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    • 专利摘要:
    The subject of the invention is a hydrolyzate of Pichia anomala for its application as an active ingredient intended to combat the phenomenon of chronic inflammation of the skin, in particular a Pichia anomala hydrolyzate comprising at least 30% of biopeptides and less than 10% of sugars by weight relative to the total weight of solids, part of the biopeptides of which are biopeptides with molecular masses of less than 5000 Da. The invention also relates to cosmetic compositions including this active ingredient.
    公开号:FR3016521A1
    申请号:FR1450565
    申请日:2014-01-23
    公开日:2015-07-24
    发明作者:Jean Paufique
    申请人:Societe Industrielle Limousine dApplication Biologique SA SILAB；
    IPC主号:
    专利说明:

    [0001] ACTIVE SUBSTANCE OBTAINED FROM ANOMALA PICHIA AND USE TO COMBAT THE CHRONIC SKIN INFLAMMATION PHENOMENON The present invention relates to a hydrolyzate of Pichia anomala and to its use as an active ingredient for combating the phenomenon of chronic inflammation of the skin. the skin. The invention also relates to cosmetic compositions including such an active ingredient. Inflammation is a phenomenon that allows the body to protect itself against the various aggressions and stress that threaten it daily. Indeed, whether the source of stress is intrinsic (lack of sleep, unbalanced diet, etc.) or extrinsic (UV rays, pollution, pathogenic microorganisms, etc.), the human body responds by triggering a cascade of local reactions: the inflammatory process. It is characterized by four manifestations: redness, pain, swelling and heat, and allows the recognition, destruction and elimination of foreign substances, before repairing the damaged tissue. When properly controlled, this phenomenon is a real weapon that offers the body an effective defense, but with age the body loses its ability to modulate and regulate perfectly the inflammatory response that becomes excessive in relation to the needs. real defense. The inflammatory cascade is then triggered without being the response to aggression and latent inflammation gradually settles silently and not visibly. This results in an increase in the production of pro-inflammatory mediators such as, for example, interleukins 6 and 8 and the establishment of a level that can be low but chronic inflammation. This phenomenon, called "inflammaging", can have several origins such as the accumulation of tissue damage, the failure of the immune system or even the accumulation of senescent cells and their ability to secrete pro-inflammatory cytokines. Indeed, following stress, cells can enter senescence. However, senescent cells, through their secretions, exert a wide variety of deleterious effects on their environment and cause an inflammation that propagates in the tissue leading to the modification of the cell and matrix environment and consecutively to the accelerated skin aging. The objective of the present invention is to provide an active ingredient which makes it possible to limit the deleterious effects of inflammation on the skin, in particular by modulating the communication between the senescent cells and their environment. In order to meet them, the invention aims to use a hydrolyzate of Pichia anomala. By acting on the communication between the cells and their environment, the active principle according to the invention limits the pro-inflammatory exchanges between the epidermis and the dermis and thus preserves the functionality of the tissue. It limits the damage caused by the phenomenon of chronic inflammation and thus allows the skin to appear younger. Pichia anomala is a yeast belonging to the Saccharmoycetes family. Its use in cosmetics for the treatment of the skin has already been described but never to fight against the phenomenon of inflammation. The invention relates to a hydrolyzate of Pichia anomala for its application as an active ingredient to fight against the phenomenon of chronic inflammation of the skin, the use of a hydrolyzate of Pichia anomala as a cosmetic active ingredient to fight against the phenomenon of inflammation chronic skin. The subject of the invention is also a particular Pichia anomala hydrolyzate, namely a Pichia anomala hydrolyzate comprising at least 30% of biopeptides and less than 10% of sugars by weight relative to the total weight of dry matter, at least a portion of biopeptides being biopeptides of molecular masses lower than 5000Da. The invention also relates to a process for obtaining this hydrolyzate and compositions comprising it. The present invention is now described in detail.
    [0002] According to a first aspect, the invention therefore relates to a hydrolyzate of Pichia anomala for its application as an active ingredient for combating the phenomenon of chronic inflammation of the skin, in particular the use of a hydrolyzate of Pichia anomala as a cosmetic active ingredient in a cosmetic composition intended for topical application, to combat the phenomenon of chronic inflammation of the skin.
    [0003] The senescence of keratinocytes exerts direct effects on keratinocyte differentiation but also indirect effects on the dermal-epidermal junction and the dermis via pro-inflammatory mediators. Indeed, the manufacture of reconstructed skin (see test results point II.A.1) with senescent and inflamed keratinocytes leads to the production of modified skins presenting a refined epidermis as well as a modified epidermal junction and dermis. The use of a Pichia anomala hydrolyzate on the skin as an active ingredient in particular makes it possible to standardize the secretome produced by the skin in a state of chronic inflammation, in particular by limiting the release of interleukins 6 and interleukins 8; - to protect the tissue environment from chronic inflammation, in particular: o to ensure the maintenance of an effective barrier function by promoting the construction of the epidermis: in particular by increasing the thickness of the epidermis and by activating the synthesis of fillagrin, o to ensure good epidermal-dermal cohesion by increasing the synthesis of collagen VII, o to preserve the dermal compartment thanks to the synthesis of procollagen I, hyaluronic acid and the expression of tropoelastin. The active ingredient according to the invention applied to the skin, by limiting the harmful pro-inflammatory exchanges, controls the language established between the senescent cells and their environment and preserves the functionality of the tissue. It can be used as an active ingredient, especially as a cosmetic active ingredient to: - promote the restructuring of the dermal matrix - improve the elasticity and tonicity of the skin - smooth the cutaneous microrelief of the crow's feet by reducing the roughness of the skin skin, and reduce wrinkles. It can be used especially to allow the skin to look younger. According to a particularly suitable embodiment, the invention relates to the use of an active ingredient obtained from Pichia anomala, as described below. The active ingredient useful according to the invention is preferably a Pichia anomala hydrolyzate, comprising biopeptides with molecular masses of less than 5000Da. For the purposes of the invention, the term "biopeptide" is intended to mean a peptide, that is to say a protein fragment, which is biologically active. The peptides of the active principle according to the invention are biopeptides and the terms peptides and biopeptides will be used in the present application. In particular the invention relates to a hydrolyzate of Pichia anomala comprising less than 10% of sugars and at least 30% of biopeptides by weight relative to the total weight of dry matter, a portion of the biopeptides being biopeptides of molecular masses less than 5000 Da . Preferably at least 50% of the biopeptides are biopeptides of molecular masses less than 5000 Da, and even more preferably at least 85% of the biopeptides are biopeptides with molecular masses of less than 5000 Da.
    [0004] According to a suitable embodiment, the Pichia anomala hydrolyzate according to the invention comprises at least 50% of biopeptides by weight of solids. By "hydrolyzate" within the meaning of the invention is meant any extract obtained from Pichia anomala, comprising at least one step of hydrolyzing Pichia anomala. Preferably, it is an enzymatic hydrolyzate of Pichia anomala. By "enzymatic hydrolyzate" is meant any extract obtained from Pichia anomala, comprising at least one enzymatic hydrolysis step of Pichia anomala. According to a preferred embodiment of the invention, the Pichia anomala hydrolyzate is obtained by a process comprising at least one basic hydrolysis and at least one enzymatic hydrolysis of the proteins.
    [0005] The active ingredient preferably has a yellow color. It may be in clear liquid form and may be defined by at least one of the features set out below, preferably all. Dry matter: The dry matter content of an active ingredient according to the invention (measured by passing in an oven at 105 ° C. in the presence of sand of a sample of initial weight given until a constant weight is obtained ) may be between 5 and 50 g / l, preferably between 10 and 18 g / l. PH measurement: The pH (measured by the potentiometric method at room temperature) can be between 5.5 and 8, preferably between 6.5 and 7.5.
    [0006] Protein content (peptides): The protein content of an active ingredient according to the invention can be determined by the method of KJELDHAL (reference: Official method of analysis of the AOC 1975, 12th ed W. Horwitz, ED, New York , p 15-60), and expressed as a percentage of weight relative to the total weight of the dry matter of the active ingredient. The active ingredient according to the invention preferably comprises 3 to 36 g / l of proteins, in particular between 6 and 13 g / l.
    [0007] The protein content (peptides) is therefore preferably greater than 30%, even more preferably greater than 50% by weight of dry matter. The majority of the proteins are preferably peptides with a molar mass of less than 5000 Da. Preferably at least 85% of the peptides (proteins) are peptides with a molar mass of less than 5000 Da.
    [0008] Crude ash content: The raw ash content is determined by the weighing of residues from incineration at 550 ° C in an electric muffle furnace (VULCAN®). The raw ash content is determined as a percentage of weight relative to the total weight of the dry matter of the active ingredient. It is between 10 and 25% by weight of dry matter. Carbohydrate content The DUBOIS method is used. In the presence of concentrated sulfuric acid and phenol, the reducing sugars give an orange-yellow compound. From a standard range, it is possible to determine the level of carbohydrates of an active ingredient according to the invention.
    [0009] The level of carbohydrates of an active ingredient according to the invention measured by the DUBOIS method is preferably less than 10% by weight of solids, more preferably less than 6%. Carbohydrate Characterization The HPLC analysis of an active ingredient according to the invention makes it possible to determine the distribution of the molecular weights of the carbohydrate moiety. The characterization by gas chromatography (GC) of the carbohydrate moiety of the active ingredient according to the invention shows that it is characterized by a carbohydrate moiety consisting predominantly of oligosaccharides of degree of polymerization between 2 and 40, these oligosaccharides being mainly glucose and galactose compounds.
    [0010] Identification of the active fraction In order to determine the active fraction, the molecular species of the active principle according to the invention were fractionated: a purified fraction A into sugars a purified B fraction into peptides a fraction C containing the ashes of the active principle . Fraction B has an efficiency comparable to that of the active ingredient according to the invention (efficacy evaluated according to the tests of point 11). The peptide fraction therefore plays a key role in the effectiveness of the active ingredient. This peptide fraction preferably comprises at least 50% and even more preferably at least 85% of peptides with molar masses less than 5000Da. By way of example, the peptide fraction (fraction B) of an active ingredient of Example 1 was analyzed by FPLC molecular filtration with UV detection. The results obtained are presented below: fraction 1 (molecular weight greater than 5000 Da): 13% (1.45 g / l) fraction 2 (molecular weight between 2000 and 5000 Da): 38.4% (4) 29 g / l) - fraction 3 (molecular weight between 243 and 2000 Da): 46.3% (5.17 g / l) - fraction 1 (molecular mass between 0 and 243 Da): 2.3% (0) , 26 g / 1). In this example, the peptide fraction of the active principle according to the invention comprises 87% of peptides with a molar mass of less than 5000 Da. The active ingredient according to the invention is obtained by a process for concentrating the active agent in terms of biopeptides, in particular biopeptides with a molecular mass of less than 5000 Da. It is preferably obtained by a process comprising at least one basic hydrolysis and at least one enzymatic hydrolysis of the proteins. A particularly suitable process comprises at least the following succession of steps: solubilization of Pichia anomala in water, preferably at the rate of 20 g / l - basic hydrolysis of the solution of Pichia anomala, - separation of the soluble and insoluble phases, by by filtration, centrifugation or decantation, - recovery of the soluble phase - filtration and purification to recover the molecules - enzymatic hydrolysis of the proteins, preferably by means of a protease, - filtration and purification to recover biopeptides smaller than 5000 Da .
    [0011] Additional steps of filtration and sterilizing filtration can be considered. Deodorization and bleaching or concentration steps can be added. The parameters of the various steps must be adjusted in order to obtain active ingredients having the characteristics of the invention. These steps are usual in the field of active extractions from plants or yeasts and the skilled person is able to adjust the reaction parameters on the basis of his general knowledge. The present invention also covers cosmetic compositions including a Pichia anomala hydrolyzate according to the invention, in different galenic forms, suitable for topical administration to the skin.
    [0012] These compositions may especially be in the form of oil-in-water emulsions, water-in-oil emulsions, multiple emulsions (Water / Oil / Water or Oil / Water / Oil) which may be optionally microemulsions or nanoemulsions, or in the form of solutions, suspensions, hydrodispersions, aqueous gels or powders. They can be more or less fluid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste or a foam, or in solid form. It may be compositions comprising between 0.1 and 3% of active principle (s) obtained (s) from Pichia anomala according to the present invention. These compositions comprise, in addition to the active agent, a physiologically acceptable and preferably cosmetically acceptable medium, that is to say which does not cause unacceptable sensations of discomfort for the user such as redness, tightness or tingling. The compositions according to the invention may contain as adjuvant 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 carnauba wax; silicone elastomers; surfactants, preferably emulsifiers, whether they are nonionic, anionic, cationic or amphoteric; co-surfactants, such as linear fatty alcohols; thickeners and / or gelling agents, humectants, such as polyols such as glycerine; organic filters, inorganic filters, dyes, preservatives, fillers, tensors, sequestering agents, perfumes, and mixtures thereof, without this list being limiting. Examples of such adjuvants are cited in particular in the CTFA Dictionary (International Cosmetic Ingredient Dictionary and Handbook published by the Personal Care Product Council). Of course, those skilled in the art will take care to choose any additional compounds, active or non-active, and their amount, so that the advantageous properties of the mixture are not, or not substantially, impaired by the addition 20 envisaged . These compositions are especially intended to combat the phenomenon of inflammation in particular to limit skin damage induced by the phenomenon of chronic inflammation. They can be used in particular to promote the restructuring of the dermal matrix, improve the elasticity of the skin, smooth the microrelief of the crow's feet by reducing the roughness and reduce wrinkles. They can therefore be used to fight against the phenomenon of chronic inflammation especially to allow the skin to look younger. In order to illustrate the cosmetic effects of a Pichia anomala hydrolyzate according to the invention, the following examples with their test results are presented including comparative results with other active ingredients obtained from Pichia anomala, not presenting not the features of the invention.
    [0013] I. EXAMPLES 1.1 Example 1 Active Principle According to the Invention An example of a process for obtaining an active ingredient according to the invention comprises the following steps: solubilization of 20 g of Pichia anomala biomass in 11 of water, - basic hydrolysis of the Pichia anomala solution, with soda for at least 30 minutes, - separation of the soluble and insoluble phases, for example by centrifugation, - recovery of the soluble phase, - filtration to recover the molecules by ultrafiltration, - enzymatic hydrolysis of proteins, preferably by means of a protease, in particular by means of a protease for 1 hour, - filtration to recover biopeptides smaller than 5000Da, - sterilizing filtration on 0.22u filter.
    [0014] The active ingredient obtained has the following characteristics: - appearance: clear aqueous solution - color: light yellow - dry matter content: 12.7 g / l - protein content of 7.2 g / l, ie 57%, of which 86% are biopeptides with a molecular weight of less than 5000 Da, - carbohydrate content of less than 10%, - pH: 7.4 1.2 Example 2: Active principle obtained from Pichia anomala outside the invention The active principle of the invention example 2 corresponds to the extract A of the application FR2897266. It is obtained by a process comprising the implementation of the following steps: - culture of yeasts of Pichia anomala in a medium adapted to their development - centrifugation to recover the biomass - solubilization of biomass - enzymatic hydrolysis at basic pH - phase separation soluble and insoluble - heat treatment - filtration, and - sterilizing filtration. The active ingredient obtained has the following characteristics: - solids content: between 48 and 84 g / l - pH between 4 and 9 - protein content between 22 and 100% by weight of solids - sugar content greater than 10% ( between 12 and 87%, preferably between 20 and 50% by weight of solids. 1.3 Example 3 Active ingredient obtained from Pichia anomala outside the invention The active ingredient of Example 3 corresponds to the extract B of application FR2897266.
    [0015] It is obtained by a process comprising the implementation of the following steps: - culture of Pichia anomala yeasts in a medium adapted to their development - centrifugation to recover biomass - solubilization of biomass - enzymatic hydrolysis at acidic pH - phase separation soluble and insoluble - heat treatment - filtration, and - sterilizing filtration. The active ingredient obtained has the following characteristics: - solids content: between 58 and 95 g / l - pH between 4 and 9 - protein content between 24 and 93% by weight of solids, - sugar content greater than 10% (Between 12 and 55%, preferably between 20 and 34%) by weight of solids. 1.4 Example 4 Active ingredient obtained from Pichia anomala outside the invention The active ingredient of Example 4 corresponds to the extract C of application FR2897266. It is obtained by a process comprising the implementation of the following steps: - yeast culture of Pichia anomala in a medium adapted to their development - centrifugation to recover biomass - solubilization of biomass - successive enzymatic hydrolysis in basic medium, - separation soluble and insoluble phases - heat treatment - filtration, and - sterilizing filtration. The active ingredient obtained has the following characteristics: - dry matter content: between 91 and 195 g / l - pH between 4 and 9 - protein content between 18 and 100% by weight of dry matter, - sugar content preferably between 19 and and 33% by weight of dry matter. 1.5 EXAMPLE 5 Active Principle Obtained from Pichia Anomala Apart from the Invention The active principle of Example 5 corresponds to the extract D of Application FR2897266. It is obtained by a process comprising the implementation of the following steps: - culture of Pichia anomala yeasts in a medium adapted to their development - centrifugation to recover the biomass - solubilization of the biomass - simultaneous enzymatic hydrolysis of at least two enzymes at acid pH, - separation of soluble and insoluble phases - heat treatment - filtration, and - sterilizing filtration. The active ingredient obtained has the following characteristics: dry matter content: between 5 and 53 g / l, pH between 4 and 9, protein content between 4 and 100% by weight of dry matter, sugar content preferably between 20 and 45%. and 34% by weight of dry matter. 1.6 Example 6: Active ingredient obtained from Pichia anomala outside the invention The active ingredient of Example 6 corresponds to the extract E of application FR2897266. It is obtained by a process comprising the implementation of the following steps: - culture of Pichia anomala yeasts in a medium adapted to their development - centrifugation to recover biomass - solubilization of biomass in a hydroglycolic medium, - simultaneous enzymatic hydrolysis of at least two acidic pH enzymes; separation of the soluble and insoluble phases; heat treatment; filtration; and sterilizing filtration. The active ingredient obtained has the following characteristics: - dry matter content: between 172 and 300 g / l - pH between 4 and 9 - protein content between 23 and 57% by weight of dry matter, - sugar content of between 11 and 58%, preferably between 19 and 33% by weight of solids. 1.7 Example 7 Active ingredient obtained from Pichia anomala outside the invention The active principle of Example 7 corresponds to an active ingredient according to Application FR2938768. It is obtained by a process comprising the implementation of the following steps: - solubilization of Pichia anomala in water - enzymatic hydrolysis to cut the proteins and obtain a protein fraction of molecular mass less than 5000 Da, - enzymatic inactivation by heat treatment separation of the soluble and insoluble phases elimination of the protein fraction of molecular mass less than 5000 Da by ultrafiltration - filtrations. The active ingredient obtained has the following characteristics: - dry matter content: between 26 and 40 g / l - pH between 6 and 7 - protein content of less than 45%, preferably less than 30%, consisting of higher molecular weight peptides at 5000 Da, - sugar content greater than 30%, preferably greater than 50%. 13 QSP 100% 1.8 Example 8: Use of an active ingredient according to the invention in a cream Phase A. Water Glycerin 3% Phase B. Lanol 99 (Seppic) 5% DUB ZENOATE (Stéarinerie DUBOIS) 6% DUB MCT5545 (Stéarnerie DUBOIS) 5% DUB RGAE (Stéarinerie DUBOIS) 2% DUB SC15P (Stéarinerie DUBOIS) 5% Lanol wax (Seppic) 3% Phase C. Preservative 0.7% Active principle according to the invention (example 1) 3% Phase D Iron oxide (CI 77491, C177492, CI 77499) 0.06% Kaolin and iron oxides (CI Y43) 0.1% Kaolin and iron oxides (Cl R102) 0.4% TiO2 3.8% Amounts indicated are given as a percentage of weight. This ocher pink emulsion clear, unctuous and brilliant, odorless has a pH of 5.5. In topical application, it presents an easy and soft application, a smooth and slippery spreading, a very fast penetration, a soft and dry finish, a good coverage and a good-looking effect. It can be obtained by carrying out the following steps: - heating A in a water bath at 80 ° C. with magnetic stirring, - mixing B, heating in a water bath at 80 ° C. with magnetic stirring, - emulsifying B in A under rotor sator at 1200 rpm, - at 30 ° C, add C, in the order indicated, under a stator rotor at 1500 rpm, - let cool and add one by one the dyes of phase D and allow to agitate until complete homogenization. 1.9 EXAMPLE 9 Use of an Active Principle According to the Invention in a Sublimation Serum Phase A. Water QSP 100% Carbopol Aqua SF1 (Novéon) 3% Phase B. DC345 (Dow Corning) 3% DC5200 (Dow Corning) 3% Phase C. Preservative 0.7% Active ingredient according to the invention (example 1) 3% Phase D. Mica and iron oxide (Cl 77019, C177491) 0.5% Phase E. NaOH QSP pH6 The amounts indicated are given in percentage weight. This light, shiny, copper-colored gel with a slight odor has a pH of 6. It is easy to grip, with a smooth, silky spread, immediate penetration, a fresh feel, a soft, dry finish and an anti-imperfection effect. immediate.
    [0016] It can be obtained by carrying out the following steps: - mix A, B and C - stir under mechanical blade at 1000 rpm - add D, little by little, still stirring - adjust the pH with E and shake until complete homogenization. 1.10 Example 10: Use of an active ingredient according to the invention in a light cream Phase A. Water QSP 100% Glycerin 6% Carbopol Ultrez 10 (Novéon) 0.6% Montanox 60 (Seppic) 3.5% Phase B. DC5200 (Dow Corning) 4% Heliocrystal DUB (Stearinerie Dubois) 10% C. Phase Preservative 0.7% Active ingredient according to the invention (example 1) 3% Phase D. NaOH QSP pH 5.2 The amounts indicated are given in percentage weight. This emulsified gel, white, unctuous and glossy, odorless has a pH of 5.2. In topical application, it has a soft and light application, a smooth and slippery spread, a fast penetration and a soft and dry finish. It can be obtained by carrying out the following steps: - mix A and heat in a water bath at 60 ° C. with magnetic stirring, - mix B and heat in a water bath at 60 ° C. with magnetic stirring, - emulsify B in A under rotor sator at 1800 rpm, - at 30 ° C, add C, in the order indicated, under a stator rotor at 1500 rpm, - adjust the pH with E and allow to cool with stirring, checking the homogenization. 1.11 EXAMPLE 11 Use of an Active Principle According to the Invention in a Gentle Balm Phase A. Water QSP 100% Blanose 7M31CF (Hercules) 1.5% Viscogum BE (Degussa) 1.5% Phase B. Sterol CC5595 (Cesalpinia ) 6% Sorbitol L (Cesalpinia) 3% Phytowax 12L44 (Sophim) 3% Phytowax 18L57 (Sophim) 3% DUB GMS AE (Stéarinerie Dubois) 2% Phase C. Preservative 0.7% Active ingredient according to the invention (Example 1 ) 3% The quantities indicated are given as a percentage of weight.
    [0017] This gel emulsion, white, thick and unctuous, with a slight pyrazine odor, has a pH of 6.3. In topical application, it has a light application, easy and slippery spreading, a fairly fast penetration, a cool effect, an ultra soft feeling, and a dry and matte finish. It can be obtained by carrying out the following steps: - mix A taking care to disperse the gel with mechanical stirring - heat A in a water bath at 80 ° C. with mechanical stirring, - mix B and heat in a bain-marie at 70 ° C. with magnetic stirring, - emulsify B in A under rotor at 1600 rpm, - at 30 ° C., add C, in the order indicated, under a stator rotor at 1000 rpm, - allow to cool lowering the stirring speed regularly until complete homogenization. II. EVALUATION OF THE COSMETIC EFFECTIVENESS OF AN ACTIVE INGREDIENT ACCORDING TO THE INVENTION A. In vitro tests II. A.1 - Characterization of the chronic inflammatory phenotype The objective is to model: - in a first step: the effect of repeated solar irradiations (UVB) on the induction of the senescence of the keratinocytes and consecutively on the production of their secretome inflammatory - in a second step: the construction of a skin from senescent and inflammed keratinocytes (SASP - secretory phenotype associated with senescence) The characterization of the senescence of the keratinocytes was carried out by measurement of the 3-galactosidase activity and of proliferation (Ki-67). In parallel, the analysis of the inflammatory profile was made using ELISA assays: Interleukin-6 and Interleukin-8, mediators involved in SASP.
    [0018] The characterization of the reconstructed skin was carried out by the study of: - Morphological changes by HES staining, - Secretometry by ELISA assay of IL-6 and IL-8, - Changes in the epidermal compartment by immunohistological labeling of filaggrin, - modifications of the dermo-epidermic junction by immunohistological labeling of collagen VII, modifications of dermal matrix markers by immunohistological labeling of procollagen I or hyaluronic acid and by the study of tropoelastin expression by PCR quantitative. at. Characterization of the keratinocyte SASP phenotype For the characterization of the keratinocyte SASP phenotype, the operating protocol is as follows: at OJ, the human keratinocytes are inoculated and incubated at 37 ° C. For several days, senescence is activated by repeated irradiation : o Irradiations (UVB) are carried out using a UV lamp "Bio-Sun" (Vilber-Lourmat, France). After 6 hours of incubation, the secretomes of keratinocytes are recovered and frozen pending the characterization of their inflammatory profile by ELISA assay of IL-6 (BioLegend) and IL-8 (R & D Systems). The keratinocytes are trypsinized and seeded to characterize their state of senescence (Ki-67 / β-galactosidase activity). - The last day: The visualization of the 3-galactosidase activity is carried out using the kit: "Senescence cells histochemical staining" (Sigma). The 3-galactosidase activity is visualized on a microscope coupled to an image analysis system. Its activity is proportional to the intensity of blue staining present in the cell cytoplasm. The more intense the blue color, the higher the pga lactosidase activity is important. In addition, a quantitative analysis of the images was performed using software. This quantification is expressed in Arbitrary Unit (AU). o Cell proliferation is visualized and quantified by immunocytological labeling of Ki-67. Realization of the immunocytological marking then visualization carried out on a microscope coupled to an image analysis system.
    [0019] The nuclei of the cells appear colored in blue. The Ki-67 labeling is visualized by a yellow fluorescence present at the level of the cell nuclei. Quantitative analysis of the images was performed using software to determine a percentage of Ki-67 positive cells.
    [0020] Repeated irradiations on keratinocytes lead to the acquisition of a SASP phenotype characterized by: - a 156% increase in 3-galactosidase activity - a decrease in cell proliferation - an increase in the secretion of pro-inflammatory cytokines: 135% interleukin-6 and 93% interleukin-8. b. Characterization of reconstructed SASP skins For the characterization of reconstructed skin, the operating protocol is as follows: At OJ, the normal human keratinocytes and SASP are trypsinized and seeded on a lattice of collagen containing normal human fibroblasts to carry out the construction of a skin respectively normal and SASP - The last day: o Tissue subnapeants are recovered and frozen at -80 ° C before assay where the reconstructed skin is recovered: ^ for histological studies, the reconstructed skin is: * included in Tissue -Tek® then frozen. Sections (4um) are then made using a cryostat * either fixed, dehydrated and included in paraffin. Sections (4um) are made using a microtome For quantitative PCR studies, the reconstructed skin is recovered and the total RNA extracted.
    [0021] The analysis of the reconstructed skin is performed by Hematoxylin Eosine Saffron (HES) staining. The visualization is performed on a microscope coupled to an image analysis system. The thickness of the epidermis (living cell layers) was measured on histological sections after HES staining. The secretome assay is performed by IL-6 and IL-8 ELISA assay using dedicated kits.
    [0022] The analysis of the modifications of the different compartments by immunohistology, is done by means of: - immunostaining: o filaggrin for the epidermal compartment o collagen VII for the dermo-epidermal junction o pro-collagen I and hyaluronic acid for the dermal compartment - the visualization performed on a microscope coupled to an image analysis system, and - the quantitative analysis of the images is carried out using software. The synthesis of the different markers studied is proportional to the intensity of the fluorescence (green color) present on the sections of reconstructed skin The study of the expression of tropoelastin by quantitative PCR is carried out in parallel. The RNAs are reverse transcripts and the complementary DNAs obtained are analyzed by the quantitative PCR technique. The mRNAs of the ribosomal protein S27, an internal reference standard, are also produced in parallel with the tropoelastin mRNAs. quantification of fluorescence incorporation is measured continuously using a thermocycler and Ct analysis (relative quantization) is performed using software.
    [0023] The results show that in response to repeated irradiation, human keratinocytes acquire a SASP phenotype characterized by a state of cellular senescence and secretion of pro-inflammatory cytokines. Integrated in a reconstructed skin, these allow to obtain a modified model with: - an inflammatory level characterized by a 39% increase in interleukins IL-6 and 47% of IL-8 interleukins, - at epidermal level a 52% decrease in epidermal thickness and filaggrin synthesis - at the level of the epidermal junction a 51% decrease in collagen VII synthesis - in the dermis, a 14% decrease in synthesis of procollagen I, a sharp decrease in hyluronic acid synthesis and a 75% decrease in tropoelastin transcription.
    [0024] II. A.2 Efficacy of the active principle according to the invention on chronic inflammation The objective of this study is to evaluate the effect of an active ingredient according to the invention on its ability to limit chronic tissue inflammation and compare this effect to other active ingredients from Pichia anomala. For this, the study focused on: - the morphological changes of the skin reconstructed by HES staining, - the secretome of the skin reconstructed by IL-6 and IL-8 ELISA assay, - the changes in the epidermal compartment by immunohistological staining of the skin. filaggrin, - modifications of the dermo-epidermic junction by immunohistological labeling of collagen VII, - modifications of the dermal matrix by immunohistological labeling of procollagen I and hyaluronic acid, and by the study of the gene expression of the tropoelastine by quantitative PCR.
    [0025] The operating protocol is described following.
    [0026] At 0, normal human keratinocytes and SASP are trypsinized and seeded on a collagen lattice containing normal human fibroblasts to achieve the construction of respectively normal and SASP skin. For several days, the normal reconstructed skin and SASP are treated with the active ingredient according to the invention of Example 1 at 0.25% (V / V) or with the active principle of Example 7 at 0.25%. (V / V). On the last day: - the tissue sub-swimmers are recovered and frozen at -80 ° C before the assay - for histological studies: the reconstructed skin is: * included in Tissue-Tek® and then frozen. Sections (4um) are then made using a cryostat. * fixed, dehydrated and included in paraffin. Sections (4um) are made using a microtome. - For molecular biology studies: reconstructed skin is recovered and total RNA extracted. The protocols for the study of reconstructed skin are those described in II.A.1. The results obtained are shown in the following tables: Measurement of thickness Efficacy / PR SASP control of the epidermis (gm) (%) Normal reconstructed skin Control 154 Active principle of Example 1 at 0.25% 152 Reconstructed skins SASP Control 74 Active principle of Example 1 at 0.25% 109 + 47% Active principle of Example 7 at 0.25% 78 + 5% Table 1: Morphological analysis of skin reconstructed by HES staining These results show that tested 0.25% on reconstructed skin made from human keratinocytes SASP, the active ingredient according to the invention can increase the thickness of 47%. In addition, it is found that extracts of Pichia anomala not having the characteristics of the active ingredient according to the invention do not have this effectiveness. Level of IL-6 Efficacy / PR SASP (pg / ml) control (%) Normal reconstructed skin Control 508 Active principle of Example 1 at 0.25% 436 Reconstructed skin SASP Control 708 Active principle of Example 1 to 0 25% 469 -34% Active principle of Example 7 at 0.25% 1034 + 46% Table 2: Secretometry analysis by ELISA-IL-6 assay IL-8 level Efficacy / PR SASP (μg / ml) control (%) Normal reconstructed skin Control 797 Active ingredient of Example 1 at 0.25% 922 Reconstructed skin SASP Control 1171 Active ingredient of Example 1 at 0.25% 790 -33% Active principle of Example 7 at 0.25% 1869 + 60% Table 3: Analysis of the secretome by ELISA-IL-8 assay These results show that tested at 0.25% on reconstructed skins made from human keratinocytes SASP, the active ingredient according to the invention reduces the synthesis of IL-6 by 34% and IL-8 by 33%, thus limiting the phenomenon of inflammation. In addition, it is found that extracts of Pichia anomala not having the characteristics of the active ingredient according to the invention do not have this effectiveness.
    [0027] Efficiency Intensity Mean / PR SASP Control Filaggrin Marking (%) (x 104 AU) Normal Reconstructed Skin Control 1750 Active Principle of Example 1 at 0.25% 1,946 Reconstructed Skin SASP Control 264 Active Principle Example 1 at 0.25% 505 + 91% Table 4: Analysis of the changes in the epidermal compartment These results show that tested at 0.25% on reconstructed skin manufactured from human keratinocytes SASP, the active ingredient according to the invention allows to increase the synthesis of filaggrin by 91%. Efficiency Intensity Mean / PR SASP Collagen Labeling Control (%) VII (x 106 AU) Normal Reconstructed Skin Control 391 Active Principle of Example 1 at 0.25% 572 Reconstructed Skin SASP Control 191 Active Principle Example 1 at 0.25% 304 + 59% Table 5: Analysis of the changes in the dermal-epidermal junction These results show that tested at 0.25% on reconstructed skin manufactured from human keratinocytes SASP, the active ingredient according to US Pat. The invention makes it possible to increase the synthesis of collagen VII by 59%.
    [0028] Efficiency intensity average / PR SASP control staining of (%) procollagen I (x105 AU) Normal reconstructed skin Control 316 Active principle of Example 1 at 0.25% 331 Reconstructed skin SASP Control 271 Active ingredient of Example 1 at 0.25% 307 + 13% Table 6: Analysis of modifications of the dermal matrix by labeling procolenes The results of immunohistological labeling of hyaluronic acid being qualitative, three levels of fluorescent labeling were defined: Low detection (low green color) - Medium detection (medium green color) ++ - High detection (intense green color) +++ Intensity of hyaluronic acid labeling Normal reconstructed skin Control +++ Reconstructed skin SASP Control - Active principle of Example 1 0.25% ++ Table 7: Analysis of modifications of the dermal matrix by labeling of hyaluronic acid These results show that tested at 0.25% on reconstructed skins manufactured from keratin SASP human cats, the active ingredient according to the invention makes it possible to increase the synthesis of hyaluronic acid.
    [0029] Expression of Efficacy / PR SASP control tropoelastin (%) Normal reconstructed skin Control 100 Active ingredient of Example 1 at 0.25% 96 Reconstructed skin SASP Control 25 Active principle of Example 1 at 0.25% 39 +56 Table 8: Analysis of changes in the dermal matrix by study of the expression of tropoelastin by quantitative PCR These results show that tested at 0.25% on reconstructed skin made from human keratinocytes SASP, the active ingredient according to US Pat. The invention makes it possible to increase the procollagen I synthesis by 10%, the hyaluronic acid synthesis and the tropoelastine expression by 56%. The active ingredient according to the invention therefore makes it possible to limit chronic tissue inflammation, in particular by: - limiting inflammation via IL-6 and IL-8 - increasing the thickness of the epidermis - now functional the epidermal compartment via the synthesis of filaggrin - now functional dermal-epidermal junction via collagen VII synthesis - now functional dermal compartment via the synthesis of procollagen I, hyaluronic acid and tropoelastin expression. In addition, it is found that these efficiencies are related to the specificity of the active ingredient according to the invention, since the comparative tests show that with another Pichia anomala extract, the same effects are not obtained.
    [0030] II. B. In Vivo Tests The objective of these tests is to evaluate the effect of an active principle according to the invention (example 1) formulated at 3% in emulsion after 28 days of twice-daily applications and in comparison with a placebo .
    [0031] The composition of the emulsion used is as follows: lsonyl isononoate (Lanol 99, Seppic) 5.0% Active ingredient of Example 1 3.0% Behenyl alcohol / arachidyl glucoside / Arachidyl alcohol (Montanov 202, Seppic) 3, 0% Cetearyl alcohol / cetearyl glucoside (Montanov 68, Seppic) 2.0% Phenoxyethanol / methylparaben / ethylparaben / propylparaben (Phenonip XB, Clariant) 0.7% Polyacrylamide / C13-14 isoparaffin / laureth7 (Sepigel 305, Seppic) 0, 3% Water qs 100% The study was conducted on 22 healthy female volunteers aged between 56 and 71 years old. The following cutaneous characteristics were studied: - the organization of the fibers of the dermal matrix - the biomechanical properties of the skin - the cutaneous relief at the level of the crow's feet II. B.1 - Modeling of the chronic inflammatory phenotype The volunteers participating in this study were selected by crossing different criteria: - the average age of the volunteers is greater than 55 years, because with the age the immune system becomes less efficient and the level circulating inflammatory cytokines increases - volunteers must have been photoexposed during their lifetime because photoexposure promotes inflammation and loss of collagen - volunteers have a high level of IL-1a, because IL-1a is a important regulator of the SASP phenotype. He. B.2 - Effect of the active principle according to the invention on the dermal matrix The objective of the study is to evaluate, in vivo, the capacity of an active ingredient according to the invention formulated at 3% in emulsion, at promote the restructuring of the dermal matrix disorganized with age and chronic inflammatory phenomena. This study was performed against placebo, in the cheeks, by confocal microscopy. In this study, fiber network acquisitions were performed by confocal microscopy (Vivascope 1500 trislaser) before and after 28 days of twice-daily treatment.
    [0032] For the observation of the papillary dermis and the evaluation of the collagen fibers, the wavelength of 785 nm was chosen. Horizontal sections were made just below the dermal papillae to determine 2 representative zones of this horizontal section. Then, on each of these 2 zones, a series of horizontal sections of the image was carried out in order to scan the dermis, descending step by step until a relative depth of 601.J.m. Images at a relative depth of 40i.J.m were selected for visual analysis using a 4-stage scoring scale by a trained panel of 3 experts. In its assessment, the jury takes into account both the dermal matrix and the fiber aspect: when the dermal matrix is not altered as in the case of young skin or protected areas, the collagen appears as fibrous, elongated fibrillar structures arranged like a spider's web. The observation of an aged skin or of photoexposed areas shows, conversely, a collagen composed of thick fibers, coarsely arranged, curled up and fragmented, having a spongy appearance. The following scale is used to visualize the classification used by the jury: Alteration of the dermis Appearance of the matrix Aspect of the fibers Network Network Network Coarse canvas and coarse web network and spongy spongy spider spider Stage 4 ++ ++ - Stage 3 + +/- + +/- Stage 2 +/- + +/- + Stage 1 - ++ - ++ The operating protocol of the study is as follows: Between D-14 and OJ, volunteers apply a placebo cream twice daily to the entire face.
    [0033] At OJ, two symmetrical cutaneous zones are determined at the level of the cheeks and acquisitions are made by confocal microscopy at these two zones. Between OJ and J27, the placebo and the emulsion containing the active ingredient according to the invention are applied in half-face. At D28, new acquisitions are made by confocal microscopy at the two zones.
    [0034] Under the conditions of this study, after 28 days of twice-daily application and in comparison with placebo, the evaluation carried out on a grade scale by a jury of experts showed an improvement in the appearance of the dermal matrix of 35, 4 % with the active ingredient according to the invention formulated at 3%. In particular, the use of the active principle according to the invention makes it possible: to reduce the stage of organization of the dermal matrix to change from an aged skin morphotype (stage 1 to 2) to a morphotype approaching that of a young skin (stage 3 to 4).
    [0035] The use of the active ingredient according to the invention thus makes it possible to improve the organization of the dermal matrix and thus to minimize the harmful effects induced by chronic inflammatory phenomena. He. B.3 Effect of the active ingredient according to the invention on the biomechanical properties of the skin The objective of the study is to evaluate, in vivo, the effect of an active ingredient according to the invention formulated at 3% in emulsion against placebo, on the improvement of the biomechanical properties of the skin. Indeed, the phenomenon of inflammation causes the imbalance between the synthesis and the degradation of the collagen fibers, making the skin less elastic and more relaxed. In this study, analysis of the biomechanical properties of the skin is performed using a cutometer in the cheeks before and after 28 days of twice-daily application. The skin is sucked into the orifice of a probe of diameter 6 mm by a constant depression for a constant duration. Several successive aspirations are made. The depth of penetration of the skin into the probe is measured by means of two optical prisms placed at the opening of the probe. From the curves obtained it is possible to calculate various characteristic parameters of the biomechanical properties of the skin. Among all the parameters calculated from the curves obtained, the following parameters are used to quantify the evolution of the biomechanical properties at the level of the cheeks: - The elasticity of the skin is appreciated by the parameter R5: Net elasticity R5 = Ur / Ue If R5 increases, the elasticity of the skin increases. - The tonicity of the skin is appreciated by the parameter X: tonicity or elastic retraction X = (Uf-Ur) = RO (1-R7) If 1 / X or -X increases, the tonicity increases. The operative protocol of the study is as follows: Between D-14 and D0, the volunteers apply twice a day a placebo cream on the whole face. At D0, two symmetrical cutaneous zones are determined at the level of the cheeks and the cutometer is made at these two zones. Between OJ and J27, the placebo and the emulsion containing the active ingredient according to the invention are applied in half-face. At D28, new cutometer measurements are made at the two zones. The results obtained show a variation at D28 between active ingredient and placebo: - 8.6% for the R5 parameter - 8.0% for the -X parameter. Under the conditions of this study, after 28 days of twice-daily application and in comparison with placebo, the active ingredient according to the invention formulated at 3% in emulsion thus increases: the elasticity of the skin of 8.6%; skin tone of 8.0%. By improving the elasticity of the skin, the active ingredient according to the invention thus makes it possible to fight against the cutaneous manifestations caused by the phenomenon of inflammation. He. B.4 - Effect of the active ingredient according to the invention on the cutaneous relief The wrinkles that appear especially at the corner of the eye are the result of a loss of collagen due to age accentuated by phenomena such as inflammation chronic. Thus, the objective of the study is to evaluate in vivo the anti-wrinkle effect of an active ingredient according to the invention formulated at 3% against placebo. In this study, the analysis of the cutaneous relief is carried out using a device of projection of fringes at the level of crow's feet before and after 28 days of applications twice daily.
    [0036] The fringe projection apparatus includes a measurement sensor combining a bright fringe projector and a high resolution CCD camera connected to an acquisition software. A system of repositioning the volunteer's head according to the 3 axes of displacement makes it possible to find the same measurement zone at the different times of the study.
    [0037] The effect of the product is measured on a region of interest of 18X18mm cut automatically on the original acquisition. The microrelief of the skin is studied by two roughness parameters in 3D - Sq: RMS of surface roughness. - Sa: arithmetic mean of surface roughness.
    [0038] The volume of the wrinkle is studied by a parameter of volume - negative volume: volume lower than the surface of the skin. A decrease in these different parameters is characteristic of a smoothing of the relief of the studied surface and a reduction of wrinkles. The operative protocol of the study is as follows: Between D-14 and OJ, volunteers apply twice a day a placebo cream on the entire face. At OJ, two symmetrical cutaneous zones are determined at the crow's feet and fringe projection measurements are made at these two zones. Between OJ and J27, the placebo and the emulsion containing the active ingredient according to the invention are applied in half-face. At D28, new fringe projection measurements are made at the two zones. Under the conditions of this study, after 28 days of twice-daily application and in comparison with placebo, the active ingredient according to the invention formulated at 3% in emulsion: - smooths the cutaneous relief at the level of the crow's feet, by decreasing the roughness parameters: -6.8% for the Sa parameter and -6.7% for the Sq parameter-reduces the wrinkles by decreasing the negative volume parameter (-14.8%) The active ingredient according to the invention thus allows to fight against the manifestations on the cutaneous relief, generated by the phenomenon of inflammaging.
    [0039] These various tests thus show the cosmetic effects of an active ingredient obtained from Pichia anomala according to the invention and a cosmetic composition including it, on the phenomenon of inflammation.
    权利要求:
    Claims (11)
    [0001]
    REVENDICATIONS1. Pichia anomala hydrolyzate comprising at least 30% of biopeptides and less than 10% of sugars by weight relative to the total weight of solids, characterized in that at least a portion of the biopeptides are biopeptides of molecular masses less than 5000Da.
    [0002]
    2. Pichia anomala hydrolyzate according to claim 1, characterized in that at least 50% of the biopeptides are biopeptides of molecular masses less than 5000Da.
    [0003]
    3. Pichia anomala hydrolyzate according to one of the preceding claims, characterized in that at least 85% of the biopeptides are biopeptides of molecular masses less than 5000Da.
    [0004]
    4. Pichia anomala hydrolyzate according to one of the preceding claims, characterized in that it comprises at least 50% of biopeptides by weight of solids.
    [0005]
    5. Pichia anomala hydrolyzate according to one of the preceding claims, characterized in that it is obtained by a process comprising at least one basic hydrolysis and at least one enzymatic hydrolysis of the proteins.
    [0006]
    6. Anomala Pichia hydrolyzate comprising biopeptides of molecular masses less than 5000Da, for its application as an active ingredient for combating the phenomenon of chronic inflammation of the skin.
    [0007]
    7. Pichia anomala hydrolyzate according to one of claims 1 to 5, for its application as an active ingredient for combating the phenomenon of chronic inflammation of the skin.
    [0008]
    8. Pichia anomala hydrolyzate according to one of the preceding claims, for its application as an active ingredient intended to limit pro-inflammatory exchanges between the epidermis and the dermis.
    [0009]
    9. Pichia anomala hydrolyzate according to one of the preceding claims, for its application as an active ingredient for modulating the communication between senescent skin cells and their environment.
    [0010]
    10. Cosmetic composition for topical application, characterized in that it comprises an active ingredient according to one of claims 1 to 9, present between 0.1 and 3% by total weight of the composition.
    [0011]
    11. Process for obtaining a Pichia anomala hydrolyzate according to one of claims 1 to 5, characterized in that it comprises at least the following succession of steps: - solubilization of Pichia anomala in water less than - basic hydrolysis of the solution of Pichia anomala - separation of soluble and insoluble phases - recovery of the soluble phase - filtration and purification to recover macromolecules - enzymatic hydrolysis of proteins - filtration and purification to recover peptides of size 5000Da
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    FR2897266A1|2006-02-16|2007-08-17|Limousine D Applic Biolog Dite|Use of Pichia anomala extract as active principle for skin treatment, to prepare the composition e.g. to treat and/or prevent skin aging and wrinkles, for cohesion favor of the skin and to restructure the cornea layer and as moisturizer|
    WO2009101503A2|2008-02-12|2009-08-20|Lesaffre Et Compagnie|Use of natural active substances in cosmetic or therapeutic compositions|
    FR2938768A1|2008-11-21|2010-05-28|Limousine D Applic Biolog Dite|Use of an extract obtained from yeast belonging to genus Pichia, comprising mannan, in a cosmetic composition for restructuring the skin|EP3332762A1|2016-12-12|2018-06-13|Johnson & Johnson Consumer Inc.|Topical composition containing glycerin and yeast extract|
    WO2018127604A1|2017-01-09|2018-07-12|Societe Industrielle Limousine D'application Biologique|Hydrolysate of pichia minuta and cosmetic use thereof for controlling hair loss and stimulating regrowth|
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    US11045416B2|2018-08-30|2021-06-29|Johnson & Johnson Consumer Inc.|Topical compositions comprising Pichia anomala and retinol|
    US11110051B2|2018-08-30|2021-09-07|Johnson & Johnson Consumer Inc.|Topical compositions comprising Pichia anomala and n-acetyl glucosamine|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1450565A|FR3016521B1|2014-01-23|2014-01-23|ACTIVE PRINCIPLE OBTAINED FROM PICHIA ANOMALA AND USE TO COMBAT THE CHRONIC SKIN INFLAMMATION PHENOMENON|FR1450565A| FR3016521B1|2014-01-23|2014-01-23|ACTIVE PRINCIPLE OBTAINED FROM PICHIA ANOMALA AND USE TO COMBAT THE CHRONIC SKIN INFLAMMATION PHENOMENON|
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