to restore epidermal markers (%) Filaggrin 100 30 41 59 +41% Desmoglein 1 100 23 25 42 +25% Aquaporin3 100 47 64 66 +36% Laminin 332 100 73 92 98 +93%

[0151] Thus, applied to aged human keratinocytes, the secretome from aged human fibroblasts pre-treated with 0.4% of the active ingredient according to the invention significantly restores the expression of the markers studied. By regulating the secretome of aged fibroblasts, the active ingredient according to the invention then significantly limits its deleterious effects on the biological functions of the epidermis.

[0152] Test 6 - Evaluation of the capacity of the active ingredient according to the invention to improve the thickness and quality of the epidermis.

[0153] The objective of this test is to evaluate in vivo the biological activities of the active ingredient according to the invention formulated at 1.7% in emulsion, on the epidermal compartment of the skin, in comparison with a placebo formula in Caucasian volunteers. In particular, this test aims to study the thickness of the layers of the epidermis from acquisitions carried out by LC-OCT after the active ingredient according to the invention has been applied twice daily, on half of the face for 28 days.

[0154] The results are presented in Table 11, below.

[0155] [Tableauxll] Variation / Placebo (%) D14 D28 Thickness of living layers +6.7 +9.0

[0156] From 14 days of twice-daily application and in comparison with the placebo, the active ingredient according to the invention formulated at 1.7% in emulsion, significantly increases the quality of the epidermis of Caucasian volunteers. This effect results in an increase in the thickness of the living layers of 6.7% in 72% of the volunteers. This effect continues after 28 days of treatment with an increase equal to +9.0% and is observed in 83% of the subjects.

[0157] Furthermore, this trial was continued with a comparison of these results with those obtained during a study conducted on 23 volunteers aged between 51 and 60 years (56 ± 3 years).

[0158] The results are presented in Table 12, below.

[0159] [Tablesl2] Thickness of living layers (pm) D0 D14 D28 Placebo on Group 62 - 70 years 43.5 43.8 43.8 Placebo on Group 50-60 years 48.6 PA at 1.7% on Group 62-70 years 42.4 45.6 46.6

[0160] As a result, the active ingredient according to the invention allows the volunteers who used it to regain an epidermis thickness comparable to that of a group of volunteers of average age 10 years younger. The difference between the two groups is no longer significant.

[0161] Test 7 - Evaluation of the capacity of the active ingredient according to the invention to improve the water content of the epidermis,

[0162] The objective of this test is to evaluate in vivo the cosmetic benefits of the active ingredient according to the invention formulated at 1.7% in emulsion, in comparison with a placebo formula, on healthy volunteers. A summary of the results corresponding to the moisturizing effect of the active ingredient according to the invention formulated at 1.7% in emulsion, is presented in Table 13, below.

[0163] [Tables 13] Variation in epidermal water content / D0 (%) D14 D28 Placebo +5.5 -0.8 PA at 1.7% +12.6 +10.0

[0164] From 14 days of twice-daily application and in comparison with placebo,

[0165] the active ingredient according to the invention formulated at 1.7% in emulsion improves the water content of the skin of Caucasian volunteers (+7.2%). This effect is observed in 78% of the volunteers. This improvement continues after 28 days of treatment with an increase in water content equal to 10.7% and is observed in 83% of the subjects.

[0166] Test 8 - Evaluation of the capacity of the active ingredient according to the invention to smooth the skin microrelief.

[0167] The objective of this trial is to evaluate in vivo the cosmetic benefits of the active ingredient according to the invention formulated at 1.7% in emulsion, in comparison with a placebo formula, on healthy volunteers.

[0168] The results are presented in Table 14, below.

[0169] [Tables 14] Variation / Placebo (%) D14 D28 Parameter Sa -6.1 -7.8 Pattern density -14.2 -20.1

[0170] From 14 days of twice-daily application by Caucasian volunteers and in comparison with the placebo, the active ingredient according to the invention formulated at 1.7% in emulsion significantly reduces fine lines and pattern density. This smoothing effect is accentuated after 28 days of treatment where a reduction in the Sa parameter (-7.8%) and pattern density (-20.1%) is measured (effect observed in 63% and 75% of subjects respectively).

[0171] Test 9 - Evaluation of the capacity of the active ingredient according to the invention to boost the radiance of the complexion.

[0172] The objective of this trial is to evaluate in vivo the cosmetic benefits of the active ingredient according to the invention formulated at 1.7% in emulsion, in comparison with a placebo formula, on healthy volunteers.

[0173] The results are presented in Table 15, below.

[0174] [Tablesl5] Variation / Placebo (%) D14 D28 Radiation +12.6 +15.2 Pink color +17 +22.9 Olive color -12.6 -15.2 Eye fatigue -6.1 -9.7

[0175] Compared to the placebo, the active ingredient according to the invention significantly improves the characteristic parameters of the radiance of the complexion of Caucasian volunteers. Indeed, from 14 days of treatment, the active ingredient according to the invention formulated at 1.7% in emulsion makes the complexion brighter, fresher and improves the healthy glow by reducing the olive color and eye fatigue (significant effects compared to the placebo).

[0176] These effects intensify after 28 days of twice-daily application with an increase of 15.2% in skin radiance and 22.9% in pink color in 78% and 67% of volunteers respectively. A decrease in olive color of 15.2% and in eye fatigue of 9.7% were also observed in 72% and 78% of volunteers respectively.

[0177] Finally, examples of composition according to the invention are described below.

[0178] Example of composition according to the invention

[0179] Example 2 - Composition according to the invention

[0180] An example of a formulation comprising the active ingredient according to the invention in the form of a light cream is presented in the table below.

[0181] [Tables 16] Ingredients % Al Purified Water qsp 100 Preservative qs Butylene Glycol 3.00 A2 Glycerin 2.00 Acacia Senegal Gum & Xanthan Gum 0.30 B Sodium Stearoyl Glutamate 0.75 Pentaerythrityl Distearate 1.50 Pentaerythrityl Tetrac apry late / Tetrac aprate 2.50 Isononyl Isononanoate 4.00 Caprylic / Capric Triglyceride 4.00 Isodecyl Neopentanoate 4.00 C Sodium Polyacrylate 0.75 D EXTRACT INVENTION 2.00

[0182] The composition of Example 2 can in particular be obtained by the following process: a. Add A2 to Al with stirring and heat to 70°C b. Place B with stirring and heat to 70°C. c. Emulsify B in A under shear stirring for 10 minutes. d. At room temperature, with moderate stirring, add C then D

[0183] The composition then appears in the form of a flexible, white and shiny emulsion.

[0184] Example 3 - Composition according to the invention

[0185] An example of a formulation comprising the active ingredient according to the invention in the form of a smooth anti-aging cream is presented in the table below.

[0186] [Tablesl7] Ingredients % Al Purified Water qsp 100 Preservative qs Butylene Glycol 3.00 A2 Glycerin 2.00 Sclerotium Gum & Xanthan Gum 0.20 B Lanol ​​Wax CTO2 4.00 Jojoba Butter3 2.00 Dipentaerythrityl Pentaisononanoate 2.00 Cetearyl Ethylhexanoate 5.00 Diisopropyl Adipate 5.00 Dimethicone 2.00 Lecithin & Helianthus Annuus (Sunflower) Seed Oil & Ascorbyl Palmitate & Tocopherol & Rosmarinus Officinalis (Rosemary) Extract 0.01 C Polyacrylate-13 & Polyisobutene & Polysorbate 20 1.00 D EXTRACT INVENTION 2.00

[0187] The composition of Example 3 can in particular be obtained by the following process: a. Add A2 to Al with moderate stirring and heat to 80°C. b. Place B under stirring and heat to 80°C. c. Emulsify B in A under shear stirring for 10 minutes. d. At room temperature, with moderate stirring, add C then D.

[0188] The composition then appears in the form of a thick, shiny emulsion, white in color.

[0189] Example 4 - Composition according to the invention

[0190] An example of a formulation comprising the active ingredient according to the invention in the form of an anti-aging BB cream is presented in the table below.

[0191] [Tablesl8] Ingredients % A Purified Water qsp 100 1,2-Hexanediol 1.00 Glycerin 1.00 Glycereth-26 1.00 Soda Solution 28% 0.45 B1 C20-22 Alkyl Phosphate & C20-22 Alcohols 3.00 Undecane & Tridecane 7.00 Isononyl Isononanoate 8.00 B2 Polyacrylate Crosspolymer-6 0.70 B3 Lauroyl Lysine 2.00 CI 77499 (Iron Oxides) & Hydrogenated Lecithin 0.10 Hydrogenated Lecithin & CI 77491 (Iron Oxides) & CI 77499 (Iron Oxides) 0.35 CI 77891 (Titanium Dioxide) & Hydrogenated Lecithin 7.00 CI 77492 (Iron Oxides) & Hydrogenated Lecithin 0.70 C Preservatives qs D EXTRACT INVENTION 2.00

[0192] The composition of Example 4 can in particular be obtained by the following process: a. Heat A to 70°C with moderate stirring. b. Heat B1 to 75°C while stirring. Add B2 then B3 while stirring. Stir for 3 minutes. c. Emulsify B in A under shear stirring for 10 minutes. d. Add C to AB and cool with gentle stirring. e. At room temperature, add D with gentle stirring.

[0193] The composition then appears in the form of a flexible and shiny emulsion, dark beige in color.

Claims

Claims

1. Cosmetic active ingredient comprising an extract of Pichia stipitis, said extract comprising peptides.

2. Cosmetic active ingredient according to the preceding claim, characterized in that said extract comprises at least 70% of peptides, by weight of the total weight of the extract.

3. Cosmetic active ingredient according to one of the preceding claims, characterized in that at least 80% of the peptides have a molecular weight of less than 3500 Da.

4. Cosmetic active ingredient according to one of the preceding claims, characterized in that the extract also comprises sugars and minerals.

5. Cosmetic active ingredient according to one of the preceding claims, characterized in that the extract is a hydrolyzate of Pichia stipitis.

6. Cosmetic active ingredient according to one of the preceding claims, characterized in that the extract is an enzymatic hydrolyzate of Pichia stipitis.

7. Cosmetic active ingredient according to one of the preceding claims, characterized in that it is obtained by a process comprising the following steps: a. solubilization of the Pichia stipitis biomass in water, b. enzymatic hydrolysis, c. enzymatic inactivation by heat treatment, d. separation of the soluble and insoluble phases by centrifugation, and recovery of the soluble phase, e. molecular sorting f. chromatographic purification.

8. Cosmetic composition comprising at least 0.1% of an active ingredient according to one of claims 1 to 7, by weight of the total weight of the composition.

9. Cosmetic use of an active ingredient according to one of claims 1 to 7 or of a composition according to claim 8, for its anti-aging effects.

10. Cosmetic use of an active ingredient according to the preceding claim, to improve dermo-epidermal communication.

11. Cosmetic use of an active ingredient according to claim 9, for its anti-aging effects on the epidermis and / or dermis.

12. Cosmetic use according to one of claims 9 to 11, for a moisturizing and / or smoothing effect, and / or to improve the radiance of the complexion.

13. Cosmetic use according to one of claims 9 to 12, for combating skin aging.