PEPTIDE AND OSIDIC HYDROLYZATION OF COCOA BEANS, COSMETIC COMPOSITIONS COMPRISING THE SAME AND THEIR

专利摘要:
The invention relates to a peptide and osidic hydrolyzate of beans of Theobroma cacao L. (cocoa), its preparation process, cosmetic compositions comprising it and their cosmetic uses, in particular for protecting the skin against blue light and for combating the appearance of signs of aging and skin photoaging.
公开号:FR3048881A1
申请号:FR1600441
申请日:2016-03-16
公开日:2017-09-22
发明作者:Corinne Coquet；Catherine Gondran；Isabelle Imbert；Joel Mantelin；Nouha Domloge；Sebastien Garnier；Jeremie Borsotto；Esmeralda Ciccheti
申请人:Jafer；ISP Investments LLC；
IPC主号:

专利说明:

The invention relates to a peptide and osidic hydrolyzate of beans of Theobroma cacao L. (cocoa), to a process for the preparation thereof, to cosmetic compositions comprising it and to their uses. cosmetics especially for skin protection vis-à-vis the blue light.
The blue light represents a portion of the visible light between 400 and 495 nm. This is the most energetic part of the visible spectrum. Most of the blue light received comes from the sun. In our indoor environment, we are increasingly exposed to artificial blue light from LEDs (Light-Emitting Diodes), present in lighting, smartphones, tablets, computers and TV screens.
The photons of visible light are captured by the photoreceptors present on the cells of the retina, initiating the photo-transduction mechanism. These photoreceptors called opsins are coupled to G proteins, and contain a chromophore, the 11-cis retinal, which undergoes a photo-isomerization reaction in all-trans retinal, resulting in a signal transduction cascade.
The perception of light acts on the body in two ways: the primary optical pathway that includes visual effects, and the retino-hypothalamic pathway that is followed by biological effects such as melatonin secretion, regulation of body temperature , the expression of circadian genes.
Studies have been conducted on the damage caused by blue light on retinal cells. Exposure to visible light produces reactive oxygen species in the retinal epithelial cells, creating damage to the mitochondrial DNA and potentially leading to apoptosis of these cells. These alterations may have been related to the development of age-related macular degeneration, during which lipofuscin accumulation occurs in retinal cells4.
In addition, it has recently been shown that blue light induces the aggregation of opsins in murine retinal cells, leading to cellular damage5.
Another effect of blue light through the use of devices such as tablets has been reported. The use of this type of device before falling asleep modifies the rate of melatonin and leads to a shift in the circadian rhythm6. To counteract the deleterious effects of blue light, several optical brands or LED-containing devices have developed lenses or screens blocking wavelengths corresponding to blue.
Blue light also has effects on the skin. It has been shown on keratinocytes and endothelial cells of human skin that blue light exerts an anti-proliferative effect and promotes the differentiation of keratinocytes7. In addition, it suppresses the maturation of dendritic cells8. These properties have led to the use of blue light in the treatment of hyper-proliferative and / or inflammatory skin diseases such as psoriasis, atopic dermatitis or eczema.
In addition, the exposure to blue light of skin fragments whose barrier function has been previously altered has shown a delay in the recovery of the cutaneous barrier, particularly with regard to lipids secreted at the interface between the stratum corneum. and stratum granulosum. This result was not observed with other wavelengths of the visible spectrum, suggesting a specific activity of blue light in relation to the homeostasis of the cutaneous barrier1.
With regard to the treatment of acne, exposure to blue light creates a photo-excitation of the bacterial porphyrin of Propionibacterium acnes, leading to the elimination of the bacterium9.
Very recently, the expression of opsin receptors OPN1-SW, OPN2, OPN3 and OPN5 has been identified in keratinocytes and melanocytes, suggesting their role in phototransduction in skin cells10. Signals that arise from the activation of opsins in skin cells generate non-visual effects including regulation of circadian genes11.
Considering the harmful effects that blue light can have on the skin, it seemed important to be able to develop a cosmetic composition that is useful for protecting human skin against blue light.
Thus, surprisingly, the Applicants have developed a novel extraction process from cocoa beans making it possible to develop an extract rich in peptides and osides which has been found to demonstrate a protective activity with respect to blue light by improving the barrier function of the skin as well as a preventive activity against the appearance of skin aging.
The cacao tree or cacao tree or also called "Cocoa" is a small evergreen tree of the genus Theobroma of the family Sterculiaceae, according to the classical classification, or Malvaceae, according to the phylogenetic classification. Cocoa is a tropical species native to Mexico domesticated about 3,000 years ago. It is a tree that is 10 to 15 meters tall, usually cut to 6 or 8 meters, cauliflora and evergreen. It flowers from 3 years and gives flowers, fruits and leaves throughout the year. The tree can produce up to 100,000 white or slightly pink flowers annually. They appear all year round on the bulges of the wood of the tree, called floral cushions. As a result, flowers and fruits are found on the tree at the same time. Its fruits, the "pods", are large elongated berries. Each pod can weigh up to 400 g for 15 to 20 cm long. They have the particularity of growing both on the main branches but also directly on the trunk of the tree. Their characteristics are very variable from one population to another but also within the same population. Depending on the genotype, fruit ripening lasts 5 to 7 months. On average one tree gives about 150 pods a year. The pods contain many seeds (between 25 and 75) grouped in ears and called cocoa beans rich in starch, fat and alkaloids.
The cocoa beans are agglomerated in the center of the pod in a mass comprising five rows corresponding to the five boxes of the egg. They have a variable shape, ovoid-flattened and measure about 25 mm long, 15 mm wide and 8 mm thick. Fresh, they are sticky because surrounded by a white pulp called mucilage. The almond of the cocoa bean is formed by a large embryo that has two cotyledons folded on themselves. It is of variable color, ranging from white to dark purple depending on the variety. It is wrapped with a pink or pale red integument. It is known that cocoa beans contain about 50% fat called cocoa butter, 5% water, 7% starch, 4% cellulose, 2% theobromine, 20% protein and 6% mineral substances.
Cocoa is known to have stimulating, tonic, nutritive and stress-relieving effects and various substances have been extracted from cocoa because of such properties. In cosmetics in particular, cocoa butter from Theobroma cacao is used for its nourishing action on the skin. The butter contains 53% fat, (triglycerides mainly containing oleic, stearic and palmitic acids) with super-nourishing properties, it has the specificity of being rich in unsaponifiables, composed of phytosterols and squalene. The first have an antioxidant, healing and soothing action (against burns and chapped skin). The second, naturally present in the skin and sebum, helps regenerate the lipid cement of the stratum corneum. This is why we find cocoa butter as an ingredient in skin massage butters as well as moisturizing, soothing and protective balms. It is also appreciated in hair care very dry and undisciplined, as well as damaged tips.
Patent document FR2810242 also discloses a cosmetic and / or dermatological composition based on cocoa protein extract comprising at least polyphenols, amino acids and an unsaponifiable fraction. The compositions are useful for treating and preventing signs of skin aging. In this document, all the cocoa bean is used, and in particular cocoa butter, to obtain, after total enzymatic hydrolysis of the proteins, a mixture comprising from 0.5 to 3% or from 0.5 to 5% of unsaponifiables, from amino acids and polyphenols.
Patent document WO20080153341 also discloses a cosmetic composition comprising a cocoa bean extract for promoting the pigmentation of the hairs and / or the hair. The process for preparing the bean extract includes the removal of lipids, nucleic acids and carbohydrates from the bean. The process for obtaining the bean extract is focused on enzymatic hydrolysis of the proteins to generate polypeptides, peptides and free amino acids.
Document US2013 / 0035291 discloses a bioactive product comprising peptides of 5 to 20 amino acids and originating from a cocoa hydrolyzate, said product being intended for feeding and for treating neuro-degenerative diseases. The method implemented in this document makes it possible to obtain specific fractions of purified peptides, free of sugars.
The extraction process according to the present invention, developed by the Applicants on cocoa beans, is new compared to the documents cited above. This method is optimized to select and guarantee in the hydrolyzate obtained cocoa beans a high content of target molecules of interest including particular peptides and osides. The peptide and osidic hydrolyzate according to the invention does not comprise unsaponifiable molecules or free amino acids and the traces of polyphenols present in the hydrolyzate confirm a profile different from that of the patent WO200195872. The invention therefore firstly relates to a peptide and osidic hydrolyzate of Theobroma cacao L. beans comprising mainly peptides and osides.
In addition, a second subject of the invention is a process for obtaining a peptide and osidic hydrolyzate of Theobroma cacao L. beans according to the invention, comprising the following steps according to which: a) the aqueous phase is dispersed in ground beans of Theobroma cacao L .; b) the enzymatic treatment of the aqueous dispersion obtained in step a) is carried out; c) the recovery of the enzymatic hydrolyzate is carried out by solid / liquid separation, d) the hydrolyzate is purified by ultrafiltration and nanofiltration, then optionally; e) lyophilization of the hydrolyzate obtained in step d). The third subject of the invention is a cosmetic composition, characterized in that it comprises, as active agent, an effective amount of a hydrolyzate of Theobroma cacao L. beans according to the invention, and a physiologically acceptable medium. .
Finally, the fourth subject of the invention is the cosmetic use of a composition according to the invention, for protecting the skin from the harmful effects of blue light, in particular the oxidative stress generated by blue light, in order to improve the barrier function of the skin. the skin and to fight against the appearance of signs of skin aging and photo-aging. The invention and the advantages which result therefrom will be better understood on reading the description and the nonlimiting embodiments which follow, written with reference to the appended figures in which: FIG. 1 represents the comparison of polyphenolic profiles of the hydrolyzate according to the invention and the hydro alcoholic extract rich in polyphenols according to FR2810242 and - the aminogram of the hydrolyzate according to the invention, produced by total hydrolysis of the peptides by a strong acid such as hydrochloric acid 6 N for 24 to 48 hours, with that given in document FR2810242. FIG. 2 represents the level of opsin-1, opsin-2 and opsin-3 after exposure to blue light (425 nm and 470 nm); FIG. 3A represents the quantification of the expression of collagen 1 by the Cocoa hydrolyzate according to the invention - Figure 3B represents the quantification of the expression of elastin by the cocoa hydrolyzate according to the invention - Figure 3C represents the quantification of the expression of fibrillin 1 by the cocoa hydrolyzate according to the invention - FIG. 3D represents the results of the cocoa hydrolyzate according to the invention on the methylation of the DNA.
In this description, unless otherwise specified in the text, it is understood that: - when an interval is given, it includes the upper and lower bounds of the said interval, - the% are expressed in weight / weight in the text, unless otherwise indicated.
It is also indifferently used in the description the expression "peptide and glycidic hydrolyzate of bean Theobroma cocoa L." or "peptide hydrolyzate and osidic" or "cacao hydrolyzate according to the invention" or "hydrolyzate".
In the present invention, the term "effective amount" means the necessary amount of active molecules to obtain the desired result, namely, in particular to allow skin protection against blue light, without this amount be toxic. - "predominantly peptides and osides" an amount greater than 50%, preferably greater than 60%, preferably still greater than 70% and up to about 90% (weight / weight) of dry matter peptides and oses, preferably 90 % of the weight of the dry matter. "Peptide and osidic hydrolyzate" is understood to mean a hydrolyzate comprising predominantly or essentially peptides and monosaccharides (monosaccharides). The proteins and polysaccharides naturally present in beans have been hydrolysed into peptides, oligosaccharides and monosaccharides, advantageously the hydrolysis is an enzymatic hydrolysis. "Topical application", the application or spreading of the active principle according to the invention, or a composition containing it, on the surface of the skin, a mucous membrane or superficial body growths. - "cosmetically acceptable", that the active ingredient according to the invention, or a composition containing it, is suitable for coming into contact with the skin or a mucosa without causing reactions of toxicity or intolerance. - "physiologically acceptable" which is suitable for topical use, in contact with human skin, or for use by other routes of administration, eg orally or injection into the skin, without risk of toxicity, incompatibility, instability, allergic response. The invention therefore firstly relates to a peptide and osidic hydrolyzate of Theobroma cacao L. beans comprising mainly peptides and osides. The hydrolyzate according to the invention is obtained from beans of Theobroma cacao L., as a starting material, which can include either the bean alone or the bean and its envelope, preferentially the beans comprising the bean and its envelope will be used.
The beans used are dried beforehand and can be fermented or unfermented. In a preferred embodiment according to the invention, the beans used are unfermented and dried beans of Theobroma cacao L.
Theobroma cacao L. beans harvested, washed and dried are preferably from Peru. They can come from a mixture of different varieties or come from at least one of the three varieties Criollo, Forastero and Trinitario.
In a preferred embodiment according to the invention, the beans will come from the Criollo variety and more particularly from the Porcelana sub variety. In addition, the cocoa beans will not undergo the essential steps of the process of obtaining the chocolate, ie fermentation and roasting because these steps have a detrimental effect on the proteins and sugars for this application.
In a preferred embodiment according to the invention, the peptide and osidic hydrolyzate according to the invention is obtained by an enzymatic treatment carried out with at least one carbohydrase and at least one protease. The enzymatic treatment is carried out in two stages, a first treatment uses the carbohydrases to lead mainly to di and monosaccharides or monosaccharides, then a second treatment uses proteinases to obtain the peptides.
In an even more preferred embodiment according to the invention, the carbohydrase is chosen from pectinases, cellulases, arabanases, hemicellulases, xylanases and β-glucanases and the protease is of the alkaline, neutral or acidic type, preferably of the alkaline type. endopeptidase type.
The carbohydrase will advantageously be a carbohydrase cocktail comprising at least one polygalacturonase, a pectin methyl esterase, a beta glucanase, a beta mannanase, a beta glucosidase, a cellulase, a hemicellulase, an arabanase and a xylanase. The non-lyophilized cocoa hydrolyzate according to the invention comprises from 20 to 70% of peptides and from 5 to 40% of osides. The freeze-dried cocoa hydrolyzate without a drying support according to the invention comprises more than 90 percent of dry matter comprising 20 to 70% of peptides and 5 to 40% of osides.
In another highly preferred embodiment according to the invention, the peptides and osides present in the hydrolyzate according to the invention have a molecular weight of between 200 Da and 10 kDa.
Advantageously, the hydrolyzate according to the invention does not contain unsaponifiables.
Advantageously, the hydrolyzate according to the invention does not comprise free amino acids.
Finally, the hydrolyzate according to the invention comprises a maximum of 0.05% of polyphenols, which are predominantly methylxanthine type molecules, as indicated in FIG. 1. The subject of the invention is a process for obtaining a hydrolyzate Peptide and osidic of Theobroma cacao L. beans according to the invention, comprising the following steps according to which: a) Water-ground beans of Theobroma cacao L. are dispersed in aqueous phase; b) the enzymatic treatment of the aqueous dispersion obtained in step a) is carried out; c) the enzymes are denatured by heat treatment d) the enzymatic hydrolyzate is recovered by solid / liquid separation, e) the hydrolyzate is purified by ultrafiltration and nanofiltration, then optionally; f) lyophilization of the hydrolyzate obtained in step e).
The crushed beans of Theobroma cacao L. used in step a) of the process according to the invention are previously harvested, washed, dried and then delipidated by pressing to obtain a cake of cocoa bean. This cake is used for the implementation of step a) of the method according to the invention.
Drying is the post-harvest phase in which the beans are dehydrated to sufficiently reduce the water content of the beans to ensure favorable conditions for storage or further processing of the beans. By way of example, the beans can be naturally dried by exposure to free air (in the sun or in the shade) for a period of not more than 10 to 20 days, or advantageously delicately dried artificially in a dryer with hot air blown, by controlling the relative humidity at a temperature between 40 ° C and 60 ° C.
Preferably, after having been advantageously washed, the beans are dried, then crushed or even cryobreaked and delipidated.
Delipidated beans are understood to mean all processes making it possible to eliminate all or part of the lipids of cocoa beans such as solid / liquid extraction by a volatile solvent, extraction by supercritical fluid, or else by pressing.
In step a) of the process according to the invention, the beans and in particular the pressing cake is preferably put at 10% of dry matter in solution in water.
The enzymatic treatment according to step b) of the process according to the invention is advantageously carried out in two stages, a first treatment uses carbohydrases to hydrolyze the walls of the plant cells, then a second treatment uses proteinases to hydrolyze the proteins.
In a preferred embodiment of the process according to the invention, the carbohydrase is chosen from pectinases, cellulases, arabanases, hemicellulases, xylanases and β-glucanases and the protease, of the alkaline, neutral or acidic type, is preferably of the alkaline type. with endopeptidase activity.
The carbohydrase will advantageously be a carbohydrase cocktail comprising at least one polygalacturonase, a pectin methyl esterase, a beta glucanase, a beta mannanase, a beta glucosidase, a cellulase, a hemi cellulase, an arabanase and a xylanase.
The enzymes used in step b) of the process according to the invention are advantageously between 0.1% and 3.0% by weight relative to the weight of the cake to be treated.
In a preferred embodiment according to the invention, more pectinases will be used than cellulases. Thus, a 2: 1 ratio of pectinases to cellulases will preferably be used. Step c) of denaturation of the enzymes is carried out at a temperature between 80 and 90 ° C for a period of between 15 minutes to 2 hours, preferably the heat treatment conditions are 85 ° C for 45 minutes.
Preferably, during the different steps a) to d) of the process according to the invention, the temperature is between 20 and 90 ° C and the pH is between 3.0 and 11.0.
The recovery of the enzymatic hydrolyzate carried out according to stage c) of the process according to the invention is carried out by carrying out a solid / liquid separation by various processes known to those skilled in the art such as centrifugation, spinning, filtration, so as to obtain an extract free of solid particles. It is advantageously by centrifugation that the liquid phase is recovered.
This solid-liquid separation step is followed by a step d) purification by ultrafiltration, in order to eliminate residual proteins and denatured enzymes, with a membrane having a cutoff threshold of between 10 and 15 kDa, preferably 10 kDa, and recovery of permeate. Diafiltration is advantageously carried out, by diluting the retentate to improve the separation performance and thus recover a maximum of compounds of interest.
By "diafiltration" is meant a rinsing of the retentate by the same volume of water as this retentate, so as to facilitate the passage and thus recover the maximum of molecules whose molecular weight is below the filtration threshold.
This step d) may also include nanofiltration to optionally remove free amino acids and mineral salts. For example, the cut-off threshold will be between 100 Da (Daltons) and 300 Da, advantageously between 130 and 300 Da, typically 200 Da.
In another embodiment, after the first 10 kDa ultrafiltration step, a second 5 kDa membrane ultrafiltration step is performed. This step makes it possible to recover the fraction of the retentate containing peptides and osides whose molecular weight is between 5 kDa and 10 kDa.
Finally, the permeate of this ultrafiltration at 5 kDa is in turn purified by nanofiltration to remove free amino acids and minerals and obtain a hydrolyzate comprising peptides and osids between 200 Da and 5 kDa. The hydrolyzate thus obtained can then be further diluted in water or in any solvent mixture containing water. Thus, the cocoa hydrolyzate according to the invention may advantageously be diluted in one or more physiologically acceptable solvents, such as water, glycerol, ethanol, propanediol, butylene glycol, dipropylene glycol or ethoxylated diglycols. or propoxylated, cyclic polyols or any mixture of these solvents.
A third object according to the invention relates to a cosmetic composition, characterized in that it comprises, as active agent, an effective amount of a hydrolyzate of beans Theobroma cacao L. according to the invention, and a medium physiologically acceptable.
In a preferred embodiment according to the invention, the cocoa hydrolyzate is present in the composition at a concentration of 0.001% to 20% relative to the total weight of the composition, preferably from 0.1 to 10%, more preferably from 0.2 to 5%, more preferably from 0.5 to 1.5%. To prepare the composition, the cacao hydrolyzate according to the invention can be in liquid or freeze-dried form.
The compositions according to the invention are intended more particularly for topical administration. These compositions must therefore contain a cosmetically acceptable medium, that is to say compatible with the skin and superficial body growths, and cover all cosmetic forms. These compositions may especially be in the form of creams, oil-in-water emulsions, or water-in-oil or multiple emulsions, solutions, suspensions, gels, milks, lotions, sticks or powders, and adapted to an application on the skin, lips and / or integuments. These compositions comprise the excipients necessary for their formulation, such as solvents, thickeners, diluents, surfactants, antioxidants, dyes, preservatives, perfumes. They can be used as a care product and / or as a make-up product for the skin.
The compositions according to the invention furthermore comprise any additive commonly used in the intended field of application and the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, colorants, filters sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, cosmetic or pharmaceutical active ingredients, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers etc. The INCI Dictionary & Handbook ("International Nomenclature of Cosmetic Ingredients 13th Ed. 2010) published by the Personal Care Products Council, Inc., Washington, DC) describes a wide variety, without limitation, of cosmetic and pharmaceutical ingredients commonly used in the industry. skin care, which are suitable for use as additional ingredients in the compositions according to the present invention.
Non-limiting examples of these additional ingredient classes include: healing agents, anti-aging agents, anti-wrinkle agents, anti-atrophy agents, moisturizing agents, softening agents, antibacterial agents, anti-parasitic agents , antifungal agents, fungicidal agents, fungistatic agents, bactericidal agents, bacteriostatic agents, antimicrobial agents, anti-inflammatory agents, anti-pruriginous agents, anesthetic agents, antiviral agents, keratolytic agents, anti-free radical agents, anti-seborrhoeic agents, anti-dandruff agents, agents for differentiating differentiation, proliferation or skin pigmentation, penetration enhancers, desquamating agents, agents stimulating or inhibiting melanin synthesis , whiteners, depigmenting agents, or brighteners, propigmenting agents, auto agents tanning agents, NO-synthase inhibiting agents, antioxidants, free radical scavengers and / or anti-air pollution agents, anti-glycation agents, firming agents, agents stimulating the synthesis of dermal or epidermal macromolecules and / or agents capable of preventing or inhibiting their degradation, agents stimulating collagen synthesis, elastin synthesis stimulating agents, decorin stimulating agents, laminin synthesis stimulating agents, agents stimulating the synthesis of defensin, agents stimulating chaperone synthesis, agents stimulating the synthesis of aquaporin, agents stimulating the synthesis of hyaluronic acid, agents stimulating the synthesis of lipids and components of the stratum comeum (ceramides, fatty acids ,. ..), agents that inhibit collagen degradation, agents that inhibit the degradation of elastin, agents that stimulate proliferation fibroblasts, keratinocyte proliferation stimulating agents, adipocyte proliferation stimulating agents, melanocyte proliferation stimulating agents, keratinocyte differentiating stimulating agents, adipocyte differentiating stimulating agents, acetylcholinesterase inhibiting agents, agents stimulating the synthesis of glycosaminoglycans, DNA repair agents, DNA protecting agents, anti-itch agents, agents for the treatment and / or care of sensitive skin, firming agents, anti-inflammatory agents stretch marks, astringent agents, sebum-regulating agents, skin-relaxing agents, healing adjuvants, re-epithelializing agents, adjuvant re-epithelialization agents, cytokine growth factors, calming agents, anti-inflammatory agents, agents acting on the circulation and / or microcircu capillary action, angiogenesis stimulating agents, vascular permeability inhibiting agents, agents acting on cellular metabolism, agents for improving the dermal-epidermal junction, agents inducing hair growth and / or hair, agents which inhibit or slow the growth of hair and / or hair, muscle relaxants, anti-pollution and / or anti-radical agents, lipolysis stimulating agents, slimming agents, anti-cellulite agents, agents acting on microcirculation, agents acting on cell metabolism, cleaning agents, hair styling agents, hair growth stimulants, sunscreens, total screens, makeup agents, detergents, pharmaceuticals , emulsifying agents, emollients, organic solvents, antiseptic agents, deodorant active agents, physiologically acceptable media, urfactants, abrasives, absorbents, aesthetic components such as perfumes, pigments, dyes, natural dyes and dyes, essential oils, touching agents, cosmetic astringents, anti-acne agents, anti-acne agents, coagulation, anti-foam agents, antioxidants, leachates, biological additives, enzymes, enzyme inhibitors, enzyme inducers, coenzymes, chelating agents, plant extracts and plant derivatives, essential oils, extracts biofermentation and / or biotechnology processes, mineral salts, cell extracts, sunscreens (organic or inorganic photoprotective agents which are active against ultraviolet A and / or B rays) ceramides , peptides, buffers, volume agents, chelating agents, chemical additives, dyes, cosmetic biocides, denaturan ts, medicinal astringents, external analgesics, film-forming agents, such as polymers, to exacerbate the film-forming properties and substantivity of the composition, quaternary derivatives, substantivity-enhancing agents, opacifying agents, pH adjusters and regulators (ex. triethanolamine), propellants, reducing agents, sequestering agents, bleaching and / or skin lightening agents, skin conditioning agents (ie, humectants, including miscellaneaous and occlusive agents), moisture retentive substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolytic enzymes, soothing and / or healing agents, skin-treating agents, anti-wrinkle agents, agents capable of reducing or treating bags under the eyes, exfoliating agents, thickeners, softeners, gelling polymers, vitamins and their derivatives, wetting agents, peeling agents, calming agents, curative agents for the skin, lignans, preservatives (iephenoxyethanol and parabens), anti-UV, cytotoxic agents, anti-neoplastic agents, viscosity modifiers, non-volatile solvents, pearling agents, antiperspirants, depilators vaccines, scented water, skin restructuring agent, excipients, fillers, minerals, anti-mycobacterial agents, anti-allergenic agents, antihistamines H1 or H2, anti-irritants, immune stimulating agents , immune system inhibitors, insect repellents, lubricants, pigmentants or dyes, hypopigmenting agents, photostabilizers, and mixtures thereof, as long as they are physically and chemically compatible with the other ingredients of the composition and especially with the assets of the present invention.
Moreover, the nature of these additional ingredients should not unacceptably alter the benefits of the assets of the invention. These additional ingredients may be synthetic or natural such as plant extracts or come from a biofermentation process. Additional examples can be found in PINCI Dictionary & Handbook.
Such additional ingredients may be selected from the group consisting of: amino sugars, glucosamine, D-glucosamine, N-acetylglucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetyl mannosamine, galactosamine, N-acetylgalactosamine, vitamin B3 and its derivatives, niacinamide, dehydro-sodium acetate, dehydroacetic acid and its salts, phytosterols, salicylic acid compounds, hexamidines, dialkanoyl dihydroxyproline compounds, extracts and soy derivatives, equol, isoflavones, flavonoids, phytantriol, famesol, geraniol, bisabolol, peptides and their derivatives, di -, tri -, tetra -, penta -, and hexapeptides and their derivatives, lys-thr-thr-lys-ser, palmitoyl-lys-thr-thr-lys -ser, camosin, amino acid compounds N-acyl, retinoids, retinyl propionate, retinol, retinyl palmitate, retinyl acetate, retinal, retinoic acid, water-soluble vitamins, ascorbates, vitamin C, ascorbyl glucoside, ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, vitamins and their salts and derivatives, provitamins and their salts and derivatives, ethyl panthenol, vitamin B and its derivatives, vitamin
Bl, vitamin B2, vitamin B6, vitamin B12, vitamin K and its derivatives, pantothenic acid and its derivatives, pantothenyl ethyl ether, panthenol and its derivatives, ethyl panthenol, dexpanthenol, biotin, amino acids and their salts and derivatives, acids water-soluble amines, asparagine, alanine, indol, glutamic acid, water insoluble vitamins, vitamin A, vitamin E, vitamin F, vitamin D and its compounds, mono-, di-, and triterpenoid, beta-ionol, cedrol, and their derivatives, water insoluble amino acids, tyrosine, tryptamine, particulate materials, butylated hydroxytoluene, butylated hydroxyanisole, allantoin, tocopherol nicotinate, tocopherol, tocopherol esters, palmitoyl-gly-his-lys, phytosterol, hydroxy acids, acid glycolic acid, lactic acid, lactobionic acid, ketoacids, pyruvic acid, phytic acid, lysophosphatidic acid, stilbenes, cinnamates, resveratrol, kinetin, zeatin, dimethylaminoethanol, natural peptides, l soya peptides, acid sugar salts, manganese gluconate, zinc gluconate, piroctone olamine, 3,4,4'-trichlorocarbanilide, triclocarban, zinc pyrithione, hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, ascorbyl glucoside, pyridoxine, aloe vera, terpene alcohols, allantoin, bisabolol, dipotassium glycyrrhizinate, glycerol acid, sorbitol, pentaerythritol, pyrrolidone and its salts, dihydroxyacetone, erythrulose, glyceraldehyde, tartaraldehyde, nail essence clove, menthol, camphor, eucalyptus essence, eugenol, menthyl lactate, witch hazel distillate, eicosene copolymer and vinyl pyrrolidone, iodopropyl butylcarbamate, polysaccharide, essential fatty acid, salicylate , a glycyrrhetinic acid, carotenoids, ceramides and pseudo-ceramides, a complex lipid, oils generally of natural origin such as shea butter, apricot oil, oil evening primrose oil, prune oil, palm oil, monoi oil, kahai oil, hydroquinone, HEPES, procysteine, O-octanoyl-6-D-maltose, disodium salt of methyl glycine diacetic acid, steroids such as diosgenin and DHEA derivatives, DHEA dehydroepiandrosterone and / or a chemical or biological precursor or derivative, N-ethylcarbonyl-4-para-aminophenol, extracts bilberry, phytohormones, yeast extracts Saccharomyces cerevisiae, extracts of algae, extracts of soya, lupine, maize and / or pea, alverine and its salts, in particular alverine citrate, extracts of small holly and chestnut and their combinations, a metalloproteinase inhibitor, Schinus soft extracts.
In all cases, those skilled in the art will ensure that these adjuvants and their proportions are chosen so as not to adversely affect the desirable properties of the composition according to the invention.
A fourth object according to the invention relates to the cosmetic use of a composition according to the invention, to protect the skin from the harmful effects of blue light and to improve the barrier function of the skin, as well as to fight against the appearance of signs of aging and photo-aging skin.
"Signs of skin aging" means any change in the appearance of the skin due to chronological aging such as, for example, lines and wrinkles, cracks, bags under the eyes, dark circles, wilting, loss. elasticity and / or tone of the skin, dullness or lack of radiance, pigmentary defects, but also any internal change in the skin that does not systematically result in a modified external appearance, such as, for example, Thinning and loss of density of the dermis, thickening of the stratum corneum.
By "signs of skin photoaging" is meant any degradation of the skin following exposure to UV radiation such as the premature appearance of fine wrinkles around the eyes and mouth and expression lines on the forehead; telangiectasia (small dilated blood vessels) on the nose, cheeks and neck, various pigment spots such as freckles and sun lentigines, irregularities of the complexion, the generalized loss of skin tone, the skin of lips that becomes more tense, loses its color and thins, the skin can also thicken and become rough. It also means any internal change in the skin that does not systematically result in a modified external appearance such as, for example, the thickening of the vessel walls, the change in the shape of fibroblasts, the slowing down of collagen synthesis and a disorganization of collagen fibrils, an accumulation of abnormal and amorphous material containing elastin (solar elastosis). As an illustration, preferred examples of embodiments of the invention are described below.
Example 1 Preparation of a cocoa hydrolyzate from cocoa beans
The beans used include their husks and are of Peruvian origin, of the variety Criollo and more particularly of the sub-variety Porcelana.
The following is carried out: a) Dissolving of the cake of cocoa beans delipidated by pressing, at 10% dry matter (DM) in water; b) Enzymatic hydrolysis of carbohydrates by the action of a cocktail of cellulases (0.5% / MS) and pectinases (1.0% / MS), comprising a polygalacturonase, a pectin methyl esterase, a beta glucanase, a beta mannanase beta glucosidase, cellulase, hemicellulase, arabanase, and xylanase at a pH of 4.5 and a temperature of 55 ° C for 2 hours; c) followed by enzymatic hydrolysis with an endopeptidase-type alkaline protease (0.5% MS) at a pH of 9.0 and a temperature of 55 ° C for 4 hours; d) Heat treatment at 85 ° C for 45 minutes to denature the enzymes; e) Centrifugation and recovery of the liquid phase; f) Ultrafiltration and diafiltration of the liquid phase on a 10 kDa membrane and recovery of the permeate; g) Nanofiltration of the ultrafiltration permeate on a 200 Da membrane and recovery of the retentate. The cocoa hydrolyzate obtained in the retentate in step g) contains between 3.0 and 4.0% of dry matter.
This dry matter comprises in particular 20 to 70% by weight of peptides and 5 to 40% of osides having a molecular weight of between 200 Da and 10 kDa. The hydrolyzate can then be diluted in water or in any solvent mixture containing water. Thus, the cocoa hydrolyzate according to the invention may advantageously be diluted in one or more physiologically acceptable solvents, such as water, glycerol, ethanol, propanediol, butylene glycol, dipropylene glycol or ethoxylated diglycols. or propoxylated, cyclic polyols or any mixture of these solvents.
Example 2 Preparation of a Cocoa Hydrolyzate from Cocoa Beans
Steps a) to g) are identical to those carried out in Example 1. Then we proceed as follows: h) Lyophilization of the nanofiltration retentate with 50% by weight of maltodextrin (drying medium) relative to the dry extract of this retentate. The cocoa peptide hydrolyzate obtained in the retentate of step h) contains between 94.0% and 98.0% of dry matter.
This dry matter comprises in particular 50% of maltodextrin, 10 to 35% by weight of peptides and 2 to 20% of osides having a molecular weight of between 200 Da and 10 kDa.
Example 3 Preparation of a cocoa hydrolyzate from cocoa beans
Steps a) to g) are identical to those performed in Example 2. Then we proceed as follows: h) Lyophilization of the nanofiltered retentate without drying support. The cocoa peptide hydrolyzate obtained in step h) contains between 94.0% and 98.0% of dry matter.
This dry matter comprises in particular 20 to 70% by weight of peptides and 5 to 40% of osides having a molecular weight of between 200 Da and 10 kDa.
Example 4: Preparation of a cocoa hydrolyzate from cocoa beans
Steps a) to f) are identical to those carried out in Example 1 for recovery of ultrafiltrate 1. This is followed as follows: g) Ultrafiltration of ultrafiltered permeate 1 on a 5 kDa membrane and recovery of the retentate. h) Lyophilization of the ultrafiltered retentate 2 without drying medium. The cocoa peptide hydrolyzate obtained in step h) contains between 94.0 to 98.0% of dry matter having a molecular weight of between 5 kDa and 10 kDa.
This dry matter comprises in particular 20 to 70% by weight of peptides and 5 to 40% of sugars.
Example 5 Preparation of a cocoa hydrolyzate from cocoa beans
Steps a) to f) are identical to those carried out in Example 1 for recovering the permeate of the ultrafiltration 10 kDa (ultrafiltrate 1). This is followed as follows: g) Ultrafiltration 2 of the ultrafiltrate 1 on a 5 kDa membrane and recovery of the permeate (ultrafiltrate 2); h) Nanofiltration of the ultrafiltrate 2 on a 200 Da membrane and recovery of the retentate. The peptide and cocoa solids hydrolyzate obtained in step h) contains between 3.0 and 4.0% of dry matter having a molecular weight of between 200 Da and 5 kDa.
This dry matter comprises in particular 20 to 70% by weight of peptides and 5 to 40% of sugars.
Example 6 Comparison of the hydrolyzate according to the invention with the extract obtained according to FR2810242
Patent FR2810242, cited as prior art, proposes a cosmetic composition which contains a combination of 0.05 to 5% by weight of amino acids extracted from poo and 0.05 to 5% by weight of polyphenols extracted from cocoa and 0.05 at 3% by weight of unsaponifiable concentrate.
As a comparative extract, a hydro-alcoholic extract rich in polyphenols (named polyphenol-rich extract in the following examples) was made according to FR2810242 in the following manner:
The polyphenols are extracted by maceration of the delipid cake at 10% solids content in 70% ethanol for 1 hour at 50 ° C., then the ethanol is evaporated under vacuum. The extract is then assayed by the method of Folin Ciocalteu which allows to assay all the polyphenols. The method uses gallic acid as standard and the results are expressed in gallic acid equivalent weight%.
The quantitative comparison of the two extracts is as follows. The peptide and osidic hydrolyzate according to the invention is characterized: by a polyphenol content of 0.5% gallic acid equivalent. The composition which contains 10% of the hydrolyzate according to the invention comprises at most 0.05% of polyphenols. This corresponds to the minimum contents of FR2810242 between 0.5 and 5% with equivalences (molar mass of gallic acid compared with molar mass of polyphenols present in the hydrolyzate). in that it contains no or few free amino acids. They are in the form of peptides (partial hydrolysis of proteins). - in that it does not include unsaponifiable compounds.
The qualitative comparison of the two extracts is as follows:
It is important to note that the peptide and osidic hydrolyzate according to the invention contains no or few free amino acids. They are in the form of peptides (partial hydrolysis of proteins). Thus, in terms of comparison, it is not justifiable to compare the amino acid content and the peptide content. However it is interesting to compare the relative aminograms (relative profile of the amino acids represented in the two extracts).
The polyphenolic and amino acid profiles of the hydrolyzate according to the invention are given in FIG. 1. This figure comprises: the polyphenolic profiles of the hydrolyzate according to the invention and the hydro alcoholic extract rich in polyphenols according to FR2810242 and - the amino acid profiles of the hydrolyzate according to the invention hydrolysed completely and the results of the aminogram given in FR2810242. The polyphenol rich extract according to FR2810242 has a complex polyphenolic profile with the potential presence of three families of molecules: flavonoids (for example catechol and derivatives), phenylpropanoids (for example ferulic acid and derivatives) and methylxanthines (by example caffeine and theobromine). In comparison, the polyphenolic profile of the hydrolyzate according to the invention no longer contains the flavonoids but always the potential methylxanthines and phenylpropanoids.
Conclusion:
The qualitative comparison of the hydrolyzate according to the invention with a hydroalcoholic extract rich in polyphenols according to FR2810242 shows significant differences in terms of composition of polyphenols and amino acids (free or not).
EXAMPLE 7 Evaluation of the Effects of a Peptide and Cocoa Osidia Hydrolysate According to Example 1 on the Rate of Cellular Reactive Oxygen Species Generated by Blue Light on Keratinocytes in Culture
The purpose of this study is to evaluate the effect of treatment with cocoa hydrolyzate according to Example 1, on the level of oxidative stress generated by exposure to blue light, on normal human keratinocytes in culture. . In parallel, a treatment is carried out by a cocoa hydrolyzate according to Example 6 rich in polyphenols.
A blue light source consisting of LEDs is used to create stress at 415 nm and 470 nm. The reactive oxygen species (EORs) resulting from this exposure are determined at the cellular level by a fluorescent probe.
Protocol:
Normal human keratinocytes in culture are pretreated with the hydrolyzate according to Example 1 diluted to 0.1% v / v (ie 1/1000) in a specific culture medium, twice a day, while cultures controls are maintained in untreated condition. After 24 hours, a portion of the control and treated cultures are exposed to blue light (415 and 470 nm, at 3 mW / cm 2 for 18 minutes), while the other part is kept away from light. After this exposure, the hydrolyzate is applied twice a day for another 24 hours. Then, the blue light exposure is repeated again, followed by the EOR detection test.
To do this, the cells are placed in the presence of a fluorescent probe (CellROX® Green Reagent, Lifetechnologies) for 30 minutes at 37 ° C. After fixation and rinsing, the cells are observed under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). A quantification of the intensity of the fluorescence, proportional to the quantity of EORs present, is carried out using the software Volocity ® image analysis software (PerkinElmer, Inc.) from the resulting photographs.
Treatments in the presence of cocoa hydrolysates obtained according to Examples 4 and 5 were also carried out. For this, the hydrolysates were previously diluted to obtain a concentration comparable to the hydrolyzate of Example 1. Results:
Treatment with the hydrolyzate according to Example 1 at 0.1% on keratinocytes makes it possible to significantly reduce the level of EORs generated by exposure to blue light (+ 90% intensity in the cells exposed to blue light against + 34% in treated and exposed cells, compared to untreated control). In addition, the effect observed is greater than that obtained with the extract according to Example 6, rich in polyphenols, on these same cells.
Treatment with the hydrolysates obtained according to Examples 4 and 5 also made it possible to observe a reduction in the level of EORs generated by exposure to blue light.
Conclusion: The application of the hydrolyzate of a cacao hydrolyzate obtained according to one of Examples 1, 4 or 5 makes it possible to limit the oxidative stress generated by exposure to blue light, as indicated by the decrease in intracellular EOR levels in the keratinocytes treated with the hydrolyzate.
EXAMPLE 8 Evaluation of the Effects of a Cocoa Hydrolyzate According to Example 1 on the Rate of Mitochondrial Reactive Oxygen Species Generated by Blue Light on Keratinocytes in Culture
The purpose of this study is to evaluate the effect of a treatment with a cocoa hydrolyzate according to Example 1, on the level of oxidative stress generated by exposure to blue light, at the mitochondrial level, on human keratinocytes. normal in culture. Indeed, mitochondria are a favored target of EORs and mitochondrial DNA damage has been observed following exposure of retinal epithelial cells to visible light13. In parallel, a treatment is carried out with a cocoa hydrolyzate according to Example 6 rich in polyphenols on these same cells.
Protocol:
The protocol for treating the cells and exposing them to blue light is identical to that described in Example 7. After these steps, the mitochondrial EORs are revealed by a fluorescent probe (MitoSOX ™ Red Mitochondrial Superoxide Indicator, Invitrogen), put in contact with the cells for 15 minutes at 37 ° C. After rinsing and fixation, the cells are observed under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). A quantification of the intensity of the fluorescence, proportional to the quantity of EORs present, is carried out using the software Volocity® image analysis software (PerkinElmer, Inc.) from the photographs obtained.
Treatments in the presence of cocoa hydrolysates obtained according to Examples 4 and 5 were also carried out. For this purpose, the hydrolysates were previously diluted to obtain a concentration comparable to the hydrolyzate of Example 1. Results: Exposure to blue light resulted in an increase in mitochondrial EORs (+ 156% compared to non-control). treaty). In the presence of the cacao hydrolyzate according to Example 1 at 0.1%, a sharp decrease in the rate of mitochondrial EORs (+ 8% compared to the untreated control) is observed. The observed effect is greater than that obtained with the cocoa hydrolyzate according to Example 6 rich in polyphenols on these same cells.
Treatment with the hydrolysates obtained according to Examples 4 and 5 also made it possible to observe a reduction in the level of EORs generated by exposure to blue light.
Conclusion:
In addition to the results reported in Example 7, this test makes it possible to conclude that the application of the cocoa hydrolyzate according to Example 1, 4 or 5 makes it possible to limit the oxidative stress generated by exposure to blue light. as indicated by the decrease in mitochondrial EORs.
Example 9 Effect of the cocoa hydrolyzate according to Example 1 on the maintenance of the level of opsin-1. -2 and -3 after exposure to blue light.
The purpose of this study is to observe the effect of blue light exposure as defined in Examples 7 and 8, on the level of opsin-1, -2 and -3 photoreceptors, on normal human keratinocytes. treated with the cocoa hydrolyzate according to Example 1. These photoreceptors originate from the expression of the OPN1-SW, OPN2 and OPN3 genes by the keratinocytes, and are capable of inducing signaling pathways in the presence of the wavelength of blue light specific to each opsine12.
Protocol:
Normal human keratinocytes in culture are treated with the hydrolyzate according to Example 1 diluted to 0.1% v / v (ie at 1 / 1000e) and then exposed to blue light according to the protocol described in Example 1. Three hours after the second exposure, detection of opsin-1, opsin-2 and opsin-3 is performed by an indirect immunofluorescence technique. To do this, the cells are rinsed, fixed with 3.7% paraformaldehyde and permeabilized with 0.1% Triton for 10 minutes. After saturation of non-specific sites with 1% bovine serum albumin for 10 min, the cells are incubated with a solution of antibodies directed against opsin-1 (OPSN Short), opsin-2 (rhodopsin). or opsine-3 (panopsin) for one hour, followed by a fluorochrome-coupled anti-rabbit secondary antibody solution (Alexa Fluor® 488, Invitrogen). The cells are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). Quantification of the fluorescence, using the software Volocity® image analysis software (PerkinElmer, Inc.) is carried out from the photographs obtained.
Treatments in the presence of cocoa hydrolysates obtained according to Examples 4 and 5 were also carried out. For this, the hydrolysates were previously diluted to obtain a concentration comparable to the hydrolyzate of Example 1. Results:
In the presence of blue light under conditions determined to mimic overexposure to keratinocytes in culture, the labeling intensity of opsin-1 is significantly decreased by exposure to 415 nm, corresponding to the wavelength at which this opsine is sensitive (-33% compared to control). In the condition where the cells have been treated with the cacao hydrolyzate according to Example 1 at 0.1%, the labeling of opsin-1 is preserved (-10% relative to the control). Under the same conditions, the cocoa hydrolyzate according to Example 6 rich in polyphenols did not make it possible to observe a protective effect on the level of opsin-1 (-33% compared to the untreated control).
Under identical conditions of treatment, the labeling intensity of opsin-2 and opsin-3 is decreased when the keratinocytes are exposed to blue light (470 nm) (-43% and -27% respectively, compared to untreated control). In the presence of the cacao hydrolyzate according to Example 1 at 0.1%, the labeling of the opsines appears preserved (-28% and -10% respectively, compared with the untreated control). Under the same conditions, the cocoa hydrolyzate according to Example 6 rich in polyphenols did not make it possible to observe a protective effect on the level of opsin-2 and opsin-3 (-42% and -30% respectively , compared to untreated control).
The results are shown in Figure 2.
Treatment with the hydrolysates obtained according to Examples 4 and 5 also made it possible to observe the maintenance of the level of opsin-1, -2 and -3 after exposure to blue light.
Conclusion: The cacao hydrolyzate obtained according to one of Examples 1, 4 or 5 has made it possible to maintain the level of opsin-1, -2 and -3 in the presence of a stress mimicking an over-exposure to light. blue. These results demonstrate that the hydrolyzate according to the invention has a protective effect vis-à-vis the blue light.
EXAMPLE 10 Effect of the cocoa hydrolyzate according to Example 1 on the level of expression of circadian CRY-1 and PER-1 proteins. on keratinocytes exposed to blue light:
The present study consisted in evaluating the effect of the treatment with the cocoa hydrolyzate according to Example 1 on the maintenance of the level of circadian proteins CRY-1 and PER-1 by using keratinocytes exposed to blue light, in previously defined conditions to generate stress on these cells.
The modulation of circadian genes is one of the consequences of photoreceptor activation at the level of retinal cells. The expression of opsins in retinal cells is itself dependent on Clock gene expression, under regulation by environmental factors such as exposure to light13.
Protocol:
Normal human keratinocytes in culture are treated with the cocoa hydrolyzate according to Example 1 diluted to 0.1% v / v (ie 1/1000) and then exposed to blue light according to the protocol described in Example 7 Cultures treated under the same conditions are kept in the dark. Six hours after the second exposure, the level of expression of circadian CRY-1 and PER-1 proteins is revealed by the immunocytochemistry technique according to a standard protocol as described in Example 9.
After rinsing, fixation and saturation of the nonspecific sites, the cells are incubated with primary antibodies directed against the CRY-1 and PER-1 proteins, then with secondary antibodies coupled to a fluorochrome (Alexa Fluor® 488, Invitrogen). The cells are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). Quantification of the fluorescence, using the software Volocity® image analysis software (PerkinElmer, Inc.) is carried out from the photographs obtained.
Treatments in the presence of cocoa hydrolysates obtained according to Examples 4 and 5 were also carried out. For this, the hydrolysates were previously diluted to obtain a concentration comparable to the hydrolyzate of Example 1. Results:
In the condition where the cells were kept in the dark, the cacao hydrolyzate according to Example 1 at 0.1% allowed to increase by 19% and + 17% the level of expression of the CRY-1 proteins and PER-1, respectively, compared to the untreated control. Exposure to blue light (415 nm) causes a significant decrease in CRY-1 (-25% compared to unexposed cells), which is counteracted by the treatment with the cocoa hydrolyzate according to Example 1 (+ 20% compared to unexposed and untreated control). Concerning the level of expression of PER-1, it is also decreased in cells exposed to blue light (415 nm) (-11% compared to unexposed cells) and appears maintained above the control in the exposed condition. and treated with the hydrolyzate (+ 27% compared to unexposed and untreated control) under these same conditions.
Treatment with the hydrolysates obtained according to Examples 4 and 5 also made it possible to observe the same results.
Conclusion:
Treatment with the cocoa hydrolyzate obtained according to one of Examples 1, 4 or 5 made it possible to maintain the level of circadian CRY-1 and PER-1 proteins in the keratinocytes exposed to blue light. These results demonstrate that the hydrolyzate according to the invention has a protective effect vis-à-vis the blue light.
EXAMPLE 11 Effect of the cocoa hydrolyzate according to Example 1 on the maintenance of the fibrillin-1 network on ex vivo skin biopsies exposed to UVB stress or blue light:
The purpose of this study is to evaluate a potential protective effect of the cocoa hydrolyzate according to Example 1 on the fibrillin-1 network in the presence of a stress generated by UVB or exposure to blue light.
The elastic fiber network appears to be a particular target of photo-aging, especially in terms of structural changes in microfibrils rich in fibrillin-1. This protein is a constituent of the oxytalan fibers present in the superficial dermis. With photo-aging, elastin microfibrils lose their characteristic candelabra shape below the dermal-epidermal junction14.
Protocol:
Human skin biopsies maintained in culture are treated with the cocoa hydrolyzate according to Example 1 diluted to 1% v / v (ie 1/100) in PBS, twice daily for 24 hours, and then irradiated with UVB (100 mJ / cm 2) or exposed to blue light (470 nm, 3 mW / cm 2 for 26 min). Biopsies are kept in parallel in the dark. The treatment is reapplied twice daily for 24 hours, then the biopsies undergo a second exposure to UVB or blue light. After another 24 hours of treatment, the biopsies are collected to proceed to the detection of fibrillin-1 by immunohistochemistry.
This technique is performed from frozen sections, incubated in the presence of anti-fibrillin-1 antibody (mouse monoclonal). After 1 hour of incubation followed by rinses, the sections are incubated in the presence of the secondary anti-mouse antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen). The sections are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). The network of elastin microfibrils present below the dermal-epidermal junction is then observed.
Treatments in the presence of cocoa hydrolysates obtained according to Examples 4 and 5 were also carried out. For this, the hydrolysates were previously diluted to obtain a concentration comparable to the hydrolyzate of Example 1. Results:
For the biopsies left in the dark, microfibrils labeled with fibrillin-1 appear longer in the presence of the cacao hydrolyzate of example 1 to 1%, compared to untreated biopsies.
In the condition where the biopsies have been exposed to UVB, the microfibrillary network appears markedly diminished and it is no longer possible to distinguish a perpendicular orientation of the fibers. In the condition where the biopsies were treated with the cacao hydrolyzate of example 1 to 1% and exposed to UVB, longer fibers perpendicular to the dermal-epidermal junction are observed, compared to the exposed non-treated biopsies. These effects were also observed in the case of exposure to blue light.
Treatment with the hydrolysates obtained according to Examples 4 and 5 also made it possible to observe the same results.
Conclusion:
These results indicate a protective effect of the cocoa hydrolyzate obtained according to one of Examples 1, 4 or 5, with respect to fibrillin-1, in the case of a UVB stress and an exposure to blue light, suggesting an interest in the prevention of photo-aging.
EXAMPLE 12 Effect of the Cocoa Hydrolyzate According to Example 1 on the Level of Expression of Svndecan-4 in Ex Vivo Skin Biopsies
The purpose of the present study is to evaluate the potential effect of the cocoa hydrolyzate according to Example 1 on the level of syndecan-4 on human skin biopsies.
Syndecan-4 is a heparan sulfate proteoglycan present on the cell surface where it mediates cellular interactions, regulating the mechanisms of cell adhesion, migration, proliferation, endocytosis and mecanotransduction15.
Protocol:
The biopsies of human skin were treated with the cocoa hydrolyzate according to Example 1 diluted to 1% v / v (ie 1 / 100cme) in PBS, twice a day, for a total of 72 hours.
Then syndecan-4 was revealed on sections of these biopsies by immunohistochemistry. For this purpose, the sections are brought into contact with the primary antibody directed against syndecan-4 (rabbit polyclonal). After incubation and rinsing, an anti-rabbit secondary antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) is applied to the sections. The sections are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). Quantification of the fluorescence, using the software Volocity® image analysis software (PerkinElmer, Inc.) is carried out from the photographs obtained.
Treatments in the presence of cocoa hydrolysates obtained according to Examples 4 and 5 were also carried out. For this purpose, the hydrolysates were previously diluted to obtain a concentration comparable to the hydrolyzate of Example 1. Results: The cocoa hydrolyzate according to Example 1 made it possible to increase the syndecan-4 level of +18 % compared to untreated control biopsies. This effect is observed to a lesser extent with the cocoa hydrolyzate prepared according to Example 6 rich in polyphenols (+ 12%).
Treatment with the hydrolysates obtained according to Examples 4 and 5 also made it possible to observe the same results.
Conclusion: The cacao hydrolyzate according to one of the examples 1, 4 or 5, has an activity on syndecan-4 in skin biopsies, suggesting a potential effect on the interactions between the keratinocytes and their extracellular environment.
EXAMPLE 13 In Vitro Tests Demonstrating the Anti-aging and Rejuvenating Activity of the Cocoa Hydrolyzate According to Example 2
The dermis provides the epidermis with a solid support, it is also its nourishing element. It consists mainly of fibroblasts and an extracellular matrix mainly composed of collagens, elastin and a substance called the fundamental substance. These components are synthesized by fibroblasts. The cohesion between the epidermis and the dermis is ensured by the dermal-epidermal junction.
Collagens are the major proteins of the extracellular matrices of the skin. To date, 20 types of collagens are identified and noted from I to XX. The collagens mainly present in the entire dermis are the type I and III collagens which form the extracellular matrix of the whole dermis (these are the collagens which constitute 70-80% of the dry weight of the dermis). With aging, the dermis gets older and wrinkles appear on the surface of the skin. Consequently, in view of the important role of collagen in terms of the integrity of the skin and its resistance to mechanical external aggression, the stimulation of the synthesis of these collagens, and in particular of type I collagen, appears as a effective way to overcome the signs of skin aging (reviewed by Tzaphlidou M., Micron 35 (2004) 173-177). 1 - Study on young cells versus aged cells The following study made it possible to study the effect of the cocoa hydrolyzate according to Example 2 (named hydrolyzate in the figures) on the expression of collagen I, Elastin and Fibrillin 1, essential components of the extracellular matrix of the dermis, to evaluate its anti-aging and anti-photo-aging as well as rejuvenating activity. This latter activity was also evaluated by studying the effect of the cocoa hydrolyzate according to Example 2 on the methylation of the DNA, which is the target of epigenetics.
During aging, the collagen fibers and the elastic fibers are altered due to reduced synthesis and increased degradation, resulting in reduced skin quality, less tonic and loss of elasticity.
To allow a claim of cellular rejuvenation we quantified the expression of the proteins involved in the structure of these fibers in intrinsically aged cells (replicative senescence) in the case of collagen I and elastin, or extrinsic by irradiation. UV repeated (photo aging) in the case of Fibrillin 1.
In order to understand the mechanism of reactivation of the expression of the proteins observed, we proceeded to an analysis of the methylation of the DNA on cells aged intrinsically. Indeed one of the signatures of the cellular senescence is the accumulation of methylated zones within the DNA, at the level of the Cytosines contained in the dinucleotides CpG. This leads to the inactivation of promoters and the decrease of the expression of certain genes. • Prior evaluation of cytotoxicity The cytotoxicity of the molecules was evaluated on NHDFs.
The cytotoxicity was performed on the maximum concentration tested soluble in 0.1% DMSO aqueous solution and 7 Vi log dilutions in% log.
The compound was contacted with the cells for 24 hours. During the last 3 hours of Roche's WST1 ready-to-use calorimetric test was introduced into the medium.
This reagent contains tetrazolium salts, a purple indicator. This reagent is cleaved into formazan, a yellow indicator, by metabolically active cells. The level of yellow coloration is therefore proportional to the number of living cells. The absorbance measurement is 450nm. The test considers that a value less than 90% of the control indicates a cytotoxicity of the possible product (symbolized by a green line in the graphs). It may also indicate that the metabolic activity of the cells is decreased. A value below 75% indicates significant cytotoxicity (symbolized by a red line in the graphs)
In addition, the cells were observed under a microscope to observe and compare their physiognomy. The cocoa hydrolyzate according to Example 2 has a high cytotoxicity at 1000 ppm and a lower but significant cytotoxicity between 200 and 20 ppm. The cocoa hydrolyzate according to Example 2 shows no significant cytotoxicity between 2 ppm and 20 ppm.
Concentrations selected for testing: The cocoa hydrolyzate according to Example 2 is tested at the following concentrations 20-10-5 ppm.
- Collagen I Targets
Collagen I is the majority collagen that gives the skin its mechanical resistance.
This protein represents 90% of the collagen of a vertebrate. It constitutes the frame of the bone (to compare with the reinforcements of the reinforced concrete), and more generally the ordinary connective tissues. It is found in bones, skin, tendons, cornea and internal organs.
Method for Evaluating the Effect of the Cocoa Hydrolyzate According to Example 2 on the Expression of Collagen I
Normal or young Human Dermal Fibroblasts (NHDFs) aged or aged by replicative senescence were seeded in 96-well plates and incubated 24h at 37 ° C., 5% CO 2.
The cells were treated 24h in the presence of the test products.
The cells were then fixed with formalin and the expression of the proteins was detected by immunofluorescence.
Fluorescent markings were imaged and quantified by automated microscopy (Arrayscan CellomicsTM). Fluorescence was quantified by the Bioapplication Compartmental Analysis.
Evaluation of the Effect of the Cocoa Hydrolyzate According to Example 2 on the Expression of Collagen I
The results are given in Figure 3A. The cocoa hydrolyzate according to Example 2 makes it possible to reactivate the expression of Collagen I with respect to the cells aged by replication. Indeed, the protein expression percentages of the target protein obtained at the three tested cocoa hydrolyzate concentrations according to Example 2 are higher than those obtained with the aged cell model.
Conclusion: The cocoa hydrolyzate according to Example 2, is an ingredient that acts on collagen I, the target of skin aging, by reviving the protein expression compared with untreated aged cells and by restoring it partially in comparison with young witnesses. - Target Elastin Elastin is a structural glycoprotein (such as laminin and fibronectin) used in the composition of the ECM. Elastin is a protein of the structural fibrous protein family. Secreted by fibroblasts mainly during the growth period, it has elastic properties. Its synthesis decreases with age and elastin is replaced by inextensible collagen. Stretch marks are a visible example of this process, which is related to mechanical stresses. Skin aging is a second example. In the extracellular matrix elastin is synthesized and secreted in the extracellular space by the fibroblasts first in proelastin, then in tropoelastin. Elastin is the major component (up to 90%) of elastic fibers to which fibrillin is added. Thus, collagen, associated with elastin and fibrillin that form elastic fibers, by covalent cross-links, are the main constituents of the extracellular matrix. The total production of elastin stops around puberty. After that, the amount of elastin available will decrease over time.
The degradation of elastin is related to the action of elastase, an enzyme secreted by fibroblasts. The enzymatic action of elastase is inhibited by al-antitrypsin. Inhibition of degradation creates a balance that increases the stability of elastin.
Distinctive features characterize elastin: Elastin allows cells to bind and allows biological tissue to form. Thus, the proper functioning of the skin, lungs, blood vessels, connective tissues, certain tendons and cartilages is closely related to the characteristics of elastin. As its name suggests, elastin is elastic. At equal diameter, it is 5 times more elastic than a rubber band. It can stretch up to 150% of its length at rest before breaking. Thus, it allows the tissues to stretch and regain their initial state after stretching, which gives them flexibility. Elastin is found in the dermis of the skin, which acts as a support. During aging, for example, the loss of elasticity and tonicity of the dermis that can no longer oppose the contraction effects of the underlying muscles gives rise to the appearance of wrinkles.
In addition, exposure to ultraviolet rays increases the degradation of elastin. Method for Evaluating the Effect of the Cocoa Hydrolyzate According to Example 2 on the Expression of Elastin
Normal or young Human Dermal Fibroblasts (NHDFs) aged or aged by replicative senescence were seeded in 96-well plates and incubated 24h at 37 ° C., 5% CO 2.
The cells were treated 24h in the presence of the test products.
The cells were then fixed with formalin and the expression of the proteins was detected by immunofluorescence.
Fluorescent markings were imaged and quantified by automated microscopy (Arrayscan CellomicsTM). Fluorescence was quantified by the Bioapplication Compartmental Analysis. Evaluation of the Effect of the Cocoa Hydrolyzate According to Example 2 on the Expression of Elastin
The results are given in Figure 3B. The cocoa hydrolyzate according to Example 2 makes it possible to slightly reactivate the expression of Elastin with respect to the aged cells. Conclusion The cocoa hydrolyzate according to Example 2 is an ingredient which acts on Elastin, a target of skin aging, by slightly raising the protein expression compared to untreated aged cells at the three concentrations tested and in partially restoring compared to young witnesses. - Target Fibrillin 1
Fibrillin 1 is a protein that constitutes microfibrils, which are associated with elastic fibers and participate in their assembly. • Method for evaluating the effect of cocoa peptide hydrolyzate on the expression of Fibrillin 1
The NHDFs (Normal Human Dermal Fibroblasts) young or aged extrinsically by irradiation were inoculated in 96-well plate and incubated 24h at 37 ° C., 5% CO 2.
The cells intended for extrinsic aging were irradiated 3 times with UVA with 24 hours between each irradiation or 2 times with UVB with 48 hours in each irradiation.
The cells were treated 24h in the presence of the test products.
The cells were then fixed with formalin and the expression of the proteins was detected by immunofluorescence.
Fluorescent markings were imaged and quantified by automated microscopy (Arrayscan CellomicsTM). Fluorescence was quantified by the Bioapplication Compartmental Analysis. Evaluation of the Effect of the Cocoa Hydrolyzate According to Example 2 on the Expression of Fibrillin 1
The results are given in Figure 3C. The cocoa hydrolyzate according to Example 2 makes it possible to reactivate the expression of Fibrillin 1 with respect to the aged cells. Conclusion The cocoa hydrolyzate according to Example 2, is an ingredient which acts on Fibrillin 1, target of skin aging, by reviving its protein expression at the three concentrations tested compared with untreated aged cells and by restoring it almost totally in comparison to the young witnesses.
- Target DNA methylation
Methylation is a modification of the N-termini of histones. It can be carried out either on lysines or on arginines and can be concretized by the addition of one, two or three methyl groups. Depending on the methylated residues and the number of added groups, it is associated with activation or repression of transcription. Long regarded as static, histone methylation appears to be a reversible modification involved in a dynamic process, although more stable than acetylation and phosphorylation. An increasing number of histone demethylases are identified. In general, this type of modification is antagonistic to acetylation, and the deacetylation of lysines must precede their methylation. This antagonism results in the establishment of a certain dynamic equilibrium between the heterochromatin (generally non-expressible and methylated on certain key amino acids) and euchromatin (usually expressible and acetylated) territories. For example, Lysine 9 of histone H3 is known to be associated with repression of surrounding chromatin when methylated. This methylation is recognized by a protein, HP1, which therefore binds to methylated H3. In turn, HP1 attracts the Suv39 protein, a Histone Methyl Transferase, which will methylate the H3 histone H3 lysine, and so on. So we see how, step by step, the histones H3 will be methylated and the chromatin will be condensed. However, this heterochromatin invasion will be stopped if the H3 lysine 9 encountered is already acetylated. Thus a competitive equilibrium is established between chromatin domains expressed and repressed. Changes in histone tails act as epigenetic "brands" that result in the recruitment of different classes of proteins, since acetylated or methylated lysines are recognized by different protein domains. In addition, the recruitment of certain factors at the chromatin level requires the prior existence of modifications of histones and already bound proteins. The histone code is therefore interpreted in the context of other factors associated with chromatin and it is the combination of interaction between the modified histones and other factors that determines whether a protein is recruited to chromatin. All tissues of organisms are affected by aging. This process is related to epigenetic modifications such as methylation changes at cytosine residues specific to DNA, as described in many publications. The role of epigenetic modifications on aging, the accumulation of damaged cell divisions and macromolecules contribute to an older phenotype. Environmental and random events can further modify this phenotype through epigenetic mechanisms such as DNA methylation and methylation and acetylation of histones. The potential reversibility of epigenetic modifications makes them attractive targets for the treatment of aging-related pathologies.
Method for Evaluating the Effect of Cocoa Hydrolyzate According to Example 2 on DNA Methylation
Normal or young Human Dermal Fibroblasts (NHDFs) aged or aged by replicative senescence were seeded in 96-well plates and incubated 24h at 37 ° C., 5% CO 2.
The cells were treated 24h in the presence of the test products.
The cells were then peeled off in the presence of trypsin and lysed. Genomic DNA was precipitated with ethanol.
The methylation rate of the DNA was assayed by ELISA using the Enzo kit: 5-Methylcytosine DNA ELISA kit. The values are represented from 50% for a better visibility of the results.
Evaluation of the Effect of the Cocoa Hydrolyzate According to Example 2 on the Methylation of DNA
The results are given in Figure 3D. The cocoa hydrolyzate according to Example 2 makes it possible to lower the methylation level in the replenished NHDS at 10 and 5 ppm to approach that observed in the young cells. Conclusion The cocoa hydrolyzate according to Example 2 is an ingredient which acts on the reduction of the methylation rate of the DNA, which is the target of epivillination, and therefore has the potential of a rejuvenating activity. General conclusion on the anti-aging and rejuvenating activity During aging the collagen fibers and the elastic fibers are altered due to reduced synthesis and increased degradation. To demonstrate that the cocoa hydrolyzate according to Example 2 acts on cell rejuvenation, the expression of the proteins involved in the structure of these fibers in intrinsically aged cells (replicative senescence) in the case of Collagen I and Elastin, or extrinsic by repeated UV irradiation (photo aging) in the case of Fibrillin 1 were quantified. The cocoa hydrolyzate according to Example 2 makes it possible to restart the expression of the 3 markers.
As part of the DNA methylation analysis, the cocoa hydrolyzate according to Example 2 shows homogeneous results in this experiment, it reduces the methylation at 2 concentrations tested out of 3. This effect gives information on the mode of action of this product to reactivate the expression of proteins of the extracellular matrix, giving it its rejuvenating effect. The cocoa hydrolyzate according to Example 2 is therefore an active ingredient or ingredient having an anti-aging, anti-aging and rejuvenating biological activity. It acts coherently on 3 skin aging targets by reviving protein expression compared to that observed in aged cells. It also has an innovative activity on epigenetics as demonstrated by its action on DNA methylation in mature cells. Finally, it demonstrates an anti-aging action in depth by its efficiency on the synthesis of proteins of the extracellular matrix.
Procedure 1. Pour phase A into the main container and begin homogenizing. Heat to 70-75 ° C. 2. Drop the rain into the UltraThix P-100 and mix well for about 30min. 3. Add phase D ingredients to a second beaker and heat to 70-75 ° C. 4. Add phase C pre-mixed in phase A until complete homogenization 5. At 70-75 ° C add phase D to the main container and mix well. The emulsion must be perfectly homogeneous. 6. Start cooling. 7. When the mixture reaches about 50 ° C, add phase E pre-mix and mix well. 8. At room temperature, add phase F and mix until uniform. 9. Stop when the mixture reaches about 25 ° C Properties:
Appearance: White cream pH: 5.2 - 5.6
Viscosity (OD) 25000 - 50000 (Brookfield RVT / Spindle B / 5 RPM / 1 minute / 25 ° C)
The preservation of this formula has been validated by a double efficacy test over 28 days. However, preservatives have not been optimized to their lowest level of efficiency

权利要求:
Claims (14)
[1" id="c-fr-0001]
1. Peptide and osidic hydrolyzate of the beans of Theobroma cacao L., characterized in that it comprises mainly peptides and osides.
[2" id="c-fr-0002]
2. Hydrolyzate according to claim 1, characterized in that it is obtained from unfermented and dried beans of Theobroma cacao L.
[3" id="c-fr-0003]
3. Hydrolysate according to claim 2, characterized in that the peptides and osides have a molecular weight between 200 Da and 10 kDa.
[4" id="c-fr-0004]
4. Hydrolysate according to claim 3, characterized in that it is obtained by an enzymatic treatment carried out with at least one carbohydrase and at least one protease, the carbohydrase being chosen from a pectinase, a cellulase, an arabanase, a hemicellulase, xylanase and β-glucanase and in that the neutral or acidic alkaline type protease is preferably of the alkaline type with endopeptidase activity.
[5" id="c-fr-0005]
5. A process for obtaining a peptide and osidic hydrolyzate of Theobroma cacao L. beans according to one of claims 1 to 4, characterized in that it comprises the following steps according to which: a) is dispersed in aqueous phase of the ground beans of Theobroma cacao L.; b) the enzymatic treatment of the aqueous dispersion obtained in step a) is carried out; c) the enzymes are denatured by heat treatment d) the enzymatic hydrolyzate is recovered by solid / liquid separation, e) the hydrolyzate is purified by ultrafiltration and nanofiltration, then optionally; f) lyophilization of the hydrolyzate obtained in step e).
[6" id="c-fr-0006]
6. Method according to claim 5 characterized in that during the various steps a) to d) the temperature is between 20 and 90 ° C and the pH is between 3.0 and 11.0.
[7" id="c-fr-0007]
7. The method of claim 5 and 6, characterized in that the solid / liquid separation of step c) is carried out by various methods such as centrifugation, spin, filtration.
[8" id="c-fr-0008]
8. Method according to one of claims 5 to 7, characterized in that in step d) the ultrafiltration is carried out with a cutoff threshold of between 5 and 15 kDa and the nanofiltration is performed with a cutoff threshold included between 100 Da and 300 Da.
[9" id="c-fr-0009]
9. Cosmetic composition, characterized in that it comprises, as active agent, an effective amount of a hydrolyzate of Theobroma cacao L. beans according to one of claims 1 to 4, and a physiologically acceptable medium.
[10" id="c-fr-0010]
10. Composition according to claim 9, characterized in that the hydrolyzate of beans Theobroma cacao L. is present in the composition at a concentration of 0.001 to 20% relative to the total weight of the composition, preferably from 0.1 to 10%, preferably 0.2 to 5%, more preferably 0.5 to 1.5%.
[11" id="c-fr-0011]
11. Composition according to one of claims 9 and 10, characterized in that it is formulated to be applied topically to the skin.
[12" id="c-fr-0012]
12. Cosmetic use of a composition according to one of claims 9 to 11 for protecting the skin from the harmful effects of blue light.
[13" id="c-fr-0013]
13. The cosmetic use according to claim 12 for protecting the skin from the oxidative stress generated by exposure to blue light.
[14" id="c-fr-0014]
14. Cosmetic use of a composition according to one of claims 9 to 11 to combat the appearance of signs of aging and skin photoaging.

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同族专利:

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公开号 | 公开日

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CN109152727B|2021-08-20|

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PE20190606A1|2019-04-23|

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EP3429696B1|2021-05-05|

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US20210106022A1|2021-04-15|

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US20190075813A1|2019-03-14|

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WO2017157998A1|2017-09-21|

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US10932477B2|2021-03-02|

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JP2019509341A|2019-04-04|

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CN109152727A|2019-01-04|

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FR3048881B1|2020-03-27|

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EP3429696A1|2019-01-23|

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2017-09-22| PLSC| Search report ready|Effective date: 20170922 |

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优先权:

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申请号 | 申请日 | 专利标题

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FR1600441A|FR3048881B1|2016-03-16|2016-03-16|PEPTIDE AND OSIDIC HYDROLYSAT OF COCOA BEANS, COSMETIC COMPOSITIONS COMPRISING SAME AND THEIR COSMETIC USES|

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FR1600441|2016-03-16|FR1600441A| FR3048881B1|2016-03-16|2016-03-16|PEPTIDE AND OSIDIC HYDROLYSAT OF COCOA BEANS, COSMETIC COMPOSITIONS COMPRISING SAME AND THEIR COSMETIC USES|

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JP2019500005A| JP2019509341A|2016-03-16|2017-03-15|Cocoa bean peptide and sugar hydrolyzate, cosmetic composition containing it, and cosmetic use thereof|

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US16/084,783| US10932477B2|2016-03-16|2017-03-15|Peptide and saccharide hydrolysate of cocoa beans, cosmetic compositions containing same, and cosmetic uses of same|

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EP17710011.2A| EP3429696B1|2016-03-16|2017-03-15|Peptide and saccharide hydrolysate of cocoa beans, cosmetic compositions containing same, and cosmetic uses of same|

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PE2018001825A| PE20190606A1|2016-03-16|2017-03-15|PEPTIDES AND OSIDIC HYDROLYSATS OF COCOA GRAINS, COSMETIC COMPOSITIONS THAT THE SAME AND THEIR COSMETIC USES INCLUDE|

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PCT/EP2017/056084| WO2017157998A1|2016-03-16|2017-03-15|Peptide and saccharide hydrolysate of cocoa beans, cosmetic compositions containing same, and cosmetic uses of same|

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CN201780028630.1A| CN109152727B|2016-03-16|2017-03-15|Peptides and sugar hydrolysates of cocoa beans, cosmetic compositions containing them and cosmetic use of the cosmetic compositions|

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US17/103,351| US20210106022A1|2016-03-16|2020-11-24|Peptide and saccharide hydrolysate of cocoa beans, cosmetic compositions containing same, and cosmetic uses of same|